Study on enzymatic hydrolysis of soybean β-conglycinin using alkaline protease from Bacillus subtilis ACCC 01746 and antigenicity of its hydrolysates

Haicheng Yin*,Xinrui Zhang,Jin Huang

College of Biological Engineering,Henan University of Technology,Zhengzhou 450001,China

ABSTRACT Due to its beneficial health effects,the use of soybean protein has shown a continuous increase,but concerns regarding the allergenicity of soybean antigenic protein have also increased.This study aimed to evaluate the hydrolytic effects of a non-commercial alkaline protease isolated from the Bacillus subtilis ACCC 01746 on soybean β-conglycinin and the allergenicity of its hydrolysates.Alkaline protease of the strain was separated by precipitation method of organic solvents,and the β-conglycinin was separated by alkali-solution and acid-isolation and purified by use of gel column.Using the degree of hydrolysis(DH)and inhibition rate as evaluation indexes,the enzymatic hydrolysis parameters of β-conglycinin was optimized by single factor and L9(34)orthogonal tests,so as to explore the effect of the protease on the hydrolysis degree and the antigenicity of β-conglycinin hydrolysates.The results showed that the native enzyme existed as an 18.3 kDa monomer with a 430 U/g maximum activity.The purity of βconglycinin was 84.8%.The single-factor test results showed that DH showed the oppostie trendency with the inhibition rate,and the increase of protein concentration caused monotone increasing and monotone decreasing of the inhibition rate and the DH,and the optimal protein concentration was 30 mg/mL.The optimization results showed that pH had the largest impacts on both DH and the inhibition rate,followed by enzyme dosage,hydrolysis temperature and hydrolysis time.Under the optimum hydrolysis conditions ofprotein concentration 30 mg/mL,enzyme dosage 0.7%,hydrolysis time 40 min,temperature 55°C and pH 8.5,the DH reached the highest of 76.28%,and the inhibition rate was the lowest of 27.03%,which was reduced greatly compared with that before optimization.These results suggested that alkaline protease appeared to show a relatively high effeciency in lowering soybean allergenicity,making it possible to produce low-allergenicity soybean protein.

Keywords:Alkaline protease Bacillus subtilis ACCC 01746 β-cConglycinin Hydrolysis degree Antigenicity

1.Introduction

Allergies can occur when the immune system overreacts to allergens[1].Food allergies are an abnormal immune response to specific food allergens,which often cause health problems in allergic patients,such as hives and anaphylactic shock.At present,the growing incidence of food allergies has resulted in the increasing interest all over the world.One of the most notable food allergens is regarded as soybean and soybean protein products.Soybeans are widely used as a significant protein source in foods and animal feed.However,it has been found that the protein in soybeans could induce an allergic response in humans and animals, especially infants and young animals, ranging from oxidative stress and inflammation to life-threatening reactions[2].According to incomplete statistics,approximately 25% of food-related allergic reactions are caused by soybean protein antigens, which generated serious health problems and constrain for further use of soy protein in foods and animal feed[3].Among the 38 known soybean allergen proteins, β-conglycinin has been identified as a major trimer allergen composed of α′, α, and β subunits with respective molecular weights of 76,72, and 53 kDa that are connected by electrostatic, hydrophobic interactions and hydrogen bonding [4].All three subunits of β-conglycinin are sensitized.The content of soybean β-conglycinin in soybean antigenic protein is high and the threshold of allergic reaction is low.Consequently,patients with soybean allergy must avoid consuming soybeans and their derivatives as much as possible[5].However,given the widespread use of soybeans,it is nearly impossible to avoid ingestion.Therefore,reducing or eliminating the antigenicity of soybean protein via effective processing is of critical importance.

