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Physicochemical properties and gluten structures of hard wheat flour doughs as affected by salt
Accepted Manuscript Physicochemical properties and gluten structures of hard wheat flour doughs as affected by salt Gengjun Chen, Laura Ehmke, Chetan Sharma, Rebecca Miller, Pierre Faa, Gordon Smith, Yonghui Li PII: DOI: Reference:
S0308-8146(18)31312-8 https://doi.org/10.1016/j.foodchem.2018.07.157 FOCH 23272
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
3 April 2018 5 July 2018 24 July 2018
Please cite this article as: Chen, G., Ehmke, L., Sharma, C., Miller, R., Faa, P., Smith, G., Li, Y., Physicochemical properties and gluten structures of hard wheat flour doughs as affected by salt, Food Chemistry (2018), doi: https:// doi.org/10.1016/j.foodchem.2018.07.157
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Physicochemical properties and gluten structures of hard wheat flour doughs as affected by salt
Gengjun Chen1, Laura Ehmke1, Chetan Sharma1, Rebecca Miller1, Pierre Faa2, Gordon Smith1, Yonghui Li1*
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Department of Grain Science and Industry, Kansas State University, Manhattan, KS
66506 2
Frito-Lay North America, Plano, TX 75024
*Correspondence to: Yonghui Li, E-mail: [email protected], Ph: 785-532-4061, fax: 785-532-7010
Submit to Food Chemistry
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Abstract Hard wheat flour doughs were prepared with five different levels of sodium chloride, and rheological properties were characterized. Zeta potential, disulfide-sulfhydryl groups, surface hydrophobicity, secondary structure, and extractable gliadin and glutenin of gluten were analyzed to elucidate gluten structure changes induced by salt. Addition of higher levels of salt (2.0 and 2.4%, fwb) in doughs resulted in larger storage and loss modulus, and elongational viscosity. Starch gelatinization temperatures increased with higher amounts of salt. The presence of salt decreased the free sulfhydryl content but increased the β-sheet structure of gluten. RP-HPLC indicated that salt enhanced the macromolecular aggregation of gluten proteins. The changes in gluten molecular conformation and network structure induced by salt significantly contributed to the improved physicochemical properties of dough. This study provides a better understanding of salt functionality in hard wheat flour dough and a valuable guide in searching for salt alternatives for bakery products.
Keywords: Sodium chloride salt, gluten, dough, rheological properties, gliadin, glutenin, hydrophobicity, secondary structure, Zeta potential, HPLC
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1. Introduction Wheat is one of the major cereal crops worldwide, and its global production reached 749 million tons in 2016 (FAO, 2016). Hard wheats (both winter and spring, red and white) account for more than 80% of total U.S. wheat production (USDA-ERS, 2018), and one of the main applications is for bakery products, including bread. Bread has been a widely consumed product in the human diet, which is served in a variety of forms (Miller, & Hoseney, 2008). The formation of dough plays a key role in breadmaking, since it incorporates all the ingredients and develops a unique viscoelastic gluten network (Letang, Piau, & Verdier, 1999). Shewry, Halford, Belton, and Tatham (2002) pointed out that gluten was associated with the water absorption, mixing time, and strength index of dough. More than 80% of gluten protein consisted of gliadins and glutenins (Shewry, 2009; Song, & Zheng, 2007). The gliadins (ω-, αand γ-type) are relatively small molecules that are soluble in alcohols, which may act as a plasticizer in dough systems (Goesaert, Brijs, Veraverbeke, Gerbruers, & Delcour, 2005). On the other hand, the glutenins are heterogeneous polymers including high molecular weight glutenin subunits (HMW-GS) and low molecular weight glutenin subunits (LMW-GS) (Shewry, et al., 2002). Moreover, glutenins contribute to the mixing resistance, elasticity, and cohesiveness of the dough network (Wieser, & Kieffer, 2001). When water is added into flour, the gluten fraction is hydrated and forms a protein network, which is considered as an important factor in determining dough properties (Autio et al., 2005). Sodium is an essential nutrient for humans, but excessive sodium intake increases blood pressure and risk of cardiovascular disease (CVD) (Bailey, et al., 2016). The average daily sodium intake of Americans is about 3,440 mg per day, which far exceeds the 2015 Dietary Guidelines for Americans recommended upper limit of 2,300 mg per day (USDA, & HHS, 2015). The largest contributor to dietary sodium is salt (known as table salt, or sodium chloride, NaCl). There is an urgent need to reduce the amount of salt in the diet. Salt is actually one of the four essential ingredients (flour, water, yeast, and salt) in any bread formula. In addition to its sensory contribution, salt plays critical technological roles throughout the 3
bread-making process (mixing and dough formation, fermentation and proofing, baking, and storage) (Miller, & Hoseney, 2008). It is well known that salt increases dough mixing resistance, decreases dough stickiness during processing, stabilizes yeast fermentation rate, leads to a more attractive crust colour, improves bread texture, retards bread staling, and inhibits microbial growth during bread storage. Bread accompanied by other cereal products are the largest sodium source, accounting for about 30% of total sodium intake (Angus, 2007). Therefore, reducing salt in bread products could be a critical step to achieve the sodium reduction goal and ultimately improve our health and well-being. However, as discussed previously, removing or reducing salt from bread formulas will have negative effects on dough properties and finished product qualities, and ultimately decrease consumer’s acceptability. Fundamental understanding of the role of salt in the formation and development of dough is necessary for developing appropriate salt reduction strategies for bakery products. Many previous researches have focused on the physical profile or quality analysis of dough with salt (He, Roach, & Hoseney, 1992; Uthayakumaran, Batey, Day, & Wrigley, 2011). A few studies were also conducted to investigate the interactions of gluten with salt in bread doughs (Tuhumury, Small, & Day, 2014; Ukai, Matsumura, & Urade, 2008). However, the mechanism of how salt impacts the gluten structure and physicochemical changes of wheat dough at a molecular level is not yet fully understood. It was hypothesized that salt affects gluten macromolecular aggregation and the non-covalent (hydrogen bonding, hydrophobic, and electrostatic) and covalent (SH oxidation and SH/SS exchanged) interactions during dough development, which contribute to the improved rheological and physical properties of doughs. Thus, the objectives of this study were to investigate the effect of salt on the rheological properties and physical processibility of hard wheat flour dough and shed light on the gluten characteristics and interactions induced by salt. A series of physical and biochemical analytical techniques, including mixograph, dynamic rheometer, Fourier transform infrared spectroscopy (FTIR), spectrophotometer, and reversed phase high-performance liquid chromatography (RP-HPLC) were used to investigate the 4
mixing and rheological properties of dough and the macromolecular characteristics as affected by salt. These were conducted to gain a better insight into the relationship between dough macroscopic properties and structure characteristics.
2. Material and methods 2.1. Materials Hard wheat flour (bread flour, protein content 12.6%, moisture content 13.9%, ash content 0.5%, and fat content 1.6%, fwb) was provided by Frito-Lay North America. All chemicals and reagents in the experiments were of analytical grade and purchased from Fisher Scientific (Fairlawn, NJ, USA) or Sigma-Aldrich (St. Louis, MO, USA). 2.2. Dough mixing Flour-water model dough systems were prepared with five different levels of sodium chloride (0.0%, 1.0%, 1.7%, 2.0%, and 2.4%, fwb). Dough pH was determined according to AACCI Approved Method 02-52.01 (AACC International, 2000). Mixograph was collected according to AACCI Approved Method 54-40.02 (AACC International, 2000). Each treatment was developed in triplicate. Farinographs were tested according to AACCI Approved Method 54-21.01 (AACC International, 2000). Water absorption was optimized, and then a single optimized Farinograph was developed for each treatment. 2.3. Dough strength and extensibility Dough strength and extensibility measurements were carried out using a TA-XTPlus texture analyzer (Stable Micro Systems, Godalming, UK) with an SMS/Kieffer rig (Stable Micro Systems) based on settings of: measure force in tension, pre-test speed of 2 mm s-1, test speed of 3.3 mm s-1, post-test speed of 10 mm s-1, distance of 75 mm, and trigger force of 5 g. Formulation was based on 100 g flour, and optimal mixing time based on mixograph results was used. The dough was mixed in a pin mixer, then was set in a closed container for 30 min. After equilibrating, the dough was gently molded and left on a Teflon former grooved section lubricated with mineral oil, and a cover block was placed on top to form the regular strips. After 5
resting in a clamping tool for 30 min, the dough strips were tested. The output quantities were the force (g) needed to rupture the dough, as an indication of strength, and the distance (mm) to rupture, as extensibility. Three separate doughs were prepared per treatment and at least five strips per dough were tested. 2.4. Dynamic oscillatory rheology Rheological properties were measured with a Bohlin CVOR 150 rheometer (Malvern Instruments, Southborough, MA, USA) with a PP 20 parallel plate with a gap size of 1200 μm. The dough was prepared fresh as described previously and left to rest for 30 min. After loading, any excessive dough sample outside the plate edge was removed. Paraffin oil was utilized to cover the lateral surface to avoid dryness. Amplitude scan was carried out at strain values of 0.01-10% at a constant frequency (1Hz, 25 °C) within the linear viscoelastic region. The strain amplitude was set constantly (0.1% strain, 25 °C), and frequency sweep was changed from 0.1 to 100 Hz. The rheological properties were assessed on the basis of the shear storage modulus (G’), loss modulus (G”) and tan (δ =G”/G’). Duplicate measurements were conducted. 2.5. Lubricated uniaxial compression Lubricated uniaxial compression was used to measure the elongational viscosity of the dough. The dough was prepared following the same procedure as in section 2.3. After mixing, dough was sheeted and left to rest for 10 min. Three 1” diameter disks were then cut from the dough. Mineral oil was used to lubricate the dough and prevent its adhesion to the probe. The TA.XTPlus (Texture Technologies, Scarsdale NY, and Stable Micro Systems, Godalming, UK) was used to compress the disks with a 2” diameter probe, which was deformed to 50% strain at a speed of 0.4 mm s-1. Three separate doughs (3 disks per dough) were tested for each treatment. Elongational viscosity (ηe) was calculated as: ηe = 2FH/R2Vz, where F is peak force for dough deformation, Vz is cross-arm speed, and R is radius, and H is height of dough after compressing. 2.6. Differential scanning calorimetry (DSC) Thermal phase transitions of dough were analyzed with a differential scanning calorimetry (DSC) instrument (Q200, TA Instruments, Schaumburg, IL, USA). The DSC 6
