Method for preparing low-sensitization and low-bitterness whey protein hydrolysate through limited enzymolysis process regulated by endopeptidase and exopeptidase

Abstract

The invention belongs to the technical field of food protein processing, and particularly relates to a method for preparing a low-sensitization and low-bitterness whey protein hydrolysate through a limited enzymolysis process regulated by endopeptidase and exopeptidase, which comprises the following steps of: properly hydrolyzing and separating whey protein through trypsin, chymotrypsin, alkaline protease and flavourzyme; the method is used for obtaining optimal enzymolysis conditions of low allergenicity, low bitterness and high processing characteristics of whey protein. The enzymolysis product with the hydrolysis degree of 5% shows excellent emulsibility and foamability and lower bitterness, when the hydrolysis degree of the alkaline protein enzymolysis product is 5%, the antigenicity is reduced by 70.51%, the bitterness of the flavor protein enzymolysis product is the lowest, and the emulsibility of the enzymolysis product with the hydrolysis degree of trypsin being 5% is improved by 6.45%.

Description

Technical Field

[0001] This invention belongs to the field of food protein processing technology, and mainly relates to a method for obtaining low-allergenicity and low-bitterness whey protein hydrolysate through a limited enzymatic hydrolysis process regulated by endopeptidase and exopeptidase. Background Technology

[0002] Whey protein accounts for approximately 20% of total bovine milk protein. It possesses a complete amino acid profile and exhibits functional properties such as gelation, emulsification, and good solubility, making it a key matrix in high-protein nutritional formulations. However, β-lactoglobulin is a major allergen. Enzymatic hydrolysis can reduce allergenicity by specifically cleaving peptide bonds and altering the three-dimensional exposure of antigenic epitopes. Simultaneously, enzymatic hydrolysis also reshapes the protein's solubility, foaming properties, and emulsifying properties. Generally, limited enzymatic hydrolysis can improve molecular flexibility and interfacial activity, thus improving processing performance. Excessive enzymatic hydrolysis, however, may break down the backbone into too many short peptides, leading to a decline in function. Due to its reduced allergenicity, whey protein hydrolysates are widely used in infant formula and other fields. However, the hydrophobic peptides released during hydrolysis easily bind to TAS2R receptors, resulting in a noticeable bitter taste and limiting product acceptability. By controlling the degree of hydrolysis and reaction procedure, moderately hydrolyzed products that reduce allergenicity and suppress bitterness can be obtained, making it a preferred protein raw material.

[0003] To balance desensitization with flavor and function, this study selected proteases with different specificities based on the approach of endopeptidase and exopeptidase: trypsin preferentially cleaves arginine and lysine residues, acting on the positively charged region of β-lactoglobulin; chymotrypsin prefers aromatic residues such as phenylalanine, tyrosine, and tryptophan, which can destroy hydrophobic antigenic epitopes; alkaline proteases provide broad-spectrum endopeptidase cleavage sites; and flavor enzymes are rich in exopeptidase activity, which can cleave hydrophobic amino acid residues at both ends, reduce the formation of bitter peptides, and finely adjust the taste.

[0004] This invention uses isolated whey protein as a substrate and systematically compares the differences in allergenicity, emulsifying properties, foaming properties, and bitterness of hydrolysates obtained by the above-mentioned enzymes at the same degree of hydrolysis. The optimal enzyme and reaction time are then screened to obtain whey protein hydrolysates with low allergenicity, low bitterness, and excellent processing performance. This strategy achieves precise control over the allergenicity and flavor of whey protein, providing an feasible process route for developing food-grade whey protein hydrolysates with low allergenicity and low bitterness. Summary of the Invention

[0005] This invention relates to a method for obtaining low-allergenicity and low-bitterness whey protein hydrolysate through a limited enzymatic hydrolysis process regulated by endopeptidase and exopeptidase. The purpose is to provide an enzymatic hydrolysis method that reduces the allergenicity and bitterness of whey protein hydrolysate.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A method for obtaining low-sensitivity, low-bitterness whey protein hydrolysate through a limited enzymatic hydrolysis process regulated by endopeptidase and exopeptidase, characterized by comprising the following steps:

[0008] (1) Enzymatic hydrolysis of isolated whey protein: isolated whey protein was dissolved in water at a concentration of 30 mg / mL. Endoenzymes and exoenzymes were used to hydrolyze the isolated whey protein. The mass ratio of enzyme to substrate was 1:100 (w / w). During the hydrolysis process, 1 mol / L NaOH or HCl was used to maintain the pH of the system at the optimal reaction conditions of the enzyme.