In order to protect consumers against possible allergic reactions,several processing methods and techniques have been developed to reduce or eliminate the antigenicity of soybean protein,such as heat treatment,enzymatic hydrolysis,fermentation,and glycation[6].Enzymatic hydrolysis is an effective method to alter the structure and conformation of soybean protein, reduce its sensitization, and improve its biochemical functions.Enzymatic hydrolysis can break the protein peptide chain, produce peptides or amino acids with lower molecular weights,change the linear and spatial epitopes of allergens,so as to reduce protein antigenicity.Peptides produced through the process of enzymatic hydrolysis also exert antihypertensive and antioxidant effects [7].For instance, Wang et al.[8] found that enzymatic hydrolysis decreased the antigenicity of soy protein hydrolysates.Coscueta et al.[9] reported that enzymatic hydrolysis of defatted soy flour protein can decrease its sensitization ability,and the most suitable enzyme for this purpose is alkaline protease.These results showed that alkaline protease had the highest degree of hydrolysis(DH)and could effectively hydrolyze soybean antigen protein.As the most effective enzyme in lowering the antigenicity of protein,alkaline protease had the highest antigen removal rate in hydrolyzing antigen protein[10].However,to the best of our knowledge,few existing studies have specifically focused on β-conglycinin hydrolysates using alkaline protease from Bacillus subtilis.Although we have reported the hydrolysis of B.subtilis ACCC 01746 enzymes,these studies mainly focused on the hydrolysis of soybean protein by fermentation and the evaluation of biological activity[11,12].In this study,β-conglycinin had been separated and extracted from Soybean Protein Isolate (SPI),then hydrolyzed using alkaline protease isolated from B.subtilis ACCC 01746.Finally,the hydrolysis conditions were studied to analyze the antigenicity of hydrolysates so as to provide experimental data for the production of lowantigenicity soybean proteins.

2.Methods

2.1.Extraction of β-conglycinin

β-conglycinin was isolated and purified from SPIs according to the following steps.The SPIs (90.31%, Henan Anyang Kunhua Biological Technology Group Co., Ltd.,China)was mixed with Tris-HCl(0.03 mol/L and pH 8.5)solution at a protein-water ratio of 1∶15 and stirred at 37 °C for 60 min,then centrifuged at 4 °C(10,000 r/min for 20 min).The supernatant was mixed with NaHSO3(0.01 mol/L) and CaCl2(5 mmol/L), adjusted to pH 6.4,and centrifuged.The pH of the separated supernatant was adjusted to 5.5 with HCl(2 mol/L)and then centrifuged.Next, the supernatant was diluted with 2-fold distilled water and stirred at 4 °C for 60 min, adjusted to pH 4.8,and centrifuged for 15 min at 10,000 r/min.The precipitates were collected and dissolved in Tris-HCl buffer(0.03 mol/L) and then adjusted to pH 7.0 using 2 mol/L HCl to obtain antigenic protein solution.Then the solution was dialyzed and freeze-dried to obtain β-conglycinin.The extracted β-conglycinin was characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)and stored at-20°C for subsequent hydrolysis tests.

2.2.Isolation and purification of alkaline protease

Coarse enzymes from B.subtilis ACCC 01746 were prepared in our laboratory following the method of Fan et al.[13].The strain was cultured in an LB medium at 150 r/min and 37°C for 24 h.Then,it was inoculated into 50 mL coarse enzyme medium containing 1.5%peptone,1.0% glucose, 0.28% MgSO4, 0.1% KCl, and 5% NaCl at pH 7.0.After treated for 72 h,the culture was centrifuged at 5,000 r/min and 4 °C for 20 min, and then the supernatant was collected as the coarse enzyme solution.Proteins in the crude culture filtrate(500 mL)were precipitated with (NH4)2SO4at an 85% saturation level.After overnight culture at 4°C,the precipitates were centrifuged at 5,000 r/min for 15 min and dialyzed overnight with 50 mmol/L Tris-HCl buffer(pH 7.2)at 4°C,during which the buffer was changed 4 times.

The dialyzed sample was loaded into a 1.2 × 20 cm diethylaminoethanol(DEAE)-Sepharosecolumn and equilibrated with 50 mmol/L Tris-HCl buffer(pH 7.2).The unconjugated proteins were washed with the same buffer,and the binding proteins were eluted with 0-1 mol/L NaCl at a flow rate of 0.5 mL/min, and each component(2.0 mL)was collected.Then,the protease activity of each component was analyzed, and the highly active components were collected,concentrated,and further subjected to a gel filtration column containing Sephadex G-75 and 50 mmol Tris-HCl buffers(pH 7.2).The protein was eluted with the same buffer at a flow rate of 0.5 mL/min,and each component(2.5 mL)was collected,and subjected to the absorbance measurement at 280 nm.Then,the protease activity of each component was analyzed, and the molecular weight of the purified protease was determined by 12%sodium dodecylsulphate-polyacrylamide gel electrophoresis(SDS-PAGE)using the standard molecular weight marker on the vertical gel device.

