Effects of Flavourzyme and Alkaline Protease Treatment on Structure and Allergenicity of Peanut Allergen Ara h 1

SUMMARY Research background Peanut allergy poses a significant threat to human health due to the increased risk of long-term morbidity at low doses. Modifying protein structure to affect sensitization is a popular topic. Experimental approach In this study, the purified peanut allergen Ara h 1 was enzymatically hydrolysed using Flavourzyme, alkaline protease or a combination of both. The binding ability of Ara h 1 to antibodies, gene expression and secretion levels of the proinflammatory factors interleukin-5 and interleukin-6 in Caco-2 cells was measured. Changes in the secondary and tertiary structures before and after treatment with Ara h 1 were analysed by circular dichroism and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Results and conclusions The results indicated a decrease of the allergenicity and proinflammatory ability of Ara h 1. The evaluation showed that the Flavourzyme and alkaline protease treatments caused particle shortening and aggregation. The fluorescence emission peak increased by 3.4-fold after the combined treatment with both proteases. Additionally, the secondary structure underwent changes and the hydrophobicity also increased 8.95-fold after the combined treatment. Novelty and scientific contribution These findings partially uncover the mechanism of peanut sensitization and provide an effective theoretical basis for the development of a new method of peanut desensitization.


INTRODUCTION
Peanuts are rich in fat and protein and contain vitamins, calcium, iron, and other minerals that are beneficial to health (1,2).Peanuts are popular in the processed food industry because of their delightful taste and nutritional value.Peanut allergy symptoms include lip and face edema, tracheal stenosis, urticaria, twitching, vomiting, diarrhoea, asthma, anaphylactic shock, etc.They can cause severe discomfort and even death (3).Peanut allergies are caused by 17 allergens collectively known as Ara h 1 to Ara h 17 (4).Notably, Ara h 1-3 and Ara h 6 are recognised by 90 % of the sera of allergic patients (5).Ara h 1 is the most abundant allergen in peanuts.Accounting for 12-16 % of the total peanut protein.It has been reported that serum specific IgE for Ara h 1 is present in 75 % of peanut allergic individuals (6).Chosen for its abundance and high specificity, Ara h 1 is currently under Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.
3 investigation for methods to mitigate its allergenicity, with existing research in this area being inconclusive.
Peanut allergy is a severe food allergy, which manifests as an IgE-mediated immune response triggered by the consumption of peanuts or peanut containing products (7,8).Protease treatment, commonly employed in food processing is a key strategy for reducing the allergenicity of peanut proteins (9-11).Protein structures undergo changes during cross-linking and aggregation (12), either disrupting the original protein epitope or generating new epitopes, thereby influencing the allergenicity of peanut proteins.Cabanillas et al. reported a 65 %, inhibition rate of peanut protein and IgE binding after flavorzyme treatment (13).In another study, Yu et al. (14) processed peanuts with alkaline protease, papain, neutral protease, and bromelain, observing varied hydrolytic effects on specific allergens.Among these, alkaline proteases most effective in reducing the concentration and allergenicity of raw peanut allergens.Flavorzymes, alkaline proteases, exoproteases and endoproteases substantially diminished protein allergenic.After treatment with flavourzyme and alkaline protease, allergenic peanut proteins are degraded into small peptides by the protease system (15).In this study, Ara h 1 was treated with flavorzyme and alkaline protease, both of which are edible enzymes that are harmless to humans.In addition to the common single-enzyme treatment, this study also attempted to use at combination of flavorzyme and alkaline protease for the enzymatic hydrolysis of peanuts.The studies suggest that hydrolysing peanut-allergenic proteins with non-specific protease can significantly reduce peanut allergenicity (16).However, the mechanism of protease treatment on peanut allergenicity is yet to be studied.
Roth Walter et al. (17) used Caco-2 cells as a model to study the allergenicity of milk protein, and the results show that the aggregation of soluble milk β-lactoglobulin can induce the production of IgE antibodies and cytokines in Caco-2 cells, such as IL-6, IL-8, IL-15 and thymic stromal lymphopoietin (TSLP).Previous studies have shown that the inflammatory factors IL-5 and IL-6 play important roles in peanut allergic reactions, which are induced by the Th2 response once susceptible individuals consume peanuts (18).Basophils can be activated and recruited by these factors to, release their cellular contents, which trigger tissue damage and inflammation in the body (19).Therefore, IL-5 and Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.

