Method for enhancing targeted degradation of major epitopes of alphas1-CN through choline chloride modified papain

Abstract

The invention provides a targeted degradation method for improving main epitopes of alpha s1-CN, which comprises the following steps: directionally modifying 82 # tyrosine and 83 # arginine residues of papain in a loop2 region through choline dihydrogen citrate, and enhancing the surface electrostatic potential of a catalytic triad through a Loop2-Loop1-alpha 3-alpha 2 path, so as to improve the main epitopes of alpha s1-CN. According to the present invention, the indole plane of the Trp177 at the inlet of the substrate binding channel is regulated and controlled to be converted from the horizontal direction to the vertical direction so as to form the narrow substrate binding channel, and the binomial probability logarithmic ratio of the papain to the tyrosine residue hydrolysis selectivity is improved from 1.4 to 8.7, and the preference to the tyrosine residue is improved by 6.15 times; therefore, the targeted degradation capacity of the papain on the main epitope Y154-T174 of alpha s1-CN is improved, and the binding capacities of hydrolysate of the papain and IgE and IgG are respectively reduced to 5.36% and 8.55% from 7.28% and 12.42%. According to the papain modified by the dihydrocholine citrate, the degradation of the papain on main epitopes of alphas1-CN is enhanced, and meanwhile, the function loss of the papain is reduced as much as possible.

Description

Technical Field

[0001] The present application relates to the field of protein and enzyme engineering technology, and in particular to a method for enhancing the targeted degradation of the main epitope of αs1-CN by modifying papain with choline chloride. Background Art

[0002] At present, proteolytic enzymes play an important role in the food industry, such as tender meat, beverage production, dairy processing, and functional food development. Moreover, proteases are of great significance in improving the nutritional value and solubility of food and improving the metabolic process. It is estimated that the protease market will reach US$3.12 billion in 2024, of which the dairy enzyme market is estimated to be US$730 million ( DS, Trends Food Sci. Tech., 2022).

[0003] The enzyme and substrate (8 amino acid residues, where the N-terminal direction of the cleavage site is P4-P1, and the C-terminal direction of the cleavage site is named P1'-P4') are combined by using the pre-existing conformation through the induced fit effect (Fernández-Lucas, J. Trends Food Sci Technol, 2017). Usually, proteases usually have significant specificity for homologous substrates, but some enzymes also show weak catalytic ability for non-homologous substrates. These unstable low catalytic functions are secondary functions of enzymes, also known as enzyme catalytic promiscuity, with continuous characteristics ranging from narrow (specific) to broad (universal) substrate preference. The existence of enzyme promiscuity greatly weakens its ability to hydrolyze specificity.

[0004] Milk protein is considered to be an important source of antihypertensive peptides. However, in traditional food processing, such as the preparation of bioactive peptides, functional peptides are usually prepared at the cost of high energy consumption, time and high hydrolysis degree. For example, Espejo-Carpio FJ, Int Dairy J, 2013) used subtilisin, trypsin and their combined enzymatic hydrolysis technology to hydrolyze milk protein at 50°C for 3 hours. The ACE inhibition rate of the hydrolyzate was 33% to 64%. After component separation and interception, the Ic50 of the smallest molecular weight component of the composite enzymatic hydrolysis was the best at 5.12ug / ml, but its hydrolysis degree was as high as about 32%. The smallest molecular weight component with a high hydrolysis degree is bound to contain bitter peptides, thus producing a bad taste. Based on this, domestic and foreign scholars began to study the technology of directional preparation of functional peptides, such as Lin K, J Agri Food Chem, 2018) based on the fact that casein is rich in aromatic amino acid residues, and the thermophilic protease-alkaline protease step-by-step enzymatic hydrolysis technology (50℃→70℃, 2h) was established by computer simulation of enzyme cutting. The ACE inhibitory activity Ic50 of the component less than 3kDa was 2.63ug / ml. Although the processing time was reduced to a certain extent, its hydrolysis degree was still very high (28.9%). In addition, Mazorra-Manzano, TC, (Food Chem. 2013) reported that whey protein was basically completely degraded in the crude extract of melon enzyme from the crude enzymes isolated from citrus flowers, silver leaf bricks and muskmelon. Among them, the hydrolyzate less than 10kDa could inhibit the angiotensin-converting enzyme by 90%, but the technology took up to 24h, indicating that the activity of the enzyme needs to be improved.

