CN114672533B - Method for removing bitter soybean peptide by utilizing aminopeptidase and alkaline protease in synergy - Google Patents
Method for removing bitter soybean peptide by utilizing aminopeptidase and alkaline protease in synergy Download PDFInfo
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- CN114672533B CN114672533B CN202210409141.3A CN202210409141A CN114672533B CN 114672533 B CN114672533 B CN 114672533B CN 202210409141 A CN202210409141 A CN 202210409141A CN 114672533 B CN114672533 B CN 114672533B
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- 102000004400 Aminopeptidases Human genes 0.000 title claims abstract description 61
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- LOUPRKONTZGTKE-UHFFFAOYSA-N cinchonine Natural products C1C(C(C2)C=C)CCN2C1C(O)C1=CC=NC2=CC=C(OC)C=C21 LOUPRKONTZGTKE-UHFFFAOYSA-N 0.000 description 3
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- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 2
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- 238000004090 dissolution Methods 0.000 description 1
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 1
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- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical compound O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 description 1
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Classifications
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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
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
- A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
- A23L5/25—Removal of unwanted matter, e.g. deodorisation or detoxification using enzymes
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Biotechnology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
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Abstract
The invention belongs to the technical field of food processing, and particularly relates to a method for removing soybean peptide by utilizing aminopeptidase and alkaline protease in a synergistic manner. The soybean peptide is subjected to debittering by adopting the complex enzyme of aminopeptidase and alkaline protease, the aminopeptidase and alkaline protease are synergistically hydrolyzed to achieve a good debittering effect, and various factors influencing debittering such as enzyme adding amount, substrate concentration, enzymolysis temperature, enzymolysis time and the like are optimized to obtain optimal enzymolysis conditions, so that the debittering effect is obvious, the bitterness score of soybean peptide hydrolysate is reduced to 1.3, the molecular weight of the soybean peptide is totally distributed below 6500Da, only 1450Da occupies 93.87%, and the soybean peptide has wide application prospect in the fields of foods and the like.
Description
Technical Field
The invention belongs to the technical field of food processing, and particularly relates to a method for removing soybean peptide by utilizing aminopeptidase and alkaline protease in a synergistic way.
Background
Soybean is one of the most important food crops in China, and not only provides plant protein, but also provides a source of soybean peptide for people. The soybean peptide can well improve the solubility, stability, fluidity and sensitization of soybean protein, and has physiological functions of reducing blood pressure, reducing blood fat, enhancing physical ability, regulating blood sugar and regulating immunity and the like as a bioactive peptide. However, during the preparation of soy peptides, the flavor and mouthfeel of the soy protein are often altered after enzymatic hydrolysis, and often produce an intolerable bitter taste.
The main reason for the bitter taste of soy protein is that hydrophobic groups coated in soy protein are exposed to contact with taste buds after hydrolysis. And the soy protein is bitter in taste from peptides rather than amino acids. Bitter peptides are mostly polypeptide chains with single or multiple hydrophobic amino acids at the amino terminus, and there are also few carboxyl-terminal hydrophobic amino acids, so bitter taste is a major obstacle to the use of soy protein hydrolysates or polypeptides in the food field. Various attempts have been made to reduce or mask the bitter taste of soy protein hydrolysates or polypeptides, such as separation and extraction, activated carbon treatment, maillard reaction, cyclodextrin encapsulation, etc., but the current removal methods suffer from the disadvantages of loss of basic functions of peptides, low recovery rate, specific equipment requirements, and additional production costs. The use of the telopeptidase is a promising method for eliminating bitter taste and changing mouthfeel, and especially the aminopeptidase has wider action range and better debittering effect.
Disclosure of Invention
According to the defects of the prior art, the invention aims to provide a method for removing the bitter soybean peptide by utilizing the cooperation of aminopeptidase and alkaline protease, which adopts compound enzyme to remove the bitter soybean peptide together, optimizes various factors influencing the bitter soybean peptide such as enzyme adding amount, substrate concentration, enzymolysis temperature, enzymolysis time and the like, obtains optimal enzymolysis conditions, has obvious bitter removing effect, ensures that the molecular weight of the soybean peptide is totally distributed below 6500Da, and has wide application prospect in food.
