Recombinant Wheat Endoplasmic Reticulum ... - ACS Publications

Feb 24, 2017 - Guangdong Province Key Laboratory for Green Processing of Natural ... China University of Technology, Guangzhou, Guangdong 510640,...
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Recombinant Wheat Endoplasmic Reticulum Oxidoreductin 1 Improved Wheat Dough Properties and Bread Quality Guang Liu,† JingJing Wang,† Yi Hou,# Yan-Bo Huang,# Ya-Ping Zhang,† Cunzhi Li,§ Lin Li,†,‡ and Song-Qing Hu*,†,‡ †

School of Food Sciences and Engineering, South China University of Technology, Guangzhou, Guangdong 510641, China Guangdong Province Key Laboratory for Green Processing of Natural Products and Product Safety, South China University of Technology, Guangzhou, Guangdong 510640, China # State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou, Guangdong 510640, China § Department of Food Science and Engineering, Jinan University, Guangzhou, Guangdong 510632, China ‡

S Supporting Information *

ABSTRACT: Recombinant wheat endoplasmic reticulum oxidoreductin 1 (wEro1) with considerable ability was expressed in Escherichia coli. The functional roles of wEro1 in flour processing quality were investigated by farinographic, rheological, texture profile analysis, electrophoresis, size exclusion chromatography, scanning electron microscopy, and Fourier transform infrared spectroscopy. wEro1 exhibited an obvious oxidation activity of sulfhydryl groups in small molecule and protein. Addition of wEro1 could strengthen the processing quality of dough, indicated by the improved mixing characteristics, viscoelastic properties, and bread qualities. These improvement effects of wEro1 could be attributed to the formation of macromolecular gluten polymers and massive gluten networks by disulfide cross-linking. Additionally, the increased β-turn structure further demonstrated the enhancement of dough strength. Moreover, the amount of peroxide in dough was improved significantly from 2.36 to 2.82 μmol/g of flour with 0.15% wEro1 treatment. Therefore, the results suggested that wEro1 is a promising novel flour improver. KEYWORDS: recombinant wheat endoplasmic reticulum oxidoreductin 1, flour processing quality, SDS-insoluble gluten, disulfide bonds, hydrogen peroxide

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INTRODUCTION It is the unique viscoelastic properties of gluten that account for the functional properties of dough, including its strength and elasticity, which in turn determine the quality of the final products.1 The viscoelastic properties of dough are attributed to the formation of gluten network stabilization by cross-linking of intermolecular disulfide bonds.2 Therefore, the interchain disulfide bonds play the dominant function in determining the quality of dough and/or flour products.2 The use of enzymes instead of chemical oxidants is a very interesting option to facilitate the formation of intermolecular disulfide bonds within glutens, because enzymes are perceived as natural and are generally recognized as safe (GRAS status).3 Among these enzymes, sulfhydryl oxidases (SOXs) are enzymes capable of oxidizing free sulfhydryl groups in proteins and thiolcontaining small molecules.4 The majority of the reported SOXs are flavin-dependent enzymes that harbor flavin mononucleotide or flavin adenine dinucleotide (FAD) molecules acting as an organic cofactor. The cofactor in SOXs involves the oxidation of free sulfhydryl groups, which are able to use molecular oxygen as an electron acceptor to generate hydrogen peroxide (H2O2).5 Because they catalyze the formation of disulfide bonds, SOXs might be expected to have positive effects on flour processing quality. Thus, the effects of SOXs on the processing quality of dough have been previously investigated, but SOXs from © XXXX American Chemical Society

different origins exerted diverse effects on dough quality. Kaufman and Fennema6 reported that a SOX isolated from skim milk membranes did not show detectable influence on dough and bread qualities. However, Faccio et al.7 indicated that addition of SOX from Aspergillus oryzae resulted in a weaker and more extensible dough, whereas opposite effects were detected in the presence of ascorbic acid. Additionally, positive results have also been obtained with SOX from Aspergillus niger, which was used in combination with glucose oxidase.8 Endoplasmic reticulum (ER) oxidoreductin 1 (Ero1), a member of the SOX family and a FAD-dependent enzyme,4 is an essential ER-resident protein that is involved in the formation of disulfide bonds for protein folding with catalytic cysteines pairs.9 Furthermore, Ero1 cannot directly catalyze the cross-linking of disulfide bonds in nascent proteins without the assistance of protein disulfide isomerase (PDI) family members, such as PDI and ER protein 57.10 The exchange of disulfide bonds from Ero1 to PDI to client necessitates electron flow in the reverse direction, from client to PDI to Ero1. In this process, Ero1 uses cofactor FAD to reduce molecular oxygen to Received: Revised: Accepted: Published: A

November 18, 2016 February 21, 2017 February 24, 2017 February 24, 2017 DOI: 10.1021/acs.jafc.6b05192 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry generate H2O2.10 Until now the effects of Ero1 on the processing quality of flour have rarely been reported in the literature and patents except for only a Japanese patent, which showed that the recombinant wheat PDI (wPDI) accompanied by recombinant wheat Ero1 (wEro1) and FAD exhibited a more significant effect on improving bread quality than just wPDI had.11 However, the functional role of wEro1 in dough quality was not evaluated alone. In the present study, recombinant wEro1 was successfully cloned and overexpressed in Escherichia coli. The improved effects of wEro1 on the functional properties of wheat dough were investigated from the perspectives of mixing resistance, viscoelasticity, and structural change of gluten including the formation of macromolecular aggregates, gluten network, disulfide bonds, and secondary structure changes. On the basis of the above facts, the potential use of wEro1 in dough and breadmaking applications will be put forward.

