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Bran-induced changes in water structure and gluten conformation in model gluten dough studied by Fourier transform infrared spectroscopy
Food Hydrocolloids 31 (2013) 146e155
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Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd
Bran-induced changes in water structure and gluten conformation in model gluten dough studied by Fourier transform infrared spectroscopy Jayne E. Bock, Srinivasan Damodaran* Department of Food Science, University of Wisconsin-Madison, Madison, WI 53706, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 August 2012 Accepted 18 October 2012
The impact of bran addition on the state of water and gluten secondary structure in gluten dough was studied using Fourier transform infrared spectroscopy to understand the underlying physical mechanism by which bran impacts dough properties. Comparison of the OH stretch band of water in gluten dough with that of H2OeD2O mixture having the same water content revealed formation of two distinct water populations in gluten dough corresponding to IR absorption frequencies at 3580 cm1 and 3180 cm1. The band intensity at 3180 cm1, which is related to water bound to gluten matrix, decreased with increase of moisture content of the dough. Addition of bran to gluten dough caused redistribution of the bound water in the gluten-bran dough system. This water redistribution affected the secondary structure of gluten in the dough as evidenced from changes in the second-derivative spectrum in the amide I region. In the hydrated state, b-turn (in the form of b-spiral) was the major secondary structure (w60%) in gluten. Addition of bran to gluten dough caused conversion of b-spirals into b-sheet and random structures. However, the extent of this conversion in the presence of bran was inversely related to the moisture content of the dough. This study revealed that when bran is added to gluten dough, water redistribution promotes partial dehydration of gluten and collapse of b-spirals into intermolecular bsheet structures; this trans-conformation might be physical reasons for the poor quality of bread containing added bran. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Dough Gluten Bran ATR-FTIR spectroscopy Gluten secondary structure State of water in dough Effect of bran addition on gluten structure in dough
1. Introduction Consumers regard taste, texture, smell, and appearance as sensory barriers to whole grain consumption (Bakke & Vickers, 2007). Literature corroborates some of these assertions with reports of deterioration of quality characteristics such as loaf volume and crumb structure in products containing added bran (Gan, Ellis, Vaughan, & Galliard, 1989; Lai, Hoseney, & Davis, 1989a, 1989b). The basis for negative effects from bran on bread quality needs to be well understood in order to develop successful and efficient processing and ingredient strategies to mitigate such quality defects. There are two schools of thought regarding the basic mechanism by which bran affects dough and bread properties. The first implicates competitive water binding by bran as the major factor affecting the loaf volume and internal crumb structure of wholewheat bread (Lai et al., 1989a). Changes in water sorption isotherms of flour with and without added bran provide indirect support for
* Corresponding author. Tel.: þ1 608 263 2012; fax: þ1 608 262 6872. E-mail address: [email protected] (S. Damodaran). 0268-005X/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodhyd.2012.10.014
competitive water uptake by bran (Cadden, 1988; Cherian & Chinachoti, 1996). In addition, pre-hydration of bran has been reported to improve the quality of wheat bread (Nelles, Randall, & Taylor, 1998; Salmenkallio-Marttila, Katina, & Autio, 2001). The second hypothesis contends that bran affects loaf volume and internal crumb structure by physically disrupting the gas cells and the gluten network as evidenced from scanning electron micrographs (SEM) (Gan et al., 1989). It is quite possible, however, that the freeze fracture step used during sample preparation for SEM may cause damage to the gluten matrix and thus create artifacts in SEM. It is plausible that both these mechanisms may be intertwined. That is, competitive water binding by bran may cause redistribution of moisture in wheat dough; this may result in partial dehydration of gluten, which may in turn cause conformational changes in gluten and adversely affect its viscoelastic properties. These events may collectively promote partial collapse of the gluten network. Recently, Fourier transform infrared (FTIR) spectroscopy has been used to probe gluten secondary structure in dough under various conditions (Li, Dobraszczyk, Dias, & Gil, 2006; Mejia, Mauer, & Hamaker, 2007; Robertson, Gregorski, & Cao, 2006; Wellner et al., 2005). However, there is no comprehensive study in the literature
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linking alterations in the structural and thermodynamic state of water caused by bran addition to dough and impact of such changes on gluten conformation in dough. FTIR spectroscopy is an ideal technique for simultaneously probing changes in the state of water in dough by monitoring changes in the OH stretch band at 3000e3800 cm1 and alterations in gluten structure by monitoring changes in the amide I band at 1600e1700 cm1. It has been shown that addition of various polar and nonpolar solutes and/or water state transitions during freezee thaw cycles cause significant changes in the OH stretch band of water (Dashnau, Nucci, Sharp, & Vanderkooi, 2006; Scott, Nucci, & Vanderkooi, 2008; Sharp, Madan, Manas, & Vanderkooi, 2001; Zelent, Bryan, Sharp, & Vanderkooi, 2009; Zelent & Vanderkooi, 2009). These studies have shown that changes in relative hydrogen bonding properties and water’s mobility as the conditions of the system are altered can be deduced from shifts in the OH stretch absorption band. This approach can be potentially used to understand changes in the state of water and secondary structure of gluten (from amide I band) in dough as a consequence of bran addition and relate such changes to the impact of bran on bread quality. Thus, the aim of this study was to study the impact of bran addition on the state of water and gluten secondary structure in model gluten dough using attenuated total reflectance (ATR) FTIR spectroscopy. 2. Materials and methods 2.1. Materials Commercial wheat gluten (Provim ESP vital wheat gluten) was obtained from Archer Daniels Midland Milling Company (Shawnee Mission, KS, USA). Gluten composition was 9.0% moisture, 78% protein, and 0.8% ash. Hard red winter wheat bran (particle size > 1400 mm) was obtained from the Kansas State University Hal Ross flour mill (Manhattan, KS, USA). D2O was purchased from SigmaeAldrich Co. (St. Louis, MO, USA). 2.2. Methodology 2.2.1. Experimental design The compositions of model gluten and gluten-bran dough investigated in this study are given in Table 1. The total moisture content of the dough was varied from 35% to 50%. The total Table 1 Model gluten and gluten-bran dough compositions. Moisture (%)
Gluten (%)
Bran (%)
35
65.0 63.4 61.8 60.2 58.5 60.0 58.5 57.0 55.5 54.0 55.0 53.6 52.2 50.8 49.5 50.0 48.8 47.5 46.2 45.0
0.0 1.6 3.2 4.8 6.5 0.0 1.5 3.0 4.5 6.0 0.0 1.4 2.8 4.2 5.5 0.0 1.2 2.5 3.8 5.0
40
45
50
147
moisture content included the moisture content of gluten (9% w/w) plus the added water. The moisture content of gluten was determined as per the American Association of Cereal Chemists (AACC) Method 44-15.02 (Air Oven Methods) (AACC International, 1999). Keeping the total solids content (i.e., gluten þ bran) constant, at each moisture content the bran content was increased from 0 to 10% of the total solids at 2.5% increments with corresponding adjustments in gluten content. 2.2.2. Gluten dough preparation Gluten and gluten þ bran doughs of the composition shown in Table 1 were prepared by mixing gluten and bran with distilled water until the consistency of the dough peaked in a Brabender Intelli-torque Plasti-corder farinograph (C.W. Brabender Instruments, Inc., South Hackensack, NJ, USA) using a 50 g mixing bowl and WinMix v. 2.0 software. A short premix was executed to distribute the bran prior to water addition. Doughs were collected at the end of mixing and ATR-FTIR analysis was performed within 30e45 min. 2.2.3. ATR-FTIR spectroscopy ATR-FTIR spectra were collected using a Bruker Equinox 55/S FTIR/NIR spectrophotometer (Bruker Optics, Inc., Billerica, MA, USA) using a horizontal multi-reflectance ZnSe crystal accessory. The instrument housed a deuterated tri-glycine sulfate (DTGS) detector and a potassium bromide (KBr) beam splitter. Spectra were collected in the 4000e400 cm1 infrared spectral range at room temperature with a N2 purge to remove interference from atmospheric moisture. A clamp was used to ensure dough contact with the crystal and consistent sample thickness. Each spectrum was an average of 32 scans at 4 cm1 resolution. A minimum of 3 spectra was used for spectral analysis. 2.2.4. ATR-FTIR reference spectra of water Reference FTIR spectra of H2O, corresponding to 35, 40, 45, and 50% water content in the dough, were obtained by mixing H2O with D2O and scanning these mixtures in the 4000e400 cm1 spectral range. These spectra were used as reference spectra to detect shifts in OH stretch absorption band of H2O (3000e3800 cm1) and/or changes in absorption intensity of the OH stretch band in gluten and gluten þ bran doughs at corresponding water content. This was accomplished by generating ATR-FTIR difference spectrum of dough samples by digitally subtracting the H2OeD2O reference spectrum from dough spectrum having the same H2O content. The H2OeD2O reference spectra were also used to digitally subtract contribution of H2O to absorption in the amide I region (1600e1700 cm1) to obtain information on gluten secondary structure in dough. D2O showed no absorption either in the 3000e 3800 cm1 or in the amide I regions. 2.2.5. Spectral analysis of gluten dough Spectral analysis of gluten dough was conducted using OPUS software v. 6.5. All spectra were vector normalized to correct for any difference in IR sample penetration depth caused by any density difference among samples. In experiments involving bran addition, the ATR-FTIR spectrum of bran powder was subtracted from the gluten dough spectrum. The vector-normalized spectra of dough samples were offset-corrected followed by digital subtraction of vector normalized H2OeD2O reference spectra of same water content. The difference spectra thus obtained in the 3000e 3800 cm1 regions were analyzed for changes in the state of water structure in dough compared to the reference state in the H2OeD2O mixture using the approach used elsewhere (Jain, Varshney, & Maitra, 1989; Sutandar, Ahn, & Franses, 1994; Zelent et al., 2009).
