Preparation method of low-sensitization hydrolyzed whey protein nano-scale microcapsule powder

Abstract

The invention relates to the technical field of food, in particular to a preparation method of low-sensitization hydrolyzed whey protein nanoscale microcapsule powder, which comprises the following steps: S1, preparing a whey protein solution, performing microfiltration, and treating with cysteine hydrochloride; s2, trypsin hydrolysis; s3, inactivating and cooling; s4, adding chymotrypsin, and synchronously applying ultrasonic waves; s5, bromelain is added, ultrafiltration is performed, and peptide fragments are collected; s6, preparing a capsule wall solution; s7, adopting a micro-fluidic chip to form a nano emulsion; s8, the mixture is added into a CaCl2 solution to be stirred, multi-layer self-assembly is conducted, and micro-capsule particles are obtained; and S9, carrying out ultrahigh-pressure micro-jet homogenization, vacuum freeze drying and low-temperature plasma assisted grinding on the micro-capsule particles to obtain the nano-scale micro-capsule powder. The stability of active ingredients is maintained through multi-enzyme cooperation and ultrasonic-assisted efficient elimination of allergens, multi-layer nano-embedding under microfluidic control and low-temperature plasma grinding, and the limitations of low single enzymolysis efficiency, insufficient embedding rate and thermal denaturation risk in the prior art are overcome.

Description

Technical Field

[0001] The present application relates to the field of food technology, and in particular to a method for preparing low-allergenic hydrolyzed whey protein nano-microcapsule powder. Background Art

[0002] Allergic reactions caused by cow's milk are also a hot topic in the field of food safety and biomedicine. α-Lactalbumin (ALA) is one of the important allergens in cow's milk. Although bovine ALA has about 75% homology with human ALA, bovine ALA is still highly allergenic. Therefore, how to reduce the allergenicity of ALA has become one of the key scientific issues being tackled at home and abroad.

[0003] Whey is a by-product of cheese production. The whey protein contained in whey is rich in nutrition and has high nutritional value. It is known as the king of protein. It has a good promoting effect on the growth and development of infants and young children and is widely used in processed foods. However, some people will have allergic reactions due to consuming whey protein. This is mainly because the β-lactoglobulin (β-lactoglobulin, β-LG) and α-lactoglobulin (α-lactoglobulin, α-LA) contained in whey protein contain potential allergens. At present, the treatment of cow milk allergy is relatively rare. If the allergy is confirmed, most of them can only be prevented by completely avoiding food. However, always avoiding the intake of cow milk and its products may lead to malnutrition and even affect the growth of infants and young children. Therefore, it is very necessary to study appropriate whey protein modification technology to degrade these allergenic whey proteins. Among the many protein modification technologies, enzymatic modification is more mature. Enzymes catalyze the hydrolysis of macromolecular proteins into small molecular peptides and amino acids, destroying the linear or spatial conformation of allergen proteins, thereby significantly reducing their allergenicity. Moreover, it has the advantages of mild action conditions, economy, and the production of functional new peptides. At present, most of my country's low-allergenic formula milk powders are imported from abroad, and there are few low-allergenic dairy products on the market in China.

[0004] In addition, the whey protein powder currently on the market is directly spray-dried. During spray drying and storage, the peptides may refold due to hydrophobic interactions, exposing allergenic epitopes and increasing the risk of allergic reactions. Summary of the invention

[0005] In view of the above-mentioned shortcomings of the prior art, the purpose of the present application is to provide a method for preparing low-allergenic hydrolyzed whey protein nano-capsule powder, which adopts a three-in-one technical method of step-by-step enzymatic hydrolysis-microfluidic embedding-low-temperature nano-ization to obtain uniform and stable low-allergenic hydrolyzed whey protein nano-capsule powder.

[0006] To achieve the above objectives, this application is implemented through the following technical solutions: The preparation method of low-allergenic hydrolyzed whey protein nano-capsule powder comprises the following steps: S1: The whey protein is prepared into a solution, sterilized by microfiltration, and then pretreated by adding cysteine ​​hydrochloride; S2: adding trypsin to the pretreated whey protein solution for hydrolysis under alkaline conditions to obtain a first enzymatic hydrolyzate; S3: After heating and inactivating the first enzymatic hydrolysate, cooling it; S4: adjusting the first enzymatic hydrolysate after inactivation and cooling to be acidic, adding chymotrypsin, and simultaneously applying ultrasonic assistance to perform hydrolysis to obtain a second enzymatic hydrolysate; S5: After adding bromelain to the second enzymatic hydrolysate for treatment, the low-allergenic peptides <3 kDa are collected through an ultrafiltration system; S6: preparing sodium alginate solution and chitosan solution; S7: using a microfluidic chip, forming a W / O / W type nanoemulsion in the cross-linking area of ​​the chip, wherein: the inner phase is the hydrolyzed peptide solution obtained in step S5, the middle phase is a sodium alginate solution; and the outer phase is a chitosan solution; S8: adding the nanoemulsion into the CaCl2 solution, performing magnetic stirring, and achieving multilayer self-assembly of the capsule wall by zeta potential regulation to obtain microcapsule particles; S9: The microcapsule particles are homogenized by ultra-high pressure microfluidization, vacuum freeze-dried, and low-temperature plasma-assisted grinding to obtain nano-scale microcapsule powder.