[0009] (2) Preparation of whey protein hydrolysate: During the hydrolysis of endonuclease and exonuclease, samples were taken at 0, 10, 20, 30, 40, 50, 60, 90, 120, 150, 180, 210, 240, 300 and 360 min respectively. After adjusting the pH of the hydrolysate to neutral, the enzyme was quickly placed in a 75 ℃ water bath for 15 min to inactivate it. Then the sample was centrifuged at 7,000×g for 20 min. The supernatant was lyophilized and stored at -20℃.

[0010] (3) Measure the hydrolysis curve of the protein sample treated in step (2) to determine the hydrolysis time of different proteases at the same degree of hydrolysis;

[0011] (4) Measure the antigenicity changes of the protein sample determined in step (3);

[0012] (5) Measure the emulsifying activity and emulsifying stability of the protein sample determined in step (3);

[0013] (6) Measure the foaming properties and foam stability of the protein sample determined in step (3);

[0014] (7) Measure the change in bitterness of the protein sample determined in step (3).

[0015] The endopeptidases in step (1) are trypsin (250 U / mg), chymotrypsin (1200 U / mg) and alkaline protease (1600 U / mg), and the exopeptidase is flavor protease (1590 U / mg).

[0016] The experimental conditions for step (3) are as follows: Weigh 200 mg SDS, dissolve it in a small amount of 0.4 mol / L borate buffer (pH 9.5), add 4 mL OPA ethanol solution (40 mg / mL) and 400 μL β-mercaptoethanol, mix well, quantitatively transfer this mixture to a 200 mL brown volumetric flask, and dilute to the mark with 0.4 mol / L borate buffer (pH 9.5). Shake well to obtain the OPA reagent. After appropriate dilution of the sample, mix it with an equal volume of OPA reagent, react in the dark for 5 min, and immediately measure the absorbance at a wavelength of 340 nm. Simultaneously process L-leucine standard solutions (concentration gradient: 0, 5, 10, 15, 20, 25, 30 μg / mL), and plot the standard curve of absorbance versus concentration according to the same operating procedure. The degree of hydrolysis (DH, %) is calculated according to the standard curve and the following formula:

[0017] DH (%) = (A0-A1) × N / (131.7×X×h tot )

[0018] Where A0 is the leucine equivalent concentration (μg / mL) corresponding to the free amino group in the unhydrolyzed sample, A1 is the leucine equivalent concentration (μg / mL) corresponding to the free amino group in the hydrolyzed sample, N is the sample dilution factor, 131.17 is the molar mass of leucine (g / mol), X is the initial protein concentration of the sample (g / L), and h tot = 8.8 (mmol / g) is the peptide bond content in isolated whey protein.

[0019] The experimental conditions for step (4) are as follows: 50 μL of the sample to be tested is added to the microwells pre-coated with β-lactoglobulin antibody. Then, 100 μL of horseradish peroxidase (HRP) labeled detection antibody is added to each well. The reaction wells are sealed with a sealing film and incubated in a constant temperature incubator at 37 °C for 60 min. After pouring out the liquid in the wells and washing 5 times, 50 μL of substrate A (hydrogen peroxide) and 50 μL of substrate B (TMB) are added to each well and incubated in the dark at 37 °C for 15 min. Finally, 50 μL of stop solution (2 mol / L H2SO4) is added to each well to terminate the reaction. The absorbance is measured at a wavelength of 450 nm. 50 μL of standards of different concentrations are processed in the same way to draw a standard curve. The blank control group contains only 50 μL of substrate A and B and 50 μL of stop solution. The antigenicity of the sample is calculated according to the standard curve.

[0020] The experimental conditions for step (5) are as follows: different samples are mixed with soybean oil at a volume ratio of 3:1, homogenized at 10000 r / min for 2 min, and 50 μL of emulsion is taken from the same position at the bottom of the container at 0 min and 10 min after homogenization and added to 5.0 mL of 1.0 mg / mL SDS solution and mixed evenly. The 1.0 mg / mL SDS solution is used as a control. The absorbance of the mixed solution at 500 nm is measured immediately. The formulas for calculating emulsifying activity and emulsifying stability are as follows:

[0021] EAI (m 2 / g) = (2×2.303×A0×n) / (C×(1-φ)×10 4 )

[0022] ESI (%) = A 10 / A0×100

[0023] Among them, A0 and A 10 The absorbance values ​​are 0 and 10 min after homogenization, respectively. n is the dilution factor, C is the initial protein concentration (g / mL), and φ is the oil phase volume fraction.