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To elucidate structural changes in gluten under different treatment conditions in dough, the difference spectrum in the amide I region (1600e1700 cm1) was second derivatized using a fivepoint SavitskyeGolay derivative function.
before sample introduction. Samples were weighed every 4 h until a constant weight was achieved (typically after 48 h) at which point equilibration was deemed to be complete. 3. Results
2.2.6. Gluten secondary structure estimation The quantitative estimation of gluten secondary structure in dough was determined from second-derivative spectra according to procedures described elsewhere (Dong, Huang, & Caughey, 1990; Dong, Caughey, Caughey, Bhat, & Coe, 1992; Kalnin & Venyaminov, 1990; Kong & Yu, 2007; Susi & Byler, 1983). According to this procedure, the secondary structure of a protein backbone is regarded as a linear sum of a-helix, b-sheet, b-turn, and aperiodic structures. The stretching vibration of C]O group of the peptide backbone in these structural elements has same molar absorptivity, but differ in absorption frequency in the amide I region (1600e 1700 cm1) (Kong & Yu, 2007). It is known that, in addition to the stretching vibration of C]O groups of the peptide backbone, the amino acid side chains of proteins also contribute to absorption in the amide I region (Venyaminov & Kalnin, 1990). However, the contribution of amino acid side chain groups to IR absorption of protein has been reported to be only w10% of the total integral intensity of the amide I band (Venyaminov & Kalnin, 1990). Since gluten is a heterogeneous mixture of several polypeptides, it is not possible to subtract the side chain contribution to the amide I band. Furthermore, since our larger objective was to elucidate net changes in secondary structure content in gluten as a function of moisture content and bran addition, the contribution from side chains was assumed to be a minor constant factor in the secondary structure estimates. The characteristic mean absorption frequencies of the secondary structural elements in proteins are listed in Table 2 (Dong et al., 1990; Kong & Yu, 2007). The secondary structure estimates were determined from the relative area of the peaks centered at these absorption bands (Dong et al., 1990; Kong & Yu, 2007). The secondary structure estimates obtained using this approach has been shown to correlate well with the crystallographic structure of several proteins (Dong et al., 1990; Kong & Yu, 2007; Vedantham, Sparks, Sane, Tzannis, & Przybycien, 2000). 2.2.7. Moisture sorption isotherm Moisture sorption isotherms of gluten and bran were studied as described elsewhere (Rao & Damodaran, 2004). Samples (w1.5 g) were brought to an initial dry state (24 h at 102 C) prior to placement in an environmentally controlled glove box. The glove box was housed in a 5 cm thick styrofoam insulation box with temperature controlled by a copper coil connected to a circulating water bath set to 25 C 0.5 C. Equilibrium relative humidity (ERH) within the glove box was controlled by saturated salt solutions (Greenspan, 1977). The glove box was flushed with nitrogen Table 2 Mean absorption frequencies of various secondary structure elements in proteins.a Mean frequencies (cm1)
Secondary structure assignment
1624 1627 1632 1638 1642 1650 1656 1666 1672 1680 1688
b-sheet b-sheet b-sheet, extended chain b-sheet b-sheet
a
Taken from Dong et al. (1990).
unordered (random)
a-helix b-turn b-turn b-turn b-turn
3.1. H2OeD2O- reference spectra As shown in Fig. 1, the intensity of the OH stretch band located at 3000e3700 cm1 increased and that of OD stretch band located at 1900e2800 cm1 range decreased with increase of H2O concentration in H2OeD2O mixtures. In all these molecularly dispersed H2O in D2O solutions, the broad OH stretch band was centered at about 3400 cm1. In contrast, the OH stretch band of pure H2O, also shown in Fig. 1, exhibited a broad bi-modal peak positions ranging from 3500e3150 cm1. The broad spectrum of the OH stretch band in pure water is attributed to the presence of various hydrogenbonded water clusters in bulk water (Jain et al., 1989; Sutandar et al., 1994). Monomeric non-hydrogen-bonded water typically has OH stretch band centered at 3616 cm1; hydrogen-bonded dimers and small hydrogen-bonded clusters exhibit OH stretch band centered at 3536 cm1 and 3424 cm1, respectively, whereas extensively hydrogen-bonded-associated chains of water molecules exhibit OH stretch band at 3246 cm1 (Jain et al., 1989; Sutandar et al., 1994). In other words, as the proportion of extensively hydrogen-bonded water clusters or associated chains of water or water hydrogen-bonded to polymeric materials in a system increase, the OH stretch band shifts to lower frequencies. On the basis of these band assignments for various states of water, it appears that the molecularly dispersed H2O in H2OeD2O mixtures exist predominantly in small hydrogen-bonded cluster state (w3400 cm1). Conversely, it can be reasoned that if water disperses itself in an ideal manner in another mixture in which D2O has been replaced by another substance, e.g., gluten, the state of water should be same as in the H2OeD2O mixture, provided there is no specific interaction between H2O and gluten. However, if water interacts with gluten and as a consequence if the state of water changes in the gluteneH2O mixture compared to the reference state in the H2OeD2O mixture, it should reveal itself from a shift in the OH stretch absorption frequency. 3.2. Gluten and bran ATR-FTIR spectra The ATR-FTIR spectrum of gluten powder exhibited a band at 2800e3600 cm1 (Fig. 2), which is in the OH stretch region of water. This band might be attributed mostly to the moisture in the gluten sample, which was about 9% (w/w). Since this moisture content was included in the total moisture content of the gluten dough samples, there was no need to subtract the OH stretch absorption band of gluten sample from the OH stretch absorption band of gluten dough. It is also likely that there might be a small contribution from the NeH stretching (amide A) and CeH stretching vibrations of gluten as well since they weakly absorb in this region (Jackson & Mantsch, 1995). However, since these contributions are very small compared to the OH stretch absorption band of water in gluten dough, no correction was deemed necessary. Furthermore, since we are more interested in shifts in OH stretch absorption frequency rather than absolute intensity, small variations in intensity will not affect the focus of this study. The gluten sample also showed a strong absorption band in the amide I (1600e1700 cm1) region. The dry bran sample showed no strong absorption neither in the OH stretch region of water nor in the amide I region. Nevertheless, the bran spectrum was subtracted from the spectra of gluten þ bran dough samples before spectral analysis.
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Fig. 1. ATR-FTIR spectra of H2OeD2O mixtures. The legend in the figure indicates the percentage of H2O in the mixture.
3.3. State of water in gluten dough The OH stretch absorption spectrum of gluten dough containing 35% moisture (w/w) is shown along with the 35% H2O in D2O reference spectrum in Fig. 3. It should be noted that the OH stretch band of the 35% gluten dough was qualitatively different from that of the 35% H2OeD2O reference spectrum. While the reference spectrum exhibited an apparent single symmetrical band centered at 3393 cm1, the band in the gluten dough spectrum was broader and red-shifted to lower frequency. This suggests that the energy state(s) of water is dramatically altered in gluten dough compared to the reference state(s) in H2OeD2O mixture as a result of interaction with the gluten network. To characterize the newly formed sub-populations of water in the gluten dough, the H2OeD2O reference spectrum was subtracted
Fig. 2. ATR-FTIR spectra of gluten (red line) and bran (blue line) samples. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
from the gluten dough spectrum. The resultant difference spectrum, shown in Fig. 3, exhibited two positive bands, one centered at 3580 cm1 and the other centered at 3180 cm1, a shoulder at 3080 cm1, and a trough centered at 3435 cm1. The area of the trough essentially reflects the fraction of water (small hydrogenbonded cluster states) in the reference spectrum that has been transformed into other energy (structural) states in gluten dough. Previously, it has been reported that while monomeric nonhydrogen-bonded water has OH stretch vibration at w3600 cm1, water entrapped in reverse micelles exists as hydrogen-bondedassociated chains with OH stretch vibration at 3290 cm1 (Jain et al., 1989), and bound water in epoxy resins exhibits OH stretch at 3129 cm1 (Cotugno, larobina, Mensitieri, Musto, & Ragosta, 2001; Liu, Wu, Ding, Chen, & Li, 2002). It also has been reported
Fig. 3. ATR-FTIR spectra of water in the OH stretch region in 35% H2O in D2O (green line) and in gluten dough at 35% moisture content (blue line). The red line is the difference spectrum of water obtained by subtracting the green line from the blue line. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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that transformation of water from a liquid state at 278 K to ice at 200 K caused the OH stretch band to shift from 3400 cm1 to about 3290 cm1 (Zelent et al., 2009). Typically, water strongly hydrogenbonded to functional groups in a polymer network requires less energy (low frequency) for OH stretch vibration. The stronger the hydrogen bond strength, the greater is the shift of OH stretch absorption to lower frequency (Liu et al., 2002). Based on those reports, the positive peak in the difference spectrum at 3580 cm1 (Fig. 3) can be assigned to OH stretch vibration of monomeric nonhydrogen-bonded water molecules and some hydrogen-bonded water dimers. The peak at 3180 cm1 and the shoulder at 3080 cm1 can be assigned to water populations hydrogen-bonded to gluten network. Thus, there appears to be two classes of bound water in gluten dough: a relatively weakly bound state with OH stretch at 3180 cm1 and the other relatively strongly bound fraction with OH stretch at 3080 cm1. The absence of OH stretch band at w3290 cm1, which is found for water entrapped in reverse micelles (Jain et al., 1989) and for ice at 200 K (Zelent et al., 2009), suggests that water trapped in the interstices of the gluten network (i.e., capillary water) apparently does not exist as hydrogenbonded-associated chains, but is present mainly as small hydrogen-bonded clusters with OH stretch absorption centered at w3400 cm1. Fig. 4 shows the ATR-FTIR difference spectra in the OH stretch region of gluten dough at 35, 40, 45, and 50% moisture levels. It should be noted that the difference spectrum displayed positive bands at w3580 cm1 and at 3180 e 3145 cm1, and a shoulder at 3080 cm1. Although the intensity of the 3580 cm1 band varied slightly with dough moisture content, the peak position of the band did not change, indicating that the energy state of this population of water (which is mainly monomeric and possibly some hydrogenbonded water dimers) was not affected by the moisture content of dough. However, both the intensity and the peak position of the 3180 cm1 band changed with moisture content of the dough: The peak intensity decreased with increase of moisture content and the peak position shifted from 3180 cm1 at 35% moisture content to 3145 cm1 at 50% moisture content (Fig. 4). Even after normalizing the spectra for differences in the gluten content, the intensity of the band in the 3180e3145 cm1 region was significantly different at various moisture contents (data not shown). This indicates that the interaction strength of water associated with the gluten network increases with increase of dough moisture content (which is reflected in the shift in absorption band to lower frequency). The decrease in the intensity of the absorption band might be due to a decrease in the molar absorptivity of bound water molecules as the absorption frequency is shifted to lower values in this range.