In order to achieve the above purpose, the technical scheme adopted is as follows:
a method for the synergistic debittering of soybean peptides using aminopeptidases and alkaline proteases, comprising the steps of: weighing Soybean Protein Isolate (SPI), dissolving in distilled water, stirring uniformly, preparing SPI solution with a feed liquid ratio of 4-14%, cooling to room temperature after constant temperature water bath for 10-60 min at 40-65 ℃, regulating pH to alkaline by using alkaline solution, placing in constant temperature water bath at 40-65 ℃, carrying out enzymolysis on SPI by using alkaline protease for 1-4 h, adding aminopeptidase at 40-65 ℃ after enzymolysis of alkaline protease is finished, carrying out enzymolysis reaction on SPI for 1-7 h, wherein the dosage of aminopeptidase is 100-600U/g SPI, inactivating hydrolysis liquid after enzymolysis is finished, cooling, centrifuging, and taking supernatant.
As a further technical scheme of the invention, the method comprises the following steps: weighing SPI, dissolving in distilled water, stirring uniformly, preparing SPI solution with the feed liquid ratio of 6-12%, cooling to room temperature after constant temperature water bath for 20-40 min at 45-55 ℃, regulating pH to alkaline by using alkaline solution, placing in constant temperature water bath at 45-55 ℃, carrying out enzymolysis on SPI by using alkaline protease for 1-3 h, adding aminopeptidase at 45-55 ℃ after enzymolysis of alkaline protease is finished, carrying out enzymolysis reaction on SPI for 3-5 h, wherein the dosage of aminopeptidase is 300-500U/g SPI, inactivating, cooling and centrifuging hydrolysate after enzymolysis is finished, and taking supernatant.
As a further technical scheme of the invention, the method comprises the following steps: weighing SPI, dissolving in distilled water, uniformly stirring, preparing SPI solution with a material-liquid ratio of 8%, cooling to room temperature after constant-temperature water bath at 55 ℃ for 20-30 min, regulating pH to be alkaline by using alkaline solution, placing in constant-temperature water bath at 55 ℃, carrying out enzymolysis on SPI by using alkaline protease for 2h, adding aminopeptidase to carry out enzymolysis reaction on SPI at 55 ℃ for 4h after the enzymolysis of the alkaline protease is finished, wherein the dosage of aminopeptidase is 500U/g SPI, inactivating, cooling and centrifuging hydrolysate after the enzymolysis is finished, and taking supernatant fluid to obtain the SPI.
As a further technical scheme of the invention, the dosage of the alkaline protease is 4000U/gSPI.
As a further technical scheme of the invention, the alkaline solution is sodium hydroxide solution.
As a further aspect of the present invention, the pH is 8.5.
As a further technical scheme of the invention, the inactivation is to inactivate for 1h at 85 ℃.
As a further technical scheme of the invention, the centrifugation is performed at 5000r/min for 10min.
As a further technical scheme of the invention, the method further comprises the following steps: the supernatant after centrifugation was stored frozen at-20 ℃.
Compared with the prior art, the invention has the beneficial effects that: the soybean peptide is subjected to debittering by adopting the complex enzyme of aminopeptidase and alkaline protease, the aminopeptidase and alkaline protease are synergistically hydrolyzed to achieve a good debittering effect, and various factors influencing debittering such as enzyme adding amount, substrate concentration, enzymolysis temperature, enzymolysis time and the like are optimized to obtain optimal enzymolysis conditions, so that the debittering effect is obvious, the bitterness score of soybean peptide hydrolysate is reduced to 1.3, the molecular weight of the soybean peptide is totally distributed below 6500Da, only 1450Da occupies 93.87%, and the soybean peptide has wide application prospect in the fields of foods and the like.