[0024] The experimental conditions for step (6) are as follows: take 20 mL of different protein samples, homogenize them for 2 min at 10000 r / min using a homogenizer, and record the volume of foam at 0 min after homogenization and after standing for 10 min. The formulas for calculating foaming properties and foam stability are as follows:

[0025] FC (%) = V0 / 10×100

[0026] FS (%) = V 10 / V0×100

[0027] Where V0 is the foam volume (mL) at 0 min, V 10 The volume of foam (mL) after standing for 10 min is given.

[0028] The experimental conditions for step (7) are as follows: the Electronic Tongue TS-5000 Z intelligent taste system is used to measure the taste of the sample. The device is equipped with 6 sensors, AAE, CA0, CT0, CO0, AE1 and GL1, which correspond to umami, salty, sour, bitter, astringent and thick taste, respectively, as well as two aftertaste signal values: aftertaste of bitter taste (aftertaste-B) and aftertaste of astringent taste (aftertaste-A). Before the test, the sensors are immersed in a reference solution containing 0.045 mg / mL tartaric acid and 2.2365 mg / mL potassium chloride for 24 h for activation. The taste value of the sample is represented by the difference between the potential value (Vs) of the sample and the potential value (Vr) of the reference solution. The aftertaste signal value is represented by the difference between the potential value (Vr') of the sample after immersion in the reference solution and the potential value (Vr) of the reference solution. After each measurement, the sensor is immersed in an ethanol solution for cleaning. Each sample is measured in parallel 4 times.

[0029] The hydrolysis times of the endonucleases and exonucleases at the same degree of hydrolysis (5% and 7%) were as follows: trypsin: 59.90 min and 234.75 min; chymotrypsin: 50.87 min and 136.58 min; alkaline protease: 5.31 min and 7.21 min; and flavor protease: 8.18 min and 42.10 min. The limited enzymatic hydrolysates of the four enzymes exhibited superior emulsifying properties, foaming properties, and lower bitterness compared to the natural protein and the fully hydrolyzed products. Among them, the antigenicity of the alkaline protease hydrolysate decreased by 70.51% at a degree of hydrolysis of 5%, the flavor protease hydrolysate had the lowest bitterness, and the emulsifying properties of the trypsin hydrolysate increased by 6.45% at a degree of hydrolysis of 5%. Attached Figure Description

[0030] Figure 1 This is a technical flowchart of the present invention;

[0031] Figure 2 It is the degree of hydrolysis curve of whey protein separated by endonuclease and exonuclease hydrolysis;

[0032] Figure 3 The antigenicity changes of endonuclease and exonuclease hydrolysates of whey protein are shown in the following: F5, F7, F360, A5, A7, A360, C5, C7, C360, T5, T7, and T360 represent flavor enzyme, alkaline protease, chymotrypsin, and trypsin in samples with a degree of hydrolysis of 5%, 7%, and complete hydrolysis, respectively.

[0033] Figure 4The emulsifying activity and emulsifying stability of the endopeptidase and exopeptidase hydrolysates of whey protein are measured. F5, F7, F360, A5, A7, A360, C5, C7, C360, T5, T7, and T360 represent flavor enzymes, alkaline proteases, chymotrypsin, and trypsin in samples with a degree of hydrolysis of 5%, 7%, and complete hydrolysis, respectively.

[0034] Figure 5 The foaming properties and foam stability of whey protein endonuclease and exonuclease hydrolysates are given by F5, F7, F360, A5, A7, A360, C5, C7, C360, T5, T7, and T360, which represent flavor enzyme, alkaline protease, chymotrypsin, and trypsin at degrees of hydrolysis of 5%, 7%, and complete hydrolysis, respectively.

[0035] Figure 6 This is a bitterness analysis of endopeptidase and exopeptidase hydrolysates of whey protein, where F5, F7, F360, A5, A7, A360, C5, C7, C360, T5, T7, and T360 represent flavor enzymes, alkaline protease, chymotrypsin, and trypsin in samples with a degree of hydrolysis of 5%, 7%, and complete hydrolysis, respectively. Detailed Implementation

[0036] The specific embodiments are further described below with reference to the accompanying drawings.

[0037] A method for obtaining low-sensitivity, low-bitterness whey protein hydrolysate through a limited enzymatic hydrolysis process regulated by endopeptidase and exopeptidase, characterized by comprising the following steps:

[0038] (1) Enzymatic hydrolysis of isolated whey protein: isolated whey protein was dissolved in water at a concentration of 30 mg / mL. Endoenzymes and exoenzymes were used to hydrolyze the isolated whey protein. The mass ratio of enzyme to substrate was 1:100 (w / w). During the hydrolysis process, 1 mol / L NaOH or HCl was used to maintain the pH of the system at the optimal reaction conditions of the enzyme.

