Method for reducing allergenicity and bitter taste of whey protein through non-thermal processing assisted composite enzymatic hydrolysis

Abstract

The invention discloses a method for reducing allergenicity and bitter taste of whey protein through non-thermal processing assisted composite enzymatic hydrolysis, and belongs to the technical field of food processing. The preparation method comprises the following steps: adding whey protein into ultrapure water, uniformly dispersing to prepare a 5% whey protein solution, carrying out ultrasonic treatment to open the internal structure of the whey protein and fully expose sensitization groups buried in the whey protein, carrying out first-step enzymolysis on the pretreated whey protein solution by using alkaline protease, and carrying out second-step enzymolysis on the pretreated whey protein solution by using alkaline protease, and carrying out second-step enzymolysis by using Flavourzyme, and carrying out freeze-drying treatment on the whey protein solution to obtain the whey protein hydrolysate. The whey protein zymolyte prepared by the processing technology disclosed by the invention is low in sensitization and low in bitterness value, meanwhile, the delicate flavor is increased, and the applicability of the whey protein is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of food processing, and particularly relates to a method for reducing the allergenicity and bitterness of whey protein by non-thermal processing-assisted composite enzymatic hydrolysis. Background Art

[0002] Whey protein isolate (WPI) is a high-quality protein extracted from whey by ultrafiltration technology. Because its amino acid composition is similar to that of human protein, it has high nutritional value and functional properties. It is widely used in various foods. It is an ideal source of bioactive peptides and one of the main allergens in cow's milk. Whey protein accounts for about 20% of the total protein content of cow's milk. Among them, the most important allergens are β-LG and α-LA. Cow's milk is one of the eight major allergic foods recognized by the Food and Agriculture Organization of the United Nations (FAO). The allergic symptoms caused by cow's milk threaten the health of infants and young children, and in severe cases can even lead to death. Therefore, the development of low-allergenic whey protein raw materials has become a current research hotspot.

[0003] In order to reduce the allergenicity of whey protein and improve its applicability, modification techniques are usually used to reduce the allergenicity of whey protein, mainly physical modification and biochemical modification. Protease hydrolysis can hydrolyze the allergenic peptides of whey protein into very small peptides or amino acids, reduce the immune activity of β-LG and α-LA, and thus reduce the allergenicity of whey protein. However, due to the unique spatial structure of β-LG molecules, enzymatic hydrolysis cannot completely destroy the antigenic epitopes in the β-LG molecules, cannot completely eliminate its allergenicity, and has the problem of poor flavor, which limits the scope of application of whey protein.

[0004] The bitterness of protein hydrolysate mainly comes from the hydrophobic peptides in the hydrolysate. In the process of protein hydrolysis into small molecule peptides, its hydrophobic amino acid residues will be exposed. Such amino acid residues stimulate the taste buds and produce bitterness. Therefore, it has become a difficult problem to balance the low allergenicity and low bitterness of whey protein hydrolysate. Therefore, the sequence of allergenic groups is explored, the enzyme cleavage sites are studied, and the allergenic groups and hydrophobic amino acids are removed to the greatest extent, thereby obtaining a low-allergenic, low-bitter whey protein and improving the comprehensive utilization rate of whey protein. Summary of the invention

[0005] The technical problem to be solved by the present invention is to improve the defects of the prior art and establish a composite enzymatic hydrolysis method for reducing the allergenicity and bitterness of whey protein. The allergenicity and bitterness value of the product after enzymatic hydrolysis under ultrasound are evaluated to obtain a whey protein with low allergenicity and good flavor.

[0006] The invention uses ultrasound to perform pretreatment to fully unfold the structure of whey protein, and assists the composite enzymatic hydrolysis to act on the whey protein solution, thereby reducing the allergenicity of the whey protein.

[0007] The object of the present invention is to provide a method for preparing a hypoallergenic whey protein, the preparation method comprising the following steps:

[0008] S1, pre-treating whey protein with ultrasound;

[0009] S2, single factor compound enzymatic hydrolysis based on ultrasonic pretreatment of whey protein;

[0010] S3. Optimize based on the most suitable single factor condition.

[0011] The whey protein in step S1 is isolated whey protein.

[0012] Preferably, the ultrasonic conditions in step S1 are: ultrasonic interval ratio of 3:7s, ultrasonic power of 200W, ultrasonic time of 5 to 20min, and ultrasonication is completed in ice bath water.