[0005] In addition, infant formula based on milk protein is the first food source for non-breastfed infants. However, the prevalence of infant food allergies caused by cow milk protein in my country is as high as 6%, and milk protein has become the main food allergen for infants (Luo J. China CDC Weekly, 2022). Deeply hydrolyzed formula is mainly recommended for infants with milk allergies, but some studies have reported that infants who consume casein-based deeply hydrolyzed formula have significant growth retardation. It is speculated that deep hydrolysis seriously destroys the casein structure and causes functional loss (Hoppe C,. Eur J Clin Nutr, 2009); thus, deep hydrolysis and reduction of milk protein antigenicity are contradictory to its functional performance, and targeted reduction is the key to solving this problem.

[0006] This shows that although combined enzymatic hydrolysis can improve the efficiency of enzymatic hydrolysis to a certain extent to increase the content of functional peptides or reduce the ability of protein antigenicity, its disadvantage is that it will bring bitterness with the increase of hydrolysis degree; and the selection of proteolytic enzymes with enzyme specificity not only has a large workload, but also has problems such as low enzyme activity. Therefore, the specificity of protease hydrolysis has become the main technical bottleneck restricting the production of high-economic value products such as biological functional active peptides and low-allergenic formula milk powder in the food industry. Improving the hydrolysis specificity of proteolytic enzymes has important significance and social and economic benefits.

[0007] At present, deeply hydrolyzed formula milk powder may cause growth retardation in infants and young children. The main reason is that the current low-allergenic infant formula food is a highly hydrolyzed casein hydrolyzate at the expense of the functional activity of milk protein. The main reason for this situation is that the current proteolytic enzyme has a low ability to target and degrade the main allergic epitopes of milk protein. Summary of the invention

[0008] The present application provides a method for enhancing the targeted degradation of the main epitope of αs1-CN by modifying papain with choline chloride, so as to solve the problem that the current proteolytic enzymes have low targeted degradation ability for the main allergic epitopes of milk protein, and achieves that the papain modified with choline dihydrogen citrate can enhance its degradation of the main epitope of αs1-CN, while minimizing its loss of function.

[0009] The present application provides a method for enhancing the targeted degradation of the main epitope of αs1-CN by modifying papain with choline chloride, comprising the following steps:

[0010] A. First, we modified papain with a charge-adjustable two-component choline-based ionic liquid based on molecular dynamics simulation to achieve the free energy remodeling of the papain conformational landscape.

[0011] B. The commercial papain purified by ammonium sulfate was placed in a 0-1M choline-based ionic liquid solution at 37-42°C for 0-60 min, and the effect of the choline-based ionic liquid on the structure of papain was characterized experimentally by Fourier transform infrared spectroscopy;

[0012] C. hydrolyzing casein with the papain modified with choline dihydrogen citrate in step B to prepare casein hydrolysate, and analyzing the effect of choline dihydrogen citrate modification on the catalytic specificity of papain by combined LC-MS / MS technology;

[0013] D. The results of step C are displayed by multiple fingerprints and combined with serological evaluation to evaluate the targeted degradation ability of papain modified with choline dihydrogen citrate on the main antigen epitope of αs1-CN;

[0014] E. The casein hydrolysate hydrolyzed by choline dihydrogen citrate modified papain in step D is ultrafiltered, and the components less than 10 kDa are freeze-dried and used in mouse animal experiments to verify the biological functionality of the hydrolysate.

[0015] Preferably, the choline dihydrogen citrate in step C and step D can directionally modify the 83 tyrosine + 82 arginine residues of papain.

[0016] Preferably, the papain modified with 0.4M dihydrogen citrate choline in step E is added to a 4% casein solution at an enzyme to substrate ratio of 1:3000-1:2000, reacted at pH 6-7, 37-42°C for 30-60 min, and after the reaction is completed, the enzyme is inactivated at 85°C-95°C for 5-10 min, and the supernatant is obtained by low-temperature centrifugation.