Drawings
FIG. 1 is a graph showing the relationship between aminopeptidase enzyme addition and SPI hydrolysis performance;
FIG. 2 is a graph showing the bitter taste of an aminopeptidase enzyme-added amount versus SPI hydrolysate;
FIG. 3 is a graph of substrate concentration versus SPI hydrolysis performance;
FIG. 4 is a graph of the bitter taste of substrate concentration versus SPI hydrolysate;
FIG. 5 is a graph showing the relationship between the hydrolysis temperature of aminopeptidase and SPI hydrolysis performance;
FIG. 6 is a graph showing the relationship between the enzyme hydrolysis temperature of aminopeptidase and the bitterness of SPI hydrolysate;
FIG. 7 is a graph showing the relationship between the hydrolysis time of aminopeptidase and SPI hydrolysis performance;
FIG. 8 is a graph showing the relationship between the enzyme hydrolysis time of aminopeptidase and the bitterness of SPI hydrolysate;
FIG. 9 is a chromatogram of a standard;
FIG. 10 is a MAP single enzyme hydrolysate molecular weight distribution plot;
FIG. 11 is a graph showing molecular weight distribution of aminopeptidase and MAP double enzyme synergistic hydrolysate.
Detailed Description
The invention is described below in connection with examples which are given solely for the purpose of illustration and are not intended to limit the scope of the invention.
1. Single factor variable test
(1) Influence of the enzyme addition amount of aminopeptidase on SPI enzymolysis
Weighing soybean protein isolate, dissolving in distilled water, stirring uniformly to prepare soybean protein isolate solution with the ratio of 10% of feed liquid, carrying out constant-temperature water bath at 55 ℃ for 20min, cooling to room temperature, regulating the pH to 8.5 by using 2M NaOH solution, placing in the constant-temperature water bath at 55 ℃, and carrying out enzymolysis for 2h by using alkaline protease (MAP) 4000U/g Soybean Protein Isolate (SPI), namely adding 4000U of alkaline protease into each gram of soybean protein isolate. After the enzymolysis of the alkaline protease is finished, 500U/g SPI of aminopeptidase is added, and the enzymolysis reaction is carried out for 4 hours at 55 ℃, namely 500U of aminopeptidase is added into each gram of isolated soy protein. After the enzymolysis is finished, the hydrolysate is quickly moved to 85 ℃ for inactivation for 1h. After the protease is inactivated, the hydrolysate is cooled to room temperature, centrifuged for 10min at 5000r/min, and the supernatant is obtained and frozen and preserved in a refrigerator at the temperature of minus 20 ℃ for standby.
The above experiments were conducted in 7 groups, except that the amounts of the other 6 groups of aminopeptidases added were 0, 100, 200, 300, 400, 600U/g SPI, and the SPI hydrolysis degree and the bitterness of the SPI hydrolysate were evaluated and analyzed, and the results are shown in FIGS. 1 and 2.
As can be seen from fig. 1 and 2, as the amount of aminopeptidase added increases, the hydrolysis degree of the SPI hydrolysate gradually increases, the bitterness score gradually decreases, i.e., the bitterness of the hydrolysate gradually decreases, and when the amount of aminopeptidase added is more than 400U/g, the variation in bitterness is no longer noticeable, and the variation in hydrolysis degree also tends to be stable. When the enzyme addition amount is small, the enzyme content in the reaction system is low, the enzymatic reaction is fast and the hydrolysis degree is gradually increased due to the fact that the enzyme content is dependent on the enzymatic reaction, and the bitter taste is reduced due to the fact that aminopeptidase participates in degradation of bitter peptide. As the amount of enzyme increases, the enzyme content increases gradually, the reaction rate and enzyme content lose linearity, and the diffusion of the substrate also interferes with the enzymatic reaction rate, so that the hydrolysis degree and bitter taste change are no longer obvious. Considering the problem of high utilization rate of enzyme, the proper adding amount of aminopeptidase is determined to be about 400U/g SPI.