2.3.Determination of protease activity

The extracted protease activity was measured according to the universal method at 55°C and pH 9.0 using casein as a substrate[14].The amount of the enzyme capable of catalyzing the cleavage of 1 μmoL tyrosine during 1 min under the experimental conditions(55°C and pH 9.0)was considered as a unit of activity.Proteolytic activity was expressed in U/mL,and specific activity was expressed in U/g protein.

2.4.Enzymatic hydrolysis of β-conglycinin

Before hydrolysis,we first prepared 30%(W/V)dispersion of β-conglycinin with deionized water.The enzymatic hydrolysis reaction was performed in shaker flasks at 150 r/min and 55°C for 40 min.The pH(the suggested optimal pH 8.5)of the mixture was adjusted using 5.0 mol/L NaOH hydrolysis.The hydrolysis reaction was started by adding the enzyme at an enzyme-to-β-conglycinin ratio of 0.6% to the mixture.After hydrolysis, samples were removed and the hydrolytic reaction was terminated by inactivating the enzymes at 95°C for 3 min.Then,the supernatant of the enzymatic hydrolysate was collected and centrifuged at 10,000 r/min and 4°C for 10 min,and the DH was determined using the modified OPA method described by Adler-Nissen[15].The analysis was performed in triplicate.The DH of the samples was calculated as follows:

where h was the molar qunatity of Serine-NH2(mmol),ODsample,ODblankand ODstandardwere absorbances of sample,blank and standard,respectively;X was sample's mass(g),P was protein content in sample(%),0.1 was the sample volume in liter(L),Heqvwas number of hydrolyzed bonds(eqv/g protein),Htotalwas total number of peptide bonds per protein equivalent(7.8 specific to soy protein),0.342 and 0.970 are specific values for β and α subunits of soy protein,respectively.

2.5.Antigenicity analysis by indirect competitive ELISA

The antigenicity of hydrolyzed β-conglycinin samples was analyzed by indirect competitive ELISA(ic-ELISA)according to the method of Bu et al.[6].First,24 pore culture plates were coated with 100 μL/well of 0.4 μg/mL β-conglycinin antigens in 50 mmol/L PBS and incubated at 4 °C for 12 h.The antigen concentration in PBS was determined earlier by ELISA (11.5 μg/mL for βconglycinin).All hydrolyzed β-conglycinin samples from the experiments were diluted to 2 mg/mL with PBS solution(pH 7.4),and then incubated separately with the selected pool sera(1∶2)diluted in 0.01 mol/L PBS(pH 7.4)containing 1%(W/V)BSA and 0.1%Tween-20(1∶1 ratio,V/V)at 4°C overnight.In the next step the plates were washed with 250 μL/well of 0.01 mol/L PBST (containing 0.01 mol/L PBS and 0.05%Tween-20,pH 7.4),the plate was blocked for 60 min at 37°C by adding 100 μL/well of 0.01 mol/L PBST containing 5% BSA.After washing, 100 μL of each mixture(β-conglycinin and pool sera)was added and incubated for 60 min at 37 °C and washed four times.Then,100 μL of HRP-labeled goat anti-rabbit IgE(Sigma-Aldrich,St.Louis,USA;1∶1,000)was added each well and incubated for 60 min at 37°C and washed four times.Finally,100 μL/well 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution was added and incubated at 37 °C for 30 min.The color reaction was terminated with the addition of 2 mol/L (50 μL) H2SO4to each well.An uninhibited serum sample(no sample)was used as the control,and the absorbance was measured at 450 nm and 630 nm by a microplate reader (TECOMP 8500, HongKong, China).The IgE binding was expressed by the inhibition rate (%),which was calculated using the following equation:

where B represented the absorbance of hydrolyzed βconglycinin samples and B0represented the absorbance of the uninhibited serum sample control.A low inhibition rate represents the low antigenicity of the hydrolyzed βconglycinin sample.