[0017] Preferably, the degree of hydrolysis of the casein hydrolysate is 10%-12%.

[0018] Preferably, the prepared hypoallergenic casein hydrolyzate has an IgE antibody binding capacity of ≤5% and an IgG antibody binding capacity of ≤10%.

[0019] Preferably, the hydrolyzate targeted to obtain a size below 10 kDa increases the expression of insulin growth factor-1 in mice by 4 times.

[0020] Beneficial effects: The present invention can modify the tyrosine residues No. 82 and arginine residues No. 83 of papain located in the loop2 region by choline dihydrogen citrate, and then enhance the surface electrostatic potential of the catalytic triad through the Loop2→Loop1→α3→α2 path, and regulate the indole plane of Trp177 at the entrance of the substrate binding channel from the horizontal direction to the vertical direction, thereby forming a narrow substrate binding channel. Finally, the binomial probability logarithm ratio of papain's hydrolysis selectivity for tyrosine residues is increased from 1.4 to 8.7, and the preference for tyrosine residues is increased by 6.15 times, thereby achieving the improvement of papain's targeted degradation ability for the αs1-CN main epitope Y154-T174, and its hydrolyzate's binding ability to IgE and IgG is reduced from 7.28% and 12.42% to 5.36% and 8.55%, respectively. Among them, the hydrolyzate below 10kDa obtained by targeting increases the expression of insulin growth factor-1 in mice by 4 times. The papain modified by choline dihydrogen citrate of the present invention realizes enhanced degradation of the main epitope of αs1-CN and simultaneously reduces the loss of its function as much as possible.

[0021] The above description is only an overview of the technical solution of the embodiment of the present application. In order to more clearly understand the technical means of the embodiment of the present application, it can be implemented in accordance with the contents of the specification. In order to make the above and other purposes, features and advantages of the embodiment of the present application more obvious and easy to understand, the specific implementation methods of the present application are listed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present application, a brief introduction will be given below to the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0023] Figure 1 It is a flow chart of the present invention;

[0024] Figure 2 Schematic diagram of the effect of choline dihydrogen citrate modification on the structure of papain;

[0025] Figure 3 Schematic diagram of the effect of choline dihydrogen citrate ILs on the conformation and conformational distribution of papain;

[0026] Figure 4 Schematic diagram of the effect of choline dihydrogen citrate IL on the water distribution of papain;

[0027] Figure 5 Schematic diagram of the effect of choline dihydrogen citrate IL on the surface potential of papain catalytic triad (A), substrate binding channel (B) and catalytic triad configuration (C);

[0028] Figure 6 Histogram of the binding capacity of casein hydrolysate prepared by choline dihydrogen citrate modified papain to (A) IgE and (B) IgG;

[0029] Figure 7 The molecular weight distribution of polypeptides in different hydrolysates (A) and the destruction of the main antigenic epitopes of αs1-CN are analyzed;

[0030] Figure 8 Schematic diagram of the effects of natural papain on the body weight of mice for the preparation of casein hydrolysate, ordinary hydrolysate and choline dihydrogen citrate modified papain for enhanced targeted degradation of the αs1-CN key epitope casein hydrolysate. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution 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.

[0032] In order to enable those skilled in the art to better understand the solution of the present application, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the accompanying drawings.

[0033] See also Figure 1 The present application discloses a method for enhancing the targeted degradation of the main epitope of αs1-CN by modifying papain with choline chloride, comprising the following steps:

[0034] A. First, we modified papain with a charge-adjustable two-component choline-based ionic liquid based on molecular dynamics simulation to achieve the free energy remodeling of the papain conformational landscape.

[0035] B. The commercial papain purified by ammonium sulfate was placed in a 0-1M choline-based ionic liquid solution at 37-42°C for 0-60 min, and the effect of the choline-based ionic liquid on the structure of papain was characterized experimentally by Fourier transform infrared spectroscopy;

[0036] C. hydrolyzing casein with the papain modified with choline dihydrogen citrate in step B to prepare casein hydrolysate, and analyzing the effect of choline dihydrogen citrate modification on the catalytic specificity of papain by combined LC-MS / MS technology;

[0037] D. The results of step C are displayed by multiple fingerprints and combined with serological evaluation to evaluate the targeted degradation ability of papain modified with choline dihydrogen citrate on the main antigen epitope of αs1-CN;

[0038] E. The casein hydrolysate hydrolyzed by choline dihydrogen citrate modified papain in step D is ultrafiltered, and the components less than 10 kDa are freeze-dried and used in mouse animal experiments to verify the biological functionality of the hydrolysate.

