Method for reducing protein sensitization based on combination of protease hydrolysis pretreatment and amyloid fibril modification

Abstract

The invention discloses a method for reducing protein sensitization based on protease hydrolysis pretreatment combined with amyloid fibril modification, and belongs to the technical field of protein modification. In order to solve the technical problem that although enzyme hydrolysis can reduce sensitization of beta-lactoglobulin, sensory characteristics and functional attributes of protein can be damaged, the invention provides a method for preparing beta-lactoglobulin through hydrolysis, protease enzymolysis, enzyme deactivation, rapid cooling and centrifugation by taking beta-lactoglobulin as a raw material. According to the method for preparing the low-allergenicity beta-lactoglobulin by modifying the amyloid fibril, enzyme hydrolysis pretreatment and an amyloid fibril modification method are combined, so that the allergenicity of the beta-lactoglobulin is reduced, and meanwhile, the functional attributes of the beta-lactoglobulin are reserved. The low-sensitization beta-lactoglobulin prepared by the method can be used for preparing low-sensitization cow milk or milk powder and formula food with special medical application.

Description

Technical Field

[0001] The invention belongs to the technical field of protein modification, and in particular relates to a method for reducing protein allergenicity based on protease hydrolysis pretreatment combined with amyloid fibril modification. Background Art

[0002] Milk is rich in a variety of proteins, fats, carbohydrates, vitamins and minerals, and is widely used as the basic raw material for infant formula for deep processing. Infant formula plays an important role in the healthy growth of infants. Cow milk protein, especially whey protein, is the most important food base for the production of infant formula. However, food allergies caused by whey protein mainly occur in infants and children under 3 years old, which poses a serious threat to the life and health of infants. β-lactoglobulin (β-Lg) accounts for more than 50% of whey protein, but breast milk does not contain β-Lg. Therefore, β-Lg is the first exogenous protein encountered by infants and young children, and it is also the main allergen in whey protein. At present, although enzymatic hydrolysis is the most effective method to reduce the allergenicity of β-Lg, it will damage the sensory properties and functional properties of the protein. Therefore, it is urgent to find a method to reduce the allergenicity of β-Lg without damaging other properties.

[0003] Modification of protein amyloid fibrils refers to the process of protein aggregation after unfolding under specific conditions (strong acid, high temperature), which changes from the original natural conformation to an aggregated form in which β-folds are tightly arranged along the direction perpendicular to the axis. After the protein is modified by amyloid fibrils, hydrolysis and internal structural unit aggregation will occur, resulting in the destruction of its original conformation. Therefore, a variety of reactive functional groups appear on its surface, giving the protein new functional properties. Due to its superior nutritional, sensory and functional properties, food protein amyloid fibrils have gradually become a hot research topic in the field of protein modification. In addition, due to the acid hydrolysis, conformational rearrangement and aggregation of internal structural units that occur during the formation of amyloid fibrils, the conformational epitopes and linear epitopes will be destroyed. Therefore, it is a potential method to reduce the allergenicity of proteins.

[0004] Enzymatic hydrolysis is the most effective method to reduce the allergenicity of milk protein. It reduces the allergenicity of milk protein by destroying the allergenic epitopes of the protein. In recent years, enzymatic hydrolysis combined with other technologies has attracted widespread attention as a new modification method to reduce the allergenicity of protein and retain other properties to the maximum extent. Some studies have shown that amyloid fibrils formed after protein hydrolysis have better functional properties. However, current research focuses on functional properties such as antioxidant, emulsifying and foaming properties, and there is less research on reducing allergenicity. Summary of the invention

[0005] In order to solve the technical problem that although enzymatic hydrolysis can reduce the allergenicity of β-Lg, it will damage the sensory characteristics and functional properties of the protein, the present invention combines enzymatic hydrolysis pretreatment with an amyloid fibril modification method, explores the allergenicity and other functional properties of β-Lg obtained by enzymatic hydrolysis pretreatment and amyloid fibril modification in sequence, and obtains a method based on protease hydrolysis pretreatment combined with amyloid fibril modification to reduce the allergenicity of β-Lg while improving the emulsification stability of the protein.

