Dietary Fiber-Induced Changes in the Structure and Thermal

Article pubs.acs.org/JAFC

Dietary Fiber-Induced Changes in the Structure and Thermal Properties of Gluten Proteins Studied by Fourier Transform-Raman Spectroscopy and Thermogravimetry Agnieszka Nawrocka,*,† Monika Szymańska-Chargot,† Antoni Miś,† Agnieszka Z. Wilczewska,§ and Karolina H. Markiewicz§ †

Bohdan Dobrzanski Institute of Agrophysics, Polish Academy of Sciences, Doświadczalna 4, 20-290 Lublin, Poland Institute of Chemistry, University of Białystok, Ciołkowskiego 1K, 15-245 Białystok, Poland

§

S Supporting Information *

ABSTRACT: Interactions between gluten proteins and dietary fiber supplements at the stage of bread dough formation are crucial in the baking industry. The dietary fiber additives are regarded as a source of polysaccharides and antioxidants, which have positive effects on human health. The fiber enrichment of bread causes a significant reduction in its quality, which is connected with changes in the structure of gluten proteins. Changes in the structure of gluten proteins and their thermal properties induced by seven commercial dietary fibers (fruit, vegetable, and cereal) were studied by FT-Raman spectroscopy and thermogravimetry (TGA), respectively. For this aim the bread dough at 500 FU consistency was made of a blend of wheat starch and wheat gluten as well as the fiber, the content of which ranged from 3 to 18% w/w. The obtained results revealed that all dietary fibers apart from oat caused similar changes in the secondary structure of gluten proteins. The most noticeable changes were observed in the regions connected with hydrogen-bonded β-sheets (1614 and 1684 cm−1) and β-turns (1640 and 1657 cm−1). Other changes observed in the gluten structure, concerning other β-structures, conformation of disulfide bridges, and aromatic amino acid microenvironment, depend on the fibers’ chemical composition. The results concerning structural changes suggested that the observed formation of hydrogen bonds in the β-structures can be connected with aggregation or abnormal folding. This hypothesis was confirmed by thermogravimetric results. Changes in weight loss indicated the formation of a more complex and strong gluten network. KEYWORDS: gluten protein, amide I band, disulfide bridges, aromatic acids, Raman spectroscopy, dietary fiber, TGA

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INTRODUCTION Nowadays, consumers demand dietary fiber-enriched products of appropriate taste, texture, smell, and appearance as a result of growing consumers’ awareness concerning the health benefits of the consumption of this kind of product.1 Addition of dietary fiber preparations to bread significantly reduces its quality, for example, decrease in loaf volume, gritty texture, and unsuitable taste and mouthfeel. Therefore, the basis for negative effects from dietary fiber on bread quality has to be well understood to develop efficient processing and ingredient strategies to eliminate quality defects. So far, there are two hypotheses describing the reduction of dough quality resulting from the addition of dietary fiber. One of them implicates competitive water binding by the dietary fiber as a major factor affecting dough quality.2 The second one assumes that the addition of the dietary fiber physically disrupted the gas cells and gluten network.3 Bock and Damodaran4 combined these two hypotheses in one. The authors claimed that competitive water binding by dietary fiber 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 phenomena may promote partial collapse of the gluten network. The wheat bread quality is related to the structure of gluten proteins (gliadins and glutenins), which participate in the © 2016 American Chemical Society

formation of a continuous viscoelastic network within dough. Glutenin polymers are made up of high and low molecular weight subunits that are attached to each other via disulfide bonds, whereas gliadins interact with the glutenin polymers via noncovalent hydrophobic interactions and hydrogen bonding.5 Addition of different chemical compounds (e.g., emulsifiers, polysaccharides) to the gluten proteins disturbs the network by creating new hydrogen bonds and causing changes in disulfide bridge conformation. It leads to folding or aggregation of protein complexes and results in the formation of a network characterized by different mechanical properties. The polysaccharides are used as molecules changing the structure of gluten proteins the most often. The polysaccharides can be used as breadmaking improvers because of their effect on dough functionality, quality characteristics, and product preservation. Linlaud et al.6 studied interactions between different polysaccharides (xanthan gum, locust bean gum, guar gum, and pectins) and gluten proteins by using FT-Raman spectroscopy. Each of the compounds caused different changes in the amide I band, disulfide bridge region, and aromatic amino acid microenvironment. Generally, addition of these Received: Revised: Accepted: Published: 2094