[0039] (2) Preparation of whey protein hydrolysate: During the hydrolysis of endonuclease and exonuclease, samples were taken at 0, 10, 20, 30, 40, 50, 60, 90, 120, 150, 180, 210, 240, 300 and 360 min respectively. After adjusting the pH of the hydrolysate to neutral, the enzyme was quickly placed in a 75 ℃ water bath for 15 min to inactivate it. Then the sample was centrifuged at 7,000×g for 20 min. The supernatant was lyophilized and stored at -20℃.

[0040] (3) Measure the hydrolysis curve of the protein sample treated in step (2) to determine the hydrolysis time of different proteases at the same degree of hydrolysis;

[0041] (4) Measure the antigenicity changes of the protein sample determined in step (3);

[0042] (5) Measure the emulsifying activity and emulsifying stability of the protein sample determined in step (3);

[0043] (6) Measure the foaming properties and foam stability of the protein sample determined in step (3);

[0044] (7) Measure the change in bitterness of the protein sample determined in step (3).

[0045] The endopeptidases in step (1) are trypsin (250 U / mg), chymotrypsin (1200 U / mg) and alkaline protease (1600 U / mg), and the exopeptidase is flavor protease (1590 U / mg).

[0046] The experimental conditions for step (3) are as follows: Weigh 200 mg SDS, dissolve it in a small amount of 0.4 mol / L borate buffer (pH 9.5), add 4 mL OPA ethanol solution (40 mg / mL) and 400 μL β-mercaptoethanol, mix well, quantitatively transfer this mixture to a 200 mL brown volumetric flask, and dilute to the mark with 0.4 mol / L borate buffer (pH 9.5). Shake well to obtain the OPA reagent. After appropriate dilution of the sample, mix it with an equal volume of OPA reagent, react in the dark for 5 min, and immediately measure the absorbance at a wavelength of 340 nm. Simultaneously process L-leucine standard solutions (concentration gradient: 0, 5, 10, 15, 20, 25, 30 μg / mL), and plot the standard curve of absorbance versus concentration according to the same operating procedure. The degree of hydrolysis (DH, %) is calculated according to the standard curve and the following formula:

[0047] DH (%) = (A0-A1) × N / (131.7×X×h tot )

[0048] Where A0 is the leucine equivalent concentration (μg / mL) corresponding to the free amino group in the unhydrolyzed sample, A1 is the leucine equivalent concentration (μg / mL) corresponding to the free amino group in the hydrolyzed sample, N is the sample dilution factor, 131.17 is the molar mass of leucine (g / mol), X is the initial protein concentration of the sample (g / L), and h tot = 8.8 (mmol / g) is the peptide bond content in isolated whey protein.

[0049] The experimental conditions for step (4) are as follows: 50 μL of the sample to be tested is added to the microwells pre-coated with β-lactoglobulin antibody. Then, 100 μL of horseradish peroxidase (HRP) labeled detection antibody is added to each well. The reaction wells are sealed with a sealing film and incubated in a constant temperature incubator at 37 °C for 60 min. After pouring out the liquid in the wells and washing 5 times, 50 μL of substrate A (hydrogen peroxide) and 50 μL of substrate B (TMB) are added to each well and incubated in the dark at 37 °C for 15 min. Finally, 50 μL of stop solution (2 mol / L H2SO4) is added to each well to terminate the reaction. The absorbance is measured at a wavelength of 450 nm. 50 μL of standards of different concentrations are processed in the same way to draw a standard curve. The blank control group contains only 50 μL of substrate A and B and 50 μL of stop solution. The antigenicity of the sample is calculated according to the standard curve.

[0050] The experimental conditions for step (5) are as follows: different samples are mixed with soybean oil at a volume ratio of 3:1, homogenized at 10000 r / min for 2 min, and 50 μL of emulsion is taken from the same position at the bottom of the container at 0 min and 10 min after homogenization and added to 5.0 mL of 1.0 mg / mL SDS solution and mixed evenly. The 1.0 mg / mL SDS solution is used as a control. The absorbance of the mixed solution at 500 nm is measured immediately. The formulas for calculating emulsifying activity and emulsifying stability are as follows:

[0051] EAI (m 2 / g) = (2×2.303×A0×n) / (C×(1-φ)×10 4 )

[0052] ESI (%) = A 10 / A0×100

[0053] Among them, A0 and A 10 The absorbance values ​​are 0 and 10 min after homogenization, respectively. n is the dilution factor, C is the initial protein concentration (g / mL), and φ is the oil phase volume fraction.