(2) Influence of substrate concentration on SPI enzymolysis
Weighing soybean protein isolate, dissolving in distilled water, stirring uniformly, preparing soybean protein isolate solution with a material-liquid ratio of 10%, cooling to room temperature in a constant-temperature water bath at 55 ℃ for 20min, adjusting pH to 8.5 with a 2M NaOH solution, placing in a constant-temperature water bath at 55 ℃, and carrying out enzymolysis for 2h with 4000U/g SPI of alkaline protease. After the enzymolysis of the alkaline protease is finished, 400U/g SPI of aminopeptidase is added, and the enzymolysis reaction is carried out for 4 hours at 55 ℃, namely 400U of aminopeptidase is added into each gram of isolated soy protein. After the enzymolysis is finished, the hydrolysate is quickly moved to 85 ℃ for inactivation for 1h. After the protease is inactivated, the hydrolysate is cooled to room temperature, centrifuged for 10min at 5000r/min, and the supernatant is obtained and frozen and preserved in a refrigerator at the temperature of minus 20 ℃ for standby.
The above experiment was conducted in 6 groups, except that the ratios of the other 5 groups of soy protein isolate solutions (substrate concentrations) were 4%, 6%, 8%, 12%, 14%, respectively, and the SPI hydrolysis degree and the bitterness evaluation analysis of the SPI hydrolysis liquid were conducted, and the results are shown in fig. 3 and 4.
From fig. 3 and 4, it was found that the bitterness of the hydrolysate did not change significantly when the substrate concentration was <10%, and the bitterness of the hydrolysate increased when the substrate concentration was >10% while maintaining the bitterness at about 2.0. Meanwhile, the degree of hydrolysis does not change much from 4% to 10% to 12% in the reaction, and in the reaction, when the substrate concentration is more than 10%, the substrate may not be sufficiently infiltrated or the effect of the enzyme on the substrate is affected due to the increase of viscosity, resulting in a significant bitter taste and a decrease in the degree of hydrolysis, so that it can be initially estimated that the proper substrate concentration of the enzyme is about 10%.
(3) Influence of the enzymatic hydrolysis temperature of aminopeptidase on SPI enzymatic hydrolysis
Weighing soybean protein isolate, dissolving in distilled water, stirring uniformly, preparing soybean protein isolate solution with a material-liquid ratio of 10%, cooling to room temperature in a constant-temperature water bath at 55 ℃ for 20min, adjusting pH to 8.5 with a 2M NaOH solution, placing in a constant-temperature water bath at 55 ℃, and carrying out enzymolysis for 2h with 4000U/g SPI of alkaline protease. After the enzymolysis of the alkaline protease is finished, 400U/g SPI of aminopeptidase is added, and the enzymolysis reaction is carried out for 4 hours at 55 ℃, namely 400U of aminopeptidase is added into each gram of isolated soy protein. After the enzymolysis is finished, the hydrolysate is quickly moved to 85 ℃ for inactivation for 1h. After the protease is inactivated, the hydrolysate is cooled to room temperature, centrifuged for 10min at 5000r/min, and the supernatant is obtained and frozen and preserved in a refrigerator at the temperature of minus 20 ℃ for standby.
The above experiment was conducted in 6 groups, except that the enzyme hydrolysis temperature of the other 5 groups aminopeptidase was 40 ℃, 45 ℃, 50 ℃, 60 ℃, 65 ℃, and the SPI hydrolysis degree and the bitterness evaluation analysis of the SPI hydrolysis liquid were conducted, and the results are shown in fig. 5 and 6.
As can be seen from FIGS. 5 and 6, the bitterness score of the hydrolysate gradually decreases with increasing enzymolysis temperature, and the bitterness value is minimal at 45-50 ℃. The bitterness score increases slightly instead by continuing to increase the temperature. This is because the optimum enzymatic hydrolysis temperature of the enzyme is about 50 ℃, the temperature is too low, and the enzyme activity does not reach the highest level; and when the temperature is too high, the aminopeptidase has an inhibition effect on the activity of aminopeptidase, so that the aminopeptidase does not reach the optimal enzymolysis effect. Meanwhile, the hydrolysis degree firstly rises along with the increase of the reaction environment temperature, the hydrolysis degree reaches the maximum at 50 ℃, then the temperature is continuously increased, and the hydrolysis degree is reduced. Therefore, it is assumed that the suitable enzymatic hydrolysis temperature of aminopeptidase is about 50 ℃.