[0007] Preferably, the amount of cysteine ​​hydrochloride used in step S1 is 0.05-0.1% of the whey protein mass.

[0008] Preferably, in step S2, the mass ratio of trypsin to whey protein is (0.8-1.5):50, the hydrolysis temperature is 40-45° C., and the hydrolysis time is 40-80 min.

[0009] Preferably, in step S4, the mass ratio of chymotrypsin to whey protein is (0.8-1.5):30, the ultrasonic frequency is 30-45 kHz, the hydrolysis temperature is 35-40° C., and the hydrolysis time is 70-110 min.

[0010] Preferably, in step S5, the mass ratio of bromelain to whey protein is (0.9-1.2):100, the treatment temperature is 35-40° C., and the treatment time is 40-60 min.

[0011] Preferably, lactoferrin and epigallocatechin gallate are added to the sodium alginate solution in step S6, the mass percentage concentration of the sodium alginate solution is 1-2%, the mass percentage concentration of the lactoferrin and epigallocatechin gallate are both 0.1-0.3%, and the mass percentage concentration of the chitosan solution is 0.6-1%.

[0012] Preferably, in step S7, maltodextrin is added to the hydrolyzed peptide solution at a mass percentage concentration of 3-6%, the flow rate of the sodium alginate solution is 0.6-1 mL / min, and the flow rate of the chitosan solution is 2-3 mL / min.

[0013] Preferably, the molar concentration of the CaCl2 solution in step S8 is 0.08-0.12 mol / L, and the magnetic stirring time is 20-40 min.

[0014] Preferably, the pressure used in the ultra-high pressure microfluidization homogenization in step S9 is 180-220 MPa, and a pre-freezing treatment is performed before vacuum freeze drying, and the pre-freezing treatment temperature is -60--90°C.

[0015] Preferably, the low-temperature plasma-assisted grinding is performed in a liquid nitrogen environment, and low-temperature argon plasma is injected simultaneously.

[0016] Compared with the known public technologies, the technical solution provided by this application has the following beneficial effects: (1) The preparation method of the low-allergenic hydrolyzed whey protein nano-capsule powder of the present application, through a three-in-one technical method of step-by-step enzymatic hydrolysis-microfluidic encapsulation-low-temperature nano-crushing, that is, a technical method of efficient allergen elimination assisted by multi-enzyme synergy and ultrasound-assisted multi-layer nano-encapsulation under microfluidic precise control-low-temperature plasma grinding to maintain the stability of active ingredients, surpasses the limitations of the existing low-allergenic whey protein preparation method of low single enzymatic hydrolysis efficiency, insufficient encapsulation rate, and thermal denaturation risk; (2) This application adopts two-stage enzymatic hydrolysis and combines it with ultrasound assistance to improve the hydrolysis efficiency and reduce allergens. Bromelain is additionally added before ultrafiltration to utilize its ability to specifically cut hydrophobic regions to further eliminate allergen fragments that have not been completely degraded, thereby ensuring the purity of the low-allergenic peptide segment of less than 3 kDa; (3) A W / O / W nanoemulsion was generated by using a microfluidic chip, and the electrostatic self-assembly of a conjugate of sodium alginate, lactoferrin and epigallocatechin gallate with chitosan was combined to improve the encapsulation rate and achieve the stable construction of a multilayer capsule wall. The sodium alginate layer was solidified by a CaCl2 solution, and the cationic properties of chitosan were used to achieve charge reversal to form a dense capsule wall, which significantly improved the mechanical strength and thermal stability of the microcapsules. (4) Microfluidization homogenization under high pressure, combined with ice crystal template formed by pre-freezing treatment, and vacuum freeze drying retain the porous nanostructure and avoid particle aggregation caused by spray drying; (5) Plasma-assisted cryogenic grinding was used in a liquid nitrogen environment, and argon plasma was simultaneously injected to inhibit thermal denaturation of the protein. The synergistic effect of low temperature and plasma maintained the biological activity of the peptide fragments, avoiding the problem of local temperature rise that is easily caused by traditional grinding. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0018] Figure 1 It is the particle size distribution diagram of Example 3, Comparative Example 6 and Comparative Example 8 of the present invention; Figure 2 It is an electron microscope image of Comparative Example 6 of the present invention; Figure 3 It is an electron microscope image of Example 3 of the present invention. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0020] The raw materials and sources used in the examples and comparative examples of the present invention are as follows: Whey protein isolate WPI purity 93.9% New Zealand Fonterra; Cysteine ​​hydrochloride monohydrate purity 99% Shanghai Yuanye Biotechnology; Trypsin 200u / mg Shanghai Yuanye Biotechnology; Chymotrypsin 1500u / mg Shanghai Yuanye Biotechnology; Bromelain 600u / mg Shanghai Yuanye Biotechnology; Sodium alginate purity 98% Shanghai Yuanye Biotechnology; Chitosan deacetylation degree 90% Shanghai Yuanye Biotechnology; Lactoferrin purity 95% Shanghai Yuanye Biotechnology; Epigallocatechin gallate purity 98% Shanghai Yuanye Biotechnology; CaCl2 purity ≥93% Shanghai Yuanye Biotechnology; Example 1 The preparation method of low-allergenic hydrolyzed whey protein nano-microcapsule powder comprises: S1: Whey protein isolate WPI was prepared into a 10% solution, sterilized by 0.22 μm microfiltration, and then pretreated with 0.1% cysteine ​​hydrochloride at 45°C for 20 min to break the disulfide bonds through sulfhydryl reduction, unfold the globular structure of the allergenic protein, and expose the hidden enzyme cleavage site; S2: adding trypsin to the pretreated whey protein solution for hydrolysis at a pH of 8.0 and a temperature of 45° C. for 80 min, with a mass ratio of trypsin to whey protein of 1.5:50, to obtain a first enzymatic hydrolyzate; S3: Instantly heat the first enzymatic hydrolysate to 60°C and maintain for 3 min to inactivate it, then immediately cool it to 35°C; S4: adjusting the pH of the first enzymatic hydrolysate that was inactivated and cooled to 35° C. to 5.5, adding chymotrypsin, the mass ratio of chymotrypsin to whey protein being 1.5:30, and simultaneously applying 45 kHz ultrasonic wave to assist hydrolysis for 110 min to obtain a second enzymatic hydrolysate; S5: adjusting the pH of the second enzymatic hydrolysate to 7.0, adding bromelain for treatment, the mass ratio of bromelain to whey protein is 1.2:100, the treatment time is 60 min, and the ultrafiltration system is used to intercept the macromolecular peptides and collect the low-allergenic peptides <3 kDa; S6: prepare a 2% sodium alginate solution, add 0.3% lactoferrin and epigallocatechin gallate to the sodium alginate solution, and prepare a 1% chitosan solution; S7: using a microfluidic chip with a channel diameter of 200 μm, forming a W / O / W type nanoemulsion in the cross-linking area of ​​the chip, wherein: the inner phase is the hydrolyzed peptide solution obtained in step S5, 6% maltodextrin is added to the hydrolyzed peptide solution as a freeze-drying protective agent, the middle phase is a sodium alginate solution, and the flow rate is 1 mL / min; the outer phase is a chitosan solution, and the flow rate is 3 mL / min; S8: adding the nanoemulsion to a CaCl2 solution with a molar concentration of 0.12 mol / L, and magnetically stirring for 30 min, and then controlling the zeta potential (-30 mV→+15 mV) to achieve multilayer self-assembly of the capsule wall to obtain microcapsule particles; S9: The microcapsule particles were homogenized by 220MPa ultrahigh pressure microfluidization (3 cycles), pre-frozen to -90°C to form an ice crystal template, and then vacuum freeze-dried; ground in a liquid nitrogen environment using a medium grinder (zirconia beads, 0.1mm in diameter), and simultaneously injected with low-temperature argon plasma to prevent thermal denaturation of the protein to obtain nanoscale microcapsule powder.