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a-b-b′-a′, and could be categorized into subfamily I according to the study by d′Aloisio et al.13 The purified proteins were stored at −80 °C for further use. Protein Electrophoresis. Electrophoresis was performed on 70 mm × 80 mm × 0.75 mm gels using a Mini Protein II cell (Bio-Rad Laboratories, Richmond, CA, USA). The discontinuous system consisted of separating gel (12%, pH 8.6) and stacking gel (5%, pH 6.8). For reducing SDS-PAGE, the protein samples were resuspended in loading buffer (62.5 mM Tris-HCl, 20% glycerol (v/v), 1% SDS (w/v), and 2% 2-mercaptoethanol (2-ME, v/v), pH 6.8) and heated at 100 °C for 5 min. The protein solution for nonreducing SDS-PAGE was prepared with the same solution but without 2-ME and heating. Assay of Oxygen Consumption and Detection of H2O2. Ero1 catalyzes the oxidation of sulfhydryl groups in small molecules or proteins accompanying oxygen consumption and H2O2 generation.10 Oxygen consumption was recorded by an Oxygraph Clark-type oxygen electrode (Hansatech Instruments, Pentney King’s Lynn, UK) following the method of Gross et al.14 For oxidation of small-molecule DTT, the reaction mixture composed of freshly prepared 975 μL of 50 mM Tris-HCl buffer (containing 0.15 M NaCl, pH 8.0) and 20 μL of 0.5 M DTT was premixed at 900 rpm using magnetic stirring, and then the reaction was initiated by injection of 5 μL of wEro1 (final concentration of 2 μM) into the reaction vessel of the oxygen electrode. The total reaction volume was 1.0 mL, and 1 unit of wEro1 activity was defined as the amount of the enzyme that consumes 1 μmol of oxygen in solution per minute under the selected conditions. To investigate the oxidation of wPDI catalyzed by wEro1, wEro1 (2 μM) and wPDI (10 μM) were added into the same buffer with/ without 100 μL of 0.1 M glutathione (GSH). Additionally, for the detection of H2O2, 2 μL of catalase (60 IU) was injected into this reaction system after 500 s, when the oxidation reaction of thiol was complete. Oxygen levels were monitored until a linear baseline was established. Microfarinograph Analysis. Dough-mixing properties were determined with a 4 g microfarinograph (Micro-DoughLAB, Perten Instrument, Sweden) according to the AACC approved method.15 Flour (4.0 g, 14.0% moisture base) was premixed at 30 ± 0.2 °C and kneaded at a speed of 63 rpm to determine the farinograph water absorption. Effects of wEro1 (0.05, 0.10, and 0.15%, w/w, flour basis) on low-gluten flour were investigated. Additionally, a wEro1 (0.15%, w/w, flour basis) and wPDI (0.025%, w/w, flour basis) mixture was added into low-gluten flour to explore whether a synergistic effect existed. Farinogram parameters including dough development time (DDT), dough stability time (DST), and degree of softening (DS) were recorded. Dough and Gluten Preparation. The procedures of preparing dough and gluten samples were based on those of Wang et al.16 The dough of low-gluten flour with wEro1 combined with wPDI treatment was prepared according to the AACC approved method.15 After mixing, the dough was stripped from the farinograph bowl and then wrapped with plastic wrap to prevent the evaporation of water in dough. The dough was divided into three portions after proofing at 30 °C for 30 min: the first one was immediately lyophilized to obtain freeze-dried dough; the second one was used for rheological measurement; the third one was water-washed to obtain gluten and then lyophilized. After lyophilization, the freeze-dried gluten was immediately ground into powder in a mortar, sieved through a 180 μm mesh screen, and stored at −20 °C until analysis. Dynamic Rheological Determination. The dough samples were used for rheological measurement after proofing at 30 °C for 30 min. The procedure of rheological measurement was according to the method of Balestra et al.17 Dynamic rheological analysis was performed using a controlled stress rheometer (RheoStress 1, Thermo Haake, Germany) with parallel plate geometry (40 mm diameter). The dough was placed between parallel plates, then the gap was adjusted to 1 mm, and the excess dough was removed. To prevent drying at the edges, a thin layer of Vaseline oil was applied to cover the exposed dough surfaces. Before measurement, the dough was rested for 5 min to allow relaxation after sample handling. The linear viscoelastic region was initially confirmed from a frequency sweep test. Then, the