4
IL-6 were used as inflammatory biomarkers to evaluate allergenicity in the Caco-2 cell model.
In this study, we investigated the structural changes in Ara h 1 and its ability to bind to antibodies following hydrolysis with proteases (flavorzyme, alkaline protease, flavorzyme and alkaline protease).
In addition, we investigated changes in IL-5 and IL-6 gene expression and secretion, which indicated the effects of protease treatment on Ara h 1 structure and allergenicity in on Caco-2 cells.This study provides a basis for understanding the mechanism of peanut allergy and developing new desensitization methods.

Purification of Ara h 1
Crude peanut protein extraction and Ara h 1 purification were performed as previously described by Wang et al. (2).Peanuts purchased from Wal Mart (Qiannuo, Shanghai, China).Ara h 1 was purified using a protein purification system (AKTA pure, General Electric, Boston, USA) and column (HiTrap Q HP, General Electric, Boston, USA) through anion exchange chromatography.

Enzymatic hydrolysis of Ara h 1
The alkaline protease and flavorzyme were purchased from Macklin (Shanghai, China).The hydrolysis conditions for flavorzyme were as follows: the protease concentration to protein ratio of 10:100, reaction at 60 °C in a water bath for 100 min, 100 °C for 10 min to inactivate the protease.
The hydrolysis conditions of alkaline protease are as follows: the ratio of protease to protein concentration is 4:100, 55 °C water bath for 100 min, and 100 °C for 10 min to inactivate the protease.
The hydrolysis conditions of the two proteases were as follows: the ratio of flavorzyme to protein is 5:100, 60 °C water bath for 100 min, 100 °C for 10 min to inactivate the protease, then add 2 % alkaline protease, 55 °C water bath for 100 min, 100 °C for 10 min to inactivate the protease.

SDS-PAGE
Proteins were separated using a kit (Yamei, Shanghai, China, cat number: PG112).The Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.experimental procedures have been described by Wang et al (2).We used a gel with a thickness of 1.0 mm for electrophoresis.An upper gel voltage of 80 V was applied for a duration of 20 min, and a lower gel voltage of 120 V was applied for a duration of 60 min.The gel was stained with Coomassie bright blue R-250 (Solarbio, Shanghai, China) for 30 min, decolorized overnight for 12 h, and the electrophoretic image of protein was obtained.

Observation by atomic force microscope
An atomic force microscope (AFM5500, Agilent, California, USA) was used to observe the surface morphology of Ara h 1. 10 μL of Ara h 1 (0.1 mg/mL) solution was dropped on the surface of the sample carrier.After standing for 10 min, the solution was gently blown dry with nitrogen.An AFM image of the sample was obtained using the AFM contact mode, a CSC17/AI BS/50 probe and a scanning speed of 1.72 In/s.

Infrared spectrometric determination
To observe changes in protein molecular structure, a solution of Ara h 1 (0.1 mg/mL) was analyzed using Fourier transform infrared spectroscopy (Vertex70V, Vertex, Boston, USA).200 mg KBr (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) and 200 μL Ara h 1 (0.2 mg/mL) solution were ground together in an agate mortar, then transferred to tablet mold and sent to tablet press.In the tablet press, the pressure was raised to 520 then dropped to zero and the wafer was removed from the mold.The sample wafer was inserted into the sample placement of the infrared spectrometer, and the infrared spectrum was obtained by beam scanning from 4000-400 cm -1 .