[0021] Example 2 The preparation method of low-allergenic hydrolyzed whey protein nano-microcapsule powder comprises: S1: Whey protein isolate WPI was prepared into a 6% solution, sterilized by 0.22 μm microfiltration, and then pretreated with 0.05% cysteine ​​hydrochloride at 40°C for 15 min to break the disulfide bonds through sulfhydryl reduction, unfold the globular structure of the allergenic protein, and expose the hidden enzyme cleavage site; S2: adding trypsin to the pretreated whey protein solution for hydrolysis at a pH of 8.0 and a temperature of 40° C. for 40 min, with a mass ratio of trypsin to whey protein of 0.8:50, to obtain a first enzymatic hydrolyzate; S3: Instantly heat the first enzymatic hydrolysate to 60°C and maintain for 3 min to inactivate it, then immediately cool it to 40°C; S4: adjusting the pH of the first enzymatic hydrolysate which is inactivated and cooled to 40° C. to 5.5, adding chymotrypsin, wherein the mass ratio of chymotrypsin to whey protein is 0.8:30, and simultaneously applying 30 kHz ultrasonic wave to assist hydrolysis for 70 min to obtain a second enzymatic hydrolysate; S5: adjusting the pH of the second enzymatic hydrolysate to 7.0, adding bromelain for treatment, the mass ratio of bromelain to whey protein is 0.9:100, the treatment time is 20 min, and the ultrafiltration system is used to intercept the macromolecular peptides and collect the low-allergenic peptides <3 kDa; S6: prepare a 1% sodium alginate solution, add 0.1% lactoferrin and epigallocatechin gallate to the sodium alginate solution, and prepare a 0.6% chitosan solution; S7: using a microfluidic chip with a channel diameter of 200 μm, forming a W / O / W type nanoemulsion in the cross-linking area of ​​the chip, wherein: the inner phase is the hydrolyzed peptide solution obtained in step S5, 3% maltodextrin is added to the hydrolyzed peptide solution as a freeze-drying protective agent, the middle phase is a sodium alginate solution, and the flow rate is 0.6 mL / min; the outer phase is a chitosan solution, and the flow rate is 2 mL / min; S8: adding the nanoemulsion to a CaCl2 solution with a molar concentration of 0.08 mol / L, and magnetically stirring for 40 min, and then controlling the zeta potential (-30 mV→+15 mV) to achieve multilayer self-assembly of the capsule wall to obtain microcapsule particles; S9: The microcapsule particles were homogenized by 180MPa ultra-high pressure microfluidization (3 cycles), pre-frozen to -60°C to form an ice crystal template, and then vacuum freeze-dried; ground in a liquid nitrogen environment using a medium grinder (zirconia beads, 0.1mm in diameter), and simultaneously injected with low-temperature argon plasma to prevent thermal denaturation of the protein to obtain nano-scale microcapsule powder.