[0039] In the present invention, the choline dihydrogen citrate in step C and step D can directionally modify the 83 tyrosine + 82 arginine residues of papain.

[0040] In the present invention, the papain modified with 0.4M dihydrogen citrate choline in step E is added to a casein solution with a concentration of 4% at an enzyme-to-substrate ratio of 1:3000-1:2000, and the reaction is carried out at pH 6-7 and 37-42°C for 30-60 minutes. After the reaction is completed, the enzyme is inactivated at 85°C-95°C for 5-10 minutes, and a supernatant is obtained by low-temperature centrifugation. The results of liquid chromatography-mass spectrometry analysis show that the selectivity of the papain modified with dihydrogen citrate choline for tyrosine residues is increased from 1.4 to 8.7, and the preference for tyrosine residues is increased by 6.15 times, thereby achieving the enhancement of the targeted degradation ability of papain for the main epitope Y154-T174 of αs1-CN, and the binding ability of its hydrolyzate to IgE and IgG is further reduced from 7.28% and 12.42% to 5.36% and 8.55%, respectively.

[0041] In the present invention, the hydrolyzate below 10 kDa obtained by targeted hydrolysis in step F increases the expression level of insulin growth factor-1 in mice by 4 times.

[0042] In addition, in the present invention, the hydrolysis degree of the casein hydrolysate is 10%-12%; the prepared low-allergenic casein hydrolysate has an IgE antibody binding capacity of ≤5% and has the ability to promote the growth and development of mice.

[0043] Embodiment 2:

[0044] Analyze the effect of choline dihydrogen citrate on the structure of papain at the molecular theoretical level and experimental level.

[0045] Firstly, the shortest path analysis based on molecular dynamics simulation was used to analyze the effect of choline-based ionic liquids on the network structure of papain, in order to determine whether choline dihydrogen citrate has a regulatory effect on the potential distal regulatory pathway of papain hydrolysis specificity; the interaction between choline dihydrogen citrate and papain was deeply analyzed at the molecular level, mainly including conformation, electrostatic potential and hydration layer analysis, aiming to analyze the regulatory mechanism of choline dihydrogen citrate on the hydrolysis specificity of papain from a theoretical level.

[0046] Characterization of the effect of choline dihydrogen citrate ionic liquid on the structure of papain based on the shortest path and hydrogen bond network structure. Figure 2 As shown in (AB), the three potential regulatory pathways given by SPM (shortest path) in the control group have a strong correlation with the hydrogen bond network structure analysis, which just confirms the rationality of the pathway analyzed by SPM. Compared with the control group, the addition of choline dihydrogen citrate ionic liquid greatly disrupted the original network structure of papain. The most intuitive thing is that SPM has changed a lot, but choline dihydrogen citrate perturbation retains the 82-83 regulatory pathway.

[0047] Effects of choline dihydrogen citrate ILs on the surface area solvation accessibility, hydrogen bonding and salt bonding occupancy of papain residues

[0048]

[0049]

[0050] As can be seen from the table above, the solvation surface area of ​​papain residues changed significantly after the addition of choline dihydrogen citrate. The reason may be that the electric field polarization formed by the tail aggregation effect of choline dihydrogen citrate ILs caused the electrostatic potential of papain surface to change, thereby causing fine-tuning of the structure. In addition, from the results of the residue solvation accessible surface area and hydrogen bond occupancy in Table 3, it can be seen that choline cation (CHO) has a strong targeted modification effect on Tyr82 of papain. The probability of hydrogen bond formation between CHO and Tyr82 is nearly 30%, and the addition of acid anions has a regulatory effect on the targeted modification intensity of choline-based ionic liquids. Among them, the overall hydrogen bond formation occupancy of CHO-CIT is as high as 65%. Studies have shown that when the hydrogen bond formation occupancy is greater than 10%, it indicates that there is an important interaction between hydrogen acceptors and hydrogen donors. The formation of salt bonds between protein molecules is not conducive to the modification of residues. From the circular dichroism structure (3C), the circular dichroism configuration of papain modified with choline citrate is similar to that of natural papain, and the displacement of the peaks and troughs is not obvious, indicating that the modification with choline citrate has no effect on the higher-order structure of papain. Further, from the Fourier transform infrared spectroscopy results (3D), the papain modified with choline citrate has a peak at 957cm -1 The characteristic peak of choline ion appeared at 1714cm -1 A new shoulder peak of lower intensity appeared at the 40°C, which was the carbonyl vibration peak of citrate.