Taken together, the results shown in Figs. 3 and 4 reveal that water undergoes a significant structural transformation in gluten dough compared to its reference state in H2OeD2O mixtures. This transformation involves a significant amount of water becoming hydrogen bonded to the gluten polymer, and a significant amount of water becoming released from small hydrogen-bonded cluster states to the monomeric non-hydrogen-bonded state. 3.4. State of water in gluten-bran dough The effect of bran addition on the difference spectrum of the OH stretch band of water in gluten dough at various moisture contents is shown in Fig. 5. As in the case of gluten only dough, the difference spectrum of gluten þ bran dough also exhibited OH stretch bands at 3580 cm1 and 3180 cm1 and a shoulder at 3080 cm1. However, at any given moisture content, the intensities of these bands were dependent on the bran content. In low moisture dough (35%) bran addition caused an increase in the amount of water hydrogenbonded to the polymer matrix as evidenced from an increase in the intensity of the OH stretch band at 3180 cm1 in the difference spectrum without any spectral shift (Fig. 5A). At higher moisture content (40 and 45%), however, bran addition decreased the amount of water hydrogen-bonded to the polymer matrix as evidenced from a decrease in the intensity of the OH stretch band at 3160 cm1 and an apparent shift of absorption frequency by about þ20 cm1 with increase of bran content (Fig. 5B, C). At 50% moisture content, bran addition essentially had no effect on water hydrogen-bonded to the polymer matrix as evidenced from no change in the OH stretch absorption band at w3180 cm1 (Fig. 5D). These bran-induced changes in the OH stretch band might be fundamentally due to redistribution of water in the bulk versus bound state in the gluten matrix. This redistribution may involve partial dehydration of the gluten matrix as a consequence of partitioning of water between bran and gluten. The extent of structural changes in gluten at various moisture contents (as discussed below in Sections 3.7 And 3.8) also might impact bran-induced water redistribution in the dough. In other words, the increase in the intensity of the peak at 3180 cm1 at 35% moisture content and the decrease in the intensity of the peak at 40% and 45% moisture content with the addition of bran might be a result of complex interplay of changes in gluten structure and bran-induced moisture redistribution in the dough at various moisture contents. 3.5. Gluten and bran moisture sorption isotherms The moisture sorption experiments were conducted in an attempt to further identify any competition between bran and gluten for water (Fig. 6). The isotherms indicated that bran bound more waters than gluten at any given water activity and, at any given water content the water activity in bran was lesser than in gluten. The data apparently confirms that addition of bran to gluten dough might cause redistribution of water in the system. 3.6. Secondary structure of gluten in unhydrated state
Fig. 4. ATR-FTIR difference spectra of water in the OH stretch region in gluten dough at 35e50% moisture content.
The secondary structure changes in gluten as a function of moisture content of dough and bran addition to dough was studied by second-derivative ATR-FTIR spectroscopy as described elsewhere for other proteins and polypeptides (Byler & Susi, 1986; Dong et al., 1992; Dong et al., 1990; Kalnin & Venyaminov, 1990; Kong & Yu, 2007; Kendrick, Dong, Allison, Manning, & Carpenter, 1996; Susi & Byler, 1983). Several studies have shown that the second-derivative FTIR band frequencies centered at 1624, 1627, 1632, 1638, and 1642 cm1 belonged to b-sheet structure and the band frequencies centered at 1666, 1672, 1680, and 1688 cm1
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Fig. 5. Effect of bran addition on the difference spectrum of water in the OH stretch region in gluten dough at (A) 35%, (B) 40%, (C) 45%, and (D) 50% moisture content.
Fig. 6. Moisture sorption isotherms of bran (-) and gluten (C). The equations shown are the polynomial curve-fitting equations of the data.
belonged to b-turns (Dong et al., 1990; Kong & Yu, 2007; Vedantham et al., 2000). On the other hand, the unordered (random) and a-helix structures exhibited only one frequency each centered at 1650 and 1656 cm1, respectively (Dong et al., 1990). The second-derivative ATR-FTIR spectrum of gluten powder (9% moisture content) in the amide I region (1600e1700 cm1) is shown in Fig. 7. This spectrum was generated after subtracting the ATR-FTIR spectrum of 10% H2O in D2O from the ATR-FTIR of gluten powder (w9% moisture). In the spectral region 1620e 1690 cm1 where C]O stretching vibrations of polypeptide chains occur, the second-derivative FTIR spectrum of gluten exhibited bands at 1622, 1627, 1641, 1652, 1663, 1668, and 1680 cm1, which are in agreement with bands identified for various secondary structural elements in proteins. Based on the band frequency assignments in the literature (Dong et al., 1990; Kong & Yu, 2007; Vedantham et al., 2000), the secondary structure estimates of b-sheet, random (unordered), a-helix, and b-turn contents were determined from the relative band areas in the spectral regions 1620e1644 cm1, 1644e1652 cm1, 1652e 1660 cm1, and 1660e1685 cm1, respectively. Since these bands were extensively overlapped, no attempt was made to deconvolute these bands. A similar approach has been used by Dong et al. (1990). The secondary structure of gluten in the unhydrated state is estimated as 39% b-sheet, 30% random, 17% a-helix, and 14%
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Fig. 7. Second-derivative ATR-FTIR spectrum of gluten powder (9% moisture) in the amide I region. The spectral bands in the regions between the dotted lines correspond to the secondary structures as indicated. The secondary structure estimates were calculated from the fractional area of the bands. No attempts were made to deconvolute the bands.
b-turn. These estimates are accurate within 5% (Dong et al., 1990). Thus, it appears that at low moisture level (9%) b-sheet and random structures are the major secondary structure elements in gluten. 3.7. Secondary structure of gluten in dough The second-derivative ATR-FTIR spectrum of gluten dough (derived from H2OeD2O blank-corrected ATR-FTIR difference spectrum of gluten dough) in the amide I region (1600e1700 cm1) at 35%, 40%, 45%, and 50% moisture content is shown in Fig. 8A. As in the case of unhydrated gluten, the second-derivative FTIR spectrum of gluten dough exhibited bands at 1622, 1629, 1649, 1656, 1662, 1670, and 1680 cm1, which are within 2 cm1 of the peak positions found in the unhydrated gluten (Fig. 7), but the relative areas of these bands were different compared to those in the unhydrated state. The b-sheet, random, a-helix, and b-turn secondary structure contents of gluten in dough, estimated from relative band areas in the spectral regions 1620e1644 cm1, 1644e 1652 cm1, 1652e1660 cm1, and 1660e1685 cm1, are plotted in Fig. 8B as a function of moisture content of gluten dough. The 9% moisture content refers to gluten powder. The secondary structure of gluten changed dramatically as the moisture content was increased from 9% to 50%: The b-turn content increased from about 14% to about 65% as the moisture content was increased from 9% to 40%. This dramatic increase in b-turn content was at the expense of a decrease in the amount of both b-sheet and random structures as the gluten was hydrated. These results indicate that b-turn is the preferred secondary structure of gluten in the hydrated state. 3.8. Effect of bran addition on secondary structure of gluten in dough
Fig. 8. (A) Effect of moisture content of dough on the second-derivative spectrum of gluten in the amide I region. (B) Effect of moisture content on a-helix ( ), b-sheet ( ), b-turn (:), and unordered ( ) secondary structure content of gluten in gluten dough.
content of the dough. At the 35% moisture level, addition of 10% bran decreased the b-turn content from about 54% to 33% while increasing the b-sheet content from 18% to about 32% and the random structure content from 18% to 25% (Fig. 10A). A similar trend also was observed at 40% moisture level (Fig. 10B). However, at 50% moisture content, the extent of inter-conversion of b-turn into b-sheet and random structures was minimal: The b-turn content decreased from 54% to 47% while the b-sheet content increased from 20% to 26% with no change in the random and ahelix contents (Fig. 10D). The results indicate that although b-turn is the preferred structure of gluten in the fully hydrated state, addition of bran to dough forces gluten to adopt b-sheet and random configurations, potentially as a result of water redistribution and partial dehydration of gluten by bran. 4. Discussion
The effect of bran addition on the second-derivative ATR-FTIR spectrum of gluten in gluten dough at various moisture and bran contents is shown in Fig. 9 and the secondary structure estimates are given in Fig. 10. At all moisture contents, the b-turn content decreased, the b-sheet and random structures increased, and the ahelix content remained unchanged as the bran content was increased from 0 to 10%. However, the bran-induced changes in secondary structures content were dependent on the moisture
The OH stretch region (w3500e3000 cm1) in FTIR spectra has not been closely investigated in the past as a source of information for food systems in general, and wheat dough in particular. In the present investigation, we have used ATR-FTIR spectra of H2OeD2O mixtures as reference state of H2O to elucidate changes in the state of water in gluten dough at corresponding H2O content. It is well established that hydrogen bonding broadens the OH stretch band
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Fig. 9. Effect of bran addition at various moisture contents on the second-derivative spectrum of gluten in dough: (A) 35%, (B) 40%, (C) 45%, and (D) 50% moisture content.
of water (Sutandar et al., 1994; Walrafen, 1972). The broadening of the OH stretch band toward lower absorption frequency in gluten dough compared to the reference H2OeD2O spectrum at corresponding water content (Fig. 3) reveals that there is a significant change in the structural (energy) state of water in gluten dough. The difference spectra clearly indicates that, in addition to the normal state with OH stretch absorption frequency centered at 3400 cm1, interaction of gluten with water creates two new subpopulations of water, one strongly hydrogen-bonded to gluten with absorption frequencies at 3180 cm1 and 3080 cm1 and the other non-hydrogen-bonded monomeric water population with absorption frequency at 3580 cm1 (Fig. 3). It has been reported that when water at 4 C is converted into ice at 15 C, the OH stretch band of water shifts from 3400 cm1 to only about 3306 cm1; further decrease in the temperature of ice to 73 C shifts the absorption frequency to 3290 cm1 (Zelent et al., 2009). Since the stronger the hydrogen bonding interaction, the greater is the extent of shift in OH stretch band toward lower frequency (Liu et al., 2002; Sutandar
et al., 1994), the absorption frequencies 3180 and 3080 cm1 of bound water in gluten dough implicitly suggests that the interaction of water with hydrogen bonding groups in gluten is much stronger than waterewater hydrogen bonding interaction in ice at 73 C. The addition of bran to gluten dough changed the amount of water bound to the gluten matrix (Fig. 5AeD). Although quantitative reasons for these changes are not clear, the results clearly indicate that bran affects glutenewater interaction in the 35e45% moisture content range and redistributes the released water into other energy states as evidenced from a decrease in the intensity of the band at 3180 cm1 as well as the negative trough at 3400 cm1 (Fig. 5AeD). The effect of bran, however, becomes muted at higher (50%) moisture content. Previously, it has been observed that the loaf volume of wheat dough with added bran could be improved with the addition of extra formulation water (Lai et al., 1989a). Presoaking of the bran in water prior to mixing with flour also resulted in almost complete restoration of loaf volume (Lai et al., 1989a). The
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Fig. 10. Effect of bran addition on the secondary structure contents of gluten in dough at (A) 35%, (B), 40%, (C) 45%, and (D) 50% moisture content.