was calibrated using indium reference material. Approximately 20 mg of each sample was accurately weighed and sealed in a high volume stainless steel pan containing an o-ring to avoid leaking water vapour. Then the sample was scanned from 10 °C to 180 °C at a heating rate of 10 °C/min in an inert environment using nitrogen with a gas flow rate of 50 ml/min. Starch retrogradation was also determined by re-scanning the pan using the DSC after storing at room temperature for 48 hours. Each sample was analyzed in duplicate. A sealed empty pan was used as a reference for all measurements. 2.7. Extraction of gluten from dough Gluten fractions were isolated and collected from dough prepared with the different salt levels, following AACCI Approved Method 38-10.01 (AACC International, 2000). Gluten samples were lyophilized, ground to powder, and stored at -20 °C until further analysis. The nitrogen content of samples was analyzed using a LECO TruMac nitrogen analyzer (LECO Corp., St Joseph, MI, USA). A conversion factor of 5.7 was used for the calculation of protein contents according to AACCI Approved Method 46-19.01 (AACC International, 2000). 2.8. Zeta potential The zeta potential of samples was measured following the method described by Chen, and Zhang (2006). Gluten suspension was prepared with a solid content of 0.1% for each sample. Zeta potential of samples was tested on a ZetaPALS zeta potential analyzer (Brookhaven Instruments Co., Holtsville, NY, USA) with hydrodynamic light scattering and laser doppler electrophoresis. Five runs comprising two cycles were carried out for each measurement and duplicate tests were conducted. 2.9. RP-HPLC analysis of gluten fraction Gluten sample (100 mg) was extracted stepwise twice with 1.0 ml of 60% (v/v) aqueous ethanol for 12 min at room temperature (gliadin fraction), and twice with 1.0 ml solution containing 50% (v/v) propan-1-ol, 2 mol l-1 urea, 1% (w/v) dithioerythritol and 0.05 mol l-1 Tris-HCl for 45 min at 60°C (glutenin fraction). Each extraction step started with vortex mixing for 1 min at room temperature. The suspensions were centrifuged (15,000 xg, 10 min, 20°C), and the corresponding 7
extracts were combined and filtered (Phenex™ filter membranes, Phenomenex, Torrance, CA, USA) before analyzing. Each sample was performed in duplicate. Gliadin and glutenin extracts were analyzed with an HP1050 Series HPLC (Agilent Technologies, Santa Clara, CA, USA) coupled with a diode array detector (DAD), according to the method from Wieser, Antes, and Seilmeier (1998) with some modification. A reversed-phase Aeris WIDEPORE XB-C18 column (3.6 μm, 150 x 4.6 mm, Phenomenex, Torrance, CA, USA) was utilized to separate the protein fractions. The elution solvents were water containing 0.1% trifluoroacetic acid (A), and acetonitrile containing 0.1% trifluoroacetic acid (B), with a flow rate of 0.6 ml min-1 at 60°C. The injection volume was 10 μl, and the linear gradient was 0 min 24 % B, 30 min 56 % B. The gliadins and glutenins were identified at 210 nm by comparison of their retention times and spectra with protein standards while quantitative analysis was based on their peak areas from the chromatograms. 2.10. Sulfhydryl (SH) and disulfide (SS) group contents The concentration of free SH was determined according to the method reported by Rombouts, Jansens, Lagrain, Delcour, and Zhu (2014) with some modifications. Gluten samples (30 mg) were suspended in 3.0 ml of reaction buffer A (8 mol l-1 urea, 3 mmol l-1 EDTA, 1% SDS, and 0.2 mol l-1 Tris-HCl, pH 8.0). Samples were vortexed for 30 s and mixed at room temperature for 60 min, and then 0.3 ml of buffer B (10 mmol l-1 DTNB in 0.2 mol l-1 Tris-HCl, pH 8.0) was added to each buffer A and mixed for another 60 min followed by centrifuging at 13,600 xg for 15 min at room temperature. Total SH group content was determined by the previous methods (Chan, & Wasserman, 1993) with some modification. Ten milligrams of sample were mixed with 1.0 ml reaction buffer (3 mmol l-1 EDTA, 1% SDS, 0.2 mol l-1 Tris-HCl, 0.1 mol l-1 sodium sulfite, pH 9.5, and 0.5 mmol l-1 2-nitro-5-thiosulfobenzoate (NTSB)). The samples were vortexed for 30 s and mixed in a shaker in the dark for another 60 min. Next, the samples were centrifuged at 13,600 xg for 15 min followed by diluting the supernatant (0.3 ml) with 2.7 ml reaction buffer without NTSB. Absorbance was tested at 412 nm using a double beam spectrophotometer (VWR UV-6300PC, Radnor, 8
PA, USA). The SH content (CSH) was calculated using: CSH =
(where A is the absorbance, ε
is the extinction coefficient of 13,600, b is the cell path length) (Tuhumury et al., 2014). The SS content (Css) was calculated according to the equation: Css = (where CTSH is the total SH content, and CFSH is the free SH content). 2.11. FTIR and secondary structures Extracted gluten samples were used for secondary structural characterization. FTIR spectra of gluten were collected via a PerkinElmer Spectrum 400 FT-IR/FT-NIR Spectrometer (PerkinElmer, Inc., Waltham, MA, USA) equipped with an ATR accessory. A total of 64 scans were run for each analysis at an interval of 4 cm-1 in the range of 400-4000 cm-1. Gluten secondary structures were deconvoluted from the amide I region (1600-1700 cm-1) via the secondary derivation. Gaussian curve fitting was used by OriginPro 2016 software to measure the relative areas of the selected amide I region. 2.12. Surface hydrophobicity The sodium dodecyl sulfate (SDS) binding capacity was measured following a previous method (Kato, Matsuda, Matsudomi, & Kobayshi, 1984) with some modification. A sample of 10 mg was dissolved in 40 ml of SDS (0.1 mmol l-1), then mixed for 1 hour. After being dialyzed in distilled water for 48h, 1 mg of inner dialyzates was added to 20 ml CHCl3 in a test tube, and 5 ml of methylene blue (0.024 g l-1) was added into the CHCl3 layer followed by centrifuging at 2500 xg for 15 min. The absorbance of the mixture in the lower layer was measured at 655 nm (VWR UV-6300PC, Radnor, PA, USA). The calibration data showed a linearity (R2 > 0.995) for the SDS standard solution within the range of 0.01–0.1 mmol l-1. Protein hydrophobicity was calculated according to the equation: Hydrophobicity value (H) = (where C is the concentration of sample represented by the SDS standard solution). 2.13. Statistical analysis Data were analyzed by one-way analysis of variance (ANOVA) and means were 9
compared by Tukey’s test. When significant differences were found, statistical presence was determined at a P value of 0.05. A statistical discovery software (version 9.4, SAS Institute, Cary, NC, USA) was used for the purpose.
3. Results and discussion 3.1. Mixing properties of doughs In the Mixograph test, water absorption was kept constant at 65% based on the optimized absorption for control dough to investigate the effect of salt additions on dough mixing properties, and typical mixograms are presented in Fig. S1 (supplementary material). The mixing time gradually increased from 2.75 min for the control dough (0% salt) to 3.75, 4.00, 4.50, and 4.75 mins for the doughs with 1.0, 1.7, 2.0, and 2.4% salt, respectively. It was also obvious that doughs became stronger as the mixograms were wider and exhibited more and wider arcs in the presence of NaCl (Fig. S1, supplementary material). In the Farinograph test, the mixing time also increased dramatically from 6.2 min for the control dough to 11.1 min with the addition of 2.4% NaCl, while dough stability increased from 13.1 min for the control dough to 27.7 min for the dough with 2.4% NaCl. Similar trends in Mixograph and Farinograph evaluations with salt addition were also observed in other studies (Beck, Jekle, & Becker, 2012a; He et al., 1992). 3.2. Large deformation properties of doughs The Keiffer testing data can be interpreted in terms of stress and strain. Dough strain hardening behaviour, during which the stress increases more than proportionally to the strain, is associated with the gluten network (Kindelspire, Glover, Caffe-Treml, & Krishnan, 2015; McCann, & Day, 2013). Based on the data illustrated in Table 1, the rupture force of the control was 16.1 g, while the dough containing 2.4% NaCl exhibited the highest average rupture force of 28.0 g. Nevertheless, the force values of 1.0%, 1.7% or 2.0% NaCl were not significantly different (P > 0.05). The distance of doughs containing 1.7% (92.2 mm) and 2.0% NaCl (97.6 mm) significantly differed from the control (66.8 mm); whereas the value was greatly reduced to 79.9 mm for the 2.4% NaCl dough. This indicated that the 10
dough became stiffer and less elastic with 2.4% NaCl addition. The elongational viscosity (resistance) of the dough containing 2.4% salt also significantly increased (P < 0.05) compared to the control and the other treatment groups (Table 1), although adding 1.0, 1.7 and 2.0% NaCl did not significantly change the elongational viscosity. This phenomenon may be attributed to the stronger interaction induced by the gluten polymer cross-links and aggregation with the addition of the high level of NaCl (2.4%), which will be presented and discussed in the next several sections. 3.3. Dynamic rheological measurements of doughs Oscillational rheological properties of the dough, as known by the small deformation test, were measured using strain and frequency sweeps, respectively (Fig. 1). Doughs treated with NaCl exhibited higher storage (G´) and loss modulus (G”) compared with the control. The tan δ (=G”/G’) values were not significantly different (P > 0.05) among the groups at 0.1% strain (Fig. 1C). McCann and Day (2013) found that adding more salt led to less reduction of G´ of the wheat doughs, and it enhanced the strength and elasticity. Larsson (2002) reported a significant increase of the G’ of dough when increasing NaCl levels from 0 to 1%, which may be attributed to the stronger intermolecular cross-links of glutens (i.e. SS bonds). Conversely, Lynch, Dal Bello, Sheehan, Cashman, and Arendt (2009) found both modulus reduced with the addition of NaCl in the wheat flour dough. The lack of consistency in the rheology tests might be due to the different wheat varieties, dough systems, and/or gluten quantity and quality. 3.4. Thermal phase transition properties of doughs Typical phase transition temperatures and enthalpies of fresh doughs and gelatinized doughs after storage for 48 h at room temperature were summarized in Table S1 & Fig. S2 (supplementary material). It was reported that salt competes with starch for water in the dough; therefore, it delayed starch gelatinization (Beck, Jekle, & Becker, 2012b), which is in agreement with our findings that the high levels (1.7, 2.0 and 2.4%) of NaCl significantly increased starch gelatinization temperatures (i.e., TG,max) and melting temperatures (i.e., TM,max) in the doughs (P < 0.05). Moreover,
