Physicochemical and structural characteristics of the Venn components of wheat gliadin

Feng Jia,Changfu Zhang,Qi Wang,Jinhe Li,Yu Chen,Jinshui Wang*

College of Bioengineering,Henan University of Technology,Zhengzhou 450001,China

Keywords:

Wheat gliadin

Venn components

Physicochemical properties

Structural characteristics

ABSTRACT

The aim of this study was to analyze the physicochemical and structural characteristics of the Venn components of wheat gliadin to provide theoretical basis of gliadin for processing in dough and Chinese steamed bread.Eight Venn components,Gli-8,Gli-9,Gli-10,Gli-11,Gli-12,Gli-13,Gli-14,and Gli-15,were extracted from wheat gliadin based on their solubility.The results of physicochemical characteristics showed that the differences in the contents,TDS,electrical conductivity,particle size and zeta potential of Venn components were significant,respectively.The content of Gli-15 in gliadin was the highest,and the content of Gli-9 was the lowest.The TDS value of Gli-9 was the highest(336.0),and the TDS value of Gli-15 was the lowest(52.0).The electrical conductivity of Gli-9 was the highest,which was 7.54 times the lowest value of Gli-11.The zeta potential of Gli-9 was-25.2 mV,and the zeta potential of the Gli-15 was-7.64 mV.However,the difference in the pH values was not significant.The results of UV spectrum and FTIR analysis showed that the secondary structures of the Venn components had significant differences.The results of the XRD patterns indicated that the Venn components might not be a single substance.The results of CLSM images implied that the molecular interactions among the components were varied.Hence,the results could provide research materials and basic data for deep processing and utilization of gliadin.

1.Introduction

The proteins of wheat can be divided into four groups based on their solubility:glutenin,gliadins,globulins,and albumins[1].Gliadins,which constitute approximately 50% of the total storage proteins, have high heterogeneity and complexity in their composition.The gluten network structures are closely related to the quality and quantity of gliadins present in wheat flour,which are the main factors that affect the viscoelasticity and the baking quality of the wheat dough[2].Gliadin exhibits an affinity for a variety of hydrophobic compounds.It was proposed that gliadin could be applied as a carrier for hydrophobic compounds,due to its hydrophobic nature[3].Gliadin is also associated with gluten protein diseases and is responsible for changing the redox balance of the cell[4].

Venn diagrams are commonly used to display gluten with different solubility by different circles,and each circle contains members with similar solubility.Our previous researches showed that wheat flour proteins could be divided into 15 types with Venn classification diagram based on the differences in solubility, including unitary-soluble protein, binarysoluble protein,ternary-soluble protein and quaternary-soluble protein[5,6].Gliadin could be divided into eight components[6],among them,Gli-8 could significantly increase the tensile breaking force of dough;Gli-9 had no significant effect on dough characteristics;Gli-10 could increase the content of free sulfhydryl groups of dough;Gli-11 could significantly reduce the gluten index of dough and increase the content of wet gluten;Gli-15 could significantly reduce the gluten index of dough and increase the stretch distance of dough[7].Previous studies indicated that eight Venn components isolated from wheat gliadin were quite different in the physicochemical properties,which might decide their functional properties exhibited in dough and Chinese steamed bread.However, their physicochemical and structural characteristics were not clear.Therefore,the present study aimed to check the physicochemical and structural characteristics of Venn components isolated from gliadin.The results of this study could provide research materials and basic data for the deep processing and utilization of gliadin.Moreover,the results of this study were to provide theoretical basis of gliadin for processing in dough and Chinese steamed bread.

2.Materials and methods

2.1.Materials

Wheat gluten was purchased from the local market (Feitian, Henan Feitian Agricultural Development Co., Ltd.).The contents of crude protein, moisture, fat, and ash were 85.68 g/100 g, 8.7 g/100 g,3.97 g/100 g, and 1.65 g/100 g (dry basis,W/W), respectively.Gliadin was extracted by the method previously described by Jia et al.[6].Briefly, wheat gluten (10 g) was stirred in 70%V/Vethanol(100 mL) for 2 h at room temperature, followed by centrifugation at 4000 × g for 20 min.The supernatant was collected and ethanol was removed using a rotary evaporator at 45 °C.Wheat gliadin was isolated after freeze-drying.