[0022] Example 3 The preparation method of low-allergenic hydrolyzed whey protein nano-microcapsule powder comprises: S1: Whey protein isolate WPI was prepared into an 8% solution, sterilized by 0.22 μm microfiltration, and then pretreated with 0.1% cysteine ​​hydrochloride at 50°C for 15 min to break the disulfide bonds through sulfhydryl reduction, unfold the globular structure of the allergenic protein, and expose the hidden enzyme cleavage site; S2: adding trypsin to the pretreated whey protein solution for hydrolysis at a pH of 8.0 and a temperature of 45° C. for 60 min, with a mass ratio of trypsin to whey protein of 1:50, to obtain a first enzymatic hydrolyzate; S3: Instantly heat the first enzymatic hydrolysate to 60°C and maintain for 3 min to inactivate it, then immediately cool it to 37°C; S4: adjusting the pH of the first enzymatic hydrolysate that has been inactivated and cooled to 37° C. to 5.5, adding chymotrypsin, wherein the mass ratio of chymotrypsin to whey protein is 130, and simultaneously applying 40 kHz ultrasonic wave to assist hydrolysis for 90 minutes to obtain a second enzymatic hydrolysate; S5: adjusting the pH of the second enzymatic hydrolysate to 7.0, adding bromelain for treatment, the mass ratio of bromelain to whey protein is 1:100, the treatment time is 60 min, and the ultrafiltration system is used to intercept the macromolecular peptides and collect the low-allergenic peptides <3 kDa; S6: preparing a 1.5% sodium alginate solution, adding 0.2% lactoferrin and epigallocatechin gallate to the sodium alginate solution, and preparing a 0.8% chitosan solution; S7: using a microfluidic chip with a channel diameter of 200 μm, forming a W / O / W type nanoemulsion in the cross-linking area of ​​the chip, wherein: the inner phase is the hydrolyzed peptide solution obtained in step S5, 5% maltodextrin is added to the hydrolyzed peptide solution as a freeze-drying protective agent, the middle phase is a sodium alginate solution, and the flow rate is 0.8 mL / min; the outer phase is a chitosan solution, and the flow rate is 2.5 mL / min; S8: adding the nanoemulsion to a CaCl2 solution with a molar concentration of 0.1 mol / L, and magnetically stirring for 30 min, and then controlling the zeta potential (-30 mV→+15 mV) to achieve multilayer self-assembly of the capsule wall to obtain microcapsule particles; S9: The microcapsule particles were homogenized by 200MPa ultrahigh pressure microfluidization (3 cycles), pre-frozen to -80℃ to form an ice crystal template, and then vacuum freeze-dried; ground in a liquid nitrogen environment using a medium grinder (zirconia beads, 0.1mm in diameter), and simultaneously injected with low-temperature argon plasma to prevent thermal denaturation of the protein to obtain nano-scale microcapsule powder.

[0023] Comparative Example 1 The difference between this comparative example and Example 3 is that trypsin is used for single enzyme hydrolysis, and the specific steps are as follows: S1: Whey protein isolate WPI was prepared into an 8% solution, sterilized by 0.22 μm microfiltration, and then pretreated with 0.1% cysteine ​​hydrochloride at 50°C for 15 min to break the disulfide bonds through sulfhydryl reduction, unfold the globular structure of the allergenic protein, and expose the hidden enzyme cleavage site; S2: adding trypsin to the pretreated whey protein solution for hydrolysis at a pH of 8.0 and a temperature of 45° C. for 60 min, with a mass ratio of trypsin to whey protein of 1:50, to obtain a first enzymatic hydrolyzate; S3: Instantly heat the first enzymatic hydrolysate to 60°C and maintain for 3 min to inactivate it, then immediately cool it to 37°C; S4: adjusting the pH of the first enzymatic hydrolysate to 4.2, adding bromelain for treatment, the mass ratio of bromelain to whey protein is 1:100, the treatment time is 30 min, passing through a 10 kDa membrane ultrafiltration system, intercepting large molecular peptides, and collecting low-allergenic peptides <3 kDa; S6: preparing a 1.5% sodium alginate solution, adding 0.2% lactoferrin and epigallocatechin gallate to the sodium alginate solution, and preparing a 0.8% chitosan solution; S7: using a microfluidic chip with a channel diameter of 200 μm, forming a W / O / W type nanoemulsion in the cross-linking area of ​​the chip, wherein: the inner phase is the hydrolyzed peptide solution obtained in step S5, 5% maltodextrin is added to the hydrolyzed peptide solution as a freeze-drying protective agent, the middle phase is a sodium alginate solution, and the flow rate is 0.8 mL / min; the outer phase is a chitosan solution, and the flow rate is 2.5 mL / min; S8: adding the nanoemulsion to a CaCl2 solution with a molar concentration of 0.1 mol / L, and magnetically stirring for 30 min, and then controlling the zeta potential (-30 mV→+15 mV) to achieve multilayer self-assembly of the capsule wall to obtain microcapsule particles; S9: The microcapsule particles were homogenized by 200MPa ultrahigh pressure microfluidization (3 cycles), pre-frozen to -80℃ to form an ice crystal template, and then vacuum freeze-dried; ground in a liquid nitrogen environment using a medium grinder (zirconia beads, 0.1mm in diameter), and simultaneously injected with low-temperature argon plasma to prevent thermal denaturation of the protein to obtain nano-scale microcapsule powder.