[0054] The experimental conditions for step (6) are as follows: take 20 mL of different protein samples, homogenize them for 2 min at 10000 r / min using a homogenizer, and record the volume of foam at 0 min after homogenization and after standing for 10 min. The formulas for calculating foaming properties and foam stability are as follows:

[0055] FC (%) = V0 / 10×100

[0056] FS (%) = V 10 / V0×100

[0057] Where V0 is the foam volume (mL) at 0 min, V 10 The volume of foam (mL) after standing for 10 min is given.

[0058] The experimental conditions for step (7) are as follows: the Electronic Tongue TS-5000 Z intelligent taste system is used to measure the taste of the sample. The device is equipped with 6 sensors, AAE, CA0, CT0, CO0, AE1 and GL1, which correspond to umami, salty, sour, bitter, astringent and thick taste, respectively, as well as two aftertaste signal values: aftertaste of bitter taste (aftertaste-B) and aftertaste of astringent taste (aftertaste-A). Before the test, the sensors are immersed in a reference solution containing 0.045 mg / mL tartaric acid and 2.2365 mg / mL potassium chloride for 24 h for activation. The taste value of the sample is represented by the difference between the potential value (Vs) of the sample and the potential value (Vr) of the reference solution. The aftertaste signal value is represented by the difference between the potential value (Vr') of the sample after immersion in the reference solution and the potential value (Vr) of the reference solution. After each measurement, the sensor is immersed in an ethanol solution for cleaning. Each sample is measured in parallel 4 times.

[0059] The hydrolysis times of the endonucleases and exonucleases at the same degree of hydrolysis (5% and 7%) were as follows: trypsin: 59.90 min and 234.75 min; chymotrypsin: 50.87 min and 136.58 min; alkaline protease: 5.31 min and 7.21 min; and flavor protease: 8.18 min and 42.10 min. The limited enzymatic hydrolysates of the four enzymes exhibited superior emulsifying properties, foaming properties, and lower bitterness compared to the natural protein and the fully hydrolyzed products. Among them, the antigenicity of the alkaline protease hydrolysate decreased by 70.51% at a degree of hydrolysis of 5%, the flavor protease hydrolysate had the lowest bitterness, and the emulsifying properties of the trypsin hydrolysate increased by 6.45% at a degree of hydrolysis of 5%.

[0060] Example 1

[0061] (1) Enzymatic hydrolysis of isolated whey protein: isolated whey protein was dissolved in water at a concentration of 30 mg / mL. The isolated whey protein was enzymatically hydrolyzed using trypsin (250 U / mg) at a mass ratio of enzyme to substrate of 1:100 (w / w). During the hydrolysis process, 1 mol / L NaOH or HCl was used to maintain the pH of the system at the optimal reaction conditions of the enzyme (37 ℃, pH 8.0).

[0062] (2) Preparation of whey protein hydrolysate: During the hydrolysis process, samples were taken at 0, 10, 20, 30, 40, 50, 60, 90, 120, 150, 180, 210, 240, 300 and 360 min respectively. After adjusting the pH of the hydrolysate to neutral, it was quickly placed in a 75 ℃ water bath for 15 min to inactivate the enzyme. Then the sample was centrifuged at 7,000×g for 20 min. The supernatant was lyophilized and stored at -20 ℃.

[0063] (3) The hydrolysis curves of the protein samples treated in step (2) were measured. The degree of hydrolysis of trypsin reached 5% and 7% at 59.02 min and 174.85 min, respectively.

[0064] (4) Measurement of antigenicity changes in protein samples in step (3): The antigenicity of trypsin decreased by 10.07% and 30.22% at 5% and 7% degree of hydrolysis, respectively;

[0065] (5) Measurement of emulsifying activity and emulsifying stability of protein samples in step (3): trypsin emulsifying activity increased by 6.45% at a degree of hydrolysis of 5%;

[0066] (6) Measurement of foaming properties and foam stability of protein samples in step (3): trypsin foaming properties increased by 78.29% at a degree of hydrolysis of 7%.

[0067] (7) Measurement of bitterness changes in protein samples in step (3): Trypsin has a lower bitterness at 5% degree of hydrolysis.

[0068] Example 2

[0069] (1) Enzymatic hydrolysis of isolated whey protein: isolated whey protein was dissolved in water at a concentration of 30 mg / mL. Enzymatic hydrolysis of isolated whey protein was carried out using chymotrypsin (1200 U / mg). The mass ratio of enzyme to substrate was 1:100 (w / w). During hydrolysis, 1 mol / L NaOH or HCl was used to maintain the pH of the system at the optimal reaction conditions of the enzyme (37 ℃, pH 8.0).