(4) Influence of the enzymolysis time of aminopeptidase on SPI enzymolysis
Weighing soybean protein isolate, dissolving in distilled water, stirring uniformly, preparing soybean protein isolate solution with a material-liquid ratio of 10%, cooling to room temperature in a constant-temperature water bath at 55 ℃ for 20min, adjusting pH to 8.5 with a 2M NaOH solution, placing in a constant-temperature water bath at 55 ℃, and carrying out enzymolysis for 2h with 4000U/g SPI of alkaline protease. After the enzymolysis of the alkaline protease is finished, 400U/g SPI of aminopeptidase is added, and the enzymolysis reaction is carried out for 4 hours at 50 ℃, namely 400U of aminopeptidase is added into each gram of isolated soy protein. After the enzymolysis is finished, the hydrolysate is quickly moved to 85 ℃ for inactivation for 1h. After the protease is inactivated, the hydrolysate is cooled to room temperature, centrifuged for 10min at 5000r/min, and the supernatant is obtained and frozen and preserved in a refrigerator at the temperature of minus 20 ℃ for standby.
The above experiment was performed in 6 groups, with the difference that the enzymatic hydrolysis times of the other 5 groups of aminopeptidases were 1h, 2h, 3h, 5h, 6h, 7h, respectively. The SPI hydrolysis degree and the bitterness of the SPI hydrolysate were evaluated and analyzed, and the results are shown in fig. 7 and 8.
As can be seen from fig. 7 and 8, the bitterness score gradually decreases with the prolongation of the enzymolysis time of aminopeptidase, the bitterness hardly changes at 5-7 hours, the degree of hydrolysis increases with the increase of the enzymolysis time, the degree of hydrolysis reaches 5 hours, and the change of the degree of hydrolysis is not obvious. Therefore, the suitable enzymolysis time range is determined to be about 5 hours.
2. Orthogonal assay for optimal enzymatic hydrolysis conditions
The optimal debittering condition of the aminopeptidase for enzymolysis of the isolated soy protein is determined by adopting four factors and three levels L 9 (3 4 ) And (3) designing an orthogonal test, taking a bitter taste value as an evaluation index, examining the influence of four factors of aminopeptidase enzyme adding amount, substrate concentration, enzymolysis time of aminopeptidase and enzymolysis temperature of aminopeptidase on the bitter taste value of the hydrolysate, and exploring the optimal combination condition of enzymolysis. The level design of the orthogonal test factors is shown in Table 1, and the orthogonal testThe results of the tests are shown in Table 2.
TABLE 1L 9 (3 4 ) Orthogonal test factor level design
TABLE 2 orthogonal test results and analysis
As can be seen from the extremely poor analysis of Table 2, the primary and secondary relation of the influence of each factor on the bitterness score is C > A > D > B, namely the substrate concentration > enzymolysis temperature > enzyme addition amount > enzymolysis time, and meanwhile, the optimal debitterizing process condition is A3B2C1D3, namely the enzymolysis temperature is 55 ℃, the enzymolysis time is 4 hours, the substrate concentration is 8%, and the enzyme addition amount is 500U/g SPI, namely the 8 th group of tests. The optimal result of the orthogonal test is not completely consistent with the optimal condition of single factor enzymolysis, which indicates that interaction exists between the conditions.
In order to verify the optimal enzymolysis combination of the orthogonal tests, the 8 th group of tests are carried out in three parallel tests, and the obtained SPI hydrolysate has a bitter taste value reduced to 1.3 and a hydrolysis degree up to 44.43%, so that the aminopeptidase enhances the hydrolysis degree of the isolated soy protein hydrolysate, reduces the bitter taste of the hydrolysate and has potential application in food production.