[0013] Furthermore, in step S2, the complex enzyme is alkaline protease and flavor protease.

[0014] Preferably, in step S2, the alkaline protease enzyme-substrate ratio is 6%, the pH is 10, the enzymatic hydrolysis time is 4 hours, and the enzymatic hydrolysis temperature is 60°C.

[0015] Furthermore, the enzyme inactivation temperature in step S2 is 100° C. and the time is 10 min.

[0016] Preferably, in step S2, the ratio of alkaline protease to flavor protease is 1:0.5-1:0.9, the temperature is 40-60°C, the pH is 6.5-8.5, the substrate concentration is 3-11%, and the time is 30-150 min.

[0017] Furthermore, the enzyme inactivation temperature in step S2 is 100° C. and the time is 20 min.

[0018] Preferably, in step S3, the ratio of alkaline protease to flavor protease is 1:0.7-1:0.9, the temperature is 45-55° C., the pH is 6.5-7.5, the substrate concentration is 5%, and the time is 90 min.

[0019] The present invention adopts ultrasound-assisted composite enzymatic hydrolysis technology, which changes the secondary structure and tertiary structure of the protein through ultrasound, so that the internal structure of the whey protein is fully unfolded, and the allergenic groups buried inside are exposed. The protein is then cut at specific sites by enzymatic hydrolysis, and the protein peptide bonds are broken to hydrolyze the allergenic protein into small molecule peptides or amino acids, thereby reducing its molecular weight, thereby achieving the goal of reducing the allergenicity of whey protein.

[0020] In this study, alkaline protease and flavor enzyme were selected to hydrolyze whey protein, because many studies have shown that alkaline protease has the best effect in reducing allergenicity, and flavor enzyme is both an exopeptidase and an endopeptidase, which can simultaneously reduce allergenicity and improve flavor, and reduce the bitterness of the hydrolyzate by releasing hydrophobic amino acids from the N-terminus and C-terminus, thereby improving the comprehensive utilization rate of hydrolyzed whey protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the SEM image of Example 1 at different ultrasound times

[0022] Figure 2 The secondary structure changes of Example 1 at different ultrasound times

[0023] Figure 3 The endogenous fluorescence spectra of Example 1 at different ultrasound times are

[0024] Figure 4 is the thiol content of different ultrasonic times in Example 1

[0025] Figure 5 The hydrophobicity of different ultrasonic times in Example 1

[0026] Figure 6 Example 2 Effects of five factors on the hydrolysis degree and antigen inhibition rate of composite protease

[0027] Figure 7 Example 3 Antigenicity of whey protein hydrolysate

[0028] Figure 8 The molecular weight of whey protein treated differently in Example 3

[0029] Fig. 9 Example 4: Whey protein electronic tongue response value

[0030] Fig.10 Example 4 Sensory evaluation of bitterness value of whey protein DETAILED DESCRIPTION

[0031] The specific implementation modes of the present invention are described in detail below in conjunction with specific embodiments and drawings, but the present invention is not limited to the embodiments.

[0032] Example 1 Ultrasonic pretreatment changes whey protein structure

[0033] Ultrasonic pretreatment conditions were 200 W, ice water bath, 5 to 20 min;

[0034] In the present invention, the determination method of the scanning electron microscope image, secondary structure and tertiary structure of whey protein is as follows:

[0035] (1) Scanning electron microscope image

[0036] The WPI was freeze-dried and gold-sprayed using an ion sputtering apparatus. The working distance was adjusted to 5 mm, the acceleration voltage was 10 kV, and the magnification was 500×. The WPI was observed using a tungsten filament scanning electron microscope.

[0037] (2) Circular dichroism spectroscopy analysis

[0038] The WPI sample was diluted to 200 μg / mL, added to a quartz cuvette (optical path 0.1 cm), and placed on a circular dichroism spectrometer for detection. The scanning range was 190-250 nm, the step length was 0.5 nm, and the scanning speed was 200 nm / min. The composition and content of the secondary structure in whey protein were fitted using CD Pro software.

[0039] (3) Endogenous fluorescence spectrum detection

[0040] Dilute the WPI sample to 1 mg / mL, transfer an appropriate amount of the sample to a 96-well plate using a pipette, and perform endogenous tryptophan fluorescence detection using an ELISA reader. Set the excitation wavelength to 280 nm and scan the emission spectrum from 300 to 400 nm.