[0070] (2) Preparation of whey protein hydrolysate: During the hydrolysis process, samples were taken at 0, 10, 20, 30, 40, 50, 60, 90, 120, 150, 180, 210, 240, 300 and 360 min respectively. After adjusting the pH of the hydrolysate to neutral, it was quickly placed in a 75 ℃ water bath for 15 min to inactivate the enzyme. Then the sample was centrifuged at 7,000×g for 20 min. The supernatant was lyophilized and stored at -20 ℃.

[0071] (3) The hydrolysis curves of the protein samples treated in step (2) were measured. The degree of hydrolysis of trypsin reached 5% and 7% at 50.87 min and 136.58 min, respectively.

[0072] (4) Measurement of antigenicity changes in protein samples in step (3): The antigenicity of chymotrypsin decreased by 20.15% and 30.22% at 5% and 7% degrees of hydrolysis, respectively;

[0073] (5) Measurement of the emulsifying activity and emulsifying stability of protein samples in step (3). The emulsifying properties decrease after hydrolysis by chymotrypsin.

[0074] (6) Measurement of foaming properties and foam stability of protein samples in step (3): chymotrypsin showed a 61.14% increase in foaming properties at a degree of hydrolysis of 5%.

[0075] (7) Measurement of bitterness changes in protein samples in step (3): The bitterness of chymotrypsin is lower when the degree of hydrolysis is 5%.

[0076] Example 3

[0077] (1) Enzymatic hydrolysis of isolated whey protein: isolated whey protein was dissolved in water at a concentration of 30 mg / mL. Alkaline protease (1600 U / mg) was used to hydrolyze the isolated whey protein. The mass ratio of enzyme to substrate was 1:100 (w / w). During the hydrolysis process, 1 mol / L NaOH or HCl was used to maintain the pH of the system at the optimal reaction conditions of the enzyme (50 ℃, pH 9.0).

[0078] (2) Preparation of whey protein hydrolysate: During the hydrolysis process, samples were taken at 0, 10, 20, 30, 40, 50, 60, 90, 120, 150, 180, 210, 240, 300 and 360 min respectively. After adjusting the pH of the hydrolysate to neutral, it was quickly placed in a 75 ℃ water bath for 15 min to inactivate the enzyme. Then the sample was centrifuged at 7,000×g for 20 min. The supernatant was lyophilized and stored at -20 ℃.

[0079] (3) The hydrolysis curves of the protein samples treated in step (2) were measured. The degree of hydrolysis of alkaline protease reached 5% and 7% at 5.31 min and 7.21 min, respectively.

[0080] (4) Measurement of antigenicity changes in protein samples in step (3): The antigenicity of alkaline protease decreased by 70.51% and 79.58% at degrees of hydrolysis of 5% and 7%, respectively;

[0081] (5) Measurement of the emulsifying activity and emulsifying stability of protein samples in step (3). The emulsifying properties decrease after hydrolysis by alkaline protease.

[0082] (6) Measurement of foaming properties and foam stability of protein samples in step (3): Alkaline protease showed a 61.28% increase in foaming properties at a degree of hydrolysis of 7%.

[0083] (7) Measurement steps (3) Bitterness changes of protein samples. Alkaline protease has a lower bitterness at a degree of hydrolysis of 5%.

[0084] Example 4

[0085] (1) Enzymatic hydrolysis of isolated whey protein: isolated whey protein was dissolved in water at a concentration of 30 mg / mL. Flavor protease (1590 U / mg) was used to hydrolyze the isolated whey protein. The mass ratio of enzyme to substrate was 1:100 (w / w). During the hydrolysis process, 1 mol / L NaOH or HCl was used to maintain the pH of the system at the optimal reaction conditions of the enzyme (50 ℃, pH 7.0).

[0086] (2) Preparation of whey protein hydrolysate: During the hydrolysis process, samples were taken at 0, 10, 20, 30, 40, 50, 60, 90, 120, 150, 180, 210, 240, 300 and 360 min respectively. After adjusting the pH of the hydrolysate to neutral, it was quickly placed in a 75 ℃ water bath for 15 min to inactivate the enzyme. Then the sample was centrifuged at 7,000×g for 20 min. The supernatant was lyophilized and stored at -20 ℃.

[0087] (3) The hydrolysis curves of the protein samples treated in step (2) were measured. The degree of hydrolysis of the flavor protease reached 5% and 7% at 8.18 min and 42.10 min, respectively.