results of the present study seem to provide a mechanistic explanation of those empirical observations: That is, at low moisture content (35e45%) water redistribution and partial dehydration of gluten network caused by bran might affect the development of loaf volume and this affect of bran can be nullified at higher moisture (50%) content. If bran causes partial dehydration of gluten and redistribution of water in dough, then it is reasonable to expect conformational changes in gluten as a consequence. It should be pointed out that bsheet (39%) and random (30%) conformations are the major secondary structures in the unhydrated (9% moisture) gluten (Fig. 7). However, these secondary structure elements are transformed into b-turn when gluten is hydrated as in dough (Fig. 8B). If we assume that this structural transformation is reversible, then when gluten in dough is partially dehydrated by bran addition, the b-turn should revert back to b-sheet and random configurations; the extent of this reversal must be a function of the amount of bran in the system. This indeed is found to be the case as seen in Fig. 10AeD. However, in 50% moisture dough, in which the influence of bran on the water bound to gluten is minimal (Fig. 5D), the transformation of b-turn back to b-sheet is also minimal because of the excess amount of water available in the system to adequately hydrate gluten. Thus, there seems to be a direct linkage between trans-conformational changes in gluten and changes in the extent of glutenewater interaction in dough as affected by bran addition. The abundant b-turn secondary structure motif found in gluten dough may not be similar to those b-turns associated with antiparallel and parallel pleated b-sheets found in globular proteins. The b-turn structures in gluten dough might be related to the bspiral domains in glutenin polypeptides (Wellner et al., 2005), which contain hexa- and nona- and tri-peptide repeats of amino acid sequences PGQGQQ, GYYPTSLQQ, and GQQ (Belton, 1999).
These b-spiral structures can be regarded as consecutive b-turns. It has been reported that short a-helix regions flank these b-spiral domains (Shewry, Halford, Belton, & Tatham, 2002), which is consistent with 10e12% a-helix estimated in this study. The b-spiral structures are implicated as one of the structural elements responsible for the viscoelasticity of dough (Belton, 1999; Wellner et al., 2005). The greater the amount of this structural element in dough, the greater would be the ability of dough to trap gas bubbles and the greater would be the bread loaf volume. Conversely, anything that transforms the b-spiral structure into intermolecular b-sheet structure would adversely impact the quality of breadcrumb. Based on the results presented in this study, we hypothesize that one of the reasons for the adverse effect of bran addition on
Fig. 11. Schematic description of proposed hypothesis of bran-induced conformational changes in gluten in dough leading to collapse of the viscoelasticity of gluten dough.
J.E. Bock, S. Damodaran / Food Hydrocolloids 31 (2013) 146e155
loaf volume of bread (Lai et al., 1989a) is the collapse of the b-spiral structure to intermolecular b-sheets and this transformation is caused by redistribution of water in dough, which might involve partial dehydration of gluten network. This is schematically shown in Fig. 11. The loss of the elastic b-spiral structures and formation of inelastic intermolecular b-sheet aggregates in the gluten network might decrease the viscoelasticity of the dough and thus affect the loaf volume during bread making. It is also likely that in real wheat dough, interaction of other flour components, such as starch, might also modify the secondary structure of gluten. These aspects remain to be studied in detail. 5. Conclusions The ATR-FTIR difference spectroscopy is shown to be a valuable technique for studying changes in the energy state of water and secondary structure of gluten simultaneously in dough systems. The energy state of water in gluten dough is altered compared to its reference state in H2OeD2O mixtures at the same H2O content. In addition to hydrogen bonding of water to the gluten network, glutenewater interaction causes a net increase in monomeric water population in gluten dough. The moisture content as well as bran addition affects the amount of both bound and monomeric water populations in gluten dough. The water redistribution in gluten dough by bran addition alters the conformation of gluten from bspiral (consecutive b-turns) structure to b-sheet structure. This major structural change in gluten might be the physical basis for poor quality of bread made with added bran. However, the extent of bran-induced conversion of the b-spiral structure to intermolecular b-sheet structure is minimized as the moisture content of the dough is increased from 35% to 50%. This implicitly suggests that the adverse effects of bran on bread quality could be overcome by increasing the moisture content of dough. However, since wholewheat dough is a complex system consisting of bran, starch, and other phytochemicals, it is quite possible that these components may also directly or indirectly affect gluten structure as well as water distribution in the dough. Acknowledgments We thank Gary Girdaukas and the Analytical Instrumentation Center of the School of Pharmacy, University of Wisconsin-Madison, for support in obtaining the ATR-FTIR spectrophotometric data. This research was supported by a grant (#2009-35503-05181) from the National Institute for Food and Agriculture e Agriculture and Food Research Initiative (NIFA-AFRI). References AACC International. (1999). Method 44e15.02. Moisturedair oven methods. Reapproved 03.11.99 Approved methods of analysis (11th ed.). St. Paul, MN, USA: AACC International. http://dx.doi.org/10.1094/AACCIntMethod-44-15.02. Bakke, A., & Vickers, Z. (2007). Consumer liking of refined and whole wheat breads. Journal of Food Science, 72, S473eS480. Belton, P. S. (1999). On the elasticity of wheat gluten. Journal of Cereal Science, 29, 103e107. Byler, D. M., & Susi, H. (1986). Examination of the secondary structure of proteins by deconvoluted FTIR spectra. Biopolymers, 25, 469e487. Cadden, A.-M. (1988). Moisture sorption characteristics of several food fibers. Journal of Food Science, 53, 1150e1155. Cherian, G., & Chinachoti, P. (1996). 2H and 17O nuclear magnetic resonance study of water in gluten in the glassy and rubbery state. Cereal Chemistry, 73, 618e624. Cotugno, S., larobina, D., Mensitieri, G., Musto, P., & Ragosta, G. (2001). A novel spectroscopic approach to investigate transport processes in polymers: the case of water-epoxy system. Polymer, 42, 6431e6438. Dashnau, J. L., Nucci, N. V., Sharp, K. A., & Vanderkooi, J. M. (2006). Hydrogen bonding and the cryoprotective properties of glycerol/water mixtures. Journal of Physical Chemistry B, 110, 13670e13677. Dong, A., Caughey, B., Caughey, W. S., Bhat, K. S., & Coe, J. E. (1992). Secondary structure of the pentraxin female protein in water determined by infrared
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spectroscopy: effects of calcium and phosphorylcholine. Biochemistry, 31, 9364e 9370. Dong, A., Huang, P., & Caughey, W. S. (1990). Protein secondary structures in water from second-derivative amide I infrared spectra. Biochemistry, 29, 3303e3308. Gan, Z., Ellis, P. R., Vaughan, J. G., & Galliard, T. (1989). Some effects of nonendosperm components of wheat and of added gluten on wholemeal bread microstructure. Journal of Cereal Science, 10, 81e91. Greenspan, L. (1977). Humidity fixed points of binary saturated aqueous solutions. Journal of Research, National Bureau of Standards, 81A, 89e96. Jackson, M., & Mantsch, H. H. (1995). The use and misuse of FTIR spectroscopy in the determination of protein structure. Critical Reviews in Biochemistry and Molecular Biology, 30, 95e120. Jain, T. K., Varshney, M., & Maitra, A. (1989). Structural studies of aerosol OT reverse micellar aggregates by FTIR spectroscopy. Journal of Physical Chemistry, 93, 7409e7416. Kalnin, B. I. A., & Venyaminov, S. Y. (1990). Quantitative IR spectrometry of peptide compounds in water (H2O) solution. III. Estimation of the protein secondary structure. Biopolymers, 30, 1273e1280. Kendrick, B. S., Dong, A., Allison, S. D., Manning, M. C., & Carpenter, J. F. (1996). Quantitation of the area of overlap between second-derivative amide I infrared spectra to determine the structural similarity of a protein in different states. Journal of Pharmaceutical Sciences, 85, 155e158. Kong, J., & Yu, S. (2007). Mini-review: Fourier transform infrared spectroscopic analysis of protein secondary structures. Acta Biochmica et Biophysica Sinica, 39, 549e559. Lai, C. S., Hoseney, R. C., & Davis, A. B. (1989a). Effects of wheat bran in breadmaking. Cereal Chemistry, 66, 217e219. Lai, C. S., Hoseney, R. C., & Davis, A. B. (1989b). Functional effects of shorts in breadmaking. Cereal Chemistry, 66, 220e223. Li, W., Dobraszczyk, B. J., Dias, A., & Gil, A. M. (2006). Polymer conformation structure of wheat proteins and gluten subfractions revealed by ATR-FTIR. Cereal Chemistry, 83, 405e410. Liu, M., Wu, P., Ding, Y., Chen, G., & Li, S. (2002). Two-dimensional (2D) ATR-FTIR spectroscopic study on water diffusion in cured epoxy resins. Macromolecules, 35, 5500e5507. Mejia, C. D., Mauer, L. J., & Hamaker, B. R. (2007). Similarities and differences in secondary structure of viscoelastic polymers of maize a-zein and wheat gluten proteins. Journal of Cereal Science, 45, 353e359. Nelles, E. M., Randall, P. G., & Taylor, J. R. N. (1998). Improvement of brown bread quality by prehydration treatment and cultivar selection of bran. Cereal Chemistry, 75, 536e540. Rao, C. S., & Damodaran, S. (2004). Surface pressure dependence of phospholipase A2 activity in lipid monolayers is linked to interfacial water activity. Colloids and Surfaces B: Biointerfaces, 34, 197e204. Robertson, G. H., Gregorski, K. S., & Cao, T. K. (2006). Changes in secondary protein structures during mixing development of high absorption (90%) flour and water mixtures. Cereal Chemistry, 83, 136e142. Salmenkallio-Marttila, M., Katina, K., & Autio, K. (2001). Effects of bran fermentation on quality and microstructure of high-fiber wheat bread. Cereal Chemistry, 78, 429e435. Scott, J. N., Nucci, N. V., & Vanderkooi, J. M. (2008). Changes in water structure induced by the guanidinium cation and implications for protein denaturation. Journal of Physical Chemistry A, 112, 10939e10948. Sharp, K. A., Madan, B., Manas, E., & Vanderkooi, J. M. (2001). Water structure changes induced by hydrophobic and polar solutes revealed by simulations and infrared spectroscopy. Journal of Chemical Physics, 114, 1791e1796. 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 Royal Society, London Series B-Biological Science, 357, 133e142. Susi, H., & Byler, D. M. (1983). Protein structure by Fourier transform infrared spectroscopy: second derivative spectra. Biochemistry Biophysics Research Communication, 115, 391e397. Sutandar, P., Ahn, D. J., & Franses, E. I. (1994). FTIR-ATR analysis for microstructure and water uptake in poly(methyl methacrylate) spin cast and LangmuirBlodgett thin films. Macromolecules, 27, 7316e7328. Vedantham, G., Sparks, H. G., Sane, S. U., Tzannis, S., & Przybycien, T. M. (2000). A holistic approach for protein secondary structure estimation from infrared spectra in H2O solutions. Analytical Biochemistry, 285, 33e49. Venyaminov, S. Y., & Kalnin, N. N. (1990). Quantitative IR spectrophotometry of peptide compounds in water (H2O) solutions. I. Spectral parameters of amino acid residue absorption bands. Biopolymers, 30, 1243e1257. Walrafen, G. E. (1972). Water, a comprehensive treatise. In F. Franks (Ed.). The physics and physical chemistry of water, Vol. 1 (pp. 202). New York: Plenum Press. Wellner, N., Mills, E. N. C., Brownsey, G., Wilson, R. H., Brown, N., Freeman, J., et al. (2005). Changes in protein secondary structure during gluten deformation studied by dynamic Fourier transform infrared spectroscopy. Biomacromolecules, 6, 255e261. Zelent, B., Bryan, M. A., Sharp, K. A., & Vanderkooi, J. M. (2009). Influence of surface groups of proteins on water studied by freezing/thawing hysteresis and infrared spectroscopy. Biophysical Chemistry, 141, 222e230. Zelent, B., & Vanderkooi, J. M. (2009). Infrared spectroscopy used to study ice formation: the effect of trehalose, maltose, and glucose on melting. Analytical Biochemistry, 390, 215e217.
