Haicheng Yin,Feng Jia,Jin Huang,Yong Zhang,Xin Zheng,Xinrui Zhang

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

Keywords:

Soybean

Extrusion

β-Conglycinin

Structure

Antigenicity

ABSTRACT

β-Conglycinin,the main component of 7S globulin in soybean protein,is also a key soybean antigen protein that causes allergic reactions.Extrusion technologies have received considerable attention as a method for modifying soybean protein allergens.This study investigated the changes in β-conglycinin structure and antigenicity upon extrusion.Isoelectric precipitation,ammonium sulfate precipitation,and sepharose CL-6B gel filtration were used to isolate and purify βconglycinin from soybean powder,and single-factor and orthogonal tests were used to study the effects of water content,extrusion temperature,screw rotation speed,and feeding speed on the antigenicity of β-conglycinin after extrusion.Fourier transform infrared spectrometry(FTIR)was then employed to analyze the structure of β-conglycinin after extrusion under the optimal conditions determined by the orthogonal test.The results showed that extrusion significantly reduced the antigenicity of β-conglycinin(P ＜0.05),and the degree of influence of the factors studied may be ordered as extrusion temperature ＞feeding speed ＞screw rotation speed ＞water content.The optimal parameters were temperature at 130 °C,screw rotation speed at 140 r/min,and feeding speed at 35 g/min.Under these conditions,the contents of α-helix,β-pleated sheet,and β-turn structures in β-conglycinin were significantly reduced(P ＜0.05),while the contents of random coils were significantly increased(P ＜0.05).The peak absorption intensity of amides I,II,and III also decreased.Taken together,the findings suggest that extrusion could be an effective method for reducing the antigenicity of β-conglycinin.

1.Introduction

Soybean,which contains nearly 35%-40% protein,is a common plant protein source used in the food and feed industries[1].However,soybean protein contains various anti-nutritional factors,including antigenic proteins,which not only affect intestinal digestion and nutrient absorption,but also induce adverse reactions such as allergies [2].These negative influences were partly due to the anti-nutritional factors present in soybean protein,such as glycinin,β-conglycinin,and trypsin inhibitor.β-Conglycinin,an antigenic protein in soybean with strong antigenicity,accounts for about 20%-30%of the total soybean protein;this molecule is a glycoprotein formed by linking the side-chain amino group of asparagine with acetyl-glucosamine through β-N-glucosidic bonds (sugar content,about 5%) to form a total of three subunits,namely,α,α′,and β.A trimer with a molecular weight in the range of 180-200 kDa is formed between these subunits by linking with water molecules through hydrogen bonds(H bonds)[3].Therefore,an appropriate modification method must be adopted to change or disrupt the structure of β-conglycinin,reduce its content,and/or inactivate its sensitization.

The antigenicity of β-conglycinin is determined by its structure.Different functional groups in the polypeptide chain and the special composition of amino acids form the basis of the protein's antigenicity.In its natural state,for example,β-conglycinin presents a dense molecular structure and is a heat-stable protein.Conventional heating treatments can unfold,crosslink,and polymerize the molecular structure of this molecule,causing disulfide bonds(SS)to break,reducing its antigenicity to some extent.This effect,however,is not ideal[4].A previous study showed that extrusion inactivated most of the heat-sensitive anti-nutritional factors in soybean,resulting in changes in the molecular structure of the antigenic proteins[5].An analysis of extruded soybean by Yao et al.[6]showed that when the extrusion temperature was in the range of 110-120 °C,the globulin and β-conglycinin contents in the samples were decreased by 67% and 90%,respectively.Similarly,Zhao et al.[7]treated soybean meal using extrusion technology,and found that the secondary structures of 7S and antigenic proteins in the meal were modified when the extrusion temperature was increased to 60 °C.Despite these studies,research on the effect of extrusion on the structure and antigenicity of β-conglycinin is limited.