MATERIALS AND METHODS

Materials. 5,5′-Dithiobis(2-nitrobenzoic acid) (DTNB), FAD, and catalase (6000 IU/mg) were purchased from Sigma-Aldrich (St. Louis, MO, USA) at analytical or higher grade. Three kinds of commercial wheat (Triticum aestivum L.) flours, low-gluten flour (biscuit flour; 12.10% moisture, 0.40% ash, 11.00% protein, and 57% water absorption), medium-gluten flour (noodle flour; 11.95% moisture, 0.49% ash, 12.80% protein, and 61% water absorption), and highgluten flour (bread flour; 12.80% moisture, 0.47% ash, 14.20% protein, and 59% water absorption), were used in this study. All types of flours were produced by Nanfang Flour Co. Ltd., Guangzhou, China. Construction of Recombinant Expression Vector. Wild type wheat ero1 (wero1) gene sequence was acquired from GenBank (accession no. AK332376.1). The wero1 gene was synthesized by IGE Biotechnology Ltd., Guangzhou, China, and then inserted into the pGSI vector through NdeI- and XhoI-cloning sites. On the basis of the wero1 gene sequence containing 1254 bp, a pair of gene-specific primers, 5′-AGGGCGACCCATCATATGTA-3′ (forward primer) and 5′-GATATCCCCCTCGAGTTAAGC3′ (reverse primer), were designed. To clone the wero1 gene, polymerase chain reaction (PCR) was performed, and then the amplified wero1 gene was digested with NdeI and XhoI (Thermo Fisher Scientific, USA). After digestion, the wero1 gene was ligated to a re-formed pET-28a plasmid through NdeIand XhoI-cloning sites, yielding a recombinant plasmid pET-28a-wero1. Expression and Purification of wEro1. The constructed vector pET-28a-wero1 was overexpressed in E. coli BL21(DE3) strain following induction with 0.4 mM isopropy-β-D-thiogalactoside (IPTG) at 20 °C overnight. Cell pellets were resuspended in 20 mM Tris-HCl buffer (pH 8.0, containing 0.5 M NaCl and 20 mM imidazole) and sonicated for 30 min. The supernatant of cell lysates was collected after centrifugation at 8000g for 30 min at 4 °C and then incubated with a HiTrap chelating column (from GE Healthcare) at room temperature. The bound proteins were eluted with a linear gradient of 20−500 mM imidazole at a flow rate of 2 mL min−1. The eluted protein fractions were identified by sodium dodecyl sulfate− polyacrylamide gel electrophoresis (SDS-PAGE). Subsequently, the pooled target protein was incubated with FAD in a molar ratio of 1:2 at 4 °C overnight. Then the protein was loaded onto a Superdex 200 pg 26/600 column (10−600 kDa, GE Health, USA) pre-equilibrated with 20 mM Tris-HCl and 0.15 M NaCl (pH 8.0) at a flow rate of 2.6 mL min−1. Additionally, to avoid the effect of NaCl on dough strength, wEro1 was desalted into 5 mM phosphate buffer (pH 8.0) with a HiTrap desalting column (GE Healthcare). The collected protein was concentrated with a Centricon 10 kDa molecular weight cutoff device (Millipore, UK). The concentrations of protein were spectrophotometrically determined at 280 nm (εwEro1 = 1.56 mL mg−1 cm−1). Additionally, recombinant wheat PDI (wPDI) was purified as described previously.12 wPDI contained 515 amino acid residues with a molecular weight of about 56 kDa and possessed four thioredoxin-like domains, including B

DOI: 10.1021/acs.jafc.6b05192 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 1. SDS-PAGE analysis and activity assay of wEro1: (A) electrophoresis profiles of wEro1 (lane 1) and wPDI (lane 2); (B) wEro1 catalyzed oxygen consumption accompanying DTT oxidation; (C) wEro1 catalyzed oxygen consumption accompanying wPDI oxidation with/without GSH; (D) detection of H2O2 produced by wEro1 catalyzing the oxidation of wPDI. Results in each panel represent one of three independent experiments. extracted successively by 0.5 mL of 2% NaCl solution (salt-soluble protein), 1.5% SDS solution (SDS-soluble gluten), and 1.5% SDS solution with sonication (SDS-insoluble gluten). Each step was carried out with constant stirring (200 rpm, 15 min) and centrifugation at 8000g for 15 min. The ultrasonic disrupter (VCX500, Sonics, USA) was working for 15 s at 30 W. In addition, the protein concentration of each extraction fraction was measured with the BCA Protein Assay Kit (GK5013, Generay, China). Bovine serum albumin (BSA) was used to prepare a standard curve. Size Exclusion−Fast Protein Liquid Chromatography (SEFPLC) Analysis for SDS-Insoluble Gluten. SDS-insoluble gluten was analyzed by SE-FPLC as described previously.22 Briefly, SE-FPLC was performed on a fast protein liquid chromatography system (FPLC, GE Health, USA) equipped with a Superdex 200pg Increase 10/300 column (10−600 kDa, GE Health, USA). SDS-insoluble gluten extracts were centrifuged at 15000g for 10 min and filtered through a 0.22 μm member filter. Protein solution was eluted isocratically at a flow rate of 0.75 mL/min with mobile phase consisting of 20 mM phosphate buffer (pH 7.0) and 150 mM NaCl. Absorbance was monitored at 280 nm. The column was calibrated with standard proteins in the manual: thyroglobulin (669 kDa), ferritin (440 kDa), BSA (67 kDa), and β-lactoglobulin (35 kDa). Scanning Electron Microscopy (SEM). The microstructure of freeze-dried dough was determined by SEM (Evo 18, Carl Zeiss, Germany) as described previously.23 Dough samples were fractured into sizes of about 1 × 1 × 0.5 cm using a knife, mounted on the specimen holder, and sputter-coated with gold. Finally, the samples were transferred to the microscope and observed at 10.0 kV and 500× magnifications with a vacuum of 9 × 10−5 MPa. Fourier Transform Infrared Spectroscopy (FTIR). FTIR assay was performed based on the method of Wang et al.24 Briefly, an FTIR spectrum of gluten was collected by a Bruker Vertex 70v spectrometer (Bruker Optics, Ettlingen, Germany). The second derivative and