[0006] In order to solve the above technical problems and achieve corresponding technical effects, the present invention specifically provides the following technical solutions:

[0007] The first object of the present invention is to provide a method for reducing protein allergenicity based on protease hydrolysis pretreatment combined with amyloid fibril modification. The method uses β-lactoglobulin as a raw material, undergoes hydrolysis, protease hydrolysis, enzyme inactivation, rapid cooling, and centrifugation to obtain a supernatant, and then the supernatant is subjected to amyloid fibril modification to obtain low-allergenic β-lactoglobulin; the amyloid fibril modification is to adjust the pH of the supernatant to 1.8-2.5, store it at 4°C overnight, and then heat it in a magnetic stirring water bath at 80-90°C for 10-12h, and then take it out and cool it rapidly.

[0008] In one embodiment of the present invention, 6 mol / L HCl is used to adjust the pH of the supernatant.

[0009] In one embodiment of the present invention, the hydrolysis is performed by mixing β-lactoglobulin with PBS solution at a solid-liquid ratio of 1 g:100 mL, and then placing the mixture in a water bath at 50-60° C. for 15-30 min.

[0010] In one embodiment of the present invention, the pH of the PBS solution is 7.4.

[0011] In one embodiment of the present invention, the protease is flavor protease.

[0012] In one embodiment of the present invention, when the protease is flavor protease, the enzymatic hydrolysis pH is 6.8-7.5, the enzymatic hydrolysis temperature is 50-60° C., and the protein hydrolysis degree reaches 10%.

[0013] In one embodiment of the present invention, the added amount of the protease is 1% of the amount of β-lactoglobulin.

[0014] In one embodiment of the present invention, the enzyme is inactivated at 90° C. in a water bath for 10 min.

[0015] In one embodiment of the present invention, the centrifugation is performed at 4°C and 6000 rpm for 20 min.

[0016] The second object of the present invention is to provide low-allergenic β-lactoglobulin prepared by the above method.

[0017] The third object of the present invention is to provide the use of the above-mentioned low-allergenic β-lactoglobulin in the preparation of low-allergenic milk or milk powder.

[0018] The fourth object of the present invention is to provide the use of the above-mentioned low-allergenic β-lactoglobulin in the preparation of special medical formula foods.

[0019] Beneficial effects of the present invention:

[0020] The present invention combines enzymatic hydrolysis pretreatment with an amyloid fibril modification method, and explores the sensitization and emulsification stability of β-Lg obtained by enzymatic hydrolysis pretreatment and amyloid fibril modification in sequence. The results show that compared with β-Lg obtained by only enzymatic hydrolysis pretreatment or only amyloid fibril modification, the β-Lg obtained by the method of the present invention has significantly reduced sensitization and significantly improved emulsification stability, which reduces sensitization and improves the functional properties of the protein. The low-allergenic β-lactoglobulin prepared by the present invention can be used for the preparation of low-allergenic cow's milk or milk powder and special medical formula foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a graph showing the effect of different heating times on the ThT fluorescence intensity of FH1β-Lg and Fβ-Lg obtained during the modification of amyloid fibrils;

[0022] Figure 2 This is the result of SDS-PAGE detection of the effect of different heating times on the molecular weight of Fβ-Lg and FH1β-Lg obtained during the modification of amyloid fibrils; Figure 2 (a) is the result of SDS-PAGE detection of the effect of different heating times on the molecular weight of Fβ-Lg obtained during the modification of amyloid fibrils. Figure 2 (b) is the result of SDS-PAGE detection of the effect of different heating times on the molecular weight of FH1β-Lg obtained during the modification of amyloid fibrils;

[0023] Figure 3 Fourier transform infrared spectra of β-Lg, H1β-Lg, Fβ-Lg and FH1β-Lg;

[0024] Figure 4 is the fitting spectrum of the amide I band of β-Lg, H1β-Lg, Fβ-Lg and FH1β-Lg; wherein, Figure 4 (a) is the fitting spectrum of the amide I band of β-Lg. Figure 4 (b) is the fitting spectrum of the amide I band of H1β-Lg. Figure 4 (c) is the fitting spectrum of the amide I band of Fβ-Lg. Figure 4 (d) in the figure is the fitting spectrum of the amide I band of FH1β-Lg;

[0025] Figure 5 It is the secondary structure content diagram of β-Lg, H1β-Lg, Fβ-Lg and FH1β-Lg proteins;

[0026] Figure 6 It is the UV-visible absorption spectra of β-Lg, H1β-Lg, Fβ-Lg and FH1β-Lg;

[0027] Figure 7 Fluorescence spectra of protein tertiary structure detection of β-Lg, H1β-Lg, Fβ-Lg and FH1β-Lg;