2.2.Extraction of Venn components from gliadin

The separation process for each of the Venn components of gliadin is shown in Fig.1.(1) 10 mL of 70% ethanol was added to 1000 mg of the gluten, then vortexed vigorously for 30 s.The gliadin was extracted by shaking for 30 min, subsequently the extract was centrifuged at 4000 × g for 20 min.(2) The samples of gliadin were mixed with 0.5% of sodium dodecyl sulfate (SDS) solution according to the ratio of 1:10 (W/V), respectively, and then the mixture was shaked for 30 min and centrifuged at 4000 × g for 20 min.The supernatant and the precipitate were then dried.(3) The dried supernatant and precipitate were mixed with distilled water at a ratio of 1:10(W/V), respectively, shaked for 30 min, centrifuged at 4000 × g for 20 min and dried again.(4)The process was repeated using 0.5 mol/L NaCl aqueous solution according to the ratio 1:10(W/V),respectively,and then the mixture was shaked for 30 min and centrifuged at 4000×g for 20 min.After dialysis and drying,the components of the supernatant were Gli-9,Gli-10,Gli-13,and Gli-14,and the components of the precipitate were Gli-8,Gli-11,Gli-12,and Gli-15.

2.3.Determination of physicochemical properties of Venn components

2.3.1.Determination of total dissolved solids

Total dissolved solids(TDS)were detected using a TDS-3 detection pen(Shenzhen Yumei Instrument Equipment Co.,Ltd.,China).10 mL of 70%ethanol was added to 100 mg of the Venn components and shaked well.The pen was inserted into the solution, and the value was read/noted after the solution was stabilized.

2.3.2.Determination of electrical conductivity

The electrical conductivity of the Venn components was measured using a DDS-11AW conductivity meter(Shanghai Minyi Electronics Co.,Ltd.,China).The sample(100 mg)was mixed with 20 mL of 70%ethanol,and stirred with a glass rod to make the solution uniform for measuring the electrical conductivity.A calibration electrode was set constant at 0.95,the surface was washed with purified water three times,then washed twice with the solution to be measured.Next, the washed electrode was immersed in the solution to be measured to measure the conductivity.Finally,the electrode was washed twice with purified water before testing the next sample.

Fig.1.Separation process of Venn components of gliadin.

2.3.3.pH measurement

The pH of the suspension was measured by pHS-3C acid-alkalinity meter(Hangzhou Paipang Automation Technology Co.,Ltd.,China).A mixture of 100 mg of the Venn components and 20 mL of 70%ethanol was stirred with a glass rod until the pH of the suspension became constant.

2.3.4.Particle size distribution and zeta potential measurement

The particle sizes and zeta potentials of the Venn components were determined according to the method of Li et al.[8] using a Zetasizer Nano ZS90 particle size analyzer (Malvern Instruments Limited, United Kingdom).When the PDI value exceeded 0.5,the average particle size z-average of each component was recorded.

2.4.Determination of ultraviolet absorption characteristics

The ultraviolet(UV)absorption characteristics of the Venn components were determined using a Thermo Scientific NanoDrop 2000(Thermo Fisher Scientific,United States).5 mg Venn components was dissolved completely in 500 μL ethanol solution.The absorption characteristics of the Venn components were checked in the range of 260-290 nm.

2.5.Determination of the secondary structure of gliadin

The secondary structure of the Venn components was studied using a BioRad FTS 165 Fourier transform near-infrared spectroscopy(FTIR)(Varian,Inc.,United States).The spectra were recorded on the FTIR in the wavelength range of 4000-400 cm-1.Peakfit v4.12 was used to peak separation fit in the FTIR spectrum of wave number range of 1600-1700 cm-1and the structural parameters were calculated subsequently.KBr discs were prepared according to the method of Jia et al.[9].Each spectrum was baseline corrected according to the method described by Wellner et al.[10].Fourier self-deconvolution(FSD) was also carried out with an enhancement factor of 1.3 and a bandwidth of 30.The positions of the absorbance peaks located in the amide I region were determined using the second derivative[11,12].

2.6.Determination of X-ray diffraction patterns

X-ray diffraction(XRD)patterns analyses were performed using a X-ray diffractometer(model SmartLab,Rigaku,Japan)with Cu Kα radiation in the 2θ range of 10-80°at 40 mV and 40 mA.

2.7.Determination of the structural morphology of gliadin

Images of Venn components of gliadin were investigated using a confocal laser scanning microscopy(CLSM)(Model LSM 710,Germany)according to the method described by Jia et al.[13]and Wang et al.[14].