[0088] (4) Measurement of antigenicity changes in protein samples in step (3): The antigenicity of flavor protease decreased by 60.44% and 67.49% at degrees of hydrolysis of 5% and 7%, respectively;

[0089] (5) Measurement of the emulsifying activity and emulsifying stability of protein samples in step (3), and the emulsifying properties decreased after hydrolysis of flavor protease;

[0090] (6) Measurement of foaming properties and foam stability of protein samples in step (3): The foaming property of flavor protease increased by 99.63% at a degree of hydrolysis of 5%.

[0091] (7) Measurement steps (3) Bitterness changes of protein samples. Flavor protease has the lowest bitterness at a degree of hydrolysis of 5%, and produces a certain umami flavor.

Claims

1. A method for obtaining low-sensitivity, low-bitterness whey protein hydrolysates through a limited enzymatic hydrolysis process regulated by endopeptidase and exopeptidase, characterized in that... Includes the following steps: (1) Enzymatic hydrolysis of isolated whey protein: isolated whey protein was dissolved in water at a concentration of 30 mg / mL. Endoenzymes and exoenzymes were used to hydrolyze the isolated whey protein. The mass ratio of enzyme to substrate was 1:100 (w / w). During the hydrolysis process, 1 mol / L NaOH or HCl was used to maintain the pH of the system at the optimal reaction conditions of the enzyme. (2) Preparation of whey protein hydrolysate: During the hydrolysis of endonuclease and exonuclease, samples were taken at 0, 10, 20, 30, 40, 50, 60, 90, 120, 150, 180, 210, 240, 300 and 360 min respectively. After adjusting the pH of the hydrolysate to neutral, the enzyme was quickly placed in a 75℃ water bath for 15 min to inactivate it. Then the sample was centrifuged at 7,000×g for 20 min. The supernatant was lyophilized and stored at -20 ℃. (3) Measure the hydrolysis curve of the protein sample treated in step (2) to determine the hydrolysis time of different proteases at the same degree of hydrolysis; (4) Measure the antigenicity changes of the protein sample determined in step (3); (5) Measure the emulsifying activity and emulsifying stability of the protein sample determined in step (3); (6) Measure the foaming properties and foam stability of the protein sample determined in step (3); (7) Measure the change in bitterness of the protein sample determined in step (3).

2. The method for obtaining low-sensitivity, low-bitterness whey protein hydrolysate through a limited enzymatic hydrolysis process regulated by endopeptidase and exopeptidase according to claim 1, characterized in that: The endopeptidases in step (1) are trypsin (250 U / mg), chymotrypsin (1200 U / mg) and alkaline protease (1600 U / mg), and the exopeptidase is flavor protease (1590 U / mg).

3. The method for obtaining low-sensitivity, low-bitterness whey protein hydrolysate through a limited enzymatic hydrolysis process regulated by endopeptidase and exopeptidase according to claim 1, characterized in that: The experimental conditions for step (3) are as follows: Weigh 200 mg SDS, dissolve it in a small amount of 0.4 mol / L borate buffer (pH 9.5), add 4 mL OPA ethanol solution (40 mg / mL) and 400 μL β-mercaptoethanol, mix well, quantitatively transfer this mixture to a 200 mL brown volumetric flask, and dilute to the mark with 0.4 mol / L borate buffer (pH 9.5). Shake well to obtain the OPA reagent. After appropriate dilution of the sample, mix it with an equal volume of OPA reagent, react in the dark for 5 min, and immediately measure the absorbance at a wavelength of 340 nm. Simultaneously process L-leucine standard solutions (concentration gradient: 0, 5, 10, 15, 20, 25, 30 μg / mL), and plot the standard curve of absorbance versus concentration according to the same operating procedure. The degree of hydrolysis (DH, %) is calculated according to the standard curve and the following formula: DH (%) = (A0-A1) × N / (131.7×X×h tot ) Where A0 is the leucine equivalent concentration (μg / mL) corresponding to the free amino group in the unhydrolyzed sample, A1 is the leucine equivalent concentration (μg / mL) corresponding to the free amino group in the hydrolyzed sample, N is the sample dilution factor, 131.17 is the molar mass of leucine (g / mol), X is the initial protein concentration of the sample (g / L), and h tot = 8.8 (mmol / g) is the peptide bond content in isolated whey protein.