[0028] Figure 8 Fluorescence spectra of surface hydrophobicity detection of β-Lg, H1β-Lg, Fβ-Lg and FH1β-Lg;

[0029] Fig. 9 The results of indirect competitive enzyme-linked immunosorbent assay for detecting the binding rates of β-Lg, H1β-Lg, Fβ-Lg and FH1β-Lg with IgE;

[0030] Fig.10 This is a graph showing the test results of the emulsification stability of β-Lg, H1β-Lg, Fβ-Lg and FH1β-Lg. DETAILED DESCRIPTION

[0031] The present invention is further described in detail below in conjunction with specific examples and drawings. The following examples are convenient for better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The drugs, reagents, and materials used in the following examples can be purchased through commercial channels unless otherwise specified.

[0032] Example 1: A method for reducing protein allergenicity based on flavor protease hydrolysis pretreatment combined with amyloid fibril modification

[0033] Weigh 100 mg of β-Lg and dissolve it in 10 mL of PBS solution (pH 7.4), hydrolyze in a 55°C magnetic stirring water bath for 20 min; after the hydrolysis is completed, add 1 mg of flavor protease to the β-Lg solution, adjust the solution pH to 7.0, and perform enzymolysis at 55°C; after enzymolysis for 75 min (the hydrolysis degree of the hydrolyzate is determined by the o-phthalaldehyde method, and the time when the hydrolysis degree reaches 10% is recorded), place the solution in a 90°C water bath to inactivate the enzyme for 10 min, and then quickly cool it with ice water; centrifuge at 4°C, 6000rpm for 20 min, and the supernatant obtained is the β-Lg hydrolyzate; use 6 mol / L HCl to adjust the β-Lg hydrolyzate to pH 2.0, and store it at 4°C overnight; then place it in a 85°C magnetic stirring water bath and heat it for 12 h, and quickly cool it in an ice bath to obtain a solution containing low-allergenic β-Lg, which is freeze-dried and the obtained β-Lg is recorded as FH1β-Lg and stored at -20°C.

[0034] Comparative Example 1: A method for reducing protein allergenicity based on flavor protease hydrolysis

[0035] Weigh 100 mg of β-Lg and dissolve it in 10 mL of PBS solution (pH 7.4), and hydrolyze it in a 55°C magnetic stirring water bath for 20 minutes; after the hydrolysis is completed, add 1 mg of flavor protease to the β-Lg solution, adjust the solution pH to 7.0, and perform enzymolysis at 55°C; after 75 minutes of enzymolysis (the degree of hydrolysis of the hydrolyzed product is determined by the o-phthalaldehyde method, and the time when the degree of hydrolysis reaches 10% is recorded), place the solution in a 90°C water bath to inactivate the enzyme for 10 minutes, and then quickly cool it with ice water; centrifuge it at 4°C, 6000rpm for 20 minutes, and the supernatant obtained is the β-Lg hydrolyzate. The β-Lg hydrolyzate is freeze-dried, and the obtained hydrolyzate is recorded as H1β-Lg, which is stored at -20°C for later use.

[0036] Comparative Example 2:

[0037] 100 mg of β-Lg was weighed and dissolved in 10 mL of PBS solution (pH 7.4), and hydrolyzed in a 55°C magnetic stirring water bath for 20 min to obtain a β-Lg hydrolyzate; the β-Lg hydrolyzate was adjusted to pH 2.0 using 6 mol / L HCl and stored at 4°C overnight; then, it was heated in a 85°C magnetic stirring water bath for 12 h, quickly cooled in an ice bath, and freeze-dried. The obtained protein was recorded as Fβ-Lg and stored at -20°C.

[0038] (1) Determining the heating time during the modification of amyloid fibrils by detecting the ThT fluorescence intensity

[0039] The present invention prepares FH1β-Lg and Fβ-Lg by the methods of Example 1 and Comparative Example 2, respectively, and detects the effect of different heating times on the ThT fluorescence intensity of the obtained FH1β-Lg and Fβ-Lg during the modification of amyloid fibrils.