[0041] (4) Determination of thiol content

[0042] Take 200 μL of WPI sample and 1 mL of Tris-glycine buffer (pH 8.0, containing 4 mmol / L EDTA) and mix them. Then add 20 μL of Ellman's reagent (4 mg / mL). After shaking at room temperature (25°C ± 2°C) and avoiding light for 30 minutes, measure the absorbance at 412 nm. The blank control is the Ellman's reagent alone. The determination method of total thiol content is consistent with that of surface thiol content. The buffer solution used is 4 mmol / L EDTA, 8 mol / L urea, pH 8 free thiol content.

[0043]

[0044] Where A412 is the absorbance at 412 nm; D is the dilution (%); and C is the protein concentration in WPI (mg / mL).

[0045] (5) Determination of surface hydrophobicity

[0046] The surface hydrophobicity of whey protein was determined using the ANS fluorescent probe method. The whey protein powder obtained by vacuum freeze drying with different heat treatments was prepared into a whey protein solution with a concentration of 0.2 mg / mL using a phosphate buffer solution. 50 μL of 8 mM ANS solution was added to 5 mL of 0.2 mg / mL whey protein solution, and phosphate buffer was used as a blank control. The spectrum was measured using a fluorescence spectrophotometer. The area formed by the scanned spectrum and the horizontal axis is the surface hydrophobicity of the protein sample. Determination parameters: excitation wavelength 390 nm, emission wavelength 470 nm, emission and excitation slits are both 5 nm, and the measurement temperature is 25 ° C.

[0047] Changes in the secondary structure of whey protein Figure 2 As shown, the proportion of regular structure β-folds decreases, while the proportion of irregular structure β-turns and random coils increases, and the protein structure becomes loose and disordered.

[0048] The tertiary structure of whey protein is Figure 3 , 4 As shown in Figures 5 and 5, the intensity of ultrasonic intrinsic fluorescence gradually increased with the extension of ultrasonic time, and the disulfide bonds inside the protein were gradually converted into thiol groups, which gradually increased the thiol content. The thiol content of the 15-min treatment group changed little compared with that of the 20-min treatment group, and the surface hydrophobicity was similar to the thiol trend. In summary, the internal structure of the 20-min ultrasonic treatment was almost the same as that of the 15-min ultrasonic treatment. Therefore, the 15-min ultrasonic treatment time was selected as the pretreatment condition for the next enzymatic hydrolysis. At this time, the internal structure of whey protein was fully developed, which was fully prepared for further enzymatic hydrolysis.

[0049] Example 2 Composite enzyme hydrolysis based on ultrasonic pretreatment (AF)

[0050] Sample 1

[0051] (1) Prepare a 5% (w / v) whey protein solution.

[0052] (2) adding alkaline protease to the whey protein solution prepared in step (1) at an enzyme-substrate ratio of 6%, and hydrolyzing the solution at pH 8 and 60° C. for 2 h. During the hydrolysis reaction, 0.5 mol NaOH was added to maintain the pH value of the reaction system.

[0053] (3) adding flavor protease to the alkaline protease whey protein hydrolyzate of step (2) at an enzyme-substrate ratio of 4.8%, and hydrolyzing at pH 6.5-8.5 at 50° C. for 1.5 h. During the hydrolysis reaction, the pH value of the reaction system was maintained by adding 0.5 mol NaOH, the degree of hydrolysis was determined, and a hydrolysis curve was drawn to obtain a hydrolyzed sample with a degree of hydrolysis of 2.3%.

[0054] Sample 2

[0055] (1) Prepare a 5% (w / v) whey protein solution.

[0056] (2) adding alkaline protease to the whey protein solution prepared in step (1) at an enzyme-substrate ratio of 6%, and hydrolyzing the solution at pH 8 and 60° C. for 2 h. During the hydrolysis reaction, 0.5 mol NaOH was added to maintain the pH value of the reaction system.

[0057] (3) adding flavor protease to the alkaline protease whey protein hydrolyzate of step (2) at an enzyme-substrate ratio of 3% to 5.4%, and hydrolyzing at pH 7 and 50° C. for 1.5 h. During the hydrolysis reaction, the pH value of the reaction system is maintained by adding 0.5 mol NaOH, the degree of hydrolysis is determined, and a hydrolysis curve is drawn to obtain a hydrolyzed sample with a degree of hydrolysis of 2.3%.

[0058] Sample 3

[0059] (1) Prepare a 5% (w / v) whey protein solution.