evaluation was performed with a frequency of 0.1−10 Hz and deformation of 0.1%. The storage modulus (G′), loss modulus (G″), complex modulus (G* =2 G′2 + G″2 ), and loss tangent (tan δ = G″/G′) were analyzed. Breadmaking Procedure and Bread Quality Evaluation. The breadmaking procedure was performed according to that of Kim et al.18 with slight modifications. Briefly, the bread formula consisted of 100 g of high-gluten flour or medium-gluten flour, 1.6 g of NaCl, 6 g of sucrose, 3 g of blend oil (Arowana Brand, China), 1 g of compressed active dry yeast (Angle Brand, China), and the appropriate amount of water (59 g for high-gluten flour and 61 g for medium-gluten flour). wEro1 was dissolved in the water and supplemented at a level of 0.10% (w/w, flour basis). All ingredients was mixed at 100 rpm for 18 min, and then the dough was divided into three pieces of the same weight (50 g) and hand-rounded. After proofing at 30 °C (80% relative humidity (RH)) for 60 min, the dough was baked in an oven (T1L101B, Midea, China) at 185 °C for 10 min under vigorous initial steam. Loaves were removed from the pans and analyzed after they had cooled at 25 °C and 45% RH for 1 h. Quality analysis of fresh bread samples was carried out by measuring weight, volume, height, and diameter. The bread volume was determined by seed displacement according to the method of Wang et al.19 The specific volume (volume/weight ratio) and height/diameter ratio were used to evaluate bread quality. Crumb texture was determined by a Texture Analyzer (TA-XT Plus, Stable Microsystem, Surrey, UK) equipped with an 25 mm diameter cylindrical aluminum probe.19 Slices of 2 cm thickness were compressed to 50% of their original height at 1 mm/s speed test, with a 10 s delay between the first and second compressions. Primary parameters including hardness, chewiness, and springiness of the crumb were calculated from a force distance graph. Extraction and Concentration Determination of Wheat Proteins. Extraction of wheat proteins was based on a modification of previous methods.20,21 Briefly, 50 mg of freeze-dried dough was C

DOI: 10.1021/acs.jafc.6b05192 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Table 1. Effects of wEro1 and Combined with wPDI on Farinograph Characteristicsa wheat flour

group

DDT (min) ± ± ± ± ± ±

0.00 0.00c 0.07c 0.07c 0.07c 0.11c

DST (min) 1.65 3.20 6.65 7.05 1.85 6.75

± ± ± ± ± ±

0.07g 0.28f 0.07ab 0.49a 0.21g 0.07a

DS (FU) 127.45 120.00 107.50 95.00 127.45 100.00

± ± ± ± ± ±

low gluten

E0 E1 E2 E3 P E3P

1.00 1.10 1.25 1.15 1.05 1.15

3.46a 7.07ab 3.53bc 7.07c 3.46a 14.53bc

medium gluten

E0 E2

1.80 ± 0.14b 1.80 ± 0.07b

4.45 ± 0.21e 4.75 ± 0.07d

129.95 ± 7.00a 110.00 ± 7.07bc

high gluten

E0 E2 E4

4.35 ± 0.21a 4.20 ± 0.20a 4.25 ± 0.21a

5.75 ± 0.07c 5.83 ± 0.15c 6.40 ± 0.00b

97.50 ± 10.61c 98.33 ± 7.64c 95.43 ± 3.83c

a

DDT, DST, and DS represent dough development time, dough stability time, and degree of softening, respectively. E0, E1, E2, E3, and E4 indicate additive level of wEro1 (0, 0.05, 0.10, 0.15, and 0.30%), and P and E3P indicate 0.025% wPDI and 0.15% wEro1 + 0.025% wPDI, respectively. Values represent the mean ± SD of three replicate samples. Letters within a column indicate significantly different values (P < 0.05).

curve-fitting were used to analyze the results using the PeakFit version 4.12 software (SPSS Inc., Chicago, IL, USA). Quantification of Free Sulfhydryl Groups in Gluten. Changes in free sulfhydryl content of gluten samples were determined according to a previous study.25 Gluten (15 mg) treated with/without wEro1 treatment was suspended in 0.5 mL of Tris−Gly buffer (86 mM Tris, 90 mM glycine, and 4 mM EDTA, pH 8.0) with 8 M urea. The suspension was vortexed for 10 min and centrifuged at 15000g for 5 min. Then, the clear supernatant (100 μL) was added to 150 μL of Tris−Gly/urea solution and 50 μL of DTNB (4 mg/mL, dissolved in 90 mM Tris−Gly buffer). After incubation at 30 °C for 30 min, the absorbance of the supernatants was read at 412 nm against the reagent buffer as the blank. Results were calculated against the GSH standard calibration curve. Detection of H2O2 in Dough. The H2O2 content of dough was determined according to the colorimetric method reported by Pescador-Piedra et al.26 with some modifications. Half a gram of dough was weighed as it was obtained from the microfarinograph after 5 min of mixing and dispersed in 1 mL of deionized water (1 mL) and 150 μL of trichloroacetic acid (72%, w/v). The sample was vortexed for 10 min and then centrifuged at 8000g for 15 min. For the color reaction, 20 μL of supernatant was measured and then, in sequence, 1.0 mL of 50 mM HCl, 0.2 mL of 1 M KI, 0.2 mL of (NH4)6Mo7O24 in H2SO4 (1 mM (NH4)6Mo7O24 in 0.5 M H2SO4), and 0.2 mL of starch (5%, w/v) were added to the supernatant solution. A resting period of 20 min between the addition of (NH4)6Mo7O24 and the addition of the starch solution was required. Twenty minutes after the addition of the starch solution, the absorbance was measured at 570 nm. H2O2 was used to prepare a standard curve. Statistical Analysis. Data were reported as means ± standard deviations (SD) for three replicate treatments. One-way analysis of variance (ANOVA) and Duncan’s tests were performed using the SPSS software package (version 17.0, SPSS Inc., Chicago, IL, USA). Statistical significance was declared at P < 0.05.