FTIR spectroscopy is an important and advanced method for studying protein conformations.The absorption peaks of FTIR spectrum are caused by stretching changes in dipole moment and the sensitivity of CO,NH,OH,and polar groups to vibrational changes.This technique is especially helpful for determining the secondary structure of proteins through computer-aided analysis;thus,it is widely used in protein structure studies[8].ic-ELISA was used in this study to detect changes in the antigenicity of β-conglycinin treated by extrusion.For β-conglycinin treated by optimized extrusion,FTIR spectroscopy was adopted to analyze changes in the secondary structures of the antigenic protein,and determined the relationship between changes in its structure and antigenicity.The results of this work provide a scientific basis for exploring new soybean protein processing technologies and developing soybean proteins with low allergenicity.

2.Materials and metho ds

2.1.Test materials and equipment

Soybean seed(Yu-29)was bought from the Academy of Agricultural Sciences of Henan,mashed,and then filtered through 80 mesh sieves.NHexane was used to prepare defatted soybean powder through degreasing.Soybean protein powder was finally obtained by isoelectric precipitation and salting out.

A twin-screw extruder (Brabender DSE-25,Germany) with an outer screw diameter of 25 mm,length-diameter ratio of 20:1,circular die,and die mouth aperture of 4 mm was used to extrude the soybean protein powder.A high-speed freezing centrifuge(LGJ-18,Shanghai Anting Scientific Instrument Factory)was used to sample pretreatments.

2.2.Extraction and purification of β-conglycinin

2.2.1.Isolation of β-conglycinin

β-Conglycinin was isolated from Yu-29 soybean protein powder according to the method described by Bu et al.[9]with slight modifications.At room temperature,soybean protein powder was mixed with 0.03 mol/L Tris-HC1 solution(pH 8.5)at a ratio of 1:10.The mixture was then heated to 45 °C,stirred for 1 h,cooled to 4 °C,and then centrifuged for 20 min at 10,000 r/min.The supernatant was collected and mixed with 0.01 mol/L NaHSO3and 5 mmol/L CaC12.After adding 2.0 mol/L HCl to adjust the pH to 6.4,the solution was allowed to stand overnight.The solution was centrifuged for 20 min at 10,000 r/min,the supernatant was collected,and the pH of the supernatant was adjusted to 5.5 using 2.0 mol/L HCl.The solution was stirred for 1 h at 4 °C,followed by centrifugation for 20 min at 10,000 r/min to obtain the supernatant.The collected supernatant was diluted once with pure water,and its pH was adjusted to 4.8 using 2.0 mol/L HCl.The resulting solution was centrifuged at 10,000 r/min and 4 °C for 20 min.

The obtained precipitate was dissolved in 0.03 mol/L Tris-HCl buffer solution,and the pH of the resulting mixture was adjusted to 6.2 with 2.0 mol/L HCl.The solution was centrifuged for 20 min at 10,000 r/min and 4 °C,and the pH of the supernatant was adjusted to 7.6 using 2.0 mol/L HCl to obtain the crude 7S component.The 7S component was used to produce a 51%saturated solution with(NH4)2SO4,and the saturated solution was centrifuged for 20 min at 4 °C and 8500 r/min,then,the supernatant was collected.The supernatant was used to produce a 100%saturated solution with(NH4)2SO4,and this solution was centrifuged for 20 min at 4 °C and 8500 r/min.The precipitate was dissolved in phosphate buffer(pH 7.6)and then freeze-dried after dialysate desalination.Finally,a Sepharose CL-6B gel column(1.6 cm ×120 cm)was used to filter the solution at a flow rate of 0.45 mL/min.Here,the eluant was composed of 2.6 mmol/L KH2PO4,32.5 mmol/L K2HPO4,0.4 mol/L NaCl,and 10 mmol/L β-mercaptoethanol (pH 7.6,ionic strength 0.5).The elution peaks of target proteins were collected,and β-conglycinin was obtained through freeze-drying after dialytic desalination.