[0024] Comparative Example 2 The difference between this comparative example and Example 3 is that the enzymatic hydrolysis method in the patent with publication number CN113789359A is used for enzymatic hydrolysis, and the specific steps include: S1: Whey protein isolate WPI was prepared into an 8% solution, sterilized by 0.22 μm microfiltration, and then pretreated with 0.1% cysteine ​​hydrochloride at 50°C for 15 min to break the disulfide bonds through sulfhydryl reduction, unfold the globular structure of the allergenic protein, and expose the hidden enzyme cleavage site; S2: adding pepsin to the pretreated whey protein solution for hydrolysis at a pH of 2.0 and a temperature of 37° C. for 60 min, with a mass ratio of pepsin to whey protein of 1:50, to obtain a first enzymatic hydrolyzate; S3: adjusting the pH of the first enzymatic hydrolysate to 7.0, adding trypsin at a temperature of 37°C, with a mass ratio of trypsin to whey protein of 1:25, and simultaneously applying 40kHz ultrasonic wave-assisted hydrolysis for 60 minutes to obtain a second enzymatic hydrolysate; S5: adjusting the pH of the second enzymatic hydrolysate to 4.2, adding bromelain for treatment, the mass ratio of bromelain to whey protein is 1:100, the treatment time is 30 min, passing through a 10 kDa membrane ultrafiltration system, intercepting large molecular peptides, and collecting low-allergenic peptides <3 kDa; S6: preparing a 1.5% sodium alginate solution, adding 0.2% lactoferrin and epigallocatechin gallate to the sodium alginate solution, and preparing a 0.8% chitosan solution; S7: using a microfluidic chip with a channel diameter of 200 μm, forming a W / O / W type nanoemulsion in the cross-linking area of ​​the chip, wherein: the inner phase is the hydrolyzed peptide solution obtained in step S5, 5% maltodextrin is added to the hydrolyzed peptide solution as a freeze-drying protective agent, the middle phase is a sodium alginate solution, and the flow rate is 0.8 mL / min; the outer phase is a chitosan solution, and the flow rate is 2.5 mL / min; S8: adding the nanoemulsion to a CaCl2 solution with a molar concentration of 0.1 mol / L, and magnetically stirring for 30 min, and then controlling the zeta potential (-30 mV→+15 mV) to achieve multilayer self-assembly of the capsule wall to obtain microcapsule particles; S9: The microcapsule particles were homogenized by 200MPa ultrahigh pressure microfluidization (3 cycles), pre-frozen to -80℃ to form an ice crystal template, and then vacuum freeze-dried; ground in a liquid nitrogen environment using a medium grinder (zirconia beads, 0.1mm in diameter), and simultaneously injected with low-temperature argon plasma to prevent thermal denaturation of the protein to obtain nano-scale microcapsule powder.

[0025] Comparative Example 3 The difference between this comparative example and Example 3 is that cysteine ​​hydrochloride is not added for pretreatment in step S1.

[0026] Comparative Example 4 The difference between this comparative example and Example 3 is that no ultrasonic treatment is performed in step S4.

[0027] Comparative Example 5 The difference between this comparative example and Example 3 is that in step S5, bromelain is not added to the second enzymatic hydrolysate for treatment.

[0028] Comparative Example 6 The difference between this comparative example and Example 3 is that the treatment of steps S6-S9 is not performed, and the low-allergenic peptide segment solution of <3 kDa prepared in S5 is spray dried to obtain a low-allergenic hydrolyzed whey protein powder sample. The spray drying uses an atomizing nozzle with a diameter of 0.5 mm, an air inlet temperature of 180 ± 5 °C, an air outlet temperature of 90 ± 5 °C, and a sample flow rate of 5 mL / min.

[0029] Comparative Example 7 A 100 g / L whey protein solution was prepared in phosphate buffer solution (PBS) and adjusted to the optimal pH value for alkaline protease. The solution was heated to stabilize the temperature to 50 °C, 7.16 g / L alkaline protease was added, and the solution was stirred and hydrolyzed for 5.6 h. After the hydrolysis was completed, the solution was heated to 100 °C and maintained for 20 min to inactivate the protease in the system. After the hydrolysis was completed, the solution was quickly cooled and the enzymatic hydrolysis product was spray dried to obtain a low-allergenic hydrolyzed whey protein powder sample.