4. The method for obtaining low-sensitivity, low-bitterness whey protein hydrolysate by a limited enzymatic hydrolysis process regulated by endopeptidase and exopeptidase according to claim 1, characterized in that: The experimental conditions for step (4) are as follows: 50 μL of the sample to be tested is added to the microwells pre-coated with β-lactoglobulin antibody. Then, 100 μL of horseradish peroxidase (HRP) labeled detection antibody is added to each well. The reaction wells are sealed with a sealing film and incubated in a constant temperature incubator at 37 °C for 60 min. After pouring out the liquid in the wells and washing 5 times, 50 μL of substrate A (hydrogen peroxide) and 50 μL of substrate B (TMB) are added to each well and incubated in the dark at 37 °C for 15 min. Finally, 50 μL of stop solution (2 mol / L H2SO4) is added to each well to terminate the reaction. The absorbance is measured at a wavelength of 450 nm. 50 μL of standards of different concentrations are processed in the same way to plot a standard curve. The blank control group contains only 50 μL of substrate A and B and 50 μL of stop solution. The antigenicity of the sample is calculated according to the standard curve.

5. The method for obtaining low-sensitivity, low-bitterness whey protein hydrolysate by a limited enzymatic hydrolysis process regulated by endopeptidase and exopeptidase according to claim 1, characterized in that: The experimental conditions for step (5) are as follows: different samples are mixed with soybean oil at a volume ratio of 3:1, homogenized at 10000 r / min for 2 min, and 50 μL of emulsion is taken from the same position at the bottom of the container at 0 min and 10 min after homogenization and added to 5.0 mL of 1.0 mg / mL SDS solution and mixed evenly. The 1.0 mg / mL SDS solution is used as a control. The absorbance of the mixed solution at 500 nm is measured immediately. The formulas for calculating emulsifying activity and emulsifying stability are as follows: EAI (m 2 / g) = (2×2.303×A0×n) / (C×(1-φ)×10 4 ) ESI (%) = A 10 / A0×100 Among them, A0 and A 10 The absorbance values ​​are 0 and 10 min after homogenization, respectively. n is the dilution factor, C is the initial protein concentration (g / mL), and φ is the oil phase volume fraction.

6. The method for obtaining low-sensitivity, low-bitterness whey protein hydrolysate by a limited enzymatic hydrolysis process regulated by endopeptidase and exopeptidase according to claim 1, characterized in that: The experimental conditions for step (6) are as follows: take 20 mL of different protein samples, homogenize them at 10000 r / min for 2 min using a homogenizer, and record the volume of foam at 0 min after homogenization and after standing for 10 min. The formulas for calculating foaming properties and foam stability are as follows: FC (%) = V0 / 10×100 FS (%) = V 10 / V0×100 Where V0 is the foam volume (mL) at 0 min, V 10 The volume of foam (mL) after standing for 10 min is given.

7. The method for obtaining low-sensitivity, low-bitterness whey protein hydrolysate by a limited enzymatic hydrolysis process regulated by endopeptidase and exopeptidase according to claim 1, characterized in that: The experimental conditions for step (7) are as follows: the ElectronicTongue TS-5000 Z intelligent taste system is used to measure the taste of the sample. The device is equipped with 6 sensors, AAE, CA0, CT0, CO0, AE1 and GL1, which correspond to umami, salty, sour, bitter, astringent and thick taste, respectively, as well as two aftertaste signal values: aftertaste of bitter taste (aftertaste-B) and aftertaste of astringent taste (aftertaste-A). Before the test, the sensors are immersed in a reference solution containing 0.045 mg / mL tartaric acid and 2.2365 mg / mL potassium chloride for 24 h for activation. The taste value of the sample is represented by the difference between the potential value of the sample (Vs) and the potential value of the reference solution (Vr). The aftertaste signal value is represented by the difference between the potential value of the sample after immersion in the reference solution for the second time (Vr') and the potential value of the reference solution (Vr). After each measurement, the sensor is immersed in an ethanol solution for cleaning. Each sample is measured in parallel 4 times.

8. The method for obtaining low-sensitivity, low-bitterness whey protein hydrolysate by a limited enzymatic hydrolysis process regulated by endopeptidase and exopeptidase according to claim 1, characterized in that: The hydrolysis times of the endonucleases and exonucleases at the same degree of hydrolysis (5% and 7%) were as follows: trypsin: 59.90 min and 234.75 min; chymotrypsin: 50.87 min and 136.58 min; alkaline protease: 5.31 min and 7.21 min; and flavor protease: 8.18 min and 42.10 min. The limited enzymatic hydrolysates of the four enzymes exhibited superior emulsifying properties, foaming properties, and lower bitterness compared to the natural protein and the fully hydrolyzed products. Among them, the antigenicity of the alkaline protease hydrolysate decreased by 70.51% at a degree of hydrolysis of 5%, the flavor protease hydrolysate had the lowest bitterness, and the emulsifying properties of the trypsin hydrolysate increased by 6.45% at a degree of hydrolysis of 5%.

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