2.2.2.SDS-PAGE and purity analysis of β-conglycinin

The SDS-PAGE and purity analysis of β-conglycinin were tested using the method proposed by Bu et al.[9].Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) was performed with a 15%separating gel and 5%stacking gel to detect protein profiles of the βconglycinin samples.A 2.5 mg β-conglycinin sample was dissolved in pH 8.0 and 1 mol/L Tris-HCl buffer solution(containing 2%SDS,2%2-mercaptoethanol,0.2%bromophenol blue,and 10%glycerol).Electrophoresis was performed in a vertical electrophoresis unit at concentration gel 80 V,separation gel 150 V for 1.5-2.0 h.For dyeing the gel,the concentration of Coomassie brilliant blue(R-250)was 0.25%,and the gel destaining solution was composed of 50%methanol and 5.0%acetic acid solution.Quantity one software was used to analyze the purity of the obtained protein.

2.3.Extrusion experiment design

2.3.1.Single-factor extrusion operation

Approximately 500 g of β-conglycinin was used for each experimental group.The extruder was preheated for 1 h to achieve a stable state.A single-factor experiment was used to determine the best conditions for the extrusion process.Extrusion temperature,feeding speed,screw rotation speed,and water content were the variables.First,under an extrusion temperature of 140 °C,screw rotation speed of 120 r/min,and feeding speed of 40 g/min,the effects of water contents of 10.0%,12.0%,14.0%,16.0%,and 18.0%on the antigenicity of β-conglycinin were studied.Then,under a screw rotation speed of 120 r/min,feeding speed of 40 g/min,and water content of 14.0%,the effects of extrusion temperatures of 100,120,140,160,and 180 °C on the antigenicity of β-conglycinin were studied.Third,under an extrusion temperature of 140 °C,feeding speed of 40 g/min,and soybean water content of 14.0%,the effects of screw rotation speeds of 80,100,120,140,and 160 r/min on the antigenicity of βconglycinin were studied.Finally,under an extrusion temperature of 140°C,screw rotation speed of 120 r/min,and soybean water content of 14.0%,the effects of feeding speeds of 20,30,40,50,and 60 g/min on the antigenicity of β-conglycinin were studied.Experiments were conducted in triplicate and the results were expressed as the mean ±SD.All data were analyzed using SPSS17.0.Subsequent orthogonal experiment design was based on the results of the single-factor experiments.

2.3.2.Determination of the effects of extrusion on the antigenicity of βconglycinin via an orthogonal experiment design

Based on the study of the effects of extrusion conditions on the antigenicity of β-conglycinin,the factors and levels greatly affecting antigenicity were selected to create an L9(34)orthogonal experiment and determine the best conditions to reduce the antigenicity of β-conglycinin(Table 1).

2.4.Antigenic detection of β-conglycinin

96-Well and 48-well ELISA plates were used to set up control and experimental groups and detect the antigenicity of β-conglycinin obtained from single-factor and orthogonal experiments.Four wells were prepared for each group.Here,100 μL of standard β-conglycinin (5.0 μg/mL) was added to each well,and the plates were incubated at 4 °C overnight.Then,each well was washed with PBST four times for 3 min once time,and then the plates were patted dry.About 100 μL of blocking liquid was added to each well,and the plates were incubated once more for 1 h at 37 °C.The plates were subsequently washed with PBST (0.15 mol/L,pH 7.4)four times and patted dry.Equal volumes(100 μL/well)of evenlymixed antiserum and β-conglycinin sample liquid(5.0 μg/mL,not added to the control group)were added to each well and incubated for 1 h at 37 °C.The plates were washed with PBST four times and then patted dry.About 100 μL of goat anti-rabbit IgG(HRP tab,A6154)was added to each well,and the plates were further incubated for 1 h at 37 °C.The plates were washed with PBST four times and then patted dry.Finally,100 μL of TMB chromogenic liquid was added to each well,and the plates were allowed to develop for 10 min at 37 °C in the dark.About 50 μL of 2 mol/L H2SO4was added to each well to terminate the reaction.A double-wavelength microplate reader was used to detect the OD of each well at 450 and 620 nm(OD=OD450-OD620).

Table 1 Factors and levels in orthogonal experiment.