Ultraviolet absorption spectrum
To observe the absorption of Ara h 1 in the ultraviolet before and after enzymatic hydrolysis, an Ara h 1 (0.1 mg/mL) solution was measured using by UV-Vis spectrophotometer (UV759CRT, Yoke, Shanghai, China).The scanning range was 240-450 nm and the scanning speed was 100 nm/min.Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.

Fluorescence spectrum and determination of hydrophobicity
Observing changes in the internal structure of proteins by detecting surface hydrophobicity, Ara h 1 (0.2 mg/mL) solution was added with 80 μM Bis-ANS (Aladdin, Shanghai, China), mixed and transferred to a fluorescence dish for analysis by fluorescence spectrometer (FS F-2500, Shimadzu, Kyoto, Japan).The fluorescence intensity was detected at 400-600 nm by scanning at 25 °C with an excitation wavelength of 350 nm.The scanning conditions were 1200 nm/min, 0.5 s, 500 V, and 5 nm.
Ara h 1 of different concentrations (0.025, 0.05, 0.1, 0.2, and 0.4 mg/mL) were prepared and mixed with 80 μM Bis-ANS, then transferred to a fluorescence spectrophotometer (FS F-2500, Shimadzu, Kyoto, Japan) for analysis.The excitation wavelength was 350 nm, and the fluorescence intensity was detected at 400-600 nm.The maximum fluorescence intensity was plotted by protein concentration and curve fitting was performed by least square method.The slope represents the surface hydrophobicity of the protein (20).Each experiment was performed at least three times.

Circular dichroism
Ara h 1 (0.1 mg/mL) solution was analyzed by circular dichroism (J-815, JASCO, Kyoto, Japan) to detect its secondary structure.The scanning range was 190-240 nm, the scanning rate was 100 nm/min, the optical diameter was 0.1 cm, the spectral interval was 0.1 nm, and the bandwidth was 0.1 nm.The samples were tested 5 times in parallel.The data were analyzed using CD Pro software (http://lamar.colostate.edu/~sreeram/CDPro)(21), and the secondary structure content was calculated.

LC-MS/MS spectrometry
Ara h 1 (untreated, 0.1 mg/mL) and a combination of flavorzyme and alkaline protease treated Ara h 1 (flavorzyme and alkaline protease, 0.1 mg /mL) protein solution was put into a sterilization centrifuge tube and sent to Sangon Biotechnology Co., Ltd (Shanghai, China) for LC-MS/MS identification.Analyze the differences in protein peptide segments before and after enzymatic hydrolysis through LC-MS/MS.Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.

Determination of binding ability of protein to antibody by Western Bolt
To test the binding ability of anti-Ara h 1 before and after enzymatic hydrolysis was performed as described by Wang et al. (2).The protein was separated using a 10 % SDS electrophoresis kit (Yamei, Shanghai, China, cat number: PG112).The protein on the gel was transferred to a nitrocellulose membrane PVDF(HVHP02500, Millipore, Massachusetts , USA) using a Sandwich system.After blocking, incubating primary and secondary antibodies, the immunoreactive bands were detected by enhanced chemiluminescence (Tanon, Shanghai, China, cat number:180-506).

Determination of binding ability of protein to antibody by ELISA
The binding ability of Ara h 1 to antibody was determined by indirect ELISA.Ara h 1 (10 μg/mL, 200 μL/well) were added into the HRP-labeled plate.The samples were coated at 4 °C overnight.200 μL skim milk solution (5 %) was added to each well, the plate was sealed at 37 °C for 1 h, and then washed three times with phosphate buffer containing Tween 20 (PBST).The plate was incubated at 37 °C for 2 h with Mouse anti-Ara h 1 monoclonal antibody (1:400 dilution) and washed with PBST for 5 times.The plate was incubated at 37 °C for 1 h with goat anti-mouse HRP-bound anti-IgG antibody (1:2000 dilution) and washed with PBST for 5 times.Finally, TMB solution (Tanon, Shanghai, China)was added to color rendering for 10-15 min.The absorbance value at 450 nm was measured with a single function light absorption protease plate analyzer (EMax Plus, Molecular Devices, San Jose, USA).