[0030] Comparative Example 8 The difference between this comparative example and Example 3 is that in step S9, low-temperature plasma-assisted grinding treatment is not performed, but a low-allergenic hydrolyzed whey protein powder sample is obtained after vacuum freeze-drying.

[0031] Testing items and methods Determination of degree of hydrolysis The OPA method was used to determine the degree of hydrolysis of whey protein using a UV spectrophotometer. L-serine (0.1 g / L) was used as the standard. 400 μL of 0.1 mg / mL of the sample to be tested was added to a test tube containing 3 mL of L OPA solution, shaken and mixed for 5 seconds, and reacted in the dark for 2 minutes. Deionized water was used as a blank control. The absorbance was measured at 340 nm. The degree of hydrolysis was calculated according to the following formula (1): (1) Where: OD sa Indicates the absorbance of the sample; OD b Indicates the absorbance of the blank control; OD st represents the absorbance of the standard; X represents the sample weight, g; P represents the protein content in the sample; 0.1 represents the sample volume converted to L.

[0032] Detection of residual antigenicity of β-LG and α-LA Antigen coating: Prepare 2.5 μg / mL of purified bovine β-LG or α-LA with coating buffer, add 100 μL to each well of a 96-well plate, and coat at 4°C for 12 h; Blocking: Wash the plate, pat dry, and repeat the operation three times to absorb the unadsorbed coated antigen. Then, add 1% gelatin blocking solution to each well, 250 μL per well, and incubate in a 37°C incubator for 1 hour to obtain the prepared solid phase antibody.

[0033] Antigen-antibody reaction: dilute the standard with different concentration gradients, add 50μL of the standard with different concentrations to each well, add 40μL of the sample diluent and 10μL of the sample to be tested to the sample well, shake evenly, add HRP enzyme-labeled antibody to each well, set a blank well, seal the plate with a sealing film, and place it in a 37℃ constant temperature box for 60 minutes; Washing and color development: discard the liquid and spin dry. Wash the plate with washing solution, repeat several times and pat dry. Add 50μL of color developer carbamide peroxide to each well, then add 50μL of color developer TMB solution, shake gently and evenly, and color develop at 37℃ in the dark for 15 minutes; Reaction termination and measurement: Add 50 μL of stop solution to each well. Then adjust the blank well to zero and use an enzyme-labeled instrument to measure the absorbance of each sample at a wavelength of 450 nm. A standard curve is drawn based on the standard substance, and the content of the allergenic protein in the sample is quantified using the standard curve. The residual antigenicity is calculated according to the following formula (2).

[0034] (2) Where: R represents residual antigenicity, %; A xIt represents the allergenic protein content of the measured sample, g / 100g; A0 represents the allergenic protein content of the unhydrolyzed whey protein isolate, g / 100g.

[0035] 3. Hydrolysis degree / sensitization after digestion in the stomach and gastrointestinal tract In vitro simulated gastrointestinal digestion, preheat the sample protein and pepsin solution (pH2) at 37 °C for 10 min, the ratio of pepsin to protein concentration is 182:1 (units / mg protein), and mix the sample and pepsin solution. Simulate gastric digestion in a water bath at 37 °C, and the digestion time is 60 and 120 min, respectively. After the reaction time is up, quickly add an appropriate amount of 1 mol / L NaHCO3 solution to adjust the pH to 6.5 to terminate the reaction; Take the sample digestion solution after 120 minutes of gastric digestion, add trypsin solution (pH 6.5) and mix evenly, and continue gastrointestinal digestion. The concentration ratio of trypsin to gastric digestion sample is 35:1 (units / mg protein), and the digestion time is 60 and 120 minutes respectively. After the reaction time is up, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride is quickly added to make the final concentration of 4mmol / L to terminate the intestinal digestion.

[0036] The samples after gastric digestion and gastrointestinal digestion were tested for hydrolysis degree, residual antigenicity of β-LG and α-LA.

[0037] The test results of the degree of hydrolysis are shown in Table 1, and the test results of the residual antigenicity of β-LG and α-LA are shown in Tables 2 and 3.

[0038] Table 1 Degree of hydrolysis of whey protein powder prepared in Examples 1-3 and Comparative Examples 1-8

[0039] From Example 3 and Comparative Example 6, it can be concluded that due to the encapsulation of peptides during the microencapsulation process, the contact between the reagent and the peptide is affected, resulting in a low degree of hydrolysis measured in Example 3. When entering the gastric juice, the microencapsulated structure (sodium alginate / chitosan) remains stable in gastric acid (pH 2.0), the peptide release rate is low, and the further hydrolysis of the internal peptide by pepsin is limited. However, when entering the intestinal juice, the microcapsules dissolve at the intestinal pH, releasing the peptides, and pancreatic enzymes further degrade the residual macromolecular peptides, and the degree of hydrolysis is greatly improved. Comparative Example 6 is not protected by microencapsulation, and the peptides may aggregate due to high-temperature spray drying, resulting in a decrease in the actual effective degree of hydrolysis. The low degree of hydrolysis in the gastrointestinal tract may be due to the aggregation of peptides during digestion, resulting in a decrease in the degree of hydrolysis in the gastrointestinal tract.