December 2, 2015 February 29, 2016 February 29, 2016 February 29, 2016 DOI: 10.1021/acs.jafc.5b05712 J. Agric. Food Chem. 2016, 64, 2094−2104

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Journal of Agricultural and Food Chemistry

moisture basis were kneaded for 5 min using a Farinograph-E (Brabender, Germany) according to ICC Standard 114/1. The amount of added water was adjusted to optiml water absorption (500 FU), and 6 g of sodium chloride was dissolved in water. Sodium chloride (2% w/w) was added to the blend mass. The amount of added water was again adjusted to optimal water absorption (500 FU). The gluten samples were washed out from unmodified and fibermodified dough samples by using a Glutomatic 2200 (Perten Instruments, USA). Next, gluten samples were freeze-dried for 24 h, milled, and used in the FT-Raman and TGA measurements. FT-Raman Spectra Collection and Data Manipulation. The FT-Raman spectra were acquired on a FT-Raman module (NXR FT Raman) for a Nicolet 6700 FT-IR bench using an InGaAs detector and a CaF2 beamsplitter (Thermo Scientific, Madison, WI, USA). The samples placed in stainless cubes were illuminated using an Nd:YAG excitation laser operating at 1064 nm. The maximum laser power was 1 W. The spectra were recorded over the range of 3500−150 cm−1, and each spectrum was an average of five registered spectra. The gluten samples were analyzed in the form of powder. The spectra were manually corrected with linear baseline in OMNIC software (version 8.2, Thermo Fischer Scientific Inc., USA). Spectral data from the sample scans were normalized against the phenylalanine band at 1003 cm−1 using ORIGIN (version 9.0 PRO, OriginLab Corp., USA). The intensity and location of the phenylalanine band are not sensitive to the protein conformation or to the microenvironment, so it was used as internal standard to normalize the vibrational intensity.14 The disulfide bridge region (490−550 cm−1) and aromatic amino acid environment (tyrosine doublet (I(850)/ I(830)), tryptophan band (I(760)), and amide I band (1590−1720 cm−1; βS, β-sheet, pβS, pseudo-β-sheet, aβS, antiparallel-β-sheet, βT, β-turns, AGR, aggregates)) were analyzed. Structural analyses of the disulfide bridge (percentage distribution of disulfide bridge conformations: gauche−gauche−gauche (SSg‑g‑g), trans−gauche−gauche (SSt‑g‑g), trans−gauche−trans (SSt‑g‑t)) were also conducted using ORIGIN. The Gaussian components in the S−S region were determined on the basis of the second-derivative spectrum. The derivative spectrum was obtained by using a five-point, two-degree polynomial function. The S−S bands were assigned to each conformation according to the procedure of Sugeta.15 To determine changes in the secondary structure of gluten proteins (amide I band), two kinds of difference spectra were calculated according to the method of Nawrocka et al.9,10 Briefly, a spectrum of gluten washed out from model dough (control) was subtracted from the spectrum of a gluten−fiber mixture. Next, the dietary fiber spectrum was subtracted from the first difference spectrum. The second difference spectrum showed interactions between gluten proteins and dietary fiber components. The difference spectra for different concentrations of dietary fiber (3, 6, 9, 12, and 18%) were compared in pairs: the spectrum of 3% with 6%, the spectrum of 6% with 9%, etc. The Raman spectra for all five levels of fiber concentration were obtained only for two fibers, cranberry (CRR) and carob (CAR). For other dietary fibers spectra were studied for two (OAT, FLX), three (CHB, CRR), and four (CAC) fiber content levels. This resulted from the fact that the higher contents of fiber contributed to difficulties with Raman measurements (burning of the gluten−fiber sample) or with washing out of the gluten (unextractable gluten). Thermogravimetric Analysis. TGA was performed on a Mettler Toledo Star TGA/DSC1 unit (Mettler Toledo Corp., Zurich, Switzerland). Argon was used as a purge gas (mL min−1). Freezedried gluten samples between 2 and 5 mg were placed in oxide aluminum pans and heated from 50 to 900 °C at a heating rate of 10 °C/min.16 The degradation temperature (Td) and weight loss at 600 °C were calculated using ORIGIN (version 9.0 PRO, OriginLab Corp., USA). The FT-Raman and TGA measurements were performed in five replicates.