Culture of Caco-2 cells
The culture of Caco-2 cells was carried out at 37 °C and 5 % CO2 as described by Wang et al.
(2).The human colorectal cancer cell line Caco-2 (ZhongQiao, Shanghai, China) was cultured with RPMI cell culture medium (Gibco, California, USA), 10 % fetal bovine serum (FBS)(Gibco, California, USA) and 1 % Penicillin-Streptomycin Solution (Aibo, Hangzhou, China).The cells were cultured for 2-3 days to achieve fusion, and to assist in the process of cell growth and differentiation, fresh culture medium was added daily.The growing cells were digested with trypsin and passaged continuously at Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.a ratio of 1:2.

Detection of Caco-2 cytokines by RT-PCR
RNA extraction and real time polymerase chain reaction (RT-PCR) steps in Caco-2 cells were consistent with those previously described by Wang et al. (2).RT-PCR was performed on real time PCR detection system (CFX96, BioRad, Shanghai, China).The total cDNA was standardized with GAPDH as the housekeeper gene.The gene primers used in this paper are as follows: (1)IL-5: Forward: GCTTCTGCATTTGAGTTTGCTAGCT, Reverse: TGGCCGTCAATGTATTTCTTTATTAAG

Statistical analysis
The data are expressed as the mean±S.D., and all assays from every independent treatment were carried out in triplicate.Appropriate data were analyzed to determine statistical significance using ANOVA (SPSS version 13.0, SPSS Inc, Chicago, IL, USA) (2).The T test and Fisher's least significant difference (LSD) test were used to examine differences between group means.
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Effect of protease treatment on the binding ability of Ara h 1 to antibodies
In vitro tests for detecting allergens commonly include serum-specific antibody assays and cell model allergenicity evaluations (2,22,23).To detect the antigenicity and potential allergenicity of Ara h 1 after enzymatic hydrolysis, Western Blot and ELISA were used to conduct in vitro antigen-antibody binding experiment with mouse anti-Ara h 1 monoclonal antibody (Fig. 1a).The band of Ara h 1 became shallow after protease treatment, indicating that the binding ability of Ara h 1 to antibody was decreased after protease treatment.This phenomenon was verified using indirect ELISA (Fig. 1b).
Compared to the control group, the binding ability slightly decreased after single protease treatment, whereas it decreased significantly by 37.7 % after flavorzyme and alkaline protease treatment.Hence, a significant decrease in the binding ability after protease treatment can reduce the in vitro