Ero1 catalyzes the oxidation reaction of thiol with the aid of cofactor FAD, which reduces molecular oxygen generating H2O2.14 The activity of wEro1 was monitored by oxygen consumption in the presence of DTT or wPDI as a substrate. As shown in Figure 1B, a negligible amount of oxygen was consumed by a nonenzymatic oxidation reaction of DTT (black line), and the rate of oxygen consumption by wEro1 was nonsignificant in the absence of DTT (red line). However, the rate increased substantially with the addition of 10 mM DTT (blue line), suggesting that the oxidation of DTT was catalyzed by wEro1; a similar phenomenon has previously been observed with human Ero1α (hEro1α).27 Under the selected conditions, the specific activity of wEro1 was calculated as 0.25 IU/mg in oxidation of DTT. As for another small molecular reductant with free thiol, GSH was not an effective substrate for wEro1 with the fact that no significant oxygen consumption was observed when DTT was replaced by GSH (Figure 1C, red line). PDI is the most important physiological substrate for Ero1 in ER.28 To investigate whether wEro1 had activity toward its physiological substrate, wEro1 was incubated with wPDI in the presence or absence of GSH. Mixtures of wEro1 and wPDI showed no significant consumption of oxygen without GSH (Figure 1C, blue line), although a low rate of oxygen consumption had been reported between human PDI (hPDI) and hEro1α.28 However, an obvious decrease of oxygen concentration was observed in the presence of GSH (green line), and similar results were reported by Araki and Nagata,27 suggesting that GSH was able to transfer its electron to hPDI and then to hEro1α to facilitate the catalytic oxidization of hPDI by hEro1α. Moreover, when catalase was added into the wEro1-mediated oxidation of the wPDI reaction system at 500 s, it could be found that the concentration of oxygen in solution significantly increased (Figure 1D), demonstrating that H2O2 had been present and was decomposed into oxygen by catalase, which was in good accordance with previous results.14,29 Therefore, wEro1 was capable of oxidizing free sulfhydryl groups in thiol-containing small molecule (DTT) and protein (wPDI), accompanied by generation of H2O2. Effects of wEro1 on Flour Processing Quality. Microfarinograph Measurements. First, the effects of wEro1 (0.10%, w/w, flour basis) on farinograph properties of three kinds of wheat flour samples were investigated. DST is a critical

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RESULTS AND DISCUSSION Expression and Activities Assay of wEro1. To characterize the enzymatic activity, recombinant wEro1 was purified by immobilized-metal affinity chromatography and size exclusion chromatography. As shown in Figure 1A, wEro1 exhibited a main band of ∼47 kDa in SDS-PAGE profiles after size exclusion chromatography, indicating that wEro1 was purified to homogeneity and electrophoretic purity. Additionally, wPDI was also purified to homogeneity according to a previous study.12 The purified proteins were employed for activities assays. D

DOI: 10.1021/acs.jafc.6b05192 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 2. Effects of wEro1 on dynamic rheological properties of dough: (A) elastic modulus (G′) of dough; (B) viscous modulus (G″) of dough; (C) complex modulus (G*) of dough; (D) loss tangent (tan δ) of dough. Values represent the mean of three replicate samples.

Table 2. Effects of wEro1 on Qualities of Bread from High- and Medium-Gluten Floursa wheat flour

wEro1 level (%)

SV(cm3/g)

H/D

hardness (g)

chewiness (g)

springiness

high gluten

0 0.10

2.56 ± 0.09d 2.77 ± 0.05c

0.53 ± 0.01d 0.59 ± 0.02c

734.36 ± 51.33c 615.92 ± 15.04d

459.21 ± 13.17c 397.78 ± 14.41d

0.925 ± 0.006b 0.937 ± 0.001a

medium gluten

0 0.10

3.42 ± 0.04b 3.90 ± 0.06a

0.66 ± 0.02b 0.74 ± 0.01a

1132.98 ± 41.75a 881.05 ± 42.46b

695.21 ± 31.64a 576.84 ± 30.71b

0.890 ± 0.028c 0.923 ± 0.019b

SV, specific loaf volume; H/D, height/diameter ratio of loaf. Values represent the mean ± SD of three replicate samples. Letters within a column indicate significantly different values (P < 0.05).

a

indicator of flour strength, with higher values suggesting stronger dough.24 The greatest improvement of DST by 0.10% wEro1 addition was found in a sample of low-gluten flour, followed by medium-gluten flour, whereas high-gluten flour did not show any significant change (Table 1). An obvious (P < 0.05) improvement of DST of high-gluten flour was observed with 0.30% wEro1 addition. Therefore, low-gluten flour was chosen to remarkably and systematically explore the influence of wEro1 on characteristics of dough and wheat protein, whereas medium-gluten flour and high-gluten flour were employed to investigate the action of wEro1 on bread on the basis of the desired application in the wheat-processing industry.30 wEro1 significantly improved the farinograph properties of low-gluten flour. As shown in Table 1, DDT and DST were prolonged with the addition of wEro1 in wheat flour, although no significant difference (P < 0.05) was presented for DDT. With the addition of 0.15% (w/w, flour basis) wEro1, the DDT and DST were increased by 15 and 327%, respectively, indicating an increased maximum resistance to mixing and enhanced dough strength. In addition, DS generally gives the rate of breakdown and strength of dough: the higher the value,

the weaker the dough.31 The DS of dough gradually decreased as the wEro1 level increased, further demonstrating a reinforcement of dough. The strengthening effect by wEro1 was critical for the ability of dough to retain its elasticity to allow expansion during proofing.32 Dynamic Oscillatory Testing of Dough. The study on dough rheological properties is useful to predict the attributes of the end product quality.21 As shown in Figure 2A,B, both the elastic (G′) and viscous (G″) moduli increased with increasing wEro1 addition level, indicating an improvement of viscoelastic properties of dough. In all cases, the value of G′ was higher than G″ (tan δ < 1), definitely suggesting a weak gel behavior or a solid viscoelastic behavior.33 Likewise, the complex modulus (G*), another factor representing the strength of dough, also showed a similar increasing tendency with G′ and G″ (Figure 2C). Moreover, a greater value of G* suggested greater stiffness of the dough.32 Thus, the improvement of G* suggested that the dough with 0.15% (w/w, flour basis) wEro1 treatment had the greatest rigidity. Figure 2D shows that tan δ (G″/G′) decreased in the presence of the enzyme, proving that the relative contribution of the solid character (G′) increased.32 A greater G′ and a lower tan δ indicated a dough with high gluten E