2.8.Statistical analysis

All data were expressed as mean±standard deviation(SD)of three replicates.Data was analyzed using the one-way analysis of variance(ANOVA)test,and the Duncan's multiple range was used to test the differences at a value ofP＜0.05 using SPSS 16.0 software.The results were plotted using the Excel 2007 software.

3.Results and analysis

3.1.Physicochemical properties of Venn components

Gliadin was extracted from gluten and the average extraction rate of gliadin was 20.86% ± 0.53%.The contents of the eight Venn components in gliadin were shown in Fig.2A.According to Fig.2A,the Gli-15 content was as high as 54.02%, and was the major component in gliadin; and the Gli-9 content was the lowest, only 0.69%.The results of the Duncan's test showed that the contents of Venn components among the group one (Gli-15), group two (Gli-11/Gli-12/Gli-14), group three (Gli-8/Gli-10/Gli-13), and group four (Gli-9) had significant difference (P＜0.05).

TDS is water quality parameter, which is used to describe salinity level [15].The results of TDS of eight Venn components in gliadin were shown in Fig.2B.The results showed that the TDS values of the Venn components in gliadin varied widely.The results of Duncan's test showed that there were significant differences (P ＜0.05) among the six groups of Gli-9, Gli-8, Gli-10,Gli-13/Gli-14, Gli-12, and Gli-11/Gli-15.The TDS value of Gli-9 was the highest (336.0 mg/L), and the TDS value of Gli-15 was the lowest (52.0 mg/L).The highest TDS value was 6.5 times the lowest.

The results of the electrical conductivity of the Venn components were shown in Fig.2C.The results showed a big difference in electrical conductivity of the Venn components;the trend was similar to the TDS values,and their correlation coefficient was 0.9817.The results of the Duncan's test showed that there were significant differences(P＜0.05)among the electrical conductivities of five groups,namely Gli-9,Gli-8,Gli-10/Gli-14,Gli-12/Gli-13,and Gli-11/Gli-15.Among these,the electrical conductivity of Gli-9 was the highest,which was 7.54 times the lowest electrical conductivity of Gli-11.

The pH of the Venn components were shown in Fig.2D.According to Fig.2D, there was no significant difference among the pH values of the eight components, which were all weak acid solutions.

The differences in particle size of Venn components were significant (Fig.2E).The Gli-15, which could be dissolved only in ethanol solution, had the largest average particle size.The average particle sizes of components (Gli-11, Gli-12, and Gli-14) soluble in two kinds of solutions were larger than those of components (Gli-8, Gli-10, and Gli-13) soluble in three solutions.Moreover, the average particle size of Gli-9,which could be dissolved by four solutions, was the smallest.These results suggested that the smaller the particle size the better its solubility.

The size of zeta potential indicates the development trend of the stability of the colloidal system [16].When the zeta potential is larger than 30 mV or less than-30 mV,the solution is considered stable[17].In addition,pH is the most important factor that affects the zeta potential,and the zeta potential is negative when the pH of the protein solution is greater than the isoelectric point[18,19].The results of the zeta potential of the Venn components were shown in Fig.2F.The results showed that the zeta potential of Gli-9, which has good solubility and small particle size, was-25.2 mV,and the stability of the solution was the highest.The zeta potential of Gli-15, which has unitary solubility and large particle size, was-7.64 mV,and the stability of the solution was poor.The zeta potentials of the Venn components were negative,indicating that the pH values of the gliadin were greater than the isoelectric points.These results showed that the isoelectric point of the Venn components were lower than 6,and they belonged to the range of partial acidity,which was consistent with the results of the pH test.The physicochemical properties of the Venn components showed that the eight components had their own characteristics,and there was a large gap among them,which was related to the functional properties in dough and Chinese steamed bread[7].

3.2.UV absorption characteristics of Venn components

Fig.2.Physicochemical properties of Venn components.