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both △HG and △HM significantly increased in the presence of 2.0 or 2.4% salt (P < 0.05). Retrogradation of starch occurred in baked doughs (i.e., “baking” within the DSC during the first heating scan) during the 48 hours storage, resulting in a more crystalline status of gelatinized starch (Table S1, supplementary material). When adding NaCl at higher concentrations (≥ 1.5%), the two peak temperatures of melting of retrograded starch crystals (TG,max and TM,max) in the dough significantly increased (P < 0.05), suggesting that high concentration of salt may prevent the retrogradation process. 3.5. Gluten isolation and preparation Doughs with different levels of salt were prepared based on 100 g flour, and glutens were extracted, and the gluten content is shown in Table S2 (supplementary material). Purity of gluten was about 78-87%. Gluten recovery was similar for all the groups (10.5-10.8 g based on 100 g initial flour), with an average of 85% protein extractability. 3.6. Zeta potential Zeta potential is an indicator of surface charge of aggregated particles in a solution. Under a lower absolute zeta potential value, electrostatic repulsion between protein particles decreases, and proteins tend to have more interactions and coagulate (Doane, Chuang, Hill, & Burda, 2011). The pH of the control gluten sample was 5.21. As the level of NaCl increased to 2.4%, the pH value slightly increased to 5.33, implying that the dissociation behaviour of gluten protein was marginally altered, which would affect the electrostatic surface potential and interactions of gluten. As shown in Fig. 2, gluten samples without salt have a surface zeta potential of -3.4 mV. The gluten became positively charged at salt levels greater than 2.0%. High amounts of sodium (2.0 or 2.4%) greatly decreased the absolute values of gluten zeta potential compared to control sample, implying that salt addition tends to promote gluten molecular interactions and aggregation; therefore, it may contribute to the formation of the stronger doughs. 3.7. RP-HPLC of gliadin and glutenin
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Typical RP-HPLC chromatograms of glutens isolated from doughs with salt are presented in Fig. S3 (supplementary material), and quantitative data is summarized in Table 2. The limit of detection (LOD) was 0.016 mg ml-1, and the limit of quantification (LOQ) was assessed to be 0.053 mg ml-1. The linearity of the standard calibration curve ranged from 0.065 to 1.05 mg within the calculated correlation coefficient, indicating a good linearity (R2 > 0.98). Recovery experiments were conducted by spiking the control samples with 1% gliadin standard (n=3), for which the average recovery rate was 88%. The coefficient of variation (CV) for repeatability of sample determination was ±5.3% (average of five determinations), which is comparable to an earlier report (Wieser et al., 1998). The gluten extract was divided into several elution ranges, including gliadin fractions (ω-, α-, γ-gliadin), glutenin fraction of bound gliadin (ωb-gliadin), HMW-GS, and LMW-GS, respectively (Shewry, et al., 2002; Wieser, 2007). All glutenin subunits are reported to be extracted from dough in the presence of urea and dithioerythritol heating at 60 °C. The amount of extractable glutenin increased significantly from 146.7 for the control to 180.0 μg mg-1 with 2.4% salt addition; whereas the gliadin content decreased from 261.3 for the control down to 215.5 μg mg-1 at 2.4% salt addition (P < 0.05). Moreover, the amount of α-, and γ-gliadins were more significantly affected with salt additions in comparison with the ω-gliadins, where cysteine groups are rarely present (Wieser et al., 1998). Previous studies proposed that SS bonds or hydrophobic interactions might accelerate the aggregation of glutenins (Butow, Gras, Haraszi, & Bekes, 2002; Letang et al., 1999). It is noteworthy that extractable HMW-GS was increased to 32% when using 2.4% NaCl, while it was only 17% for LMW-GS. Therefore, it appears that higher salt levels induced more crosslinking reactions between gliadins (especially αand γ-gliadin) and glutenin, which increased the amount of extractable glutenin (i.e., HMW-GS, LMW-GS, ωb-gliadin). The ratio of gliadin to glutenin (gli/glu) plays an important role during the formation and development of dough, since gliadin is responsible for the viscosity and flowability of the dough, whereas glutenin contributes to the elasticity and strength through the polymeric network (Butow, et al., 2002; He, et al., 1992). The 13
ratios of gli/glu were within the reported range of common wheat varieties (Wieser, 2007). Furthermore, when the gli/glu ratio was reduced (within a reasonable range), more glutenin proteins allowed for a better elasticity and cohesiveness of dough, and improved bread loaf volume (Barak, Mudgil, & Khatkar, 2013). As presented in Table 2, with higher salt levels, the ratio of gli/glu decreased significantly from 1.7 to 1.2 (P < 0.05), suggesting that it may contribute to the improved physical and rheological properties discussed previously. 3.8. Sulfhydryl and disulfide contents of gluten Disulfide (SS) cross-links and sulfhydryl-disulfide (SH-SS) interchanges are necessary for aggregation of gluten (Wieser, 2007). Free SH content of wheat gluten was reduced from 4.9 to 3.2 nmoles mg-1 (P < 0.05) when increasing the salt concentration in the dough formula from 0 to 2.4%, while SS content changed from 29. 9 to 30.8 nmoles mg-1 (Table 3). The results indicated that salt, to some extent, may induce SH oxidation reactions in gluten during dough preparation and development. Similar results were obtained by Tuhumury and others (2014), who observed a significant difference of the free SH contents in salt-washed glutens prepared from two wheat flours. The improved physico-chemical properties of doughs with salt may also be attributed to other interaction patterns, such as non-SS cross-linking or hydrophobic interaction, in addition to SS crosslinking. 3.9. Secondary structures of gluten FTIR spectra of gluten samples were collected to elucidate the effect of sodium chloride on gluten conformation. Gluten has several characteristic band regions (Fig. 3): amide I around 1600-1700 cm-1 in terms of stretching vibration of C=O (70-85%) and C-N (10-20%) groups; and amide II around 1510-1580 cm-1 in terms of in-plane N-H bending (40-60%), C-N (18-40%) and C-C (10%) stretching (Wellner, Bianchini, Mills, & Belton, 2003). Gluten secondary structures were deconvoluted from the amide I region, and peak assignments are noted in Table 3 (Georget, & Belton, 2006). Accompanied by the addition of salt, β-sheet structure was significantly increased in the gluten sample while random coil content decreased when adding 2% NaCl (P < 0.05), which may contribute to the gluten structure and physical changes of doughs; 14
however, a significant change of β-turn structure was not observed (P > 0.05). The increased amount of β-sheet was in agreement with some earlier results (Wellner et al., 2003; Tuhumury et al., 2014). 3.10. Surface hydrophobicity The physico-chemical properties of gluten depend on the status of protein macromolecules, in which the hydrophobic residues of amino acids may contribute to the interaction and conformation. SDS binding methodology was used to evaluate the degree of hydrophobicity of gluten samples. The method has been successfully applied to determine the correlation between the hydrophobicity and binding activity of insoluble proteins, such as the ovalbumins (Kato et al., 1984). Stathopoulos, Tsiami, Schofield, and Dobraszczyk (2008) found a variety-dependent hydrophobicity change in six different wheat varieties. Meanwhile, Guerrieri, Alberti, Lavelli, and Cerletti (1996) observed a heating induced the change of gluten within the decreasing solubility. Nevertheless, there is little evidence on the remarkable dependency between the hydrophobicity of gluten and salt. According to the result in Table 3, no significant differences were found among the control and NaCl treatments (P > 0.05), which indicated that the addition of salt (up to 2.4%) may not influence the total surface hydrophobicity of wheat gluten. Earlier studies discussed the possible mechanisms through which sodium chloride impacts the polymeric structure of bread dough systems, and some conceptual models were proposed. It is believed that the hydration of gluten results in the adhesion property of dough, as the mobility of hydrated gluten increases in the network; whereas adding NaCl may mitigate the stickiness of dough via the interaction of ions and water molecules to delay the hydration (van Velzen, van Duynhoven, Pudney, Weegels, & van der Maas, 2003). That could explain the longer development time of dough in the mixing experiments previously reported. Meanwhile, Shewry (2009) proposed that increasing β-sheet structure may attribute to the effect of hydrogen bonding induced by NaCl, which was also consistent with our findings. Since wheat dough is regarded as a complex material embedding gluten protein networks, a single model perhaps cannot thoroughly explain the biochemical 15
or rheological properties of dough. For instance, changes of hydrophobic interactions with salt in doughs were not observed. It was reported that SS bonds aggravated the polymerization of gluten as a result of the oxidation of SH or interchange of SH-SS (Lagrain, Brijs, & Delcour, 2008), which was partially demonstrated by HPLC results (Table 2). However, there are other interaction patterns (e.g., β-elimination, lanthionine formation) worthy of investigation in addition to the SS cross-linking, which may also influence the microstructure of bread dough. Overall, the presence of sodium chloride appeared to unfold the macromolecules, increase the β-sheet, and enhance aggregation during dough formation, which all contributed to the changes in physical and rheological properties of dough.
4. Conclusions Sodium chloride significantly increased the mixing time and stability of doughs. Salt mitigated starch gelatinization, as well as retrogradation during 48 h of storage, and increased storage and loss modulus of wheat flour doughs. The secondary structure distributions were altered, and more β-sheet was induced by salt. The free SH content of gluten was reduced, which suggested that salt led to more SH reaction and cross-linking during the dough production. Furthermore, the absolute values of zeta potential were decreased, and therefore, may contribute to the molecular interaction and aggregation. The extractable level of gliadin greatly decreased while glutenin increased with NaCl, implying that more polymeric and less soluble protein networks were formed. In conclusion, the enhanced strength, resistance, and mixing stability of bread dough were a result of synergistic inter- and intra-molecular interactions of gluten, starch, water, and salt. Higher levels of salt induced stronger gluten interactions via SH cross-linking to some extent, but the hydrophobic interactions seemed to have no effect. However, there are probably other interactions during the formation of dough, such as non-SS reactions, which need further investigation. Furthermore, an in-depth molecular profile analysis of doughs is necessary, and sodium salt replacement or reduction strategies considering both quality and sensory considerations need to be 16
developed for healthy and quality bakery products.