Effects of protease treated Ara h 1 on Caco-2 cell cytokine expression and secretion
In order to study the expression of Ara h 1 proinflammatory factor, Caco-2 cells were incubated with the peanut allergen Ara h 1, which was treated using different methods (flavorzyme, alkaline protease, flavorzyme and alkaline protease) for 24 h.The gene expression of IL-5 and IL-6 in Caco-2 cells was determined by RT-PCR (Fig. 2a-b).Compared to the blank group which was not treated with peanut protein Ara h 1 in Caco-2 cells, the expression of cytokines IL-5 and IL-6 in Caco-2 cells was up-regulated after Ara h 1 treatment.This indicated that Ara h 1 promoted inflammation and allergic reaction in Caco-2 cells.Because the originally upregulated IL-5 and IL-6 genes were Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.
suppressed after protease treatment, the pro-inflammatory levels of Ara h 1 were effectively reduced.
The secretion of the inflammatory cytokines IL-5 and IL-6 was evaluated by ELISA, and the secretion of IL-5 and IL-6 decreased significantly in protease treated Ara h 1 group (Fig. 2c-d).These results indicate that the protease treatment effectively reduced the pro-inflammatory ability of Ara h 1.
Ara h 1 is immunogenic and can be taken up by cells to trigger allergic reactions, resulting in the release of inflammatory factors such as IL-5 (26).Yang et al. (27) emphasized the effects of simulated gastric juice digestion on the immunoreactivity and pro-inflammatory properties of recombinant Ara h 1.Their results showed that Ara h 1 led to increased cytokine secretion and inflammation through activation of the NF-κB pathway.Lin et al. (28,29) found that ovalbumin promoted the increase of IL-5 in the spleen and intestine of mice.The ovalbumin food allergy model of Harusato et al. (30) showed that the expression of IL-5 cytokines in the colon of mice increased after food allergy.After subjecting Ara h 1 to enzymatic cleavage, we observed a decrease in the expression and secretion levels of the pro-inflammatory cytokines IL-5 and IL-6.This indicated a reduction in the pro-inflammatory capability of Ara h 1.Compared to the single enzyme treatment, the expression and secretion levels of proinflammatory factors in the flavorzyme and alkaline protease treatment groups were significantly decreased.This may be due to protease treatment (especially two enzyme treatments) disrupting the antibody-binding epitopes and reducing the immunogenicity of Ara h 1.Whether protease treatment affects the expression of other pro-inflammatory factors require further research.

Effects of protease treatment on particle size and aggregation of Ara h 1
To explore the relationship between decreased allergenicity and the protein structure of Ara h 1, the secondary and tertiary structures of Ara h 1 protein (before and after protease treatment) were explored by SDS-PAGE and atomic force microscopy.Purified Ara h 1 was treated to the aforementioned enzymolysis conditions and then electrophoresed.Fig. 3a shows that the protein was degraded after enzymolysis.Flavorzyme and alkaline protease had a better effect on protein degradation than single protease.Atomic force microscopy was used to further analyze the effect of enzymolysis on the degree of aggregationand particle size of Ara h 1.Protein aggregation affects the Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.11 potential allergenicity of Ara h 1 (31).Fig. 3b shows that the average particle size of Ara h 1 and the peak height of protein decreased after enzymatic hydrolysis, which indicates that the protease treatment can reduce the peak height of protein and made the protein more dispersed, especially in flavorzyme and alkaline protease treatments.Our study showed that the structure of Ara h 1 was greatly changed after protease treatment, the protein was degraded, and the particle size and aggregation degree were reduced.