DOI: 10.1021/acs.jafc.6b05192 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry quality, which had a high elastic to prevent gas cell breakage during fermentation.32 Effect of wEro1 on Bread Quality. As known, high-gluten flour is desired for breadmaking in the baking industry.30 According to farinographic results and research strategy by Steffolani et al.,21 0.10% of wEro1 was applied to make bread using high-gluten flour. As shown in Table 2, the specific volume and height/diameter ratio of loaf increased by 8.2 and 11.3% relative to the control, respectively. The increased specific volume by wEro1 was more than that by xylanase and potassium bromate, which increased the specific volume by 6.6 and 4.5%, repectivley.31,34 In addition, the specific volume is a good indicator for fresh bread crumb softness, and the larger specific volume suggested a softer bread crumb.35 Therefore, the texture of bread crumb was further determined by TPA (Table 2). Compared with the control, the hardness and chewiness of wEro1-treated crumb decreased by 16.1 and 13.4%, respectively, whereas the springiness increased by 1.3%. These results suggested that a softer and more resilient bread crumb has been formed with wEro1 supplementation. Therefore, as expected, wEro1 acted positively in the improvement of high-gluten flour baked bread quality. Additionally, the quality improvement of weak flour always attracted great interest from the wheat-processing industry.36 It is of significance when bread is made with weak flour instead of strong flour with the aid of enzyme. Thus, medium-gluten flour was also employed to bake bread with 0.10% of wEro1 supplementation. As shown in Table 2, the medium-gluten flour was weak as expected for making bread; the breadbaking qualities were poorer than those of high-gluten flour. After the addition of wEro1, the bread qualities were significantly improved, which was supported by the 14.0% increase of specific volume, 12.1% increase of height/diameter ratio, and 3.7% increase of springiness and the 22.2% decrease of hardness and 17.0% decrease of chewiness. Thus, the improving effects of wEro1 on the breadmaking quality of medium-gluten flour were better than that of high-gluten flour. Moreover, the crumb qualities of enzyme-treated bread baked from medium-gluten flour were approximately those of the control from high-gluten flour. Effects of wEro1 on the Protein Compositions of Dough and Gluten Aggregates. Previous research suggested that gluten polymerization led to a significant rise in elastic modulus of gluten network, which contributed to the reinforcement of dough quality.37 As the improved processing quality of flour with wEro1 should result from the change of the gluten structures,32 the gluten aggregates and microstructural properties of dough were investigated. SDS-PAGE Analysis and Quantitation of Protein Fractions. In this study, wheat proteins were extracted from control and wEro1-treated freeze-dried dough, yielding the following fractions: salt-soluble protein (albumins and globulins), SDSsoluble gluten (gliadins and SDS-soluble glutenins), and SDSinsoluble gluten (gluten macropolymer, GMP), based on the definition according to solubility (Osborne fractions).38 The electrophoretic result of salt-soluble wheat protein did not show significant differences with wEro1 treatment, although its content was increased due to the addition of wEro1 (Figure S1). However, obvious changes in electrophoretic and quantitative results were obtained for SDS-soluble gluten and GMP (Figures 3 and 4). The nonreducing and reducing SDS-PAGE profiles of SDSsoluble gluten are shown in Figure 3A. The intensity of bands

Figure 3. Analysis of electrophoretic patterns (A) and protein concentrations (B) of SDS-soluble gluten with wEro1 treatment. Lanes: 1−4, nonreducing electrophoretic patterns; 5−8, reducing electrophoretic patterns; M, marker; 1 (5), control; 2 (6), with 0.05% wEro1 treatment; 3 (7), with 0.10% wEro1 treatment; 4 (8), with 0.15% wEro1 treatment.

Figure 4. Analysis of electrophoretic patterns (A), protein concentrations (B), and size exclusion chromatography (C) of SDSinsouble gluten with wEro1 treatment. Lanes: 1−4, nonreducing electrophoretic patterns; 5−8, reducing electrophoretic patterns; M, marker; 1 (5), control; 2 (6), with 0.05% wEro1 treatment; 3 (7), with 0.10% wEro1 treatment; 4 (8), with 0.15% wEro1 treatment.

of high molecular weight (HMW) polymers (lanes 1−4, shown by box) was weakened gradually with increased wEro1 level, indicating that wEro1 addition could decrease the amount of HMW polymers in SDS-soluble gluten. Likewise, the lessened intensity of bands especially near ∼40 kDa (shown by arrow) corresponding to low molecular weight glutenin subunit (LMW-GS) was examined in reducing SDS-PAGE profiles (Figure 3A, lanes 5−8), which also implied a decreased SDSsoluble gluten content. These results were further confirmed by the quantification of SDS-soluble gluten. As shown in Figure 3B, the SDS-soluble gluten content decreased from 5.13 to 3.99 mg/mL with increasing dosage of wEro1. The decrease was significant (P < 0.05) for a wEro1 level of 0.05% (w/w, flour basis) or beyond. Similar phenomena were observed in dough samples treated with two other biological flour improvers, glucose oxidase and transglutaminase.21 The decreased SDS-soluble gluten content implied the increase in GMP content.39 As shown in Figure 4A, the enhanced bands intensity in the ranges of HMW polymers (lanes 1−4, shown by box) and LMW-GS (lanes 5−8, shown by arrow) could be inspected, suggesting that wEro1 could increase the amount of GMP. In agreement with electroF