[0051] Combining the interaction at the molecular level and the experimental results, it can be seen that choline dihydrogen citrate may modify the tyrosine residue 82 and the arginine residue 83 of papain in a targeted manner.

[0052] Depend on Figure 3 The results showed that choline dihydrogen citrate ILs significantly changed the preset conformational distribution of papain. Figure 3 B), in papain induced by choline citrate ILs, the conformation of the catalytic triad barely changed, but the indole plane of Trp177 at the entrance of the substrate binding channel changed from horizontal to vertical; at the same time, the amino acid residues in the substrate binding channel moved upward toward the center, thus forming a narrow substrate binding channel; these changes were beneficial to improve the selectivity of papain for substrates to a certain extent.

[0053] The spatial distribution function was further used to briefly analyze the effect of choline dihydrogen citrate ionic liquid on the surface of papain, especially the distribution of water molecules on the surface of the catalytic triad, in order to reveal the effect of ILs on the first hydration shell of papain. The results are shown in Figure 4 As shown in the figure, CIT increases the number of water molecules on the surface of the catalytic triad; in addition, statistics show that there are 33 choline cations and 15 citrate ions distributed on the surface of papain, respectively, but basically none of them are located near the catalytic triad. Therefore, the increase in the number of water molecules distributed near the catalytic triad may help improve the catalytic properties of papain.

[0054] Mastering the charge density distribution helps to better understand the ability of catalytic residues to donate or accept protons. The step-by-step hydrolysis mechanism of cysteine ​​peptide bonds has been widely recognized, that is, by transferring protons from cysteine ​​to histidine to form an ion pair state, the substrate is then attacked by the cysteine ​​anion, and then the histidine is repositioned to allow the proton to be transferred to the substrate. Generally, the charge distribution of the papain catalytic site is both positive and negative. Asn175 contributes a higher negative potential, which stabilizes the protonated histidine in the catalytic cycle, while His159 contributes a positive potential in the active site. Near neutral pH, the catalytic cysteine ​​is deprotonated. In the dihydrogen citrate choline ionic liquid system, a net charge density gradient was observed in the proton transfer region between cysteine ​​and histidine ( Figure 5 A), which is conducive to the formation and stabilization of the Cys25(-)---His159(+) catalytic ion pair, indicating that choline dihydrogen citrate may act as an electrostatic regulator to regulate the mechanism (Cys25(-)---His159(+) attenuation effect) and thus control the reactivity of the ion pair.

[0055] In addition, changes in the geometric configuration of the catalytic triad, especially changes in the substrate binding channel, will affect substrate binding. Figure 5 (BC) The results show that in the phosphate system, the distance between the amide oxygen of the Asn175 side chain and the Nε2H of His159 is Significantly increased to However, in the choline dihydrogen citrate ionic liquid, the hydrogen bond is still maintained, and the distance is reduced to According to previous studies, an important role of Asn175 is to stabilize the form of thiol-imidazole ion pairs to keep the imidazole ring of His159 in a favorable orientation. Studies have shown that stable carbon cations can maintain higher specificity. The unstable thiol-imidazole ion pairs in PBS may increase side reactions, which may explain the broad substrate specificity; while the narrow substrate binding groove and stable thiol-imidazole ion pairs after modification with choline dihydrogen citrate may be the reason for the improved specificity. In general, the distance between catalytic residues in the CHO-CIT system is shorter than that in other systems, which may help transfer protons from cysteine ​​to histidine when nucleophilic attack is carried out. This may explain the changes in papain activity.