Acknowledgements This is contribution no. 18-315-J from the Kansas Agricultural Experimental Station. Financial support was provided by Frito-Lay North America and the Kansas State University Department of Grain Science and Industry.
References AACC International (2000). Approved methods of the AACC (10th ed.). St. Paul, Minnesota, USA. Angus, F. (2007). Dietary salt intake: sources and targets for reduction. Reducing salt in foods: practical strategies (pp. 3-17). Woodhead Publishing. Autio, K., Kruus, K., Knaapila, A., Gerber, N., Flander, L., & Buchert, J. (2005). Kinetics of transglutaminase-induced cross-linking of wheat proteins in dough. Journal of Agricultural and Food Chemistry, 53(4), 1039-1045. Bailey, R. L., Parker, E. A., Rhodes, D. G., Goldman, J. D., Clemens, J. C., Moshfegh, A. J., Thuppal, S. V., & Weaver, C. M. (2016). Estimating Sodium and Potassium Intakes and Their Ratio in the American Diet: Data from the 2011-2012 NHANES. Journal of Nutrition, 146(4), 745-750. Barak, S., Mudgil, D., & Khatkar, B. S. (2013). Relationship of gliadin and glutenin proteins with dough rheology, flour pasting and bread making performance of wheat varieties. Lwt-Food Science and Technology, 51(1), 211-217. Beck, M., Jekle, M., & Becker, T. (2012a). Impact of sodium chloride on wheat flour dough for yeast-leavened products. I. Rheological attributes. Journal of the Science of Food and Agriculture, 92, 585-592. Beck, M., Jekle, M., & Becker, T. (2012b). Impact of sodium chloride on wheat flour dough for yeast-leavened products. II. Baking quality parameters and their relationship. Journal of the Science of Food and Agriculture, 92, 299-306. Butow, B. J., Gras, P. W., Haraszi, R., & Bekes, F. (2002). Effects of different salts on mixing and extension parameters on a diverse group of wheat cultivars using 17
2-g mixograph and extensigraph methods. Cereal Chemistry, 79(6), 826-833. Chan, K. Y., & Wasserman, B. P. (1993). Direct Colorimetric assay of free thiol-groups and disulfide bonds in suspensions of solubilized and particulate cereal proteins. Cereal Chemistry, 70(2), 240-240. Chen, P., & Zhang, L. (2006). Interaction and properties of highly exfoliated soy protein/montmorillonite
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http://www.fao.org/worldfoodsituation/csdb/en/. Georget, D. M. R., & Belton, P. S. (2006). Effects of temperature and water content on the secondary structure of wheat gluten studied by FTIR spectroscopy. Biomacromolecules. 7(2), 469-475. Goesaert, H., Brijs, K., Veraverbeke, W. S., Courtin, C. M., Gebruers, K., & Delcour, J. A. (2005). Wheat flour constituents: how they impact bread quality, and how to impact their functionality. Trends in Food Science & Technology, 16(1-3), 12-30. Guerrieri, N., Alberti, E., Lavelli, V., & Cerletti, P. (1996). Use of spectroscopic and fluorescence techniques to assess heat-induced molecular modifications of gluten. Cereal Chemistry, 73(3), 368-374. He, C. M., & Roach, M. R. (1994). The composition and mechanical properties of abdominal aortic aneurysms. Journal of Vascular Surgery, 20(1), 6-13. He, H., Roach, R. R., & Hoseney, R. C. (1992). Effect of Nonchaotropic Salts on Flour Bread-Making Properties. Cereal Chemistry, 69(4), 366-371. Kato, A., Matsuda, T., Matsudomi, N., & Kobayashi, K. (1984). Determination of protein hydrophobicity using sodium dodecyl sulfate binding method. Journal of Agricultural and Food Chemistry, 32(2), 284-288. Kindelspire, J. Y., Glover, K. D., Caffe-Treml, M., & Krishnan, P. G. (2015). Dough Strain 18
Hardening Properties as Indicators of Baking Performance. Cereal Chemistry, 92(3), 293-301. Lagrain, B., Brijs, K., & Delcour, J. A. (2008). Reaction Kinetics of Gliadin-Glutenin Cross-Linking in Model Systems and in Bread Making. Journal of Agricultural and Food Chemistry, 56(22), 10660-10666. Larsson, H. (2002). Effect of pH and sodium chloride on wheat flour dough properties: Ultracentrifugation and rheological measurements. Cereal Chemistry, 79(4), 544-545. Letang, C., Piau, M., & Verdier, C. (1999). Characterization of wheat flour-water doughs. Part I: Rheometry and microstructure. Journal of Food Engineering, 41(2), 121-132. Lynch, E. J., Dal Bello, F., Sheehan, E. M., Cashman, K. D., & Arendt, E. K. (2009). Fundamental studies on the reduction of salt on dough and bread characteristics. Food Research International, 42(7), 885-891. McCann, T. H., & Day, L. (2013). Effect of sodium chloride on gluten network formation, dough microstructure and rheology in relation to breadmaking. Journal of Cereal Science, 57(3), 444-452. Miller, R. A., & Hoseney, R. C. (2008). Role of salt in baking. Cereal Foods World, 53(1), 4-6. Rombouts, I., Jansens, K. J. A., Lagrain, B., Delcour, J. A., & Zhu, K. X. (2014). The impact of salt and alkali on gluten polymerization and quality of fresh wheat noodles. Journal of Cereal Science, 60(3), 507-513. Shewry, P. R. (2009). Wheat. Journal of Experimental Botany, 60(6), 1537-1553. Shewry, P. R., Halford, N. G., Belton, P. S., & Tatham, A. S. (2002). The structure and properties of gluten: an elastic protein from wheat grain. Philosophical Transactions of the Royal Society of London Series B-Biological Sciences, 357(1418), 133-142. Song, Y., & Zheng, Q. (2007). Dynamic rheological properties of wheat flour dough and proteins. Trends in Food Science & Technology, 18(3), 132-138. Stathopoulos, C. E., Tsiami, A. A., Schofield, J. D., & Dobraszczyk, B. J. (2008). Effect of 19
heat on rheology, surface hydrophobicity and molecular weight distribution of glutens extracted from flours with different bread-making quality. Journal of Cereal Science, 47(2), 134-143. Tuhumury, H. C. D., Small, D. M., & Day, L. (2014). The effect of sodium chloride on gluten network formation and rheology. Journal of Cereal Science, 60(1), 229-237. Ukai, T., Matsumura, Y., & Urade, R. (2008). Disaggregation and reaggregation of gluten proteins by sodium chloride. Journal of Agricultural and Food Chemistry, 56(3), 1122-1130. United States Department of Agriculture-Economic Research Service (USDA-ERS). (2018).
https://www.ers.usda.gov/data-products/wheat-data/Wheat
Data
Recent.xls. United States Department of Agriculture (USDA), & United Stated Department of Health and Human Services (HHS). (2015). 2015-2020 Dietary Guidelines for Americans. https://health.gov/dietaryguidelines/2015/resources/2015-2020_Dietary_Gui delines.pdf. Uthayakumaran, S., Batey, I. L., Day, L., & Wrigley, C. W. (2011). Salt reduction in wheat-based foods - technical challenges and opportunities. Food Australia, 63(4), 137-140. van Velzen, E. J. J., van Duynhoven, J. P. M., Pudney, P., Weegels, P. L., & van der Maas, J. H. (2003). Factors associated with dough stickiness as sensed by attenuated total reflectance infrared spectroscopy. Cereal Chemistry, 80(4), 378-382. Wellner, N., Bianchini, D., Mills, E. N. C., & Belton, P. S. (2003). Effect of selected Hofmeister anions on the secondary structure and dynamics of wheat prolamins in gluten. Cereal Chemistry, 80(5), 596-600. Wieser, H. (2007). Chemistry of gluten proteins. Food Microbiology, 24(2), 115-119. Wieser, H., Antes, S., & Seilmeier, W. (1998). Quantitative determination of gluten protein types in wheat flour by reversed-phase high-performance liquid chromatography. Cereal Chemistry, 75(5), 644-650. 20
Wieser, H., & Kieffer, R. (2001). Correlations of the amount of gluten protein types to the technological properties of wheat flours determined on a micro-scale. Journal of Cereal Science, 34(1), 19-27.
21
Tables and Figures Table 1. Dough properties of bread dough with different NaCl additions. NaCl (% fwb) 0 1.0 1.7 2.0 2.4
Keiffer test Force (g) 16.1c 24.6ab 21.8bc 21.4bc 28.0a
Distance (mm) 66.8c 82.1bc 92.2ab 97.6a 79.9bc
abc
Elongational Viscosity (Pa.s) 6268ab 6476ab 6354ab 5696a 8407c
Means with different superscripts within the same column are significantly different at P < 0.05.
22
Table 2. Gliadin and glutenin contents of glutens isolated from wheat flour dough at the different levels of sodium addition. Samples
HPLC elution range concentration (μg/mg) ωgliadins
αgliadins
γgliadins
Total gliadin
ωbgliadins
113.4±4.5a
261.3±12.7a 0.5±0.2a
HMWGS
LMWGS
Total glutenin
41.2±1.8a
104.9±2.7a 146.7±4.8a
1.79±0.15a 1.63±0.02a
gli:glu
Control
18.3±1.6a 130.0±6.6a
NaCl 1.0%
17.3±2.4a 119.5±4.1ab 109.3±1.4ab 246.1±5.1ab
0.5±0.3a
42.1±0.1a
107.9±1.1a 150.6±1.4ab
NaCl 1.7%
15.5±0.6a 111.3±3.2ab 95.2±0.9b
222.1±3.5ab
0.6±0.2a
44.2±1.7a
112.3±3.6a 157.2±5.1abc 1.42±0.06ab
NaCl 2.0%
15.6±0.8a 106.3±2.6ab 97.7±3.0b
219.7±4.7ab
1.3±0.3b 53.9±2.5b 127.7±2.2a 183.1±4.3c
1.20±0.01b
NaCl 2.4%
15.2±0.5a 105.3±4.2b
215.5±7.0b
1.8±0.3b 54.2±0.5b 123.7±9.0a 180.0±8.8bc
1.20±0.09b
94.5±2.4b
abc
Means with different superscripts within the same column are significantly different at P < 0.05. Value is represented as the mean ± standard deviation (n=2).