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Effects of wEro1 on the Microstructure of Dough and Gluten. Effects of wEro1 on Gluten Network. Microstructural observations can explain the quality properties of dough.23 The microstructure of freeze-dried dough with different amounts of wEro1 was observed by SEM (Figure 5). The control dough

phoretic results, the content of GMP in wEro1-treated dough increased from 4.13 to 4.53 mg/mL upon addition of up to 0.15% (w/w, flour basis) (Figure 4B). The increase was significant only for a wEro1 level of 0.15% (w/w, flour basis). The total amount of SDS-soluble protein and GMP with 0.15% wEro1 (8.52 mg/mL) was ∼92% of that of control (9.26 mg/ mL); similar effects were found in the dough samples treated by transglutaminase and xylanase.34 The remaining unextracted proteins (∼8%) might be involved in the formation of macroaggregates that could not be dissolved in SDS combined with ultrasonic processing. The facts about the increase in GMP content, accompanied by the decrease in SDS-soluble gluten with wEro1 addition, were consistent with the effects of glucose oxidase and pentosanase on gluten of dough.21 The amount of GMP in flours is strongly correlated with elastic behavior of dough and breadbaking performance, specifically volume.40 A shift of soluble gluten fraction toward GMP might be associated with cross-linking, which contributed to strengthening of dough facilitating the improvement of bread quality. The modified bread quality was approved in the present study (Table 2). SE-FPLC Analysis of GMP. Typical SE-FPLC profiles from GMP are shown in Figure 4C. In the SE-FPLC profiles, the peaks could be mainly divided into four fractions, including macromolecular weight glutenin polymers (F1, >669 kDa), large molecular weight glutenin polymers (F2, 440−669 kDa), medium molecular weight glutenin polymers (F3, 67−440 kDa), and low molecular weight glutenin (F4, 35−67 kDa), respectively.41 Compared with the control, the peak area of SE-FPLC profiles of GMP unmistakably increased with wEro1 addition (Table S1), indicating an increased content of GMP, which was in accordance with the results from quantification of GMP (Figure 4B). Moreover, the changes of peak and area are shown in Figure 4C and Table S1 with increasing wEro1 level. In the control sample, two main peaks at fractions F2 and F4 could be obviously observed, which represented large molecular weight glutenin polymers and low molecular weight gluten, respectively. After the addition of wEro1 (0.05 and 0.10%, w/w, flour basis), the absorbance of peaks of F2 and F4 reduced gradually, whereas that of peaks of F1 and F3 corresponding to macromolecular gluten polymers and medium-gluten polymers increased. Furthermore, for 0.15% (w/w, flour basis) wEro1 treatment, the peaks of fractions F2 and F4 almost disappeared, and the profile of SE-FPLC mainly consisted of the peaks at fraction F3 and especially F1. Hence, these results suggested that the addition of wEro1 facilitated the formation of macromolecular glutenin polymers in GMP and were in agreement with the nonreducing electrophoresis results of GMP (Figure 4A, lanes 1−4). It is generally accepted that both disulfide bond covalent cross-linking and hydrophobic interaction contributed greatly to the formation of large gluten aggregates,16 although nondisulfide cross-linking such as ε-N-(γ-glutamyl)lysine cross-linking was also recognized to promote the aggregation of gluten.1 In the experiments, the hydrophobic interactions in the extracted GMP were disrupted by SDS,42 and the large polymers in GMP were reduced completely by 2-ME (Figure 4A). Taking the increased GMP, especially the increased larger polymers in GMP after wEro1 treatment, into consideration, it could be concluded that the disulfide bond cross-linking was facilitated in the macromolecular glutenin polymers by the addition of wEro1.

Figure 5. Effect of wEro1 on microstructure of dough: (A) control dough; (B) dough treated with 0.05% wEro1; (C) dough treated with 0.10% wEro1; (D) dough treated with 0.15% wEro1. S and G represent starch granules and gluten matrix, respectively. Results represent one of three independent experiments.

displayed the typical structure of starch granules (S) embedded in the formed gluten matrix (G) (Figure 5A), and the gluten network was sparse, resulting in the exposed starch granules in low-gluten dough, which was consistent with previous results.43 For 0.05% wEro1 treatment, massive network structures on the gluten matrix were formed (Figure 5B), and the exposed starch granules in dough were covered by gluten network. These changes were further strengthened with the increased wEro1 levels (Figure 5C,D). Similar effects were reported by Wang et al. and Zhang et al., who found that the addition of tannin and lipoxygenase had modified the microstructure of gluten network.22,31 GMP played an important role in the formation and interaction of the protein network.40 The enhanced gluten network was in line with the increased GMP content. Additionally, the gluten network was mainly stabilized by intermolecular disulfide bonds.2 Thus, the formation of a more continuous gluten network was mainly related to the interchain disulfide cross-links. Effects of wEro1 on Secondary Structures of Gluten. The secondary structures of gluten proteins provide a biochemical basis of the intermolecular interactions among wheat storage proteins and hence play important roles in the viscoelastic properties of dough.22 In the present study, FTIR spectroscopy was used for the determination of secondary structures of gluten proteins, and the results were analyzed according to the amide I band (1600−1700 cm−1) after deconvolution and secondary derivation (Figure 6A). On the basis of previous study, the peaks’ assignments to particular protein secondary structures in the amide I region were as follows: (1) the bands located at 1650−1660 cm−1 can be assigned to the α-helix; (2) the bands at 1618−1640 and 1670−1690 cm−1 can be assigned to β-sheets; (3) the bands at 1660−1670 and 1690−1700 cm−1 G