The UV absorbances of the Venn components ranging from 260 to 290 nm were shown in the Fig.3.From the results,the UV absorption spectra of Gli-8,Gli-11,or Gli-12 peaked at 277 nm,the spectra of Gli-10,Gli-14,or Gli-15 peaked at 278 nm,and Gli-9 or Gli-13 had their peak at 279 nm.The UV absorbance value of Gli-15 was the maximum(0.840),followed by Gli-14(0.812),Gli-11(0.651),Gli-12(0.551),and the UV absorbance value of Gli-9 at 279 nm was the minimum(0.171).The maximum UV absorbance value was 4.91 times the minimum,indicating that protein contents in the Venn components were different.These results are also supported by the results of nitrogen content of the Venn components using the Kjeldahl method(data not listed).The UV absorption curve of Gli-14 had the maximum curvature,followed by Gli-15,Gli-11,Gli-12,and the curve of Gli-10 was the most gradual.These results suggested that there were significant differences in the amino acid content of the Venn components,i.e., the first protein sequences of the different Venn components were quite different.

3.3.Characteristics of the protein secondary structure of Venn components

The protein secondary structure contents of the Venn components were shown in Table 1.The content of β-turn in gliadin components was as follows,from high to low,Gli-11,Gli-13,Gli-9,Gli-12,Gli-10, Gli-8, Gli-14, Gli-15.The sum of β-turn and α-helix contents in Gli-11 was the highest(67.14%),while that in Gli-15 was the lowest (34.44%).The highest content was 1.95 times the lowest.The content of β-turns in the secondary structure of a protein was associated with the content of Proline in protein.The β-turn and α-helix belonged to the ordered structure of protein, which might have more hydrogen bonds and thus make the secondary structure rigid.The random coil and β-turn belonged to the disordered structure of protein, and there was no hydrogen bond in the disordered structure.Therefore, the residues in the peptide segment have greater degree of freedom so that the protein molecules have a certain degree of flexibility [20].

Table 1 The protein secondary content of each Venn component. %

3.4.XRD patterns of Venn components

The XRD patterns of Venn components of gliadin were presented in Fig.4.According to XRD patterns of Gli-10, several crystal diffraction spectra were displayed obviously on amorphous diffraction background, indicating that some proteins in Gli-10 existed as crystals.In addition to Gli-10, the diffraction pattern of the other seven Venn components showed one broad peak with characteristic distances of 20° (Fig.4), which was characteristic of amorphous materials.Among them, the diffraction and absorption values of the waveforms from Gli-8 to Gli-15 were quite different, suggesting that there were great differences in the separated substances of the eight components.The results were similar to the one from Sharif et al.[21], in which the diffraction patterns of the pure gliadin showed a broad diffraction band around 14°.This research suggested that pure gliadin was amorphous because it displayed no Bragg reflections but only broad humps in its pattern [22,23].

3.5.Confocal laser scanning microscopy of Venn components

The confocal laser scanning microscopy results of each Venn component of gliadin were shown in Fig.5.There were great differences in the structures of the different components, in which the pure gliadin was distributed in a membrane [21].The Gli-8,Gli-9, and Gli-12 were the tightly constructed membranes; the Gli-10 was a relatively uniform membrane; the Gli-11 was a crystal; and the Gli-13, 14 and 15 were of three-dimensional spaces(Fig.5).The structure of gliadin and glutenin after extrusion would also become membrane and porous membrane structure, indicating that the protein formed the network structure or protein formation aggregates by CLSM [13,24].Different structures of protein network, certainly, have different physicochemical properties[25].The microstructures of the Venn components were quite different, implied that the molecular interactions among the components were varied.

4.Conclusion

In this study, eight Venn components of gliadin were extracted from wheat gliadin by using different solutions.Our study indicated that the content of Gli-15 in gliadin was the highest, and the content of Gli-9 was the lowest.The TDS value of Gli-9 was the highest,and the TDS value of Gli-15 was the lowest.The electrical conductivity of Gli-9 was the highest, and that of Gli-11 was the lowest.The zeta potential of Gli-9 was -25.2 mV, and the zeta potential of the Gli-15 was -7.64 mV.The differences in the contents, TDS and electrical conductivity, particle size and zeta potential of Venn components were significant, respectively.However, there was no significant difference between the pH values of the eight components.UV spectrum and FTIR analysis showed that there were significant differences in the secondary structures of the Venn components.The results of the XRD patterns indicated that all of the Venn components might not be a single substance.CLSM images results implied that the molecular interactions among the components were varied.

Conflicts of interest

The authors declare no conflicts of interest in relation to this work.

Acknowledgements

The authors thanks for the financial support of the National Key Research and Development Program (2016YFD0400203), the National Natural Science Foundation of China (Project No.31771897,31871852, and 31772023).

Fig.4.XRD patterns of Venn components.

Fig.5.2D elaboration of CLSM images of Venn components.