[0040] The specific detection method of ThT fluorescence intensity is as follows:

[0041] Dissolve 8 mg of thiazole orange (ThT) in 10 mL of PBS solution (pH 7.4) to prepare a ThT stock solution. Filter the ThT stock solution using a 0.22 μm water filter and store it at 4°C in the dark. Before use, dilute the stock solution 50 times to prepare a ThT working solution. Mix 40 μL of sample (10 mg / mL) with 4 mL of ThT working solution and react for 1 min in the dark. Use a fluorescence spectrophotometer to measure the ThT fluorescence intensity of FH1β-Lg and Fβ-Lg at an excitation wavelength of 440 nm and an emission wavelength of 482 nm, respectively.

[0042] Protein amyloid fibrils are characterized by a common cross-β structure (building block) at the molecular level, and pairs of β-sheets are intertwined through side chains to form a specific "three-dimensional zipper" arrangement. ThT fluorescence is a standard for detecting the formation of amyloid fibrils, mainly observing the fluorescence intensity of the surface binding sites of the cross-β structure. In the present invention, the fluorescence intensity changes with the heating time and increases during the formation of amyloid fibrils (such as Figure 1 As shown). As the heating time increases, the ThT fluorescence intensity of Fβ-Lg increases rapidly. In the last 10 to 12 hours of incubation, the increase in fluorescence intensity slows down, indicating that the amyloid fibrilization of Fβ-Lg begins to enter a plateau phase. The ThT fluorescence intensity of FH1β-Lg also increases with the extension of heating time, but the increase is small. This is because the conformation of FH1β-Lg is destroyed after protease hydrolysis and a smaller peptide is formed. The degree of amyloid fibrilization is small and the degree of binding with ThT is low, resulting in a lower fluorescence intensity. Therefore, according to the ThT fluorescence intensity detection results, it can be determined that the effect of amyloid fibril modification is better when the heating time during the amyloid fibril modification process is 10-12 hours.

[0043] (2) SDS-PAGE detection to determine the heating time during the modification of amyloid fibrils

[0044] The molecular weights of Fβ-Lg and FH1β-Lg prepared by the methods described in Comparative Example 2 and Example 1 were analyzed using SDS-PAGE experiments, and the effects of different heating times during the modification of amyloid fibrils on the molecular weights of the obtained Fβ-Lg and FH1β-Lg were analyzed.

[0045] The SDS-PAGE detection method is as follows:

[0046] The gel was prepared according to the instructions of the SDS-PAGE kit, and the thickness of the vertical gel was 1.5 mm. The gel consisted of 4% acrylamide stacking gel and 15% acrylamide separation gel. The sample (1 mg / mL) and 5× protein loading buffer were mixed in equal volumes and heated in a boiling water bath for 5 min. The sample loading volume was 10 μL. The initial voltage was set to 80 V, and it was increased to 120 V after the stained band entered the separation gel. The band stopped when it ran to 0.5 cm from the edge, and it was stained for 30 min and then destained.

[0047] Figure 2 The results of SDS-PAGE detection of the effect of different heating times on the molecular weight of Fβ-Lg and FH1β-Lg obtained during the modification of amyloid fibrils. Figure 2 As shown in A, the band of Fβ-Lg is located near the molecular weight of 18.4 kDa. Figure 2 A and Figure 2 B in the figure shows that the band gradually becomes lighter as the heating time increases, and the band of FH1β-Lg becomes lighter than that of Fβ-Lg due to enzymatic hydrolysis. Since the self-assembly process of amyloid fibrils does not involve irreversible strong chemical bonds, the band pattern of amyloid fibrils after reaction is not much different. It is worth noting that Fβ-Lg shows a darker band than FH1β-Lg at 12h, indicating that Fβ-Lg is more fibrillized than FH1β-Lg. The SDS-PAGE results are consistent with the ThT fluorescence intensity results.

[0048] (3) Fourier transform infrared spectroscopy was used to detect the contents of each secondary structure in the β-Lg, H1β-Lg, Fβ-Lg and FH1β-Lg protein digests.

[0049] The specific method of Fourier transform infrared spectroscopy is as follows: referring to the protein and potassium bromide tablet method, a small amount of β-Lg, H1β-Lg, Fβ-Lg and FH1β-Lg dry samples are mixed and pressed with potassium bromide, and then full-wavelength scanning is performed using a Fourier transform infrared spectrometer, and the measurement wavelength is selected from 400 to 4000 cm -1 , scanning resolution is 4cm -1 , 64 scans, wave number accuracy 0.01cm -1 Origin Pro 8.5 software was used to collect spectra and mark characteristic peaks. Peakfit Version 4.12 software was used to perform baseline correction within the corresponding spectral band, perform function smoothing and deconvolution, and finally fit the second-order derivative curve multiple times. The content of each secondary structure in the protein digest was calculated based on the peak area of ​​each secondary structure unit.