2.6.Single-factor experiments

Single-factor experiments were conducted for exploring the effects of five factors(protein concentration,enzyme dosage,hydrolysis time,pH and hydrolysis temperature)on DH and antigen inhibition rate of β-conglycinin hydrolysates.During implementing the single-factor experiment of a certain factor, the other factors were set as a fixed value,i.e.protein concentration of 30 mg/mL,enzyme dosage of 0.6%,hydrolysis time of 40 min,pH of 8.5,and hydrolysis temperature of 55 °C.The gradient level set for each factor was shown in Table 1.

Table 1 Factors and levels of the single-factor test.

2.7.Orthogonal experiments

Based on the results of single-factor experiments, four main influencing factors were further explored by the orthogonal design to determine the their optimum synergy on DH and antigen inhibition rate.Thus, a four-factor,three-level orthogonal array design L9(34)was employed under the protein concentration of 30 mg/mL for optimzing the following factors on the enzymatic hydrolysis.Factors and levels tested were shown in Table 2.

2.8.Statistical analysis

Each experiment was performed three times, and the data were presented as mean values±standard deviations.Significant differences between measurements for the control and treated samples were analyzed using a One-Way Analysis of Variance and Duncan's multiple range tests with SPSS Statistics 19.0 software (IBM SPSS, Armonk,NY,USA).The significance level was set at P ＜0.05.

3.Results

3.1.Purification of β-conglycinin and protease

Fig.1.SDS-PAGE of purified β-conglycinin and protease.

The electrophoretic bands of Protein Marker and extracted β-conglycinin were shown in Fig.1.SDS-PAGE showed that the purity of β-conglycinin was 84.8%using Gel Analyzer software.An alkaline protease was isolated from the B.subtilis ACCC 01746 and purified according to the aforementioned method.The native enzyme existed as an 18.3 kDa monomer(Fig.1)with a 430 U/g maximum activity at 55°C and pH 8.5.

3.2.Single-factor experiments

3.2.1.Effect of protein concentration on the DH and inhibition rate of β-conglycinin hydrolysates

The effect of protein concentration on the DH and inhibition rate of β-conglycinin hydrolysates was shown in Fig.2(A).The DH decreased slightly with increasing of protein concentration from 10 to 30 mg/mL and decreased sharply above 30 mg/mL.With increased protein concentration,the inhibition rate of β-conglycinin hydrolysates increased,and the inhibition rate at 30 mg/mL had no significant difference(P ＜0.05)with those at 20,40 and 50 mg/mL.Comprehensively considering the DH and the inhibition rate,the optimal protein concentration was selected as 30 mg/mL.

3.2.2.Effect of enzyme dosage on the DH and inhibition rate of β-conglycinin hydrolysates

Fig.2(B) showed the DH and inhibition rate of βconglycinin hydrolysates at different enzyme dosages(0.2%, 0.4%, 0.6%, 0.8% and 1.0%).The DH of βconglycinin hydrolysates increased significantly(P ＜0.05)with enzyme dosage while no significant differences were found when the enzyme dosage increased from the 0.6%to1.0%.With increased enzyme dosage,the inhibition rate of β-conglycinin hydrolysates decreased,reached the lowest at the enzyme dosage of 0.8%, then increased slightly in the late stage of hydrolysis.So the optimal enzyme dosage was 0.6%for DH and 0.8%for the inhibition rate.Considering the cost,the recommended dosage was 0.6%.

Table 2 Factors and levels of orthogonal tests.

Fig.2.The effects of protein concentration(A),enzyme dosage(B),time(C),pH(D)and temperature(E)on DH and inhibition rate of hydrolysates of β-conglycinin.

3.2.3.Effect of hydrolysis time on the DH and inhibition rate of β-conglycinin hydrolysates

Fig.2(C) showed the DH and inhibition rate of βconglycinin hydrolysates at different hydrolysis time(10,20,30,40 and 50 min).The DH of β-conglycinin hydrolysates increased significantly(P ＜0.05)within hydrolysis time 10 min to 30 min, while no significant differences were found when the hydrolysis time increased from the 40 to 50 min.With increased hydrolysis time,the inhibition rate of β-conglycinin hydrolysates decreased sharply from 10 to 40 min,then increased slightly in the late stage of hydrolysis.So the optimal hydrolysis time was 40 min for both DH and the inhibition rate.