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Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd
Bran-induced changes in water structure and gluten conformation in model gluten dough studied by Fourier transform infrared spectroscopy Jayne E. Bock, Srinivasan Damodaran* Department of Food Science, University of Wisconsin-Madison, Madison, WI 53706, USA
a r t i c l e i n f o
a b s t r a c t
Article history: Received 23 August 2012 Accepted 18 October 2012
The impact of bran addition on the state of water and gluten secondary structure in gluten dough was studied using Fourier transform infrared spectroscopy to understand the underlying physical mechanism by which bran impacts dough properties. Comparison of the OH stretch band of water in gluten dough with that of H2OeD2O mixture having the same water content revealed formation of two distinct water populations in gluten dough corresponding to IR absorption frequencies at 3580 cm1 and 3180 cm1. The band intensity at 3180 cm1, which is related to water bound to gluten matrix, decreased with increase of moisture content of the dough. Addition of bran to gluten dough caused redistribution of the bound water in the gluten-bran dough system. This water redistribution affected the secondary structure of gluten in the dough as evidenced from changes in the second-derivative spectrum in the amide I region. In the hydrated state, b-turn (in the form of b-spiral) was the major secondary structure (w60%) in gluten. Addition of bran to gluten dough caused conversion of b-spirals into b-sheet and random structures. However, the extent of this conversion in the presence of bran was inversely related to the moisture content of the dough. This study revealed that when bran is added to gluten dough, water redistribution promotes partial dehydration of gluten and collapse of b-spirals into intermolecular bsheet structures; this trans-conformation might be physical reasons for the poor quality of bread containing added bran. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Dough Gluten Bran ATR-FTIR spectroscopy Gluten secondary structure State of water in dough Effect of bran addition on gluten structure in dough
1. Introduction Consumers regard taste, texture, smell, and appearance as sensory barriers to whole grain consumption (Bakke & Vickers, 2007). Literature corroborates some of these assertions with reports of deterioration of quality characteristics such as loaf volume and crumb structure in products containing added bran (Gan, Ellis, Vaughan, & Galliard, 1989; Lai, Hoseney, & Davis, 1989a, 1989b). The basis for negative effects from bran on bread quality needs to be well understood in order to develop successful and efficient processing and ingredient strategies to mitigate such quality defects. There are two schools of thought regarding the basic mechanism by which bran affects dough and bread properties. The first implicates competitive water binding by bran as the major factor affecting the loaf volume and internal crumb structure of wholewheat bread (Lai et al., 1989a). Changes in water sorption isotherms of flour with and without added bran provide indirect support for
* Corresponding author. Tel.: þ1 608 263 2012; fax: þ1 608 262 6872. E-mail address: [email protected] (S. Damodaran). 0268-005X/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodhyd.2012.10.014
competitive water uptake by bran (Cadden, 1988; Cherian & Chinachoti, 1996). In addition, pre-hydration of bran has been reported to improve the quality of wheat bread (Nelles, Randall, & Taylor, 1998; Salmenkallio-Marttila, Katina, & Autio, 2001). The second hypothesis contends that bran affects loaf volume and internal crumb structure by physically disrupting the gas cells and the gluten network as evidenced from scanning electron micrographs (SEM) (Gan et al., 1989). It is quite possible, however, that the freeze fracture step used during sample preparation for SEM may cause damage to the gluten matrix and thus create artifacts in SEM. It is plausible that both these mechanisms may be intertwined. That is, competitive water binding by bran may cause redistribution of moisture in wheat dough; this may result in partial dehydration of gluten, which may in turn cause conformational changes in gluten and adversely affect its viscoelastic properties. These events may collectively promote partial collapse of the gluten network. Recently, Fourier transform infrared (FTIR) spectroscopy has been used to probe gluten secondary structure in dough under various conditions (Li, Dobraszczyk, Dias, & Gil, 2006; Mejia, Mauer, & Hamaker, 2007; Robertson, Gregorski, & Cao, 2006; Wellner et al., 2005). However, there is no comprehensive study in the literature
J.E. Bock, S. Damodaran / Food Hydrocolloids 31 (2013) 146e155
linking alterations in the structural and thermodynamic state of water caused by bran addition to dough and impact of such changes on gluten conformation in dough. FTIR spectroscopy is an ideal technique for simultaneously probing changes in the state of water in dough by monitoring changes in the OH stretch band at 3000e3800 cm1 and alterations in gluten structure by monitoring changes in the amide I band at 1600e1700 cm1. It has been shown that addition of various polar and nonpolar solutes and/or water state transitions during freezee thaw cycles cause significant changes in the OH stretch band of water (Dashnau, Nucci, Sharp, & Vanderkooi, 2006; Scott, Nucci, & Vanderkooi, 2008; Sharp, Madan, Manas, & Vanderkooi, 2001; Zelent, Bryan, Sharp, & Vanderkooi, 2009; Zelent & Vanderkooi, 2009). These studies have shown that changes in relative hydrogen bonding properties and water’s mobility as the conditions of the system are altered can be deduced from shifts in the OH stretch absorption band. This approach can be potentially used to understand changes in the state of water and secondary structure of gluten (from amide I band) in dough as a consequence of bran addition and relate such changes to the impact of bran on bread quality. Thus, the aim of this study was to study the impact of bran addition on the state of water and gluten secondary structure in model gluten dough using attenuated total reflectance (ATR) FTIR spectroscopy. 2. Materials and methods 2.1. Materials Commercial wheat gluten (Provim ESP vital wheat gluten) was obtained from Archer Daniels Midland Milling Company (Shawnee Mission, KS, USA). Gluten composition was 9.0% moisture, 78% protein, and 0.8% ash. Hard red winter wheat bran (particle size > 1400 mm) was obtained from the Kansas State University Hal Ross flour mill (Manhattan, KS, USA). D2O was purchased from SigmaeAldrich Co. (St. Louis, MO, USA). 2.2. Methodology 2.2.1. Experimental design The compositions of model gluten and gluten-bran dough investigated in this study are given in Table 1. The total moisture content of the dough was varied from 35% to 50%. The total Table 1 Model gluten and gluten-bran dough compositions. Moisture (%)
Gluten (%)
Bran (%)
35
65.0 63.4 61.8 60.2 58.5 60.0 58.5 57.0 55.5 54.0 55.0 53.6 52.2 50.8 49.5 50.0 48.8 47.5 46.2 45.0
0.0 1.6 3.2 4.8 6.5 0.0 1.5 3.0 4.5 6.0 0.0 1.4 2.8 4.2 5.5 0.0 1.2 2.5 3.8 5.0
40
45
50
147
moisture content included the moisture content of gluten (9% w/w) plus the added water. The moisture content of gluten was determined as per the American Association of Cereal Chemists (AACC) Method 44-15.02 (Air Oven Methods) (AACC International, 1999). Keeping the total solids content (i.e., gluten þ bran) constant, at each moisture content the bran content was increased from 0 to 10% of the total solids at 2.5% increments with corresponding adjustments in gluten content. 2.2.2. Gluten dough preparation Gluten and gluten þ bran doughs of the composition shown in Table 1 were prepared by mixing gluten and bran with distilled water until the consistency of the dough peaked in a Brabender Intelli-torque Plasti-corder farinograph (C.W. Brabender Instruments, Inc., South Hackensack, NJ, USA) using a 50 g mixing bowl and WinMix v. 2.0 software. A short premix was executed to distribute the bran prior to water addition. Doughs were collected at the end of mixing and ATR-FTIR analysis was performed within 30e45 min. 2.2.3. ATR-FTIR spectroscopy ATR-FTIR spectra were collected using a Bruker Equinox 55/S FTIR/NIR spectrophotometer (Bruker Optics, Inc., Billerica, MA, USA) using a horizontal multi-reflectance ZnSe crystal accessory. The instrument housed a deuterated tri-glycine sulfate (DTGS) detector and a potassium bromide (KBr) beam splitter. Spectra were collected in the 4000e400 cm1 infrared spectral range at room temperature with a N2 purge to remove interference from atmospheric moisture. A clamp was used to ensure dough contact with the crystal and consistent sample thickness. Each spectrum was an average of 32 scans at 4 cm1 resolution. A minimum of 3 spectra was used for spectral analysis. 2.2.4. ATR-FTIR reference spectra of water Reference FTIR spectra of H2O, corresponding to 35, 40, 45, and 50% water content in the dough, were obtained by mixing H2O with D2O and scanning these mixtures in the 4000e400 cm1 spectral range. These spectra were used as reference spectra to detect shifts in OH stretch absorption band of H2O (3000e3800 cm1) and/or changes in absorption intensity of the OH stretch band in gluten and gluten þ bran doughs at corresponding water content. This was accomplished by generating ATR-FTIR difference spectrum of dough samples by digitally subtracting the H2OeD2O reference spectrum from dough spectrum having the same H2O content. The H2OeD2O reference spectra were also used to digitally subtract contribution of H2O to absorption in the amide I region (1600e1700 cm1) to obtain information on gluten secondary structure in dough. D2O showed no absorption either in the 3000e 3800 cm1 or in the amide I regions. 2.2.5. Spectral analysis of gluten dough Spectral analysis of gluten dough was conducted using OPUS software v. 6.5. All spectra were vector normalized to correct for any difference in IR sample penetration depth caused by any density difference among samples. In experiments involving bran addition, the ATR-FTIR spectrum of bran powder was subtracted from the gluten dough spectrum. The vector-normalized spectra of dough samples were offset-corrected followed by digital subtraction of vector normalized H2OeD2O reference spectra of same water content. The difference spectra thus obtained in the 3000e 3800 cm1 regions were analyzed for changes in the state of water structure in dough compared to the reference state in the H2OeD2O mixture using the approach used elsewhere (Jain, Varshney, & Maitra, 1989; Sutandar, Ahn, & Franses, 1994; Zelent et al., 2009).