[0056] Embodiment 3:

[0057] Effect of choline dihydrogen citrate modification on the catalytic specificity of papain.

[0058] Peptidomics technology was used to analyze the effect of choline citrate ILs modification on the specificity of papain cleavage sites, and 294 and 219 specific peptides were identified from choline citrate and phosphate buffer systems (control group), respectively. The results in the table below show that the log-odds values ​​of the preference for Tyr at the P1 and P1' positions of papain modified with choline citrate were increased to 4.70 (PBS: 1.41) and 3.97 (PBS: 0.53), respectively. Compared with PBS, Tyr was not the first cleavage target in CHO-CIT, but its selectivity was increased by 6.15 times overall, indicating that papain modified with choline citrate improved the cleavage specificity for Tyr. The improvement of Tyr selectivity helps to improve the targeted degradation of papain for the main antigen epitope of αs1-CN (the main antigen epitope Y154-T174 is rich in 23.8% Tyr residues).

[0059] Effect of ionic liquid modification on the probability of papain cleavage sites

[0060]

[0061] Embodiment 4:

[0062] Choline dihydrogen citrate modified papain to enhance its ability to bind to the main antigen epitope of αs1-CN

[0063] In order to explore the effect of papain modified with choline dihydrogen citrate ionic liquid on its ability to target and degrade the main antigenic epitope of αs1-CN, the serological experiment results of papain modified with choline dihydrogen citrate on the preparation of casein hydrolysate were as follows: Figure 6 As shown,

[0064] Depend on Figure 6 It can be seen that compared with natural papain, the binding ability of casein hydrolysate prepared by papain modified with dihydrogen choline citrate to IgE and IgG is further reduced, from the original 7.28% and 12.42% to 5.36% and 8.55%, respectively. Among them, the content of tyrosine residues in the key binding region Y154-T174 of αs1-CN and antibodies is as high as 23.8%, and the selectivity of papain modified with dihydrogen choline citrate to tyrosine hydrolysis is increased by 6.15 times. It can be seen that after CHO-CIT modified papain, the degradation of the main epitope binding region Y154-T174 of αs1-CN is increased, thus causing its ability to bind to antibodies to be further reduced.

[0065] Depend on Figure 7 It can be seen that the molecular weight distribution of peptides in targeted hydrolysates and ordinary papain hydrolysates is mainly concentrated below 3kDa, especially in ordinary papain hydrolysates, where the peptides are mainly oligopeptides; while there are still many peptides with molecular weight greater than 3kDa in targeted hydrolysates, indicating that the degree of fragmentation of targeted hydrolysates is lower than that of ordinary hydrolysates. The damage of the main antigenic epitopes of αs1-CN by targeted hydrolysis and ordinary hydrolysis was further analyzed. Figure 7 B and the table below show that targeted hydrolysis greatly improved the degradation of the Y154-T174 of the main epitope region of αs1-CN, from the original 23.8% to 57.1%, and the number of hydrolysis sites increased from the original 6 to 12, especially the cleavage of the key binding amino acid residues Trp164 and Tyr165 of αs1-CN and antibody, which may play a vital role in the reduction of its antigenicity. Among them, the cleavage of tyrosine residues increased from the original 9.5% to 19.1%, which also shows that papain modified with dihydrocholine citrate enhances the selectivity of papain for tyrosine residues, that is, the purpose of targeted degradation of the key epitope of αs1-CN is achieved.

[0066]

[0067] Embodiment 5:

[0068] Choline dihydrogen citrate modified papain enhances the growth-promoting function of casein hydrolysate by targeted degradation of αs1-CN key epitope

[0069] Depend on Figure 8It can be seen that, in general, the expression regulation level of insulin growth factor-1 in the intestine of mice by targeted hydrolysate was significantly higher (p<0.05) than that of the control group (casein was not hydrolyzed), and also higher than that of ordinary casein hydrolysate. Although there was no significant difference (p>0.5), the expression multiple of insulin growth factor-1 in targeted hydrolysate was about 1 times higher, indicating that targeted hydrolysate has a promoting effect on the expression of insulin growth factor-1 in mice. Figure 8 It can be seen that the targeted hydrolyzate group showed a higher weight gain in mice than the blank group (non-hydrolyzed casein), which also shows that the targeted hydrolyzate has a certain promoting effect on the growth and development of mice.