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cross-linking of disulfide bonds in gluten and hence promoted the formation of GMP, which was in line with the results from electrophoresis (Figure 4A) and SE-FPLC analysis (Figure 4C) of GMP. Moreover, the gluten polymers by cross-linking of disulfide bonds would contribute to a three-dimensional protein network,50 which in turn improves the processing quality of flour. These were identical with the enhanced gluten network and improved bread qualities (Figure 5 and Table 2). Quantification of H2O2 in Dough. It was proved that wEro1 could catalyze the cross-linking of disulfide bonds following the generation of H2O2 (Figure 1). Therefore, the amount of H2O2 in dough was determined with wEro1 treatment. As shown in Figure 6C, the content of H2O2 in wEro1-treated dough increased from 2.36 to 2.82 μmol/g of flour upon addition of up to 0.15% (w/w, flour basis). The increase was significant only for a wEro1 level of 0.15%. Glucose oxidase and pyranose oxidase, the FAD-dependent enzymes, were proved to improve dough strength through H2O2 generation.51 Therefore, the generation of H2O2 should contribute to the formation of disulfide bonds in the gluten and then to the improvement on the flour processing quality with the addition of wEro1. Furthermore, as a member of the SOX family, Ero1 can assist PDI in catalyzing the formation of disulfide bonds in nascent proteins,10 and it is reasonable to suggest that the improvement of wEro1 on dough might be involved in the mechanism of interaction with the endogenous wPDI.11 The results showed that addition of wEro1 (0.15%, w/w, flour basis) and wPDI (0.025%, w/w, flour basis) did not show synergistic actions on improvement of the farinograph properties (Table 1), but decreased the viscoelastic properties of dough (Figure S2) relative to the addition of wEro1 alone. Further studies are necessary to elucidate the catalytic mechanism of wEro1 involved in dough improvement.

Figure 6. Effects of wEro1 on secondary structure (A) and free sulfhydryl groups (B) in gluten and H2O2 in dough (C). Values represent the mean ± SD of three replicate samples. Different letters above the bars indicate significant difference (P < 0.05).

can be assigned to β-turns; and (4) the band at 1645 cm−1 can be assigned to random coils.44 As expected, the β-sheet structure was found to dominate in wheat gluten (Figure 6A).41,45 Although the addition of 0.15% wEro1 slightly decreased the content of β-sheet structure (from 33.5 to 31.9%), it was still the largest proportion of secondary structures in gluten. Additionally, the addition of wEro1 (≤0.10%) significantly affected the content of α-helix and βturn structures, but did not obviously change that of β-sheet and random coli structures (Figure 6A). Compared with the control, the α-helix content was reduced, whereas β-turn content was increased in dough treated with wEro1 (≤0.10%). Therefore, wEro1 supplementation has facilitated the transition of α-helix and β-sheet structures to β-turn structure in gluten samples, which was identical with results from previous studies reporting that the improved flour quality presented a decrease in β-sheet structure and an increase in β-turn structure.22,46 The increase of β-turn structure indicated stronger hydration interactions, which was demonstrated to provide the gluten with elastic properties. Moreover, Wellner et al. reported that the increased level of β-turn structure strengthened the molecular rigidity and dough strength.47 Furthermore, according to loop−train theory, β-sheet structures (trains) are inherently less elastic than β-turn structures (loops) in gluten protein.48,49 In conclusion, the present results indicated that adding wEro1 promoted the increase of β-turn structure in gluten, an indication of a more viscoelastic dough, which was in accordance with the improved mixing and rheological properties (Table 1 and Figure 2). Preliminary Study on the Mechanism of Dough Improvement by wEro1. Determination of Free Sulfhydryl Groups in Gluten. To further identify the formation of disulfide bonds in gluten by wEro1 treatment, the effect of wEro1 on free sulfhydryl groups in gluten was determined (Figure 6B). The free sulfhydryl contents of gluten decreased significantly (P < 0.05) with increasing levels of wEro1. After the addition of 0.15% (w/w, flour basis) wEro1, the amount of free sulfhydryl groups decreased by ∼19%. Changes in free sulfhydryl content are a persuasive indicator of variation in disulfide bonds, which play a critical role in gluten polymerization.2 Accordingly, the present results indicated that the addition of wEro1 led to the

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b05192. Table S1: Effect of wEro1 on peak area of SEC profiles of SDS-insoluble gluten. Figure S1: Analysis of electrophoretic patterns and protein concentrations of saltsoluble wheat protein with wEro1 treatment. Figure S2: Effects of wEro1 combined with wPDI on viscoelastic properties of dough (PDF)

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AUTHOR INFORMATION

Corresponding Author

*(S.-Q.H.) School of Food Sciences and Engineering, South China University of Technology, Guangzhou 510641, China. Phone/fax: 86-20-87113848. E-mail: [email protected]. ORCID

Song-Qing Hu: 0000-0003-3262-8911 Funding

We appreciate financial support from the National Natural Science Foundation of China (Grants 31471691, 31171630, and 31130042), the Specialized Research Fund for the Doctoral Program of Higher Education of China (No. 20130172110018), the Science and Technology Planning Project of Guangdong Province, China (Grant 2014A010107002), and the Science and Technology Planning Project of Foshan city, Guangdong province, China (Grant 2015AG10011). H

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Prof. Min Yao, Hokkaido University, for providing a re-formed pET-28a plasmid as a kind gift. We also thank Director Yishun Zhang, Teaching Center of Biology Experiment, Sun Yat-Sen University, for his assistance in providing the Oxygraph Clark-type oxygen electrode.

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