[0050] Proteins are biological macromolecules that are connected by amino acid peptide bonds and have a specific structure. The secondary structure is at a wave number of 1600-1700 cm -1 The Fourier transform infrared spectroscopy (FT-IR) results of the protein are shown in Figure 3 The obtained curve was processed by second-order derivative IR deconvolution and spectrum fitting using PeakFit software to obtain the amide I band spectrum of the modified protein, as shown in Figure 4 According to the sub-peak position obtained after deconvolution, the integrated area is calculated to analyze the secondary structure components and obtain the quantitative information of the secondary structure of the protein, that is, β-folding corresponds to 1610-1640 cm -1 ; Irregular curl is 1640~1650cm -1 ; α-helix is ​​1650~1658cm -1 ; β-angle is 1658~1700cm -1 .

[0051] Depend on Figure 5 It can be seen that the secondary structure of β-Lg is mainly composed of β-fold and β-turn. The relative content of β-turn in the secondary structure of H1β-Lg increased to 49.31±0.24%. The β-fold content of H1β-Lg increased, and the irregular curl disappeared in the secondary structure of H1β-Lg. This is because under the action of proteases, the hydrogen bonds and electrostatic interactions that stabilize the secondary structure of the protein change, and at the same time, the types of amino acids and peptide chain structures of the protein change, thereby affecting the spatial conformation of the protein, causing the secondary structure of β-Lg to change. The relative content of β-fold of Fβ-Lg and FH1β-Lg increased significantly compared with β-Lg and H1β-Lg, respectively (p<0.05). This is because the modification of amyloid fibrils will change the protein from its original natural conformation to an aggregated form in which β-folds are tightly arranged along the direction perpendicular to the axis, so the relative content of β-fold of β-Lg modified by amyloid fibrils will increase.

[0052] (4) UV-visible absorption spectroscopy was used to detect the optical activity of β-Lg, H1β-Lg, Fβ-Lg and FH1β-Lg. The specific method of UV-visible absorption spectroscopy was as follows: β-Lg, H1β-Lg, Fβ-Lg and FH1β-Lg were diluted to 0.1 mg / mL with PBS (pH 2.0), and the samples were added to a quartz cuvette with an optical path length of 1 cm. The scanning wavelength range was set to 190-400 nm, the slit width was 2 nm, and the scanning rate was 100 nm / min. PBS (pH 2.0) should be used for baseline correction before detection.

[0053] The UV-visible absorption spectrum can reflect the optical activity information of proteins at 270-340nm. The characteristic absorption peaks of the conjugated double bonds of tryptophan and tyrosine residues are located at 280nm. If the position or height of such peaks changes after enzymatic hydrolysis and amyloid fibril modification, it can be explained that the microenvironment of aromatic amino acid residues has changed, and the effects of the two treatment methods on proteins can be further inferred. Figure 6 It can be seen that the absorbance of H1β-Lg at 280nm is lower than that of the original protein, accompanied by a slight red shift. The absorbance of Fβ-Lg is lower than that of the original protein, accompanied by a blue shift. The absorbance of FH1β-Lg is lower than that of H1β-Lg, and a blue shift or red shift occurs. It is not difficult to see that enzymatic hydrolysis and amyloid fibril modification have a certain effect on β-Lg. The changes in protein structure caused by enzymatic hydrolysis and amyloid fibril modification have a certain loosening effect on the protein structure, so that the tryptophan and tyrosine chromophores exposed to the outside are covered, and the ultraviolet absorption intensity is weakened, indicating that the amino acid environment shifts to a more hydrophilic environment.

[0054] (5) Fluorescence spectroscopy was used to detect the polarity changes of β-Lg, H1β-Lg, Fβ-Lg and FH1β-Lg

[0055] The fluorescence spectroscopy method is as follows: β-Lg, H1β-Lg, Fβ-Lg and FH1β-Lg were diluted to 0.1 mg / mL with PBS (pH 2.0), respectively. The fluorescence spectrophotometer was used for determination, and the specific measurement conditions were an excitation wavelength of 280 nm, a scanning emission wavelength range of 300-450 nm, a slit width of 5 nm for both excitation and emission, and a scanning speed of 200 nm / min.