[0070] In summary, the present invention can modify the tyrosine residues No. 82 and arginine residue No. 83 of papain located in the loop2 region by choline dihydrogen citrate, and then enhance the surface electrostatic potential of the catalytic triad through the Loop2→Loop1→α3→α2 path, and regulate the indole plane of Trp177 at the entrance of the substrate binding channel from the horizontal direction to the vertical direction, thereby forming a narrow substrate binding channel. Finally, the binomial probability logarithm ratio of papain's hydrolysis selectivity for tyrosine residues is increased from 1.4 to 8.7, and the preference for tyrosine residues is increased by 6.15 times, thereby achieving the improvement of papain's targeted degradation ability for the αs1-CN main epitope Y154-T174, and its hydrolyzate's binding ability to IgE and IgG is reduced from 7.28% and 12.42% to 5.36% and 8.55%, respectively. Among them, the hydrolyzate below 10kDa targetedly increases the expression of insulin growth factor-1 in mice by 4 times. The papain modified by choline dihydrogen citrate of the present invention realizes enhanced degradation of the main epitope of αs1-CN and simultaneously reduces the loss of its function as much as possible.

[0071] As described above, 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, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A method for enhancing the targeted degradation of the main epitope of αs1-CN by modifying papain with choline chloride, characterized in that: The following steps are involved: A. First, we modified papain with a charge-adjustable two-component choline-based ionic liquid based on molecular dynamics simulation to achieve the free energy remodeling of the papain conformational landscape; B. The commercial papain purified by ammonium sulfate was placed in a 0-1M choline-based ionic liquid solution at 37-42°C for 0-60 min, and the effect of the choline-based ionic liquid on the structure of papain was characterized experimentally by Fourier transform infrared spectroscopy; C. hydrolyzing casein with the papain modified with choline dihydrogen citrate in step B to prepare casein hydrolysate, and analyzing the effect of choline dihydrogen citrate modification on the catalytic specificity of papain by combined LC-MS / MS technology; D. The results of step C are displayed by multiple fingerprints and combined with serological evaluation to evaluate the targeted degradation ability of papain modified with choline dihydrogen citrate on the main antigen epitope of αs1-CN; E. The casein hydrolysate hydrolyzed by choline dihydrogen citrate modified papain in step D is ultrafiltered, and the components less than 10 kDa are freeze-dried and used in mouse animal experiments to verify the biological functionality of the hydrolysate.

2. The method for enhancing the targeted degradation of the main epitope of αs1-CN by choline chloride-modified papain according to claim 1, characterized in that: The choline dihydrogen citrate in step C and step D can modify the 83 tyrosine+82 arginine residues of papain in a targeted manner.

3. The method of claim 1 for enhancing the targeted degradation of the main epitope of αs1-CN by modifying papain with choline chloride, characterized in that: In the step E, the papain modified with 0.4M dihydrocholine citrate is added to a 4% casein solution at an enzyme to substrate ratio of 1:3000-1:2000, and reacted at pH 6-7 and 37-42°C for 30-60 minutes. After the reaction, the enzyme is inactivated at 85°C-95°C for 5-10 minutes, and a supernatant is obtained by low-temperature centrifugation.

4. The method of claim 1 for enhancing the targeted degradation of the main epitope of αs1-CN by modifying papain with choline chloride, characterized in that: The degree of hydrolysis of casein hydrolysate is 10%-12%.

5. The method of claim 1 for enhancing the targeted degradation of the main epitope of αs1-CN by modifying papain with choline chloride, characterized in that: The prepared low-allergenic casein hydrolyzate has an IgE antibody-binding capacity of ≤5% and an IgG antibody-binding capacity of ≤10%.

6. The method of claim 1 for enhancing the targeted degradation of the main epitope of αs1-CN by modifying papain with choline chloride, characterized in that: Among them, the targeted hydrolyzate below 10kDa increased the expression of insulin growth factor-1 in mice by 4 times; the prepared low-allergenic casein hydrolyzate has a certain promoting effect on the growth and development of mice.

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