Effect of protease treatment on the secondary and tertiary structure of Ara h 1
The infrared spectrum of Ara h 1 before and after enzymatic hydrolysis are shown in Fig. 4a.By infrared spectrum, we can observe the changes of groups in the structure of Ara h 1, which shows that the peak positions of Ara h 1 had little influence after enzymatic treatment.The UV absorption spectrum of proteins mainly depends on the presence of side chain groups containing residues such as Tyr and Trp (32), therefore, a large number of amino acid residues are produced after enzymatic hydrolysis of proteins, especiallyTyr and Trp residues.In itsnatural status, most hydrophobic amino acid residues constituting the protein are located in the interior, forming a hydrophobic core that is crucial for maintaining the compact three-dimensional structure of the protein.In order to further observe the structural changes of Ara h 1, we performed the detection of UV spectrum, and the results are shown in Fig. 4b.Compared with the control group, the UV absorption of peanut protein after enzymatic hydrolysis was increased, especially after flavorzyme and alkaline protease treatment.This finding also coincides with the result reported by Zhang et al. (33,34).Fig. 4c exhibits the fluorescence spectrum of Ara h 1 after protease treatment.After protease treatment, the emission peak of Ara h 1 appeared at 470 nm, and the peak intensity was the highest after flavorzyme and alkaline protease treatment, indicating that the amino acid residues increased and the protein structure changed significantly after protease treatment, resulting in an increase in the binding ability of protein to the fluorescent probe.These results indicated that the protease treatment changed the spatial structure of the protein, resulting in the exposure of hydrophobic groups originally located inside the protein, especially aromatic amino acids containing conjugated double bonds.
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Ara h 1 is a trimeric complex, and its monomers have a typical cupin structure.Most IgE binding epitopes (e.g.11-13, 18-22) of Ara h 1 overlap in the α-helix region of the C-terminal of the cupin structure, and its allergenicity may be related to the stability of the cupin structure.The protein was characterized by circular dichroism (Fig. 4d-e).The effect of protease treatment on the secondary structure of the proteins was analyzed using CD.From Fig. 4d, the left and right rotational absorbances of the proteins after protease treatment decreased significantly.Random coils increased, while the other secondary structures showed a trend of decline (Fig. 4e), among them, regular β-turn decreased the most.Among the three treatments, the flavorzyme and alkaline protease treatments had the greatest influence on protein secondary structure.After enzymolysis, the content of secondary structures declined overall, and the regular β-turn decreased greatly, indicating that enzymatic hydrolysis disrupted the original arrangement of protein and made its structure more unstable and quite disordered.As the change in secondary and tertiary structures also leads to the destruction of conformational epitopes, the binding ability to antibodies and allergenicity also decrease.Compared to protease treatment alone, flavorzyme and alkaline protease treatments had a more significant effect on the allergenicity and structure of protein.Previous studies also showed that the secondary structure of the protein changed after enzyme treatment, Wang et al. (35) treated tropomyosin by transglutaminase and tyrosinase, its structure was changed, α-helices decreased by 20.1% and 15.2%, β-turn increases by 5.8% and 6.2%.
The change in surface hydrophobicity was also measured (Fig. 4f), and the results shown that the hydrophobicity of Ara h 1 increased after the protease treatment, indicating that the conformational structure of the protein was destroyed and the hydrophobic groups inside the protein were exposed to the surface.Notably, the flavorzyme and alkaline protease treatment had the greatest effect on the hydrophobicity of the protein surface, which was 8.95 folds of untreated Ara h 1. Different from our results, through laccase/caffeic acid and transglutaminase in alleviating shrimp tropomyosin, Ahmed et al. (36) found that the surface hydrophobicity decreased through laccase/caffeic acid treatment, which might be due to the aggregation of protein by hydrophobic interactions, creation of hydrophilic groups (containing −NH2 and −OH), and partial unfolding of protein.
Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.13 Fig. 4 Effect of protease treatment on the total ion efflux spectrum and structure of Ara h 1 Because of the significant influence of flavorzyme and alkaline protease treatment on protein structure and allergenicity, further exploration of the site of enzyme treatment, Ara h 1 (untreated and after flavorzyme and alkaline protease treatment), was performed by LC-MS/MS (Fig. 5).As the result, 14 different peptides were found in Ara h 1 (untreated and treated), and these sites were mainly hydrolyzed by flavorzyme and alkaline protease, resulting in conformational changes in the protein.
In Rao's studies (37), the epitopes located in Ara h 1 393-402 and Ara h 1 498-508 were recognized in 60 % and 90 % of Chinese patients with peanut allergy, respectively.Hu et al. (38) also found that Twenty-seven integral sequential epitopes were identified in newly produced peptides after flavorzyme proteolysis with β-conglycinin.In our study, both linear epitopes were effectively destroyed by protease treatment.These results indicate that protease treatment of the secondary structure of the protein, destroys its the original conformational structure and may affect the allergenicity of Ara h 1.This protein was identified as the main peanut allergen Ara h 1.By comparing the obtained peptide sequences, 14 peptides were found to differ from those of Ara h 1 after enzymolysis (Table .1).Except for peptide 1, the other 13 peptides were located in the core region of Ara h 1.It can be speculated that flavorzyme and alkaline protease changed the spatial structure of Ara h 1, which might affect its allergenicity by the enzymolysis of these main fragments.
These results provide a theoretical basis for reducing peanut allergenicity.The mechanism through which enzymatic hydrolysis diminishes allergenicity involves protein uptake and transport within cells.Additionally, understanding the pathways associated inflammatory factors and employing animal models is crucial.