[0056] Fluorescence spectroscopy can reflect the polarity changes in the microenvironment of tryptophan (Trp) and tyrosine (Tyr) residues in the aromatic amino group located in the hydrophobic region of the protein. This method is usually used to monitor changes in the tertiary structure of proteins. The fluorescence intensity of β-Lg is mainly based on tryptophan, and the changes in the protein microenvironment can be determined by detecting tryptophan residues. Figure 7 It can be seen that the fluorescence intensity of H1β-Lg increased significantly compared with β-Lg, accompanied by a blue shift, and the fluorescence spectrum of Fβ-Lg underwent the same change. This result shows that enzymatic hydrolysis and amyloid fibril modification caused the destruction of the globular structure of β-Lg, exposing the Trp residues and increasing the fluorescence intensity. The fluorescence intensity of FH1β-Lg was significantly lower than that of H1β-Lg, and a blue shift or red shift occurred, indicating that amyloid fibril modification caused the protein structure of enzymatic hydrolysis to aggregate, masking the Trp residues.

[0057] (6) Detection of the surface hydrophobicity of β-Lg, H1β-Lg, Fβ-Lg and FH1β-Lg by fluorescence spectroscopy

[0058] The specific detection method of surface hydrophobicity is as follows: weigh 23.95 mg ANS and dissolve it in 10 mL PBS (pH 7.4) to prepare 8 mmol / L ANS stock solution, and store it in a brown centrifuge tube in the dark. Dilute β-Lg, H1β-Lg, Fβ-Lg and FH1β-Lg with PBS (pH 2.0) to 0.1 mg / mL, then take 4 mL of the diluted solution and mix it with 20 μL ANS stock solution, place it in the dark at room temperature for 15 minutes, and use a 1 cm quartz cuvette to detect its fluorescence intensity under a fluorescence spectrophotometer. The specific parameters are: excitation wavelength 380 nm, scan at 200 nm / min in the emission wavelength range of 400-650 nm, and the excitation and emission slit widths are both 5 nm.

[0059] The hydrophobicity of the protein surface can reflect the changes in the hydrophobic groups on the protein surface. The hydrophobic interaction between the non-polar side chain groups of the amino acid residues of the protein plays an important role in the conformational stability of its tertiary structure. Changes in the tertiary structure of the protein often change the surface hydrophobicity of the protein. Therefore, the surface hydrophobicity value is often used to evaluate the changes in the tertiary structure of the protein. Figure 8 As shown in the figure, the surface hydrophobicity of H1β-Lg increased significantly, which may be because the enzymatic hydrolysis destroyed the structure of β-Lg, causing the original structure to open and the protein molecules to unfold, exposing the hydrophobic groups originally embedded in the lactosphere molecules, thereby increasing the surface hydrophobicity. The surface hydrophobicity of Fβ-Lg and FH1β-Lg is higher than that of β-Lg and H1β-Lg. The change of hydrophobic groups is related to their participation in the formation of protein amyloid fibrils. When protein amyloid fibrils are formed, the interaction of internal hydrophobic groups can promote the formation of β-folding. When the amyloid fibril conformation is reorganized, the extended peptide may expose more hydrophobic areas, thereby increasing the surface hydrophobicity.

[0060] (7) Indirect competitive enzyme-linked immunosorbent assay to detect the sensitization of H1β-Lg, Fβ-Lg and FH1β-Lg

[0061] Indirect competitive enzyme-linked immunosorbent assay was used to detect the binding ability of H1β-Lg, Fβ-Lg and FH1β-Lg with IgE. The specific method is as follows:

[0062] H1β-Lg, Fβ-Lg and FH1β-Lg were diluted to 5μg / mL with coating solution (0.05mol / L sodium bicarbonate buffer, pH 9.6), and 100μL was coated in each well of a 96-well high adsorption ELISA plate at 4°C overnight. Pour the coating solution in the ELISA plate, add 300μL of PBST washing solution to each well, shake and wash for 5min, wash three times, add 250μL gelatin (3%, w / v) to each well to block the residual binding sites, and incubate in a 37°C incubator for 1h. Pour the blocking solution and wash three times, and add 100μL of diluted serum to each well of the blocked ELISA plate. After incubation at 37°C for 1h, wash, pat dry, add 100μL of diluted HRP-sheep anti-human IgE to each well, and incubate at 37°C for 1h. After washing 4 times, add 100 μL of single-component TMB colorimetric solution to each well and color at 37°C in the dark for 15 min. Add 50 μL of 2 mol / L H2SO4 to each well to terminate the reaction. Place the ELISA plate in an ELISA reader and read the absorbance at 450 nm.