The antigen inhibition rate was measured to determine the antigenicity of β-conglycinin;this value can be calculated as antigen inhibition rate(%)=(1-OD/OD0)×100%,where OD refers to the absorbance of the detected sample and OD0refers to the absorbance of a sample in the control group.The antigenicity was expressed by the antigen inhibition rate.The lower the inhibition rate,the lower is the antigenicity of β-conglycinin in the sample,and there is a positive relationship between them.

2.5.Determination of the secondary structure of β-conglycinin by FTIR

A sample of about 2 mg β-conglycinin was treated by optimized extrusion and mixed with potassium bromide at a ratio of 1:100.After grinding,thin transparent slices were placed in an FTIR sample room (Shimadzu FTIR-8400S,Japan).The wavelength range was set to 400-4000 cm-1within 32 scans,and the resolution was 4 cm-1.Peak FIT 4.12 software was used for second-derivative convolution fitting to determine amide band I of β-conglycinin and calculate the α-helix,β-pleated sheet,β-turn,and random coil contents of this band.

2.6.Statistical analysis

After the experimental data were sorted by Excel 2007,SPSS17.0 software was used for one-way ANOVA.If a significant difference was detected,Duncan's multiple comparison test was performed.P ＜0.05 was considered to indicate a statistically significant difference.The single-factor test was repeated three times(n=3),and the results reported are the mean value ±standard deviation of these tests(mean ±SD,n=3).

3.Results and discussion

3.1.Extraction of β-conglycinin

The separation and purification of soybean protein antigens has been studied for many years.With the evolution and improvement of protein separation technology,alkali-solution and acid-isolation,phytase and pepsin degradation,and liquid chromatography methods of separation technology have become essential measures in soybean protein antigen separation and purification[10].The difficulties associated with β-conglycinin isolation have been gradually solved,and the related processes are widely used in the industry.However,the β-conglycinin obtained using these methods is of low purity,and their applicability to studies on the immune response leads to sub-optimal results.In this study,a method involving Tris-HCl extraction,alkali solution,and acid isolation was used to extract β-conglycinin from soybean protein powder.SDS-PAGE was used to clearly show the α,α′,and β subunits of soybean β-conglycinin.This technique remains the most popular method for isolating soybean protein antigens.

Quantity One software analysis showed that the purity of β-conglycinin isolated in this paper was 85.4%(W/W);such high purity can satisfy the requirements of subsequent antigenicity analysis.The α,α′,and β subunit contents of β-conglycinin were 44.1%,24.4%,and 16.9%,respectively.The results of this analysis were shown in Fig.1.

3.2.Effects of single-factor extrusion on the antigenicity of β-conglycinin

The effects of different extrusion temperatures,screw rotation speeds,and feeding speeds on the antigenicity of β-conglycinin after extrusion were shown in Fig.2(A,B,C,and D).Compared with the control group,extrusion could significantly reduce the antigenicity of β-conglycinin(P ＜0.05).This significant reduction in the antigenicity of β-conglycinin might be a result of the large shearing force produced by high temperatures and pressures during extrusion,which could cause depolymerization of the polypeptides of β-conglycinin,fracture peptide chains,and destroy epitopes on the molecular surface of the antigen protein,thereby reduced its antigenicity[5].Fig.2A showed the effects of soybean water content on the antigenicity of β-conglycinin.As the water content increases,antigenicity decreased.The lowest antigenicity was observed in the group with a water content of 16%,although differences among groups were not significant(P ＞0.05).This result indicated that the effect of water content on the antigenicity of β-conglycinin was limited.The main reason behind the reduction in the antigenicity of β-conglycinin by water content was the related increase in viscosity.As the viscosity of the sample increasing,the shearing force caused by extrusion also increased and antigenicity reduced[11].