[0040] In Comparative Example 1, single trypsin hydrolysis was performed without the use of chymotrypsin and ultrasound assistance, and the hydrolysis efficiency was low; in Comparative Example 2, pepsin + trypsin was used for hydrolysis, and pepsin preferentially cleaved the hydrophobic region under acidic conditions (pH 2.0), but the solubility of whey protein in an acidic environment was low, resulting in a decrease in the degree of hydrolysis. Comparative Example 3 lacked cysteine ​​hydrochloride to reduce disulfide bonds, the globular structure of the sensitizing protein was not fully unfolded, and the enzyme cleavage site was insufficiently exposed, resulting in a decrease in the degree of hydrolysis. Comparative Example 4 was not ultrasonically treated, which reduced the contact efficiency between the enzyme and the substrate, resulting in a decrease in the degree of hydrolysis. Comparative Example 5 lacked further cleavage of the hydrophobic peptide segment by bromelain, and the degree of hydrolysis decreased. Comparative Example 7 was hydrolyzed by alkaline protease alone, and the degree of hydrolysis was low. Comparative Example 8 was not subjected to low-temperature plasma-assisted grinding treatment, but was spray-dried after vacuum freezing, with a larger particle size, slow peptide release, and a decrease in the degree of hydrolysis.

[0041] Table 2 β-LG residual antigenicity of whey protein powder prepared in Examples 1-3 and Comparative Examples 1-8

[0042] Table 3 Residual α-LA antigenicity of whey protein powder prepared in Examples 1-3 and Comparative Examples 1-8

[0043] It can be concluded from Table 2 and Table 3 that the present invention prepares hydrolyzed whey protein nano-microcapsule powder by step-by-step enzymatic hydrolysis, ultrasonic assistance, ultrafiltration and microcapsule encapsulation. The residual antigenicity of β-LG is 2.97-3.98 after gastrointestinal digestion, and the residual antigenicity of α-LA is 0.84-1.87 after gastrointestinal digestion, and the sensitization is significantly reduced. Comparative Example 1 uses a single trypsin hydrolysis to completely destroy the sensitizing epitope, resulting in a high residual antigenicity. Comparative Example 2 uses pepsin and trypsin for enzymatic hydrolysis. Pepsin cannot effectively destroy the disulfide bond structure of β-LG under acidic conditions, and the residual antigenicity is high. Comparative Example 3 does not use cysteine ​​hydrochloride for pretreatment, the globular structure of the sensitizing protein is not fully unfolded, and the enzyme cleavage site is insufficiently exposed. The hidden sensitizing epitope is not destroyed, and the residual antigenicity is high. Comparative Example 4 has not been ultrasonically treated, which reduces the contact efficiency between the enzyme and the substrate, and the residual antigenicity is high. Comparative Example 5 does not use bromelain, resulting in some sensitizing peptides not being degraded, and the residual antigenicity is high. Comparative Example 6 was not microencapsulated and directly spray-dried, resulting in the peptide segment being exposed to the environment and possibly recombining the sensitizing epitope, and the residual antigenicity was high. Comparative Example 7 used alkaline protease single hydrolysis method to completely degrade the sensitizing protein, and the residual antigenicity was high. In addition, the degree of hydrolysis is also an important factor affecting the residual antigenicity. The hydrolysis degrees of Comparative Examples 1-8 in the gastrointestinal tract are far lower than those of the hydrolyzed whey protein nano-microcapsule powder prepared by the present invention, so the residual antigenicity is high.

[0044] 4. Particle size analysis The particle size and particle size distribution were characterized using the Mastersizer 2000 laser particle size analyzer from Malvern Instruments. Figure 1 .from Figure 1 It can be seen that the particle size distribution of Example 3 of the present application is uniform, while the particle size of Comparative Example 8, which has not been subjected to low-temperature plasma assisted grinding treatment, is small; Comparative Example 6 has not been subjected to microencapsulation treatment and is directly spray-dried, so the particle size is larger, and the particle size distribution is messy and uneven, with two peaks.

[0045] 5. Microscopic morphology The whey protein powder was subjected to electron microscopic analysis, and the electron microscopic images of Comparative Example 6 and Example 3 are as follows: Figure 2 , Figure 3 .from Figure 2 , Figure 3 It can be seen that the surface of the product prepared in Comparative Example 6 without microencapsulation treatment and directly spray-dried is concave, rough or even cracked, and cannot present a standard spherical shape. The surface of the product prepared in Example 3 of the present invention is flat and smooth, presenting a uniform and stable spherical structure.