3.2.4.Effect of pH on the DH and inhibition rate of βconglycinin hydrolysates

Fig.2(D) showed the DH and inhibition rate of βconglycinin hydrolysates at different pH(7.5,8.0,8.5,9.0 and 9.5).The DH increased significantly(P ＜0.05)with the increase of pH from 7.5 to 8.5 and then decreased significantly(P ＜0.05).With increased pH,the inhibition rate of β-conglycinin hydrolysates decreased sharply from pH 7.5 to 9.0,then increased significantly in the late stage of hydrolysis.So the optimal pH was 8.5 for DH and 9.0 for the inhibition rate.

3.2.5.Effect of hydrolysis temperature on the DH and inhibition rate of β-conglycinin hydrolysates

Fig.2(E) showed the DH and inhibition rate of βconglycinin hydrolysates at different hydrolysis temperatures(40,45,50,55,and 60°C).The DH of β-conglycinin hydrolysates increased significantly(P ＜0.05)with hydrolysis temperature increasing from 40 to 55°C,while no significant differences were found when the hydrolysis temperature increased from the 55 to 60°C.With increased hydrolysis temperature,the inhibition rate of β-conglycinin hydrolysates decreased greatly from 40 to 55°C,then increased slightly in the late stage of hydrolysis.So the optimal hydrolysis temperature was 55 °C for DH and the inhibition rate.

3.3.Orthogonal experiments

Based on the results of the single factor assays,the hydrolysis conditions,including enzyme dosage,time,pH,and temperature,were further optimized by an L9(34)orthogonal layout, with the fixed protein concentration of 30 mg/mL.The results of orthogonal experiments were shown in Table 3.By comparing R values in Table 3, the order of influence of each factor on the DH of soybean βconglycinin was pH(C)＞enzyme dosage(A)＞hydrolysis temperature(D)＞hydrolysis time(B).The optimal combination of the hydrolysis conditions was A3B3C2D2.Thus,the optimal hydrolysis conditions were enzyme dosage 0.7%,hydrolysis time 50 min,pH 8.5,and temperature 55°C for alkaline protease hydrolysis of soybean β-conglycinin.

The inhibition rate of β-conglycinin hydrolysates was also investigated using orthogonal tests(Table 3).The lowest inhibition rate of the hydrolysates was 27.28%.After comparing the R values of the inhibition rate,the effect of the four influencing factors on the inhibition rate ranked as pH(C)＞enzyme dosage(A)＞hydrolysis temperature(D) ＞hydrolysis time (B).The optimal combinations of the four factors on the inhibition rate were as follows:pH 8.5, hydrolysis temperature 55 °C, enzyme dosage 0.7%,and hydrolysis time 50 min.

3.4.Variance analysis and validation experiment

The variance analysis results of the orthogonal design(Table 4) showed that enzyme dosage, time, pH and the temperature had no significant effect on the inhibition rate,but all had significant effect on DH except for hydrolysis time.Therefore,the effect of enzymatic hydrolysis conditions on the DH of β-conglycinin was given priority in determining the optimal conditions (Table 4).Accordingto the optimal scheme of these three indexes,the optimal enzyme dosage was 0.7%,and the optimal hydrolysis temperature was 55 °C, and the optimal pH value was 8.5.The effect of hydrolysis time on each index was not significant,therefore 40 min was selected as the optimal time for efficient hydrolysis.Under the optimal conditions,the DH of β-conglycinin was 76.28%±2.36%and the inhibition rate was 27.03% ± 0.25%, all of which were similar to the values in the ninth group of orthogonal tests.

Table 3 Results of orthogonal experiments.

Table 4 Analysis of variance of the results of orthogonal design.