23
Table 3. Sulfhydryl and disulfide content, secondary structure and surface hydrophobicity of glutens separated from wheat doughs with NaCl addition.
Total SH content (nmoles mg )
Samples (sodium chloride %fwb) Control NaCl 1.0% NaCl 1.7% a 64.7±1.1 64.5±1.7a 64.8±1.1a
NaCl 2.0% 65.4±1.1a
NaCl 2.4% 64.9±1.4a
Free SH content (nmoles mg-1) SS content (nmoles mg-1) Extended chains (%) (1600-1615 cm-1) β-Sheet (%) (1624-1640, 1681 cm-1) Random coil (%) (1640-1650 cm-1) α-helix (%) (1650-1660 cm-1) β-turn (%) (1660-1670,1694 cm-1) Surface hydrophobicity
4.9±0.2a 29.9±0.5a 7.5±0.7ab 21.0±2.2b 34.4±5.5a 25.8±1.1ab 8.0±0.6a 18.9±2.4a
3.0±0.1b 31.2±0.6a 8.6±0.1a 27.3±0.1a 25.3±0.2cd 22.4±0.2b 8.6±0.1a 19.2±1.3a
3.2±0.1b 30.8±0.7a 5.3±0.2c 25.8±1.5ab 29.2±0.8b 31.4±2.9ab 7.3±0.1a 19.0±1.2a
-1
abcd
3.4±0.1b 30.5±0.9a 5.6±1.3c 23.9±0.2ab 23.6±1.1d 33.2±0.2a 8.2±0.2a 18.8±1.8a
3.1±0.1b 30.9±0.6a 4.3±0.2c 27.1±0.4ab 27.5±0.8bc 28.4±1.9ab 7.2±0.8a 18.0±1.6a
Means with different superscripts within the same row are significantly different at P < 0.05. Value is represented as the mean ± standard deviation (n=2).
24
Modulus G', G" (Pa)
1.0E+05
A
control salt 1.0% salt 1.7% salt 2.0% salt 2.4%
1.0E+04
1.0E+03 0.1
Modulus G', G" (Pa)
1.0E+05
1
Frequency (Hz)
10
control salt 1.0% salt 1.7% salt 2.0% salt 2.4 %
B
1.0E+04
1.0E+03 0.01%
0.10%
1.00%
10.00%
Strain 1.0
C
tan δ
0.8 0.6
control salt 1.0% salt 1.7% salt 2.0% salt 2.4%
0.4 0.2 0.0 0.1
1
Frequency (Hz)
10
Fig 1. Storage modulus G’ (solid symbols), and loss modulus G” (open symbols) of the doughs prepared from wheat flour, (A) as a function of frequency at a strain of 0.1%, (B) as a function of strain at a frequency of 1Hz, and (C) tan δ as a function of frequency at a strain of 0.1%.
25
Fig. 2. Zeta potential values of gluten isolated from hard wheat flour doughs with different salt content in DI water with the solid content of 0.1%.
26
Fig. 3. Gluten secondary structures deconvolved from amide band I. Full assignment is given as Extended chains (1600-1615 cm-1), β-Sheet (1624-1640, 1681 cm-1), Random coil (1640-1650 cm-1), α-helix (1650-1660 cm-1), β-turn (1660-1670, 1694 cm-1).
27
Highlights:
Doughs were prepared with different levels of salt (0 - 2.4%, fwb).
Doughs with higher levels of salt exhibited greater strength and viscosity.
Salt decreased free SH content but increased β-sheet structure of gluten.
Salt enhanced the macromolecular aggregation of gluten proteins.
28
S0308-8146(18)31312-8 https://doi.org/10.1016/j.foodchem.2018.07.157 FOCH 23272
To appear in:
Food Chemistry
Received Date: Revised Date: Accepted Date:
3 April 2018 5 July 2018 24 July 2018
Please cite this article as: Chen, G., Ehmke, L., Sharma, C., Miller, R., Faa, P., Smith, G., Li, Y., Physicochemical properties and gluten structures of hard wheat flour doughs as affected by salt, Food Chemistry (2018), doi: https:// doi.org/10.1016/j.foodchem.2018.07.157
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Physicochemical properties and gluten structures of hard wheat flour doughs as affected by salt
Gengjun Chen1, Laura Ehmke1, Chetan Sharma1, Rebecca Miller1, Pierre Faa2, Gordon Smith1, Yonghui Li1*
1
Department of Grain Science and Industry, Kansas State University, Manhattan, KS
66506 2
Frito-Lay North America, Plano, TX 75024
*Correspondence to: Yonghui Li, E-mail: [email protected], Ph: 785-532-4061, fax: 785-532-7010
Submit to Food Chemistry
1
Abstract Hard wheat flour doughs were prepared with five different levels of sodium chloride, and rheological properties were characterized. Zeta potential, disulfide-sulfhydryl groups, surface hydrophobicity, secondary structure, and extractable gliadin and glutenin of gluten were analyzed to elucidate gluten structure changes induced by salt. Addition of higher levels of salt (2.0 and 2.4%, fwb) in doughs resulted in larger storage and loss modulus, and elongational viscosity. Starch gelatinization temperatures increased with higher amounts of salt. The presence of salt decreased the free sulfhydryl content but increased the β-sheet structure of gluten. RP-HPLC indicated that salt enhanced the macromolecular aggregation of gluten proteins. The changes in gluten molecular conformation and network structure induced by salt significantly contributed to the improved physicochemical properties of dough. This study provides a better understanding of salt functionality in hard wheat flour dough and a valuable guide in searching for salt alternatives for bakery products.
Keywords: Sodium chloride salt, gluten, dough, rheological properties, gliadin, glutenin, hydrophobicity, secondary structure, Zeta potential, HPLC
2
1. Introduction Wheat is one of the major cereal crops worldwide, and its global production reached 749 million tons in 2016 (FAO, 2016). Hard wheats (both winter and spring, red and white) account for more than 80% of total U.S. wheat production (USDA-ERS, 2018), and one of the main applications is for bakery products, including bread. Bread has been a widely consumed product in the human diet, which is served in a variety of forms (Miller, & Hoseney, 2008). The formation of dough plays a key role in breadmaking, since it incorporates all the ingredients and develops a unique viscoelastic gluten network (Letang, Piau, & Verdier, 1999). Shewry, Halford, Belton, and Tatham (2002) pointed out that gluten was associated with the water absorption, mixing time, and strength index of dough. More than 80% of gluten protein consisted of gliadins and glutenins (Shewry, 2009; Song, & Zheng, 2007). The gliadins (ω-, αand γ-type) are relatively small molecules that are soluble in alcohols, which may act as a plasticizer in dough systems (Goesaert, Brijs, Veraverbeke, Gerbruers, & Delcour, 2005). On the other hand, the glutenins are heterogeneous polymers including high molecular weight glutenin subunits (HMW-GS) and low molecular weight glutenin subunits (LMW-GS) (Shewry, et al., 2002). Moreover, glutenins contribute to the mixing resistance, elasticity, and cohesiveness of the dough network (Wieser, & Kieffer, 2001). When water is added into flour, the gluten fraction is hydrated and forms a protein network, which is considered as an important factor in determining dough properties (Autio et al., 2005). Sodium is an essential nutrient for humans, but excessive sodium intake increases blood pressure and risk of cardiovascular disease (CVD) (Bailey, et al., 2016). The average daily sodium intake of Americans is about 3,440 mg per day, which far exceeds the 2015 Dietary Guidelines for Americans recommended upper limit of 2,300 mg per day (USDA, & HHS, 2015). The largest contributor to dietary sodium is salt (known as table salt, or sodium chloride, NaCl). There is an urgent need to reduce the amount of salt in the diet. Salt is actually one of the four essential ingredients (flour, water, yeast, and salt) in any bread formula. In addition to its sensory contribution, salt plays critical technological roles throughout the 3
bread-making process (mixing and dough formation, fermentation and proofing, baking, and storage) (Miller, & Hoseney, 2008). It is well known that salt increases dough mixing resistance, decreases dough stickiness during processing, stabilizes yeast fermentation rate, leads to a more attractive crust colour, improves bread texture, retards bread staling, and inhibits microbial growth during bread storage. Bread accompanied by other cereal products are the largest sodium source, accounting for about 30% of total sodium intake (Angus, 2007). Therefore, reducing salt in bread products could be a critical step to achieve the sodium reduction goal and ultimately improve our health and well-being. However, as discussed previously, removing or reducing salt from bread formulas will have negative effects on dough properties and finished product qualities, and ultimately decrease consumer’s acceptability. Fundamental understanding of the role of salt in the formation and development of dough is necessary for developing appropriate salt reduction strategies for bakery products. Many previous researches have focused on the physical profile or quality analysis of dough with salt (He, Roach, & Hoseney, 1992; Uthayakumaran, Batey, Day, & Wrigley, 2011). A few studies were also conducted to investigate the interactions of gluten with salt in bread doughs (Tuhumury, Small, & Day, 2014; Ukai, Matsumura, & Urade, 2008). However, the mechanism of how salt impacts the gluten structure and physicochemical changes of wheat dough at a molecular level is not yet fully understood. It was hypothesized that salt affects gluten macromolecular aggregation and the non-covalent (hydrogen bonding, hydrophobic, and electrostatic) and covalent (SH oxidation and SH/SS exchanged) interactions during dough development, which contribute to the improved rheological and physical properties of doughs. Thus, the objectives of this study were to investigate the effect of salt on the rheological properties and physical processibility of hard wheat flour dough and shed light on the gluten characteristics and interactions induced by salt. A series of physical and biochemical analytical techniques, including mixograph, dynamic rheometer, Fourier transform infrared spectroscopy (FTIR), spectrophotometer, and reversed phase high-performance liquid chromatography (RP-HPLC) were used to investigate the 4
mixing and rheological properties of dough and the macromolecular characteristics as affected by salt. These were conducted to gain a better insight into the relationship between dough macroscopic properties and structure characteristics.