[0046] From the above test and result, it can be concluded that the preparation method of the low allergenic hydrolyzed whey protein nano-scale microcapsule powder of the present invention generally adopts single enzyme or compound enzyme continuous hydrolysis relative to the prior art, adopts staged use of trypsin and chymotrypsin, combined with ultrasonic assistance, significantly improves the efficiency of allergen elimination, and adds bromelain before ultrafiltration, and utilizes its ability to specifically cut hydrophobic regions to further eliminate allergen fragments that are not completely degraded. Adopt glass microfluidic chip to generate W / O / W type nanoemulsion, combine the electrostatic self-assembly of sodium alginate-lactoferrin conjugate and chitosan, improve the embedding rate, realize the stable construction of multilayer capsule wall, solidify the sodium alginate layer by CaCl2 solution, and realize charge reversal by the cationic property of chitosan, form dense capsule wall, and improve the mechanical strength and thermal stability of microcapsule. Carry out circulation homogenization under ultrahigh pressure, make particle size distribution more uniform. Combined with the ice crystal template formed by pre-freezing, vacuum freeze drying retains porous nanostructure, avoids the aggregation of particles caused by spray drying, and grinds in liquid nitrogen environment at the same time, and synchronously injects argon plasma to suppress protein thermal denaturation. Compared with the defect of traditional grinding that easily leads to local temperature rise, the present invention maintains the biological activity of the peptide segment through the synergistic effect of low temperature and plasma.

[0047] The above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, a person skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. Such modifications or replacements will not cause the essence of the corresponding technical solutions to deviate from the protection scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for preparing low-allergenic hydrolyzed whey protein nano-capsule powder, characterized in that: The following steps are involved: S1: The whey protein is prepared into a solution, sterilized by microfiltration, and then pretreated by adding cysteine ​​hydrochloride; S2: adding trypsin to the pretreated whey protein solution for hydrolysis under alkaline conditions to obtain a first enzymatic hydrolyzate; S3: After heating and inactivating the first enzymatic hydrolysate, cooling it; S4: adjusting the first enzymatic hydrolysate after inactivation and cooling to be acidic, adding chymotrypsin, and simultaneously applying ultrasonic assistance to perform hydrolysis to obtain a second enzymatic hydrolysate; S5: After adding bromelain to the second enzymatic hydrolysate for treatment, the low-allergenic peptides <3 kDa are collected through an ultrafiltration system; S6: preparing sodium alginate solution and chitosan solution; S7: using a microfluidic chip, forming a W / O / W type nanoemulsion in the cross-linking area of ​​the chip, wherein: the inner phase is the hydrolyzed peptide solution obtained in step S5, the middle phase is a sodium alginate solution; and the outer phase is a chitosan solution; S8: adding the nanoemulsion into the CaCl2 solution, performing magnetic stirring, and achieving multilayer self-assembly of the capsule wall by zeta potential regulation to obtain microcapsule particles; S9: The microcapsule particles are homogenized by ultra-high pressure microfluidization, vacuum freeze-dried, and low-temperature plasma-assisted grinding to obtain nano-scale microcapsule powder.

2. The method for preparing the low-allergenic hydrolyzed whey protein nano-capsule powder according to claim 1, characterized in that: The amount of cysteine ​​hydrochloride used in step S1 is 0.05-0.1% of the whey protein mass.

3. The method for preparing the low-allergenic hydrolyzed whey protein nano-capsule powder according to claim 1, characterized in that: In step S2, the mass ratio of trypsin to whey protein is (0.8-1.5):50, the hydrolysis temperature is 40-45° C., and the hydrolysis time is 40-80 min.

4. The method for preparing the low-allergenic hydrolyzed whey protein nano-capsule powder according to claim 3, characterized in that: In step S4, the mass ratio of chymotrypsin to whey protein is (0.8-1.5):30, the ultrasonic frequency is 30-45 kHz, the hydrolysis temperature is 35-40° C., and the hydrolysis time is 70-110 min.

5. The method for preparing the low-allergenic hydrolyzed whey protein nano-capsule powder according to claim 3, characterized in that: In step S5, the mass ratio of bromelain to whey protein is (0.9-1.2):100, the treatment temperature is 35-40° C., and the treatment time is 40-60 min.

6. The method for preparing the low-allergenic hydrolyzed whey protein nano-capsule powder according to claim 1, characterized in that: In step S6, lactoferrin and epigallocatechin gallate are added to the sodium alginate solution, the mass percentage concentration of the sodium alginate solution is 1-2%, the mass percentage concentration of the lactoferrin and epigallocatechin gallate are both 0.1-0.3%, and the mass percentage concentration of the chitosan solution is 0.6-1%.

7. The method for preparing the low-allergenic hydrolyzed whey protein nano-capsule powder according to claim 6, characterized in that: In step S7, maltodextrin with a mass percentage concentration of 3-6% is added to the hydrolyzed peptide solution, the flow rate of the sodium alginate solution is 0.6-1 mL / min, and the flow rate of the chitosan solution is 2-3 mL / min.

8. The method for preparing the low-allergenic hydrolyzed whey protein nano-capsule powder according to claim 1, characterized in that: The molar concentration of the CaCl2 solution in step S8 is 0.08-0.12 mol / L, and the magnetic stirring time is 20-40 min.

9. The method for preparing the low-allergenic hydrolyzed whey protein nano-capsule powder according to claim 1, characterized in that: The pressure used for ultra-high pressure microfluidization homogenization in step S9 is 180-220 MPa, and a pre-freezing treatment is performed before vacuum freeze drying, and the pre-freezing treatment temperature is -60--90°C.

10. The method for preparing the low-allergenic hydrolyzed whey protein nano-capsule powder according to claim 9, characterized in that: The low-temperature plasma-assisted grinding is performed in a liquid nitrogen environment, and low-temperature argon plasma is injected simultaneously.

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