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To elucidate structural changes in gluten under different treatment conditions in dough, the difference spectrum in the amide I region (1600e1700 cm1) was second derivatized using a fivepoint SavitskyeGolay derivative function.
before sample introduction. Samples were weighed every 4 h until a constant weight was achieved (typically after 48 h) at which point equilibration was deemed to be complete. 3. Results
2.2.6. Gluten secondary structure estimation The quantitative estimation of gluten secondary structure in dough was determined from second-derivative spectra according to procedures described elsewhere (Dong, Huang, & Caughey, 1990; Dong, Caughey, Caughey, Bhat, & Coe, 1992; Kalnin & Venyaminov, 1990; Kong & Yu, 2007; Susi & Byler, 1983). According to this procedure, the secondary structure of a protein backbone is regarded as a linear sum of a-helix, b-sheet, b-turn, and aperiodic structures. The stretching vibration of C]O group of the peptide backbone in these structural elements has same molar absorptivity, but differ in absorption frequency in the amide I region (1600e 1700 cm1) (Kong & Yu, 2007). It is known that, in addition to the stretching vibration of C]O groups of the peptide backbone, the amino acid side chains of proteins also contribute to absorption in the amide I region (Venyaminov & Kalnin, 1990). However, the contribution of amino acid side chain groups to IR absorption of protein has been reported to be only w10% of the total integral intensity of the amide I band (Venyaminov & Kalnin, 1990). Since gluten is a heterogeneous mixture of several polypeptides, it is not possible to subtract the side chain contribution to the amide I band. Furthermore, since our larger objective was to elucidate net changes in secondary structure content in gluten as a function of moisture content and bran addition, the contribution from side chains was assumed to be a minor constant factor in the secondary structure estimates. The characteristic mean absorption frequencies of the secondary structural elements in proteins are listed in Table 2 (Dong et al., 1990; Kong & Yu, 2007). The secondary structure estimates were determined from the relative area of the peaks centered at these absorption bands (Dong et al., 1990; Kong & Yu, 2007). The secondary structure estimates obtained using this approach has been shown to correlate well with the crystallographic structure of several proteins (Dong et al., 1990; Kong & Yu, 2007; Vedantham, Sparks, Sane, Tzannis, & Przybycien, 2000). 2.2.7. Moisture sorption isotherm Moisture sorption isotherms of gluten and bran were studied as described elsewhere (Rao & Damodaran, 2004). Samples (w1.5 g) were brought to an initial dry state (24 h at 102 C) prior to placement in an environmentally controlled glove box. The glove box was housed in a 5 cm thick styrofoam insulation box with temperature controlled by a copper coil connected to a circulating water bath set to 25 C 0.5 C. Equilibrium relative humidity (ERH) within the glove box was controlled by saturated salt solutions (Greenspan, 1977). The glove box was flushed with nitrogen Table 2 Mean absorption frequencies of various secondary structure elements in proteins.a Mean frequencies (cm1)
Secondary structure assignment
1624 1627 1632 1638 1642 1650 1656 1666 1672 1680 1688
b-sheet b-sheet b-sheet, extended chain b-sheet b-sheet
a
Taken from Dong et al. (1990).
unordered (random)
a-helix b-turn b-turn b-turn b-turn
3.1. H2OeD2O- reference spectra As shown in Fig. 1, the intensity of the OH stretch band located at 3000e3700 cm1 increased and that of OD stretch band located at 1900e2800 cm1 range decreased with increase of H2O concentration in H2OeD2O mixtures. In all these molecularly dispersed H2O in D2O solutions, the broad OH stretch band was centered at about 3400 cm1. In contrast, the OH stretch band of pure H2O, also shown in Fig. 1, exhibited a broad bi-modal peak positions ranging from 3500e3150 cm1. The broad spectrum of the OH stretch band in pure water is attributed to the presence of various hydrogenbonded water clusters in bulk water (Jain et al., 1989; Sutandar et al., 1994). Monomeric non-hydrogen-bonded water typically has OH stretch band centered at 3616 cm1; hydrogen-bonded dimers and small hydrogen-bonded clusters exhibit OH stretch band centered at 3536 cm1 and 3424 cm1, respectively, whereas extensively hydrogen-bonded-associated chains of water molecules exhibit OH stretch band at 3246 cm1 (Jain et al., 1989; Sutandar et al., 1994). In other words, as the proportion of extensively hydrogen-bonded water clusters or associated chains of water or water hydrogen-bonded to polymeric materials in a system increase, the OH stretch band shifts to lower frequencies. On the basis of these band assignments for various states of water, it appears that the molecularly dispersed H2O in H2OeD2O mixtures exist predominantly in small hydrogen-bonded cluster state (w3400 cm1). Conversely, it can be reasoned that if water disperses itself in an ideal manner in another mixture in which D2O has been replaced by another substance, e.g., gluten, the state of water should be same as in the H2OeD2O mixture, provided there is no specific interaction between H2O and gluten. However, if water interacts with gluten and as a consequence if the state of water changes in the gluteneH2O mixture compared to the reference state in the H2OeD2O mixture, it should reveal itself from a shift in the OH stretch absorption frequency. 3.2. Gluten and bran ATR-FTIR spectra The ATR-FTIR spectrum of gluten powder exhibited a band at 2800e3600 cm1 (Fig. 2), which is in the OH stretch region of water. This band might be attributed mostly to the moisture in the gluten sample, which was about 9% (w/w). Since this moisture content was included in the total moisture content of the gluten dough samples, there was no need to subtract the OH stretch absorption band of gluten sample from the OH stretch absorption band of gluten dough. It is also likely that there might be a small contribution from the NeH stretching (amide A) and CeH stretching vibrations of gluten as well since they weakly absorb in this region (Jackson & Mantsch, 1995). However, since these contributions are very small compared to the OH stretch absorption band of water in gluten dough, no correction was deemed necessary. Furthermore, since we are more interested in shifts in OH stretch absorption frequency rather than absolute intensity, small variations in intensity will not affect the focus of this study. The gluten sample also showed a strong absorption band in the amide I (1600e1700 cm1) region. The dry bran sample showed no strong absorption neither in the OH stretch region of water nor in the amide I region. Nevertheless, the bran spectrum was subtracted from the spectra of gluten þ bran dough samples before spectral analysis.
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Fig. 1. ATR-FTIR spectra of H2OeD2O mixtures. The legend in the figure indicates the percentage of H2O in the mixture.