2. Material and methods 2.1. Materials Hard wheat flour (bread flour, protein content 12.6%, moisture content 13.9%, ash content 0.5%, and fat content 1.6%, fwb) was provided by Frito-Lay North America. All chemicals and reagents in the experiments were of analytical grade and purchased from Fisher Scientific (Fairlawn, NJ, USA) or Sigma-Aldrich (St. Louis, MO, USA). 2.2. Dough mixing Flour-water model dough systems were prepared with five different levels of sodium chloride (0.0%, 1.0%, 1.7%, 2.0%, and 2.4%, fwb). Dough pH was determined according to AACCI Approved Method 02-52.01 (AACC International, 2000). Mixograph was collected according to AACCI Approved Method 54-40.02 (AACC International, 2000). Each treatment was developed in triplicate. Farinographs were tested according to AACCI Approved Method 54-21.01 (AACC International, 2000). Water absorption was optimized, and then a single optimized Farinograph was developed for each treatment. 2.3. Dough strength and extensibility Dough strength and extensibility measurements were carried out using a TA-XTPlus texture analyzer (Stable Micro Systems, Godalming, UK) with an SMS/Kieffer rig (Stable Micro Systems) based on settings of: measure force in tension, pre-test speed of 2 mm s-1, test speed of 3.3 mm s-1, post-test speed of 10 mm s-1, distance of 75 mm, and trigger force of 5 g. Formulation was based on 100 g flour, and optimal mixing time based on mixograph results was used. The dough was mixed in a pin mixer, then was set in a closed container for 30 min. After equilibrating, the dough was gently molded and left on a Teflon former grooved section lubricated with mineral oil, and a cover block was placed on top to form the regular strips. After 5
resting in a clamping tool for 30 min, the dough strips were tested. The output quantities were the force (g) needed to rupture the dough, as an indication of strength, and the distance (mm) to rupture, as extensibility. Three separate doughs were prepared per treatment and at least five strips per dough were tested. 2.4. Dynamic oscillatory rheology Rheological properties were measured with a Bohlin CVOR 150 rheometer (Malvern Instruments, Southborough, MA, USA) with a PP 20 parallel plate with a gap size of 1200 μm. The dough was prepared fresh as described previously and left to rest for 30 min. After loading, any excessive dough sample outside the plate edge was removed. Paraffin oil was utilized to cover the lateral surface to avoid dryness. Amplitude scan was carried out at strain values of 0.01-10% at a constant frequency (1Hz, 25 °C) within the linear viscoelastic region. The strain amplitude was set constantly (0.1% strain, 25 °C), and frequency sweep was changed from 0.1 to 100 Hz. The rheological properties were assessed on the basis of the shear storage modulus (G’), loss modulus (G”) and tan (δ =G”/G’). Duplicate measurements were conducted. 2.5. Lubricated uniaxial compression Lubricated uniaxial compression was used to measure the elongational viscosity of the dough. The dough was prepared following the same procedure as in section 2.3. After mixing, dough was sheeted and left to rest for 10 min. Three 1” diameter disks were then cut from the dough. Mineral oil was used to lubricate the dough and prevent its adhesion to the probe. The TA.XTPlus (Texture Technologies, Scarsdale NY, and Stable Micro Systems, Godalming, UK) was used to compress the disks with a 2” diameter probe, which was deformed to 50% strain at a speed of 0.4 mm s-1. Three separate doughs (3 disks per dough) were tested for each treatment. Elongational viscosity (ηe) was calculated as: ηe = 2FH/R2Vz, where F is peak force for dough deformation, Vz is cross-arm speed, and R is radius, and H is height of dough after compressing. 2.6. Differential scanning calorimetry (DSC) Thermal phase transitions of dough were analyzed with a differential scanning calorimetry (DSC) instrument (Q200, TA Instruments, Schaumburg, IL, USA). The DSC 6
was calibrated using indium reference material. Approximately 20 mg of each sample was accurately weighed and sealed in a high volume stainless steel pan containing an o-ring to avoid leaking water vapour. Then the sample was scanned from 10 °C to 180 °C at a heating rate of 10 °C/min in an inert environment using nitrogen with a gas flow rate of 50 ml/min. Starch retrogradation was also determined by re-scanning the pan using the DSC after storing at room temperature for 48 hours. Each sample was analyzed in duplicate. A sealed empty pan was used as a reference for all measurements. 2.7. Extraction of gluten from dough Gluten fractions were isolated and collected from dough prepared with the different salt levels, following AACCI Approved Method 38-10.01 (AACC International, 2000). Gluten samples were lyophilized, ground to powder, and stored at -20 °C until further analysis. The nitrogen content of samples was analyzed using a LECO TruMac nitrogen analyzer (LECO Corp., St Joseph, MI, USA). A conversion factor of 5.7 was used for the calculation of protein contents according to AACCI Approved Method 46-19.01 (AACC International, 2000). 2.8. Zeta potential The zeta potential of samples was measured following the method described by Chen, and Zhang (2006). Gluten suspension was prepared with a solid content of 0.1% for each sample. Zeta potential of samples was tested on a ZetaPALS zeta potential analyzer (Brookhaven Instruments Co., Holtsville, NY, USA) with hydrodynamic light scattering and laser doppler electrophoresis. Five runs comprising two cycles were carried out for each measurement and duplicate tests were conducted. 2.9. RP-HPLC analysis of gluten fraction Gluten sample (100 mg) was extracted stepwise twice with 1.0 ml of 60% (v/v) aqueous ethanol for 12 min at room temperature (gliadin fraction), and twice with 1.0 ml solution containing 50% (v/v) propan-1-ol, 2 mol l-1 urea, 1% (w/v) dithioerythritol and 0.05 mol l-1 Tris-HCl for 45 min at 60°C (glutenin fraction). Each extraction step started with vortex mixing for 1 min at room temperature. The suspensions were centrifuged (15,000 xg, 10 min, 20°C), and the corresponding 7
extracts were combined and filtered (Phenex™ filter membranes, Phenomenex, Torrance, CA, USA) before analyzing. Each sample was performed in duplicate. Gliadin and glutenin extracts were analyzed with an HP1050 Series HPLC (Agilent Technologies, Santa Clara, CA, USA) coupled with a diode array detector (DAD), according to the method from Wieser, Antes, and Seilmeier (1998) with some modification. A reversed-phase Aeris WIDEPORE XB-C18 column (3.6 μm, 150 x 4.6 mm, Phenomenex, Torrance, CA, USA) was utilized to separate the protein fractions. The elution solvents were water containing 0.1% trifluoroacetic acid (A), and acetonitrile containing 0.1% trifluoroacetic acid (B), with a flow rate of 0.6 ml min-1 at 60°C. The injection volume was 10 μl, and the linear gradient was 0 min 24 % B, 30 min 56 % B. The gliadins and glutenins were identified at 210 nm by comparison of their retention times and spectra with protein standards while quantitative analysis was based on their peak areas from the chromatograms. 2.10. Sulfhydryl (SH) and disulfide (SS) group contents The concentration of free SH was determined according to the method reported by Rombouts, Jansens, Lagrain, Delcour, and Zhu (2014) with some modifications. Gluten samples (30 mg) were suspended in 3.0 ml of reaction buffer A (8 mol l-1 urea, 3 mmol l-1 EDTA, 1% SDS, and 0.2 mol l-1 Tris-HCl, pH 8.0). Samples were vortexed for 30 s and mixed at room temperature for 60 min, and then 0.3 ml of buffer B (10 mmol l-1 DTNB in 0.2 mol l-1 Tris-HCl, pH 8.0) was added to each buffer A and mixed for another 60 min followed by centrifuging at 13,600 xg for 15 min at room temperature. Total SH group content was determined by the previous methods (Chan, & Wasserman, 1993) with some modification. Ten milligrams of sample were mixed with 1.0 ml reaction buffer (3 mmol l-1 EDTA, 1% SDS, 0.2 mol l-1 Tris-HCl, 0.1 mol l-1 sodium sulfite, pH 9.5, and 0.5 mmol l-1 2-nitro-5-thiosulfobenzoate (NTSB)). The samples were vortexed for 30 s and mixed in a shaker in the dark for another 60 min. Next, the samples were centrifuged at 13,600 xg for 15 min followed by diluting the supernatant (0.3 ml) with 2.7 ml reaction buffer without NTSB. Absorbance was tested at 412 nm using a double beam spectrophotometer (VWR UV-6300PC, Radnor, 8
PA, USA). The SH content (CSH) was calculated using: CSH =
(where A is the absorbance, ε
is the extinction coefficient of 13,600, b is the cell path length) (Tuhumury et al., 2014). The SS content (Css) was calculated according to the equation: Css = (where CTSH is the total SH content, and CFSH is the free SH content). 2.11. FTIR and secondary structures Extracted gluten samples were used for secondary structural characterization. FTIR spectra of gluten were collected via a PerkinElmer Spectrum 400 FT-IR/FT-NIR Spectrometer (PerkinElmer, Inc., Waltham, MA, USA) equipped with an ATR accessory. A total of 64 scans were run for each analysis at an interval of 4 cm-1 in the range of 400-4000 cm-1. Gluten secondary structures were deconvoluted from the amide I region (1600-1700 cm-1) via the secondary derivation. Gaussian curve fitting was used by OriginPro 2016 software to measure the relative areas of the selected amide I region. 2.12. Surface hydrophobicity The sodium dodecyl sulfate (SDS) binding capacity was measured following a previous method (Kato, Matsuda, Matsudomi, & Kobayshi, 1984) with some modification. A sample of 10 mg was dissolved in 40 ml of SDS (0.1 mmol l-1), then mixed for 1 hour. After being dialyzed in distilled water for 48h, 1 mg of inner dialyzates was added to 20 ml CHCl3 in a test tube, and 5 ml of methylene blue (0.024 g l-1) was added into the CHCl3 layer followed by centrifuging at 2500 xg for 15 min. The absorbance of the mixture in the lower layer was measured at 655 nm (VWR UV-6300PC, Radnor, PA, USA). The calibration data showed a linearity (R2 > 0.995) for the SDS standard solution within the range of 0.01–0.1 mmol l-1. Protein hydrophobicity was calculated according to the equation: Hydrophobicity value (H) = (where C is the concentration of sample represented by the SDS standard solution). 2.13. Statistical analysis Data were analyzed by one-way analysis of variance (ANOVA) and means were 9
compared by Tukey’s test. When significant differences were found, statistical presence was determined at a P value of 0.05. A statistical discovery software (version 9.4, SAS Institute, Cary, NC, USA) was used for the purpose.