The antigenicity of β-conglycinin decreased with increasing extrusion temperature.Temperature is an important factor in protein denaturation.As the extrusion temperature increasing,H and SS bonds between protein molecules are partially broken and the spatial structure of the original protein spreads,resulting in destruction of its molecular structure.Further increases in extrusion temperature destroy the CN,CO,NH,and other chemical bonds holding the spatial structure of the protein molecule together,and the secondary structure of the protein is destroyed.This action also destroys the epitopes of the protein antigen,thereby reduces its antigenicity[12].When the extrusion temperature was set to 100 °C,the antigenicity of β-conglycinin was significantly higher than that at extrusion temperatures above 140 °C(P ＜0.05).However,the result of this group did not significantly differ from that of the group treated at 120 °C(P ＞0.05).Additionally,differences in antigenicity among groups treated at 120 °C and higher were not significant(P ＞0.05)(Fig.2B).These results revealed that β-conglycinin was a thermally stable protein.When the extrusion temperature was about 100-120 °C,epitopes on the molecular surface of the antigen were partially blocked or destroyed by aggregation and crosslinking of β-conglycinin molecules,which resulted lower in antigenicity than that of the control group[13].When the extrusion temperature was continuously increased to 140 °C and above,complete destruction of the molecular structures and epitopes of β-conglycinin occurred,causing irreversible changes in intermolecular recombination or interconnections.These changes,in turn,significantly reduced the antigenicity of βconglycinin.These results are similar to the findings of Ohishi et al.[14],who demonstrated that double-screw extrusion could reduce the antigenicity of soybean protein.When the screw rotation speed was increased,or the feeding speed was decreased during extrusion,the shearing force on the sample increased and the antigenicity of β-conglycinin decreased.When the screw rotation speed was increased from 80 r/min to 140 r/min,the antigenicity of β-conglycinin was gradually reduced.When screw rotation speed was increased to 160 r/min,the antigenicity was significantly reduced (P ＜0.05),although this result did not significantly differ from that at 140 r/min(P ＞0.05)(Fig.2C).

Fig.1.SDS-PAGE for soybean β-conglycinin.

Fig.2.Effect of different extrusion factors on the antigenicity of β-conglycinin.

The antigenicity of β-conglycinin at feeding speeds of 20 and 30 g/min was significantly lower than that at 50 g/min and 60 g/min(P ＜0.05)(Fig.2D).The reduced antigenicity of β-conglycinin caused by extrusion was related to reductions in the ordered structures of the α-helices,βpleated sheets,and β-turns of the antigen,as well as increased in random coil content,which promoted the destruction of epitopes in its molecular structure [14].Taken together,the results demonstrated that extrusion temperature,screw rotation speed,and feeding speed greatly affected the antigenicity of β-conglycinin.In contrast,water content played only a slight effect.

3.3.Optimization of the extrusion conditions of β-conglycinin by an orthogonal experiment

L9(34)orthogonal experiment was used to study the effects of extrusion temperature,screw rotation speed,feeding speed and water content on the antigenicity of β-conglycinin,as shown in Table 2.Extrusion temperature was the most important factor in affecting the antigenicity of β-conglycinin,followed by feeding speed and screw rotation speed,and the final factor was water content.The optimum combination was A3C1B2D1,which corresponded to the results of extrusion temperature of 130 °C,a feeding speed of 35 g/min,a screw rotation speed of 140 r/min,and a water content of 12%.Confirmation of optimal extrusion conditions revealed that the antigenicity of β-conglycinin under these conditions was 20.06%,which was less than the minimum inhibition rate(20.89%)in Table 2.

3.4.FTIR analysis of the secondary structures of β-conglycinin before and after extrusion