[0060] (2) adding alkaline protease to the whey protein solution prepared in step (1) at an enzyme-substrate ratio of 6%, and hydrolyzing the solution at pH 8 and 60° C. for 2 h. During the hydrolysis reaction, 0.5 mol NaOH was added to maintain the pH value of the reaction system.

[0061] (3) adding flavor protease to the alkaline protease whey protein hydrolyzate of step (2) at an enzyme-substrate ratio of 4.8%, and hydrolyzing at pH 7 and 50° C. for 30 min-150 min. During the hydrolysis reaction, 0.5 mol NaOH was added to maintain the pH value of the reaction system, the degree of hydrolysis was determined, and a hydrolysis curve was drawn to obtain a hydrolyzed sample with a degree of hydrolysis of 2.3%.

[0062] Sample 4

[0063] (1) Prepare a 5% (w / v) whey protein solution.

[0064] (2) adding alkaline protease to the whey protein solution prepared in step (1) at an enzyme-substrate ratio of 6%, and hydrolyzing the solution at pH 8 and 60° C. for 2 h. During the hydrolysis reaction, 0.5 mol NaOH was added to maintain the pH value of the reaction system.

[0065] (3) adding flavor protease to the alkaline protease whey protein hydrolyzate of step (2) at an enzyme-substrate ratio of 4.8%, and hydrolyzing at pH 7 at 40-60° C. for 1.5 h. During the hydrolysis reaction, the pH value of the reaction system was maintained by adding 0.5 mol NaOH, the degree of hydrolysis was determined, and a hydrolysis curve was drawn to obtain a hydrolyzed sample with a degree of hydrolysis of 2.3%.

[0066] Sample 5

[0067] (1) Prepare a 3-11% (w / v) whey protein solution.

[0068] (2) adding alkaline protease to the whey protein solution prepared in step (1) at an enzyme-substrate ratio of 6%, and hydrolyzing the whey protein at pH 8 and 60° C. During the hydrolysis reaction, the pH value of the reaction system was maintained by adding 0.5 mol NaOH.

[0069] (3) adding flavor protease to the alkaline protease whey protein hydrolyzate of step (2) at an enzyme-substrate ratio of 4.8%, and hydrolyzing at pH 7 and 50° C. for 1.5 h. During the hydrolysis reaction, the pH value of the reaction system was maintained by adding 0.5 mol NaOH, the degree of hydrolysis was determined, and a hydrolysis curve was drawn to obtain a hydrolyzed sample with a degree of hydrolysis of 2.3%.

[0070] The degree of hydrolysis of whey protein hydrolysate was determined by OPA method. Figure 6 The results show that with the increase of substrate concentration, pH, compound ratio and temperature, the degree of hydrolysis increases first and then decreases. With the increase of hydrolysis time, the degree of hydrolysis also increases gradually, but the trend changes slowly.

[0071] The antigen inhibition rate of whey protein hydrolysate was determined by ELISA. Figure 6 As shown, the antigen inhibition rate showed a trend of first decreasing and then increasing, among which the enzyme compound ratio, pH and temperature had a more significant effect on the antigen inhibition rate, and the lowest antigen inhibition rate was 9.95%.

[0072] The bitterness of whey protein hydrolysate was evaluated by sensory scoring. Figure 7 As shown, the sensory score showed a trend of first increasing and then decreasing, with the highest score being 8.53, which was closest to the sensory score of untreated whey protein.

[0073] The bitterness and astringency of whey protein hydrolysate were measured by electronic tongue. Figure 8 As shown in the figure, bitterness and astringency are the main evaluation indicators, among which enzyme compound ratio, pH and temperature have a greater impact on the electronic tongue results. The minimum bitterness and astringency values ​​are closer to untreated whey protein.

[0074] Example 3 Ultrasonic pretreatment improves whey protein hydrolysate

[0075] Alkaline protease and flavor protease were used for enzymatic hydrolysis. Alkaline protease and flavor protease were compounded in a mass ratio of 1:0.8, the alkaline protease enzyme-substrate ratio was 6%, the whey protein was preheated at 60°C for 20 minutes, the pH value was adjusted to 8, the hydrolysis time was 2 hours, and the enzyme was inactivated at 100°C for 10 minutes after the enzymatic hydrolysis was completed. Then, the second step of enzymatic hydrolysis was carried out, the flavor protease enzyme-substrate ratio was 4.8%, pH 7, the enzymatic hydrolysis temperature was 50°C, the enzymatic hydrolysis time was 1.5 hours, and the enzyme was inactivated at 100°C for 10 minutes after the enzymatic hydrolysis was completed.