[0063] Depend on Fig. 9 It can be seen that compared with untreated β-lactoglobulin, H1β-Lg has a lower ability to bind to IgE, which can reduce the allergenicity of the protein. The binding ability of Fβ-Lg to IgE is also greatly reduced, which is 72.98±1.75%, and it has a better effect in reducing allergenicity than H1β-Lg. This is because the modification of amyloid fibrils causes the protein to hydrolyze and aggregate its internal structural units, resulting in the destruction of its allergenic epitopes. The degree of binding of FH1β-Lg to IgE is significantly lower than that of H1β-Lg (p<0.05), from 90.36±2.99% to 70.99±0.60%, and it has a better effect in reducing allergenicity than Fβ-Lg.

[0064] (8) Emulsion stability test of β-Lg, H1β-Lg, Fβ-Lg and FH1β-Lg

[0065] The protein sample (1 mg / mL) and olive oil were mixed in a ratio of 3:1 (v / v) and homogenized at 14000 rpm for 60 s using an IKA high-speed disperser. 0.05 mL of the bottom solution was taken at 0 min and 10 min, respectively, and diluted to 5 mL with 0.1% (w / v) SDS solution, and the absorbance was measured at 500 nm. The calculation formula for the emulsion stability (ESI) value is as follows:

[0066] ESI (%) = (A0 × 10) / (A0 - A 10 )×100

[0067] In the formula, A0 and A 10 are the absorbance values ​​at 0 min and 10 min respectively.

[0068] Fig.10 The results of the protein emulsification stability test are shown. It can be seen from the figure that the emulsification stability of H1β-Lg is significantly improved compared with the original protein, and the emulsification stability of Fβ-Lg is lower than that of the original protein. However, the emulsification stability of FH1β-Lg obtained by enzymatic hydrolysis pretreatment and amyloid fibril modification is significantly improved compared with the original protein, and is significantly higher than H1β-Lg obtained only by enzymatic hydrolysis treatment.

[0069] Based on the results of indirect competitive enzyme-linked immunosorbent assay for sensitization and emulsification stability, it can be seen that the method of the present invention combines enzymatic hydrolysis pretreatment with amyloid fibril modification, which not only reduces the sensitization of β-Lg, but also improves the emulsification stability of the protein.

[0070] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A method for reducing protein allergenicity based on protease hydrolysis pretreatment combined with amyloid fibril modification, characterized in that: Beta-lactoglobulin is used as a raw material, and is subjected to hydrolysis, protease hydrolysis, enzyme inactivation, rapid cooling, and centrifugation to obtain a supernatant, which is then subjected to amyloid fibril modification to obtain low-allergenic beta-lactoglobulin; the amyloid fibril modification comprises adjusting the pH of the supernatant to 1.8-2.5, storing it at 4°C overnight, heating it in a magnetic stirring water bath at 80-90°C for 10-12 hours, and rapidly cooling it after taking it out.

2. The method according to claim 1, characterized in that The hydrolysis is performed by mixing the β-lactoglobulin with the PBS solution at a solid-liquid ratio of 1 g:100 mL, and placing the mixture in a water bath at 50-60° C. for 15-30 minutes.

3. The method according to claim 1, characterized in that The protease is flavor protease.

4. The method according to claim 3, characterized in that: The protease enzymatic hydrolysis pH is 6.8-7.5, the enzymatic hydrolysis temperature is 50-60° C., and the protein hydrolysis degree reaches 10%.

5. The method according to claim 3, characterized in that: The added amount of the flavor protease is 1% of the amount of β-lactoglobulin.

6. The method according to claim 1, characterized in that The enzyme inactivation is carried out at 90° C. in a water bath for 10 min.

7. The method according to claim 1, characterized in that The centrifugation was carried out at 4°C and 6000 rpm for 20 min.

8. Hypoallergenic β-lactoglobulin prepared by the method according to any one of claims 1 to 7.

9. Use of the hypoallergenic β-lactoglobulin according to claim 8 in the preparation of hypoallergenic cow's milk or milk powder.

10. Use of the low-allergenic β-lactoglobulin according to claim 8 in the preparation of special medical purpose formula foods.

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