The vibrational form of each group in a molecule corresponds to its absorption peak in an FTIR spectrum.Figs.3 and 4 showed the FTIR spectra of β-conglycinin without and with optimal extrusion treatment,respectively.The absorption peak located at 3600-3500 cm-1refers to the extensional swing of NH and OH bonds in the protein molecule,as well as stretching vibrations of the NH group of amide A and B bands.The absorption peak at 3000-2800 cm-1refers to the extensional swing of the CH bond.Absorption peaks at 1700-1600,1550-1530,and 1330-1260 cm-1refer to the characteristic peaks of amide I,amide II,and amide III,all of which are commonly used in studies on the secondary structures of protein.In particular,amide I is strongly related to the secondary structure of a protein[15].The following parameters were used in the current manuscript based on literature review:amide I,the CN and CO stretching vibrations;amide II,the NH bending vibrations or CN stretching vibration;amide III,the NH bending vibrations or CN stretching vibrations[16].Compared with the FTIR spectrum of untreated β-conglycinin,the FTIR spectrum of β-conglycinin obtained after extrusion revealed changes in the absorption peaks and absorbance of the amides.The intensities of the absorption peaks of amides I,II,and III of β-conglycinin treated by extrusion were significantly reduced those before extrusion.In particular,the absorption peaks of amides I and II moved toward shorter wavelengths while the absorption peak of amide III moved toward higher wavelengths.These findings demonstrated that the stretching vibration intensities of CN,CO,and NH bonds of β-conglycinin decreased after extrusion,and that changes in the absorption peak of amide II can cause corresponding changes in the H bonds between/or in protein molecules,thus and the secondary structures of β-conglycinin[17].The absorption peak of α-helices is 1654 cm-1while those of βpleated sheets are 1630 and 1245 cm-1,thus demonstrating significantly reduced peak intensity.This result revealed that α-helix and β-pleated sheet contents could be decreased in the treated β-conglycinin.

Table 2 L9(34)orthogonal experimental results.

Fig.3.FTIR of unextruded β-conglycinin.

PeakFIT 4.12 was used to achieve second-derivative convolution fitting of amide I of β-conglycinin before and after extrusion to enable calculation of the changes in α-helix,β-pleated sheet,β-turn,and random coil contents in the secondary structure of the antigenic protein.The results were shown in Table 3.The secondary structure of β-conglycinin without extrusion showed a β-pleated sheet content of 39.21%,a β-turn content of 35.77%,an α-helix content of 12.55%,and a random coil content of 12.47%.By comparison,the secondary structure of β-conglycinin treated by extrusion revealed an α-helix content of 7.33%,a β-pleated sheet content of 34.69%,and a β-turn content of 30.71%,which significantly reduced relative to the untreated β-conglycinin.In contrast to these results,a significant increase in random coil content of 27.27%was observed(P ＜0.05).These results indicated that extrusion could greatly change the secondary structure of β-conglycinin,likely because extrusion can change the original rigid structure of β-conglycinin,produce expanded protein molecules and cut-off peptide chains.The α-helices,β-pleated sheets,and β-turns in the secondary structure of the antigen changed into random coils,consistent with the findings of Achouri et al.[18].The single-factor extrusion experiment results revealed that increasing the extrusion temperature and screw rotation speed,decreasing the feeding speed decreased the antigenicity of β-conglycinin.Such changes may be attributed to a decrease in the ordered structures of α-helices,β-pleated sheets,and β-turns in the secondary structure of the antigen,as well as an increase in the order of random coils,which changes the spatial structure of β-conglycinin and conceals or destroys its epitopes.

Fig.4.FTIR of extruded β-conglycinin.

Table 3 Secondary structures content of β-conglycinin after extrusion(%).

4.Conclusion

The results showed that the antigenicity of β-conglycinin was reduced by increasing the extrusion temperature,and screw rotation speed at low feeding speeds.Compared with the other factors tested,soybean water con

tent exerted a small influence on the antigenicity of the protein.The optimal extrusion parameters included a temperature of 130 °C,screw rotation speed of 140 r/min,and feeding speed of 35 g/min.Under these conditions,the antigenicity of β-conglycinin was only 20.06%,which was lower than the results of the orthogonal experiment(20.89%).FTIR spectroscopy revealed that the contents of α-helix,β-pleated sheet,and β-turn of β-conglycinin significantly decreased,while the content of random coils significantly increased when the protein was extruded under optimal conditions.The results of this work provide a theoretical basis for selecting suitable processing technologies to reduce the antigenicity of soybean protein.

Conflicts of interest

The authors declare that there are no conflicts of interest.

Acknowledgments

We thank NSFC-Joint Research Fund of Henan(U1404323),Grain&Corn Engineering Technology Research Center,State Administration of Grain(GA2017004)and Science and Technology Research Project of Henan(172102110205 and 182102310676)for providing funds.