Testing
The bitterness evaluation method and hydrolysis degree measurement method involved in the above test were as follows:
(1) bitter taste evaluation method
The quinine sulfate standard solution is used as a standard substance to prepare a standard solution, and the concentration of the quinine sulfate standard solution is 3 multiplied by 10 -6 M (defined as a) is just without bitter, the concentration of fixed a is the lowest bitter, the concentration of 32a is the highest bitter, the concentration is higher, and the bitter is not increased. Between a and 32a, the bitterness value increased with increasing quinine sulfate concentration and the bitterness score is shown in table 3.
The evaluation group consists of 6 persons, after the evaluation person washes with distilled water, 2-3 mL of soybean protein hydrolysate is taken and put into the mouth, and after 10s, the soybean protein hydrolysate is spitted out, and after the mouth wash, standard liquid with similar bitter degree is taken for taste, so as to determine the bitter value, and the average value of the bitter value is taken for 6 persons.
TABLE 3 bitter taste scoring criteria
(2) Measurement of degree of hydrolysis
a. Solution preparation
OPA reagent: 7.620g of sodium tetraborate decahydrate and 200mg of Sodium Dodecyl Sulfate (SDS) were dissolved in 150mL of deionized water. After complete dissolution of the reagent. 160mg of phthalic aldehyde 97% was further dissolved in 4mL of absolute ethanol. The OPA solution was then completely transferred to the above solution by rinsing with deionized water, and 176mg dithiothreitol (99%, DTT) was added to the solution. The solution was diluted to 200mL with deionized water.
Serine standard 50mg serine was fixed in 500mL deionized water (0.9516 meqv/L).
Preparation of sample solution 10g of sample was dissolved in 100mL deionized water.
b. Operating procedure
Absorbance was measured at 340nm with deionized water as a reference.
400mL serine standard was added to a tube containing 3mL OPA reagent and mixed for 5 seconds. The mixture was left for 2 minutes before being read at 340nm in a spectrophotometer. Absorbance of the standard sample was measured twice before measuring the blank and the sample, absorbance of the standard sample was measured twice after measuring all the blank and the sample, and the average value of four measurements was taken at the time of calculation.
c. Calculation of
h, determination:
wherein, serine NH 2 :meqv serine NH 2 /g protein；
X: sample weight (g);
p: protein content (%) in the sample. The protein content was determined by Kjeldahl method.
h is:
wherein α=0.970, β=0.342 of the soybean protein.
Calculation of DH:
wherein h is tot Is the total number of peptide bonds per protein equivalent, h is the number of hydrolytic bonds. H of Soy protein tot ＝7.8。
(3) Determination of molecular weight distribution of Soybean peptide
Analysis was performed using a TSKgel G2000 SWXL column (300 mm. Times.7.8 mm) with a mobile phase consisting of acetonitrile: water: trifluoroacetic acid (45:55:0.1, v/v/v), flow rate 0.5mL/min, column temperature 30 ℃, detection wavelength UV220 nm, and sample volume 20. Mu.L.
According to the analysis of the soybean peptide hydrolysate obtained under the optimal combination of the enzymatic hydrolysis of aminopeptidases by chromatography, and taking only alkaline protease single-enzyme hydrolysis as a control, the chromatogram of the standard product is shown in fig. 9, the molecular weight distribution diagram of the alkaline protease (MAP) single-enzyme and double-enzyme synergistic hydrolysis product is shown in fig. 10 and 11, and the molecular weight analysis table of the alkaline protease (MAP) single-enzyme and double-enzyme synergistic hydrolysis is shown in table 4.