3. Results and discussion 3.1. Mixing properties of doughs In the Mixograph test, water absorption was kept constant at 65% based on the optimized absorption for control dough to investigate the effect of salt additions on dough mixing properties, and typical mixograms are presented in Fig. S1 (supplementary material). The mixing time gradually increased from 2.75 min for the control dough (0% salt) to 3.75, 4.00, 4.50, and 4.75 mins for the doughs with 1.0, 1.7, 2.0, and 2.4% salt, respectively. It was also obvious that doughs became stronger as the mixograms were wider and exhibited more and wider arcs in the presence of NaCl (Fig. S1, supplementary material). In the Farinograph test, the mixing time also increased dramatically from 6.2 min for the control dough to 11.1 min with the addition of 2.4% NaCl, while dough stability increased from 13.1 min for the control dough to 27.7 min for the dough with 2.4% NaCl. Similar trends in Mixograph and Farinograph evaluations with salt addition were also observed in other studies (Beck, Jekle, & Becker, 2012a; He et al., 1992). 3.2. Large deformation properties of doughs The Keiffer testing data can be interpreted in terms of stress and strain. Dough strain hardening behaviour, during which the stress increases more than proportionally to the strain, is associated with the gluten network (Kindelspire, Glover, Caffe-Treml, & Krishnan, 2015; McCann, & Day, 2013). Based on the data illustrated in Table 1, the rupture force of the control was 16.1 g, while the dough containing 2.4% NaCl exhibited the highest average rupture force of 28.0 g. Nevertheless, the force values of 1.0%, 1.7% or 2.0% NaCl were not significantly different (P > 0.05). The distance of doughs containing 1.7% (92.2 mm) and 2.0% NaCl (97.6 mm) significantly differed from the control (66.8 mm); whereas the value was greatly reduced to 79.9 mm for the 2.4% NaCl dough. This indicated that the 10
dough became stiffer and less elastic with 2.4% NaCl addition. The elongational viscosity (resistance) of the dough containing 2.4% salt also significantly increased (P < 0.05) compared to the control and the other treatment groups (Table 1), although adding 1.0, 1.7 and 2.0% NaCl did not significantly change the elongational viscosity. This phenomenon may be attributed to the stronger interaction induced by the gluten polymer cross-links and aggregation with the addition of the high level of NaCl (2.4%), which will be presented and discussed in the next several sections. 3.3. Dynamic rheological measurements of doughs Oscillational rheological properties of the dough, as known by the small deformation test, were measured using strain and frequency sweeps, respectively (Fig. 1). Doughs treated with NaCl exhibited higher storage (G´) and loss modulus (G”) compared with the control. The tan δ (=G”/G’) values were not significantly different (P > 0.05) among the groups at 0.1% strain (Fig. 1C). McCann and Day (2013) found that adding more salt led to less reduction of G´ of the wheat doughs, and it enhanced the strength and elasticity. Larsson (2002) reported a significant increase of the G’ of dough when increasing NaCl levels from 0 to 1%, which may be attributed to the stronger intermolecular cross-links of glutens (i.e. SS bonds). Conversely, Lynch, Dal Bello, Sheehan, Cashman, and Arendt (2009) found both modulus reduced with the addition of NaCl in the wheat flour dough. The lack of consistency in the rheology tests might be due to the different wheat varieties, dough systems, and/or gluten quantity and quality. 3.4. Thermal phase transition properties of doughs Typical phase transition temperatures and enthalpies of fresh doughs and gelatinized doughs after storage for 48 h at room temperature were summarized in Table S1 & Fig. S2 (supplementary material). It was reported that salt competes with starch for water in the dough; therefore, it delayed starch gelatinization (Beck, Jekle, & Becker, 2012b), which is in agreement with our findings that the high levels (1.7, 2.0 and 2.4%) of NaCl significantly increased starch gelatinization temperatures (i.e., TG,max) and melting temperatures (i.e., TM,max) in the doughs (P < 0.05). Moreover,
11
both △HG and △HM significantly increased in the presence of 2.0 or 2.4% salt (P < 0.05). Retrogradation of starch occurred in baked doughs (i.e., “baking” within the DSC during the first heating scan) during the 48 hours storage, resulting in a more crystalline status of gelatinized starch (Table S1, supplementary material). When adding NaCl at higher concentrations (≥ 1.5%), the two peak temperatures of melting of retrograded starch crystals (TG,max and TM,max) in the dough significantly increased (P < 0.05), suggesting that high concentration of salt may prevent the retrogradation process. 3.5. Gluten isolation and preparation Doughs with different levels of salt were prepared based on 100 g flour, and glutens were extracted, and the gluten content is shown in Table S2 (supplementary material). Purity of gluten was about 78-87%. Gluten recovery was similar for all the groups (10.5-10.8 g based on 100 g initial flour), with an average of 85% protein extractability. 3.6. Zeta potential Zeta potential is an indicator of surface charge of aggregated particles in a solution. Under a lower absolute zeta potential value, electrostatic repulsion between protein particles decreases, and proteins tend to have more interactions and coagulate (Doane, Chuang, Hill, & Burda, 2011). The pH of the control gluten sample was 5.21. As the level of NaCl increased to 2.4%, the pH value slightly increased to 5.33, implying that the dissociation behaviour of gluten protein was marginally altered, which would affect the electrostatic surface potential and interactions of gluten. As shown in Fig. 2, gluten samples without salt have a surface zeta potential of -3.4 mV. The gluten became positively charged at salt levels greater than 2.0%. High amounts of sodium (2.0 or 2.4%) greatly decreased the absolute values of gluten zeta potential compared to control sample, implying that salt addition tends to promote gluten molecular interactions and aggregation; therefore, it may contribute to the formation of the stronger doughs. 3.7. RP-HPLC of gliadin and glutenin
12
Typical RP-HPLC chromatograms of glutens isolated from doughs with salt are presented in Fig. S3 (supplementary material), and quantitative data is summarized in Table 2. The limit of detection (LOD) was 0.016 mg ml-1, and the limit of quantification (LOQ) was assessed to be 0.053 mg ml-1. The linearity of the standard calibration curve ranged from 0.065 to 1.05 mg within the calculated correlation coefficient, indicating a good linearity (R2 > 0.98). Recovery experiments were conducted by spiking the control samples with 1% gliadin standard (n=3), for which the average recovery rate was 88%. The coefficient of variation (CV) for repeatability of sample determination was ±5.3% (average of five determinations), which is comparable to an earlier report (Wieser et al., 1998). The gluten extract was divided into several elution ranges, including gliadin fractions (ω-, α-, γ-gliadin), glutenin fraction of bound gliadin (ωb-gliadin), HMW-GS, and LMW-GS, respectively (Shewry, et al., 2002; Wieser, 2007). All glutenin subunits are reported to be extracted from dough in the presence of urea and dithioerythritol heating at 60 °C. The amount of extractable glutenin increased significantly from 146.7 for the control to 180.0 μg mg-1 with 2.4% salt addition; whereas the gliadin content decreased from 261.3 for the control down to 215.5 μg mg-1 at 2.4% salt addition (P < 0.05). Moreover, the amount of α-, and γ-gliadins were more significantly affected with salt additions in comparison with the ω-gliadins, where cysteine groups are rarely present (Wieser et al., 1998). Previous studies proposed that SS bonds or hydrophobic interactions might accelerate the aggregation of glutenins (Butow, Gras, Haraszi, & Bekes, 2002; Letang et al., 1999). It is noteworthy that extractable HMW-GS was increased to 32% when using 2.4% NaCl, while it was only 17% for LMW-GS. Therefore, it appears that higher salt levels induced more crosslinking reactions between gliadins (especially αand γ-gliadin) and glutenin, which increased the amount of extractable glutenin (i.e., HMW-GS, LMW-GS, ωb-gliadin). The ratio of gliadin to glutenin (gli/glu) plays an important role during the formation and development of dough, since gliadin is responsible for the viscosity and flowability of the dough, whereas glutenin contributes to the elasticity and strength through the polymeric network (Butow, et al., 2002; He, et al., 1992). The 13
ratios of gli/glu were within the reported range of common wheat varieties (Wieser, 2007). Furthermore, when the gli/glu ratio was reduced (within a reasonable range), more glutenin proteins allowed for a better elasticity and cohesiveness of dough, and improved bread loaf volume (Barak, Mudgil, & Khatkar, 2013). As presented in Table 2, with higher salt levels, the ratio of gli/glu decreased significantly from 1.7 to 1.2 (P < 0.05), suggesting that it may contribute to the improved physical and rheological properties discussed previously. 3.8. Sulfhydryl and disulfide contents of gluten Disulfide (SS) cross-links and sulfhydryl-disulfide (SH-SS) interchanges are necessary for aggregation of gluten (Wieser, 2007). Free SH content of wheat gluten was reduced from 4.9 to 3.2 nmoles mg-1 (P < 0.05) when increasing the salt concentration in the dough formula from 0 to 2.4%, while SS content changed from 29. 9 to 30.8 nmoles mg-1 (Table 3). The results indicated that salt, to some extent, may induce SH oxidation reactions in gluten during dough preparation and development. Similar results were obtained by Tuhumury and others (2014), who observed a significant difference of the free SH contents in salt-washed glutens prepared from two wheat flours. The improved physico-chemical properties of doughs with salt may also be attributed to other interaction patterns, such as non-SS cross-linking or hydrophobic interaction, in addition to SS crosslinking. 3.9. Secondary structures of gluten FTIR spectra of gluten samples were collected to elucidate the effect of sodium chloride on gluten conformation. Gluten has several characteristic band regions (Fig. 3): amide I around 1600-1700 cm-1 in terms of stretching vibration of C=O (70-85%) and C-N (10-20%) groups; and amide II around 1510-1580 cm-1 in terms of in-plane N-H bending (40-60%), C-N (18-40%) and C-C (10%) stretching (Wellner, Bianchini, Mills, & Belton, 2003). Gluten secondary structures were deconvoluted from the amide I region, and peak assignments are noted in Table 3 (Georget, & Belton, 2006). Accompanied by the addition of salt, β-sheet structure was significantly increased in the gluten sample while random coil content decreased when adding 2% NaCl (P < 0.05), which may contribute to the gluten structure and physical changes of doughs; 14