3.3. State of water in gluten dough The OH stretch absorption spectrum of gluten dough containing 35% moisture (w/w) is shown along with the 35% H2O in D2O reference spectrum in Fig. 3. It should be noted that the OH stretch band of the 35% gluten dough was qualitatively different from that of the 35% H2OeD2O reference spectrum. While the reference spectrum exhibited an apparent single symmetrical band centered at 3393 cm1, the band in the gluten dough spectrum was broader and red-shifted to lower frequency. This suggests that the energy state(s) of water is dramatically altered in gluten dough compared to the reference state(s) in H2OeD2O mixture as a result of interaction with the gluten network. To characterize the newly formed sub-populations of water in the gluten dough, the H2OeD2O reference spectrum was subtracted
Fig. 2. ATR-FTIR spectra of gluten (red line) and bran (blue line) samples. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
from the gluten dough spectrum. The resultant difference spectrum, shown in Fig. 3, exhibited two positive bands, one centered at 3580 cm1 and the other centered at 3180 cm1, a shoulder at 3080 cm1, and a trough centered at 3435 cm1. The area of the trough essentially reflects the fraction of water (small hydrogenbonded cluster states) in the reference spectrum that has been transformed into other energy (structural) states in gluten dough. Previously, it has been reported that while monomeric nonhydrogen-bonded water has OH stretch vibration at w3600 cm1, water entrapped in reverse micelles exists as hydrogen-bondedassociated chains with OH stretch vibration at 3290 cm1 (Jain et al., 1989), and bound water in epoxy resins exhibits OH stretch at 3129 cm1 (Cotugno, larobina, Mensitieri, Musto, & Ragosta, 2001; Liu, Wu, Ding, Chen, & Li, 2002). It also has been reported
Fig. 3. ATR-FTIR spectra of water in the OH stretch region in 35% H2O in D2O (green line) and in gluten dough at 35% moisture content (blue line). The red line is the difference spectrum of water obtained by subtracting the green line from the blue line. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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that transformation of water from a liquid state at 278 K to ice at 200 K caused the OH stretch band to shift from 3400 cm1 to about 3290 cm1 (Zelent et al., 2009). Typically, water strongly hydrogenbonded to functional groups in a polymer network requires less energy (low frequency) for OH stretch vibration. The stronger the hydrogen bond strength, the greater is the shift of OH stretch absorption to lower frequency (Liu et al., 2002). Based on those reports, the positive peak in the difference spectrum at 3580 cm1 (Fig. 3) can be assigned to OH stretch vibration of monomeric nonhydrogen-bonded water molecules and some hydrogen-bonded water dimers. The peak at 3180 cm1 and the shoulder at 3080 cm1 can be assigned to water populations hydrogen-bonded to gluten network. Thus, there appears to be two classes of bound water in gluten dough: a relatively weakly bound state with OH stretch at 3180 cm1 and the other relatively strongly bound fraction with OH stretch at 3080 cm1. The absence of OH stretch band at w3290 cm1, which is found for water entrapped in reverse micelles (Jain et al., 1989) and for ice at 200 K (Zelent et al., 2009), suggests that water trapped in the interstices of the gluten network (i.e., capillary water) apparently does not exist as hydrogenbonded-associated chains, but is present mainly as small hydrogen-bonded clusters with OH stretch absorption centered at w3400 cm1. Fig. 4 shows the ATR-FTIR difference spectra in the OH stretch region of gluten dough at 35, 40, 45, and 50% moisture levels. It should be noted that the difference spectrum displayed positive bands at w3580 cm1 and at 3180 e 3145 cm1, and a shoulder at 3080 cm1. Although the intensity of the 3580 cm1 band varied slightly with dough moisture content, the peak position of the band did not change, indicating that the energy state of this population of water (which is mainly monomeric and possibly some hydrogenbonded water dimers) was not affected by the moisture content of dough. However, both the intensity and the peak position of the 3180 cm1 band changed with moisture content of the dough: The peak intensity decreased with increase of moisture content and the peak position shifted from 3180 cm1 at 35% moisture content to 3145 cm1 at 50% moisture content (Fig. 4). Even after normalizing the spectra for differences in the gluten content, the intensity of the band in the 3180e3145 cm1 region was significantly different at various moisture contents (data not shown). This indicates that the interaction strength of water associated with the gluten network increases with increase of dough moisture content (which is reflected in the shift in absorption band to lower frequency). The decrease in the intensity of the absorption band might be due to a decrease in the molar absorptivity of bound water molecules as the absorption frequency is shifted to lower values in this range.
Taken together, the results shown in Figs. 3 and 4 reveal that water undergoes a significant structural transformation in gluten dough compared to its reference state in H2OeD2O mixtures. This transformation involves a significant amount of water becoming hydrogen bonded to the gluten polymer, and a significant amount of water becoming released from small hydrogen-bonded cluster states to the monomeric non-hydrogen-bonded state. 3.4. State of water in gluten-bran dough The effect of bran addition on the difference spectrum of the OH stretch band of water in gluten dough at various moisture contents is shown in Fig. 5. As in the case of gluten only dough, the difference spectrum of gluten þ bran dough also exhibited OH stretch bands at 3580 cm1 and 3180 cm1 and a shoulder at 3080 cm1. However, at any given moisture content, the intensities of these bands were dependent on the bran content. In low moisture dough (35%) bran addition caused an increase in the amount of water hydrogenbonded to the polymer matrix as evidenced from an increase in the intensity of the OH stretch band at 3180 cm1 in the difference spectrum without any spectral shift (Fig. 5A). At higher moisture content (40 and 45%), however, bran addition decreased the amount of water hydrogen-bonded to the polymer matrix as evidenced from a decrease in the intensity of the OH stretch band at 3160 cm1 and an apparent shift of absorption frequency by about þ20 cm1 with increase of bran content (Fig. 5B, C). At 50% moisture content, bran addition essentially had no effect on water hydrogen-bonded to the polymer matrix as evidenced from no change in the OH stretch absorption band at w3180 cm1 (Fig. 5D). These bran-induced changes in the OH stretch band might be fundamentally due to redistribution of water in the bulk versus bound state in the gluten matrix. This redistribution may involve partial dehydration of the gluten matrix as a consequence of partitioning of water between bran and gluten. The extent of structural changes in gluten at various moisture contents (as discussed below in Sections 3.7 And 3.8) also might impact bran-induced water redistribution in the dough. In other words, the increase in the intensity of the peak at 3180 cm1 at 35% moisture content and the decrease in the intensity of the peak at 40% and 45% moisture content with the addition of bran might be a result of complex interplay of changes in gluten structure and bran-induced moisture redistribution in the dough at various moisture contents. 3.5. Gluten and bran moisture sorption isotherms The moisture sorption experiments were conducted in an attempt to further identify any competition between bran and gluten for water (Fig. 6). The isotherms indicated that bran bound more waters than gluten at any given water activity and, at any given water content the water activity in bran was lesser than in gluten. The data apparently confirms that addition of bran to gluten dough might cause redistribution of water in the system. 3.6. Secondary structure of gluten in unhydrated state
Fig. 4. ATR-FTIR difference spectra of water in the OH stretch region in gluten dough at 35e50% moisture content.
The secondary structure changes in gluten as a function of moisture content of dough and bran addition to dough was studied by second-derivative ATR-FTIR spectroscopy as described elsewhere for other proteins and polypeptides (Byler & Susi, 1986; Dong et al., 1992; Dong et al., 1990; Kalnin & Venyaminov, 1990; Kong & Yu, 2007; Kendrick, Dong, Allison, Manning, & Carpenter, 1996; Susi & Byler, 1983). Several studies have shown that the second-derivative FTIR band frequencies centered at 1624, 1627, 1632, 1638, and 1642 cm1 belonged to b-sheet structure and the band frequencies centered at 1666, 1672, 1680, and 1688 cm1
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Fig. 5. Effect of bran addition on the difference spectrum of water in the OH stretch region in gluten dough at (A) 35%, (B) 40%, (C) 45%, and (D) 50% moisture content.
Fig. 6. Moisture sorption isotherms of bran (-) and gluten (C). The equations shown are the polynomial curve-fitting equations of the data.
belonged to b-turns (Dong et al., 1990; Kong & Yu, 2007; Vedantham et al., 2000). On the other hand, the unordered (random) and a-helix structures exhibited only one frequency each centered at 1650 and 1656 cm1, respectively (Dong et al., 1990). The second-derivative ATR-FTIR spectrum of gluten powder (9% moisture content) in the amide I region (1600e1700 cm1) is shown in Fig. 7. This spectrum was generated after subtracting the ATR-FTIR spectrum of 10% H2O in D2O from the ATR-FTIR of gluten powder (w9% moisture). In the spectral region 1620e 1690 cm1 where C]O stretching vibrations of polypeptide chains occur, the second-derivative FTIR spectrum of gluten exhibited bands at 1622, 1627, 1641, 1652, 1663, 1668, and 1680 cm1, which are in agreement with bands identified for various secondary structural elements in proteins. Based on the band frequency assignments in the literature (Dong et al., 1990; Kong & Yu, 2007; Vedantham et al., 2000), the secondary structure estimates of b-sheet, random (unordered), a-helix, and b-turn contents were determined from the relative band areas in the spectral regions 1620e1644 cm1, 1644e1652 cm1, 1652e 1660 cm1, and 1660e1685 cm1, respectively. Since these bands were extensively overlapped, no attempt was made to deconvolute these bands. A similar approach has been used by Dong et al. (1990). The secondary structure of gluten in the unhydrated state is estimated as 39% b-sheet, 30% random, 17% a-helix, and 14%
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Fig. 7. Second-derivative ATR-FTIR spectrum of gluten powder (9% moisture) in the amide I region. The spectral bands in the regions between the dotted lines correspond to the secondary structures as indicated. The secondary structure estimates were calculated from the fractional area of the bands. No attempts were made to deconvolute the bands.
b-turn. These estimates are accurate within 5% (Dong et al., 1990). Thus, it appears that at low moisture level (9%) b-sheet and random structures are the major secondary structure elements in gluten. 3.7. Secondary structure of gluten in dough The second-derivative ATR-FTIR spectrum of gluten dough (derived from H2OeD2O blank-corrected ATR-FTIR difference spectrum of gluten dough) in the amide I region (1600e1700 cm1) at 35%, 40%, 45%, and 50% moisture content is shown in Fig. 8A. As in the case of unhydrated gluten, the second-derivative FTIR spectrum of gluten dough exhibited bands at 1622, 1629, 1649, 1656, 1662, 1670, and 1680 cm1, which are within 2 cm1 of the peak positions found in the unhydrated gluten (Fig. 7), but the relative areas of these bands were different compared to those in the unhydrated state. The b-sheet, random, a-helix, and b-turn secondary structure contents of gluten in dough, estimated from relative band areas in the spectral regions 1620e1644 cm1, 1644e 1652 cm1, 1652e1660 cm1, and 1660e1685 cm1, are plotted in Fig. 8B as a function of moisture content of gluten dough. The 9% moisture content refers to gluten powder. The secondary structure of gluten changed dramatically as the moisture content was increased from 9% to 50%: The b-turn content increased from about 14% to about 65% as the moisture content was increased from 9% to 40%. This dramatic increase in b-turn content was at the expense of a decrease in the amount of both b-sheet and random structures as the gluten was hydrated. These results indicate that b-turn is the preferred secondary structure of gluten in the hydrated state. 3.8. Effect of bran addition on secondary structure of gluten in dough
Fig. 8. (A) Effect of moisture content of dough on the second-derivative spectrum of gluten in the amide I region. (B) Effect of moisture content on a-helix ( ), b-sheet ( ), b-turn (:), and unordered ( ) secondary structure content of gluten in gluten dough.