polysaccharides promoted formation of a more disordered and labile gluten network. Research by Correa et al.7 showed that modified cellulose (three types: MCC, CMC, HPMC) and pectins (low LMP and high HMP degrees of methylation) addition softened dough, which could be related to protein unfolding and matrix flexibility. Similarly, the addition of the konjac glucomannan (water-soluble polysaccharide that is used as dietary fiber supplement) induced strong intermolecular hydrogen bonding between hydroxyl groups of the polysaccharide and gluten complexes, which led to the flexible gluten conformation.8 Contrary to the above-mentioned studies, our previous studies9−11 showed that insoluble-rich fiber preparations caused the formation of a more compact gluten network. These results suggested folding or aggregation of the gluten proteins. Apart from polysaccharides, the influence of antioxidative compounds (polyphenols and anthocyanins) on the gluten structure was also studied. Addition of polyphenols’ extracts caused the appearance of a band in the amide I band corresponding to random coil structures.12 The authors postulated that small molecules of phenolic acids were incorporated into the gluten network, allowing proteins to aggregate and strengthen the dough. Studies showed that interactions between gliadins and different anthocyanins induced, contrary to polyphenols, a decrease in the amount of unordered structures.13 On the other hand, anthocyanins induced also the formation of new hydrogen bonds, which led to folding or aggregation of the gluten proteins. The mechanism of interactions between dietary fibers and gluten proteins is not fully understood. For this reason, the aim of the present studies was to determine changes in the structure of gluten proteins at the stage of bread dough developing and mixing as a result of the addition of seven dietary fiber preparations in amounts of 3−18%. The changes were studied by using FT-Raman spectroscopy and thermogravimetry (TGA) because both the conformational changes in the gluten structure and its thermal properties play key roles in the baking quality of dough.

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MATERIALS AND METHODS

Materials. Wheat gluten and sodium chloride were purchased from Sigma-Aldrich (Poland) and used as received. Wheat starch was purchased from Cargill (The Netherlands). Chokeberry (CHB), cranberry (CRB), carrot (CRR), cacao (CAC), oat (OAT), and flax (FLX) fibers were purchased from Microstructure (Warsaw, Poland). The carob fiber (CAR) was a natural extract produced from the carob pulp (Carob General Application, Valencia, Spain). Double-distilled water was used. Dough Sample Preparation. In the fiber−gluten interaction studies, a model flour reconstituted from two commercial components (wheat gluten and wheat starch) was used. The pure wheat gluten was applied to provide gluten proteins of definite structure. The starch and gluten were combined in a constant weight proportion 80:15 (at 14% moisture basis). The proportion was assumed on the basis of real contents of starch and gluten in commercial bread wheat flours. The simplified composition of the model flour was intentional because the absence of native fiber substances in wheat flour facilitates the study of structural changes in gluten proteins as a result of fiber fortification. Commercial fiber preparations produced from various plant sources were used for fortification. All studied fiber preparations were micronized to avoid influence of the nonuniform size distribution of particles on gluten−fiber interactions. Fiber−flour blends were made by substituting 3, 6, 9, 12, and 18% w/w of the model flour by a single fiber preparation (at the same moisture basis). Doughs from 300 g samples of the model flour (control) and fiber−flour blends at 14% 2095

DOI: 10.1021/acs.jafc.5b05712 J. Agric. Food Chem. 2016, 64, 2094−2104

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Figure 1. continued

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Figure 1. Difference spectra depict interactions between gluten proteins and dietary fibers at different contents (3, 6, 9, 12, and 18%): chokeberry, CHB (a, b); cranberry, CRB (c−f); carrot, CRR (g, h); carob, CAR (i−l); cacao, CAC (m−o); flax, FLX (p). The spectra were obtained by subtraction of control sample and fiber spectra from spectra of gluten−fiber mixtures.

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with β-structures, for example, β-sheet (1632 cm−1), antiparallel-β-sheet (1685 cm−1), pseudo-β-sheet (1614 cm−1), and β-turns (1640, 1658, and 1667 cm−1). Detailed analysis of the CHB difference spectra showed that the 3% addition of the fiber caused the appearance of positive bands at 1614, 1625, and 1686 cm−1 and negative bands at 1632, 1676, and 1698 cm−1, simultaneously. The positive bands could be connected with the formation of protein aggregation in the form of βsheets (1625 cm−118) or antiparallel-β-sheets (1614 cm−119 and 1686 cm−120), whereas the negative bands were assigned to nonaggregated β-sheets (1632 cm−1, parallel β-sheets; 1676 and 1698 cm−1, antiparallel-β-sheets). The band at 1677 cm−1 could be also assigned to turns and loops, but analysis of the structural changes indicates that this band was rather connected with the antiparallel-β-sheets. There were also observed significant changes in β-turns that were hydrogen-bonded. The positive bands located at 1641 and 1658 cm−1 (see Figure 1a) could be assigned to the hydrogen bonding of the carbonyl groups in β-turns, whereas the negative band at 1670 cm−1 was assigned to non-hydrogen bonding of the carbonyl groups in βturns.20 Addition of 6% of the CHB fiber caused slightly different changes in the structure of gluten proteins. Bands connected with protein aggregation in the form of β-sheets