however, a significant change of β-turn structure was not observed (P > 0.05). The increased amount of β-sheet was in agreement with some earlier results (Wellner et al., 2003; Tuhumury et al., 2014). 3.10. Surface hydrophobicity The physico-chemical properties of gluten depend on the status of protein macromolecules, in which the hydrophobic residues of amino acids may contribute to the interaction and conformation. SDS binding methodology was used to evaluate the degree of hydrophobicity of gluten samples. The method has been successfully applied to determine the correlation between the hydrophobicity and binding activity of insoluble proteins, such as the ovalbumins (Kato et al., 1984). Stathopoulos, Tsiami, Schofield, and Dobraszczyk (2008) found a variety-dependent hydrophobicity change in six different wheat varieties. Meanwhile, Guerrieri, Alberti, Lavelli, and Cerletti (1996) observed a heating induced the change of gluten within the decreasing solubility. Nevertheless, there is little evidence on the remarkable dependency between the hydrophobicity of gluten and salt. According to the result in Table 3, no significant differences were found among the control and NaCl treatments (P > 0.05), which indicated that the addition of salt (up to 2.4%) may not influence the total surface hydrophobicity of wheat gluten. Earlier studies discussed the possible mechanisms through which sodium chloride impacts the polymeric structure of bread dough systems, and some conceptual models were proposed. It is believed that the hydration of gluten results in the adhesion property of dough, as the mobility of hydrated gluten increases in the network; whereas adding NaCl may mitigate the stickiness of dough via the interaction of ions and water molecules to delay the hydration (van Velzen, van Duynhoven, Pudney, Weegels, & van der Maas, 2003). That could explain the longer development time of dough in the mixing experiments previously reported. Meanwhile, Shewry (2009) proposed that increasing β-sheet structure may attribute to the effect of hydrogen bonding induced by NaCl, which was also consistent with our findings. Since wheat dough is regarded as a complex material embedding gluten protein networks, a single model perhaps cannot thoroughly explain the biochemical 15
or rheological properties of dough. For instance, changes of hydrophobic interactions with salt in doughs were not observed. It was reported that SS bonds aggravated the polymerization of gluten as a result of the oxidation of SH or interchange of SH-SS (Lagrain, Brijs, & Delcour, 2008), which was partially demonstrated by HPLC results (Table 2). However, there are other interaction patterns (e.g., β-elimination, lanthionine formation) worthy of investigation in addition to the SS cross-linking, which may also influence the microstructure of bread dough. Overall, the presence of sodium chloride appeared to unfold the macromolecules, increase the β-sheet, and enhance aggregation during dough formation, which all contributed to the changes in physical and rheological properties of dough.
4. Conclusions Sodium chloride significantly increased the mixing time and stability of doughs. Salt mitigated starch gelatinization, as well as retrogradation during 48 h of storage, and increased storage and loss modulus of wheat flour doughs. The secondary structure distributions were altered, and more β-sheet was induced by salt. The free SH content of gluten was reduced, which suggested that salt led to more SH reaction and cross-linking during the dough production. Furthermore, the absolute values of zeta potential were decreased, and therefore, may contribute to the molecular interaction and aggregation. The extractable level of gliadin greatly decreased while glutenin increased with NaCl, implying that more polymeric and less soluble protein networks were formed. In conclusion, the enhanced strength, resistance, and mixing stability of bread dough were a result of synergistic inter- and intra-molecular interactions of gluten, starch, water, and salt. Higher levels of salt induced stronger gluten interactions via SH cross-linking to some extent, but the hydrophobic interactions seemed to have no effect. However, there are probably other interactions during the formation of dough, such as non-SS reactions, which need further investigation. Furthermore, an in-depth molecular profile analysis of doughs is necessary, and sodium salt replacement or reduction strategies considering both quality and sensory considerations need to be 16
developed for healthy and quality bakery products.
Acknowledgements This is contribution no. 18-315-J from the Kansas Agricultural Experimental Station. Financial support was provided by Frito-Lay North America and the Kansas State University Department of Grain Science and Industry.
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21
Tables and Figures Table 1. Dough properties of bread dough with different NaCl additions. NaCl (% fwb) 0 1.0 1.7 2.0 2.4
Keiffer test Force (g) 16.1c 24.6ab 21.8bc 21.4bc 28.0a
Distance (mm) 66.8c 82.1bc 92.2ab 97.6a 79.9bc
abc
Elongational Viscosity (Pa.s) 6268ab 6476ab 6354ab 5696a 8407c
Means with different superscripts within the same column are significantly different at P < 0.05.
22
Table 2. Gliadin and glutenin contents of glutens isolated from wheat flour dough at the different levels of sodium addition. Samples
HPLC elution range concentration (μg/mg) ωgliadins
αgliadins
γgliadins
Total gliadin
ωbgliadins
113.4±4.5a
261.3±12.7a 0.5±0.2a
HMWGS
LMWGS
Total glutenin
41.2±1.8a
104.9±2.7a 146.7±4.8a
1.79±0.15a 1.63±0.02a
gli:glu
Control
18.3±1.6a 130.0±6.6a
NaCl 1.0%
17.3±2.4a 119.5±4.1ab 109.3±1.4ab 246.1±5.1ab
0.5±0.3a
42.1±0.1a
107.9±1.1a 150.6±1.4ab
NaCl 1.7%
15.5±0.6a 111.3±3.2ab 95.2±0.9b
222.1±3.5ab
0.6±0.2a
44.2±1.7a
112.3±3.6a 157.2±5.1abc 1.42±0.06ab
NaCl 2.0%
15.6±0.8a 106.3±2.6ab 97.7±3.0b
219.7±4.7ab
1.3±0.3b 53.9±2.5b 127.7±2.2a 183.1±4.3c
1.20±0.01b
NaCl 2.4%
15.2±0.5a 105.3±4.2b
215.5±7.0b
1.8±0.3b 54.2±0.5b 123.7±9.0a 180.0±8.8bc
1.20±0.09b
94.5±2.4b
abc
Means with different superscripts within the same column are significantly different at P < 0.05. Value is represented as the mean ± standard deviation (n=2).
23
Table 3. Sulfhydryl and disulfide content, secondary structure and surface hydrophobicity of glutens separated from wheat doughs with NaCl addition.
Total SH content (nmoles mg )
Samples (sodium chloride %fwb) Control NaCl 1.0% NaCl 1.7% a 64.7±1.1 64.5±1.7a 64.8±1.1a
NaCl 2.0% 65.4±1.1a
NaCl 2.4% 64.9±1.4a
Free SH content (nmoles mg-1) SS content (nmoles mg-1) Extended chains (%) (1600-1615 cm-1) β-Sheet (%) (1624-1640, 1681 cm-1) Random coil (%) (1640-1650 cm-1) α-helix (%) (1650-1660 cm-1) β-turn (%) (1660-1670,1694 cm-1) Surface hydrophobicity
4.9±0.2a 29.9±0.5a 7.5±0.7ab 21.0±2.2b 34.4±5.5a 25.8±1.1ab 8.0±0.6a 18.9±2.4a
3.0±0.1b 31.2±0.6a 8.6±0.1a 27.3±0.1a 25.3±0.2cd 22.4±0.2b 8.6±0.1a 19.2±1.3a
3.2±0.1b 30.8±0.7a 5.3±0.2c 25.8±1.5ab 29.2±0.8b 31.4±2.9ab 7.3±0.1a 19.0±1.2a
-1
abcd
3.4±0.1b 30.5±0.9a 5.6±1.3c 23.9±0.2ab 23.6±1.1d 33.2±0.2a 8.2±0.2a 18.8±1.8a
3.1±0.1b 30.9±0.6a 4.3±0.2c 27.1±0.4ab 27.5±0.8bc 28.4±1.9ab 7.2±0.8a 18.0±1.6a
Means with different superscripts within the same row are significantly different at P < 0.05. Value is represented as the mean ± standard deviation (n=2).
24
Modulus G', G" (Pa)
1.0E+05
A
control salt 1.0% salt 1.7% salt 2.0% salt 2.4%
1.0E+04
1.0E+03 0.1
Modulus G', G" (Pa)
1.0E+05
1
Frequency (Hz)
10
control salt 1.0% salt 1.7% salt 2.0% salt 2.4 %
B
1.0E+04
1.0E+03 0.01%
0.10%
1.00%
10.00%
Strain 1.0
C
tan δ
0.8 0.6
control salt 1.0% salt 1.7% salt 2.0% salt 2.4%
0.4 0.2 0.0 0.1
1
Frequency (Hz)
10
Fig 1. Storage modulus G’ (solid symbols), and loss modulus G” (open symbols) of the doughs prepared from wheat flour, (A) as a function of frequency at a strain of 0.1%, (B) as a function of strain at a frequency of 1Hz, and (C) tan δ as a function of frequency at a strain of 0.1%.
25
Fig. 2. Zeta potential values of gluten isolated from hard wheat flour doughs with different salt content in DI water with the solid content of 0.1%.
26
Fig. 3. Gluten secondary structures deconvolved from amide band I. Full assignment is given as Extended chains (1600-1615 cm-1), β-Sheet (1624-1640, 1681 cm-1), Random coil (1640-1650 cm-1), α-helix (1650-1660 cm-1), β-turn (1660-1670, 1694 cm-1).
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Highlights:
Doughs were prepared with different levels of salt (0 - 2.4%, fwb).
Doughs with higher levels of salt exhibited greater strength and viscosity.
Salt decreased free SH content but increased β-sheet structure of gluten.
Salt enhanced the macromolecular aggregation of gluten proteins.
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