TABLE 4 molecular weight analysis of the isolated peaks
By combining the data in fig. 9-11 and table 4, it can be found that, compared with single-enzyme hydrolysis, the molecular weight of the soybean peptide hydrolysate obtained by the synergistic hydrolysis of double enzymes is totally distributed below 6500Da, only below 1450Da accounts for 93.87%, and the obtained sample meets the requirements of the national standard on soybean active peptides; and the bitterness score of the soybean peptide hydrolysate obtained by the aminopeptidase for enzymolysis of the soybean protein isolate is reduced from 1.3, and the hydrolysate is almost free of bitterness, so that the aminopeptidase has ideal debittering effect. According to the comparison of molecular weight distribution, the content of small peptides (451-189) and amino acids (< 189) in the soybean peptide hydrolyzed by double enzymes is obviously higher than that of single enzyme hydrolysis, the reason of the reduced bitter taste can be well explained, alkaline protease is used as endopeptidase to expose hydrophobic groups in the soybean protein isolate to stimulate taste buds to generate bitter taste, the tail ends of the bitter peptides carry hydrophobic amino acids, so that the bitter taste in hydrolysate is obvious, after aminopeptidase is added for enzymolysis, the aminopeptidase can degrade the hydrophobic amino acids carried at the tail ends of peptide chains, the free hydrophobic amino acids can not stimulate the taste buds on tongue fur to generate bitter taste after entering the hydrolysate, so that the prepared soybean peptide has reduced bitter taste value, and under the optimal enzymolysis condition, the prepared soybean peptide hydrolysate can hardly taste bitter, so that the aminopeptidase and alkaline protease can achieve good debittering effect in cooperation, and the prepared soybean peptide accords with the national standard, and application experience is provided for industrialization of high-quality soybean peptide after daily use.
The foregoing description of the preferred embodiments of the invention is not intended to limit the invention to the precise form disclosed, and any such modifications, equivalents, and alternatives falling within the spirit and scope of the invention are intended to be included within the scope of the invention.
Claims (6)
1. A method for the synergistic debittering of soybean peptides using aminopeptidases and alkaline proteases, comprising the steps of: weighing SPI, dissolving in distilled water, uniformly stirring, preparing SPI solution with the material-liquid ratio of 8%, cooling to room temperature after constant-temperature water bath at 55 ℃ for 20-30 min, regulating pH to be alkaline by using alkaline solution, placing in constant-temperature water bath at 55 ℃, carrying out enzymolysis on SPI by using alkaline protease for 2h, adding aminopeptidase to carry out enzymolysis reaction on SPI at 55 ℃ for 4h after the enzymolysis of the alkaline protease is finished, wherein the dosage of aminopeptidase is 500U/g SPI, inactivating, cooling and centrifuging hydrolysate after the enzymolysis is finished, and taking supernatant;
the alkaline protease is used in an amount of 4000U/gSPI.
2. The method according to claim 1, wherein the alkaline solution is sodium hydroxide solution.
3. The method of claim 1, wherein the PH is 8.5.
4. The method of claim 1, wherein the inactivation is for 1 hour at 85 ℃.
5. The method of claim 1, wherein the centrifugation is at 5000r/min for 10min.
6. The method of claim 1, further comprising the step of: the supernatant after centrifugation was stored at-20 ℃.
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| CN101139580A (en) * | 2007-08-07 | 2008-03-12 | 江南大学 | Method for preparing debittering aminopeptidase by fermentation of bacillus subtilis |
| CN106455625A (en) * | 2015-04-30 | 2017-02-22 | 中国食品发酵工业研究院 | Hypoallergenic, bitterness-reduced soybean oligopeptide, preparation method for same, and applications thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| EP0223560A2 (en) * | 1985-11-14 | 1987-05-27 | Imperial Biotechnology Limited | Flavour control of protein hydrolysates |
| CN101139580A (en) * | 2007-08-07 | 2008-03-12 | 江南大学 | Method for preparing debittering aminopeptidase by fermentation of bacillus subtilis |
| CN106455625A (en) * | 2015-04-30 | 2017-02-22 | 中国食品发酵工业研究院 | Hypoallergenic, bitterness-reduced soybean oligopeptide, preparation method for same, and applications thereof |
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| 枯草芽孢杆菌脱苦氨肽酶在水解大豆分离蛋白中的应用研究;魏亚娟等;食品工业科技;第29卷(第4期);149-151 * |
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