CONCLUSIONS
This study showed that the binding affinity of Ara h 1 to the antibody was reduced after protease treatment.Compared to the untreated Ara h 1 group, the expression and secretion levels of the pro-Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.14 inflammatory cytokines IL-5 and IL-6 in Caco-2 cells were significantly reduced after enzymatic digestion, demonstrating that protease treatment effectively reduced the pro-inflammatory response and potential sensitization caused by Ara h 1.Further studies showed that the protease reduced the pro-inflammatory level and potential sensitization of Ara h 1 by degrading the protein particles and changing the content of secondary structures.Our study provides an effective method for reducing peanut allergy to Ara h 1, but its mechanism needs to be studied further to provide a theoretical basis for the development of low-allergenic peanut-based products.Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.
Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.16 Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.
Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.
Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.
Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing. 25
allergenicity of Ara h 1.Similar to the results of the present study, Yang et al. (24) treated egg white with human alkaline protease.The binding ability of the hydrolyzed protein to IgG and IgE was determined by competitive ELISA using rabbit polyclonal antibodies and serum from patients with egg allergy.Their study showed that the hydrolysis of protein by alkaline protease helps to reduce the binding of IgG and IgE in the hydrolysate, thus reducing the sensitization of the protein.Mikiashvili et al. (25) treated peanut allergens Ara h 1,2,3 and 6 with alcalase and papain, they found that enzyme treatments effectively reduced overall IgE-binding of peanuts.

FUNDING
This study is supported by Science and Technology Joint Project of the Yangtze River Delta (19395810100).CONFLICT OF INTERESTThis article is an original work, and no portion of the study has been published or is under consideration for publication elsewhere.None of the authors has any potential conflicts of interest related to this manuscript.All authors have contributed to the work and have agreed to submit the manuscript.AUTHORS' CONTRIBUTIONErlian Shu contributed to the design of the work, performing the analysis, data analysis, and interpretation, and drafting the article.Shuo Wang contributed to performing the analysis, data analysis and interpretation and drafting the article.Xiangxiang Kong contributed to the interpretation of the results and the critical revision of the manuscript.Xiaodong Sun and Qiaoling Yang performed the analysis, data analysis, interpretation data and drafting the work.Bing Niu and Qin Chen contributed to the design of the work and critical revision.All authors contributed to the final approval of the version to be published.

20 https:Fig. 1 .
Fig. 1.Effect of protease treatment on in vitro sensitization of Ara h 1: a) Western blot tests the binding ability of Ara h 1 and antibody, b) indirect ELISA uses to detect the binding ability of Ara h 1 and antibody.Control: Ara h 1 without protease treatment, Fla: Ara h 1 after flavourzyme treatment, Alk: Ara h 1 after alkaline protease treatment, Fla+Alk: Ara h 1 after flavourzyme and alkaline protease treatment.Data are expressed as mean±S.D. of three independent experiments.*p<0.05,**p<0.01,***p<0.001

Fig. 4 .
Fig. 4. Effect of protease treatment on the structure of Ara h 1: a) infrared spectrogram of Ara h 1, b) ultraviolet spectrum of Ara h 1, c) fluorescence spectrogram of Ara h 1, d) circular dichroism analysis of Ara h 1, e) analysis of Ara h 1 secondary structure content, and f) surface hydrophobicity of Ara h 1. Control: Ara h 1 without protease treatment, Fla: Ara h 1 after flavourzyme treatment, Alk: Ara h 1 after alkaline protease treatment, Fla+Alk: Ara h 1 after flavourzyme and alkaline protease treatment.Data are expressed as mean±S.D. of three independent experiments.*p<0.05,**p<0.01,***p<0.001

Table 1 .
The sequence of degraded protein peptides after flavorzyme and alkaline protease treatment