4.Discussion

4.1.Degree of hydrolysis of β-conglycinin hydrolysates

It has long been known that the biological properties of peptides are determined by their molecular structures and conformations,which are affected by the DH[16].DH is a basic parameter for producing hydrolysates with important biological activities[7].Therefore,it is necessary to optimize the design and control the hydrolysis of proteins to obtain hydrolysates with ideal functional activities.Hence,soybean βconglycinin was hydrolyzed using alkaline protease from B.subtilis ACCC 01746,and the DH of the protein under different conditions was studied and shown in Fig.2.The DH decreased with an increasing concentration of βconglycinin(Fig.2(A)).This decrease in the DH may have been caused by enzymatic efficiency descended [17].As shown in Fig.2(B)-(E),the DH of β-conglycinin hydrolysates was significantly influenced by the hydrolysis time,enzyme dosage,pH,and temperature.These results indicate that suitable parameters are important factors in the enzymatic hydrolysis of β-conglycinin.With increased enzymatic dosage and time, the increased DH of β-conglycinin hydrolysates may be attributable to the highly damaged molecular structure and reduced molecular crosslinking of β-conglycinin because of enzyme application, and protein denaturation could induce further cleavage of peptide bonds when the enzymic treatment was applied,having a potential effect on hydrolysis ability of β-conglycinin and thus on its DH[18].As for the initial increase but subsequent decrease in the DH of β-conglycinin hydrolysates in response to increasing pH and temperature,this phenomenon may have resulted from the change in alkaline protease activities [19].When the pH or temperature continued to increase,both the enzyme activity and DH decreased.

4.2.Antigenicity of β-conglycinin hydrolysates

It is well known that enzymolysis has a significant effect on destroying not only the primary structure of a protein,but also its higher structure,thereby exposing a large number of hydrophobic groups and more enzymatic cutting sites.Therefore,enzymatic hydrolysis is the most promising strategy for removing the antigenicity of soybean antigenic proteins.This enzymatic treatment destroys linear and conformational epitopes,preventing IgG and/or IgE mediated responses[20].Although some proteases,such as alcalase,flavorzyme, trypsin, and papain, etc., have been widely used in this treatment,the search for new protease to improve the hydrolysis of protein is still the focus of many types of research[21].

The difference in hydrolysis effect was mainly related to the hydrolysis site of the protease.B.subtilis alkaline protease is classified as a serine peptidase,in which the hydrolysis site is the carbon end of hydrophobic amino acid[13].It destroys the spatial structure and epitope conformation of antigenic protein, thus reducing or losing the ability to bind to specific antibodies[14].In this study,we confirmed again that the alkaline protease from B.subtilis was an effective enzyme to degrade the antigenicity in β-conglycinin.Similarly, in previous studies, enzymatic treatment has been shown to play a positive role in the hydrolysis of βconglycinin,which has also been shown to decrease the antigenicity of alcalase-hydrolyzed β-conglycinin [22].The key information for the reduced antigenicity of the hydrolysates after enzyme treatment may be that the peptide containing complete epitope was cut off, and the degree of antigenicity decline depends on the extent of antigen site destruction.However,it can be seen from Fig.2C that the antigenicity of β-conglycinin was slightly increased instead of decreasing at the later stage of enzymatic hydrolysis.This may be due to the further hydrolysis of peptides by protease,which may expose some linear or recessive epitopes,or the peptides with low antigenic activity continue to hydrolyze into small fragments with high activity.Similar phenomena were observed by Sung et al.[23].The results showed that enzymatic hydrolysis was an effective way to reduce the antigenicity, but none of the enzymes could eliminate the allergenicity.

5.Conclusions

The alkaline protease isolated from the B.subtilis appeared to show a relatively effective reduction of βconglycinin antigenicity.Therefore, production of lowallergenicity soybean proteins may be possible using enzymatic hydrolysis.Although clinical trials are needed to assess the residual antigenicity of soybean allergens and to prevent soybean protein allergy,the promising results of this work pave the way to the biosafety disposal of soybean proteins.These findings suggest that the non-commercial alkaline protease can be used as a reference for the application of alkaline hydrolysis and may be helpful for the further popularizing application of this enzymolysis technology.

Author Contributions

Haicheng Yin: Conceptualization, methodology, data curation, writing- original draft preparation, editing and reviewing,project administration;Xinrui Zhang:Writingreviewing and editing; Jin Huang: Data curation,experiment.

Conflicts of Interest

The authors declare that there are no conflicts of interest.

Acknowledgments

Thanks to Grain & Corn Engineering Technology Research Center,State Administration of Grain(GA2017004),and Science and Technology Research Project of Henan(172102110205 and 182102310676)for funding support.