content of the dough. At the 35% moisture level, addition of 10% bran decreased the b-turn content from about 54% to 33% while increasing the b-sheet content from 18% to about 32% and the random structure content from 18% to 25% (Fig. 10A). A similar trend also was observed at 40% moisture level (Fig. 10B). However, at 50% moisture content, the extent of inter-conversion of b-turn into b-sheet and random structures was minimal: The b-turn content decreased from 54% to 47% while the b-sheet content increased from 20% to 26% with no change in the random and ahelix contents (Fig. 10D). The results indicate that although b-turn is the preferred structure of gluten in the fully hydrated state, addition of bran to dough forces gluten to adopt b-sheet and random configurations, potentially as a result of water redistribution and partial dehydration of gluten by bran. 4. Discussion
The effect of bran addition on the second-derivative ATR-FTIR spectrum of gluten in gluten dough at various moisture and bran contents is shown in Fig. 9 and the secondary structure estimates are given in Fig. 10. At all moisture contents, the b-turn content decreased, the b-sheet and random structures increased, and the ahelix content remained unchanged as the bran content was increased from 0 to 10%. However, the bran-induced changes in secondary structures content were dependent on the moisture
The OH stretch region (w3500e3000 cm1) in FTIR spectra has not been closely investigated in the past as a source of information for food systems in general, and wheat dough in particular. In the present investigation, we have used ATR-FTIR spectra of H2OeD2O mixtures as reference state of H2O to elucidate changes in the state of water in gluten dough at corresponding H2O content. It is well established that hydrogen bonding broadens the OH stretch band
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Fig. 9. Effect of bran addition at various moisture contents on the second-derivative spectrum of gluten in dough: (A) 35%, (B) 40%, (C) 45%, and (D) 50% moisture content.
of water (Sutandar et al., 1994; Walrafen, 1972). The broadening of the OH stretch band toward lower absorption frequency in gluten dough compared to the reference H2OeD2O spectrum at corresponding water content (Fig. 3) reveals that there is a significant change in the structural (energy) state of water in gluten dough. The difference spectra clearly indicates that, in addition to the normal state with OH stretch absorption frequency centered at 3400 cm1, interaction of gluten with water creates two new subpopulations of water, one strongly hydrogen-bonded to gluten with absorption frequencies at 3180 cm1 and 3080 cm1 and the other non-hydrogen-bonded monomeric water population with absorption frequency at 3580 cm1 (Fig. 3). It has been reported that when water at 4 C is converted into ice at 15 C, the OH stretch band of water shifts from 3400 cm1 to only about 3306 cm1; further decrease in the temperature of ice to 73 C shifts the absorption frequency to 3290 cm1 (Zelent et al., 2009). Since the stronger the hydrogen bonding interaction, the greater is the extent of shift in OH stretch band toward lower frequency (Liu et al., 2002; Sutandar
et al., 1994), the absorption frequencies 3180 and 3080 cm1 of bound water in gluten dough implicitly suggests that the interaction of water with hydrogen bonding groups in gluten is much stronger than waterewater hydrogen bonding interaction in ice at 73 C. The addition of bran to gluten dough changed the amount of water bound to the gluten matrix (Fig. 5AeD). Although quantitative reasons for these changes are not clear, the results clearly indicate that bran affects glutenewater interaction in the 35e45% moisture content range and redistributes the released water into other energy states as evidenced from a decrease in the intensity of the band at 3180 cm1 as well as the negative trough at 3400 cm1 (Fig. 5AeD). The effect of bran, however, becomes muted at higher (50%) moisture content. Previously, it has been observed that the loaf volume of wheat dough with added bran could be improved with the addition of extra formulation water (Lai et al., 1989a). Presoaking of the bran in water prior to mixing with flour also resulted in almost complete restoration of loaf volume (Lai et al., 1989a). The
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Fig. 10. Effect of bran addition on the secondary structure contents of gluten in dough at (A) 35%, (B), 40%, (C) 45%, and (D) 50% moisture content.
results of the present study seem to provide a mechanistic explanation of those empirical observations: That is, at low moisture content (35e45%) water redistribution and partial dehydration of gluten network caused by bran might affect the development of loaf volume and this affect of bran can be nullified at higher moisture (50%) content. If bran causes partial dehydration of gluten and redistribution of water in dough, then it is reasonable to expect conformational changes in gluten as a consequence. It should be pointed out that bsheet (39%) and random (30%) conformations are the major secondary structures in the unhydrated (9% moisture) gluten (Fig. 7). However, these secondary structure elements are transformed into b-turn when gluten is hydrated as in dough (Fig. 8B). If we assume that this structural transformation is reversible, then when gluten in dough is partially dehydrated by bran addition, the b-turn should revert back to b-sheet and random configurations; the extent of this reversal must be a function of the amount of bran in the system. This indeed is found to be the case as seen in Fig. 10AeD. However, in 50% moisture dough, in which the influence of bran on the water bound to gluten is minimal (Fig. 5D), the transformation of b-turn back to b-sheet is also minimal because of the excess amount of water available in the system to adequately hydrate gluten. Thus, there seems to be a direct linkage between trans-conformational changes in gluten and changes in the extent of glutenewater interaction in dough as affected by bran addition. The abundant b-turn secondary structure motif found in gluten dough may not be similar to those b-turns associated with antiparallel and parallel pleated b-sheets found in globular proteins. The b-turn structures in gluten dough might be related to the bspiral domains in glutenin polypeptides (Wellner et al., 2005), which contain hexa- and nona- and tri-peptide repeats of amino acid sequences PGQGQQ, GYYPTSLQQ, and GQQ (Belton, 1999).
These b-spiral structures can be regarded as consecutive b-turns. It has been reported that short a-helix regions flank these b-spiral domains (Shewry, Halford, Belton, & Tatham, 2002), which is consistent with 10e12% a-helix estimated in this study. The b-spiral structures are implicated as one of the structural elements responsible for the viscoelasticity of dough (Belton, 1999; Wellner et al., 2005). The greater the amount of this structural element in dough, the greater would be the ability of dough to trap gas bubbles and the greater would be the bread loaf volume. Conversely, anything that transforms the b-spiral structure into intermolecular b-sheet structure would adversely impact the quality of breadcrumb. Based on the results presented in this study, we hypothesize that one of the reasons for the adverse effect of bran addition on
Fig. 11. Schematic description of proposed hypothesis of bran-induced conformational changes in gluten in dough leading to collapse of the viscoelasticity of gluten dough.
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loaf volume of bread (Lai et al., 1989a) is the collapse of the b-spiral structure to intermolecular b-sheets and this transformation is caused by redistribution of water in dough, which might involve partial dehydration of gluten network. This is schematically shown in Fig. 11. The loss of the elastic b-spiral structures and formation of inelastic intermolecular b-sheet aggregates in the gluten network might decrease the viscoelasticity of the dough and thus affect the loaf volume during bread making. It is also likely that in real wheat dough, interaction of other flour components, such as starch, might also modify the secondary structure of gluten. These aspects remain to be studied in detail. 5. Conclusions The ATR-FTIR difference spectroscopy is shown to be a valuable technique for studying changes in the energy state of water and secondary structure of gluten simultaneously in dough systems. The energy state of water in gluten dough is altered compared to its reference state in H2OeD2O mixtures at the same H2O content. In addition to hydrogen bonding of water to the gluten network, glutenewater interaction causes a net increase in monomeric water population in gluten dough. The moisture content as well as bran addition affects the amount of both bound and monomeric water populations in gluten dough. The water redistribution in gluten dough by bran addition alters the conformation of gluten from bspiral (consecutive b-turns) structure to b-sheet structure. This major structural change in gluten might be the physical basis for poor quality of bread made with added bran. However, the extent of bran-induced conversion of the b-spiral structure to intermolecular b-sheet structure is minimized as the moisture content of the dough is increased from 35% to 50%. This implicitly suggests that the adverse effects of bran on bread quality could be overcome by increasing the moisture content of dough. However, since wholewheat dough is a complex system consisting of bran, starch, and other phytochemicals, it is quite possible that these components may also directly or indirectly affect gluten structure as well as water distribution in the dough. Acknowledgments We thank Gary Girdaukas and the Analytical Instrumentation Center of the School of Pharmacy, University of Wisconsin-Madison, for support in obtaining the ATR-FTIR spectrophotometric data. This research was supported by a grant (#2009-35503-05181) from the National Institute for Food and Agriculture e Agriculture and Food Research Initiative (NIFA-AFRI). References AACC International. (1999). Method 44e15.02. Moisturedair oven methods. Reapproved 03.11.99 Approved methods of analysis (11th ed.). St. Paul, MN, USA: AACC International. http://dx.doi.org/10.1094/AACCIntMethod-44-15.02. Bakke, A., & Vickers, Z. (2007). Consumer liking of refined and whole wheat breads. Journal of Food Science, 72, S473eS480. Belton, P. S. (1999). On the elasticity of wheat gluten. Journal of Cereal Science, 29, 103e107. Byler, D. M., & Susi, H. (1986). Examination of the secondary structure of proteins by deconvoluted FTIR spectra. Biopolymers, 25, 469e487. Cadden, A.-M. (1988). Moisture sorption characteristics of several food fibers. Journal of Food Science, 53, 1150e1155. Cherian, G., & Chinachoti, P. (1996). 2H and 17O nuclear magnetic resonance study of water in gluten in the glassy and rubbery state. Cereal Chemistry, 73, 618e624. Cotugno, S., larobina, D., Mensitieri, G., Musto, P., & Ragosta, G. (2001). A novel spectroscopic approach to investigate transport processes in polymers: the case of water-epoxy system. Polymer, 42, 6431e6438. Dashnau, J. L., Nucci, N. V., Sharp, K. A., & Vanderkooi, J. M. (2006). Hydrogen bonding and the cryoprotective properties of glycerol/water mixtures. Journal of Physical Chemistry B, 110, 13670e13677. Dong, A., Caughey, B., Caughey, W. S., Bhat, K. S., & Coe, J. E. (1992). Secondary structure of the pentraxin female protein in water determined by infrared
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