[0076] Ultrasonic pretreatment conditions were: 200 W, ice water bath, 15 min;

[0077] Under this condition, 5 mg / ml whey protein was ultrasonically pretreated and then enzymatically hydrolyzed according to the enzyme conditions in Example 3.

[0078] The antigen inhibition rate of whey protein hydrolysate was determined by ELISA. Lactoglobulin (β-lactoglobulin, β-LG) is one of the main allergens in whey protein, so the β-LG antigen inhibition rate is the main standard. Figure 6 As shown in the figure, ultrasound opens the structure of whey protein and exposes its internal allergenic epitopes, resulting in increased allergenicity, but this enables further enzymatic hydrolysis to destroy more allergenic epitopes, resulting in a more efficient and effective treatment method, which better reduces the allergenicity of whey protein. Compared with WPI, the antigen inhibition rate after ultrasound pretreatment is reduced by 85.26%. Therefore, ultrasound pretreatment is used to further develop whey protein hydrolysates with low allergenicity and excellent functional properties.

[0079] The molecular weight of whey protein hydrolysate and the degradation degree of small molecular peptides were determined by SDS electrophoresis. The small molecular weight part of whey protein hydrolyzed by enzyme was not degraded, while the protein above 10kDa in WPI pretreated by ultrasound was almost completely degraded.

[0080] Example 4 Optimization of compound enzyme hydrolysis conditions

[0081] The single factor conditions obtained in Example 3 were used for enzymatic hydrolysis, and three factors and three levels of conditions were optimized.

[0082] The enzymatic hydrolysis temperature is 45-55°C, the enzymatic hydrolysis pH is 6.5-7.5, and the alkaline protease:flavor protease=1:0.8.

[0083] Table 1 Example 4 enzymatic hydrolysis conditions and antigen inhibition rate, sensory score

[0084] Method for preparing low-allergenic, low-bitter whey protein

[0085] The ultrasonic time was 15 min, the ratio of alkaline protease to flavor protease was 10:8.1, the hydrolysis temperature was 51°C and the hydrolysis pH was 7.30. Under these optimal conditions, the actual antigen inhibition rate was 9.92% and the sensory score was 8.91 after three parallel tests.

[0086] In summary, the allergenicity of whey protein is reduced through ultrasonic pretreatment, and the bitterness of whey protein is relatively improved while the allergenicity is reduced through the combination of alkaline protease and flavor protease, thereby improving the applicability and palatability of whey protein.

Claims

1. A method for reducing the allergenicity and bitterness of whey protein by non-thermal processing assisted composite enzymatic hydrolysis, characterized in that: The following steps are involved: S1. Solution preparation: dissolving whey protein in ultrapure water and dispersing it evenly to obtain a whey protein solution; S2, pretreatment: subjecting the whey protein solution described in step S1 to ultrasonic pretreatment; S3. Single factor enzymatic hydrolysis experiment based on ultrasonic pretreatment: Single factor experiment to explore the selection of the optimal combined enzymatic hydrolysis conditions of alkaline protease and flavor protease; S4: Optimize enzymatic hydrolysis conditions: Optimize according to the optimal single factor conditions obtained in step S3.

2. The method according to claim 1, characterized in that In step S1, the concentration of the whey protein solution is 5% w / v.

3. The method according to claim 1, characterized in that In step S2, the ultrasonic treatment conditions are: ultrasonic power 200W, ultrasonic time: 5-20min.

4. The method according to claim 1, characterized in that In step S3, the ratio of alkaline protease to flavor protease is 1:0.5-1:0.

9.

5. The method according to claim 1, characterized in that: In step S3, the temperature is 40-60° C., pH is 6.5-8.5, substrate concentration is 3-11%, and time (30-150 min) is a single factor condition.

6. The method according to claim 1, characterized in that In step S4, the optimal conditions are selected as a compounding ratio of 1:0.7 to 1:0.9, a temperature of 45 to 55°C, and a pH of 6.5 to 7.

5.

7. A non-thermal processing assisted composite enzymatic hydrolysis method for reducing the allergenicity and bitterness of whey protein, which is achieved by the method according to any one of claims 1 to 6.

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