RESULTS AND DISCUSSION Changes in Secondary Structure (Amide I Band). The peptide group in proteins gives rise to a few characteristic vibrational bands in the Raman spectrum. Amide I (1570−1720 cm−1) and amide III (1230−1340 cm−1) bands can be used for characterization of the protein secondary structure. The intensity of the amide III band was relatively low and overlapped with the spectra for some components in our samples. For this reason, the changes in secondary structure of gluten proteins in a model flour induced by the addition of dietary fiber preparations were studied by analysis of only the amide I band. Although a glutamine side chain can contribute to the amide I band, it has been demonstrated that this contribution is not greater than 10% for most proteins.17 The difference spectra were calculated in a two-phase method and are shown in Figure 1. Panels a and b of Figure 1 present the interactions between gluten proteins and chokeberry fiber (CHB) used at three concentrations: 3, 6, and 9%. It was impossible to register Raman spectra for samples with 12 and 18% contents of CHB fiber because the samples were damaged during the measurements. Comparison of the difference spectra for the CHB fiber showed that changes observed in the secondary structure were concerned mainly 2097


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Journal of Agricultural and Food Chemistry (1614, 1621, and 1685 cm−1) became negative, which suggested cleavage of the hydrogen bonds inside this type of aggregates and disaggregation into antiparallel-β-sheets (positive bands at 1676 and 1694 cm−1). The appearance of a new band at 1649 cm−1 coincided with an intensity decrease of the band at 1667 cm−1 (shoulder of the band at 1658 cm−1), suggesting that fiber induced the formation of hydrogen bonds between the side-chain carbonyl groups in glutamine and polysaccharide molecules.20 With regard to β-turns, the most intense band was observed at 1659 cm−1 and was connected with hydrogen-bonded CO groups in β-turns,20 whereas bands at 1641 and 1667 cm−1 were regarded as shoulders of more intense bands. Addition of 9% of the CHB preparation may also cause aggregation of gluten proteins concerning mainly β-structures (positive bands at 1624 and 1650 cm−1). Furthermore, there was observed a band assigned to aggregates at 1602 cm−1. The cranberry (CRB) preparation as an additive caused mainly changes concerning β-like structures regardless of the preparation concentration. Bands located at 1612 cm−1 (pseudo-β-sheets), 1631 cm−1 (parallel β-sheets), 1651 cm−1 (β-turns), and 1685 cm−1 (antiparallel-β-sheets) were observed in all difference spectra (see Figure 1c−f). Similar to the chokeberry fiber preparation, other changes in the secondary structure concerned β-turns. Detailed analysis of the CRB3 spectrum showed that the most intense bands were located at 1648 and 1655 cm−1. Both bands could be assigned to the carbonyl groups in β-turns, which participated in H-bonding.20 Part of the CO groups in β-turns did not participate in the formation of hydrogen bonds due to the presence of the positive band at 1672 cm−1. The presence of a negative band at 1698 cm−1 and two positive bands at 1612 and 1687 cm−1 suggested the aggregation of gluten proteins in the form of antiparallel-β-sheets between two protein complexes19 and the formation of additional hydrogen bonds inside the antiparallelβ-sheets in the same protein complex,20 respectively. Addition of 6% of the CRB fiber caused a decrease in the content of the antiparallel-β-sheets rich in H-bonds (1686 cm−1) and nonhydrogen-bonding β-turns (1672 cm−1). Simultaneously, there was observed an increase in the intensity of the bands connected with pseudo-β-sheets (1612 cm−1), aggregated βsheets (1622 cm−1), and hydrogen-bonded β-turns (1651 cm−1) as seen in Figure 1c. The biggest changes observed in the CRB9 spectrum referred to the β-turn bands (1649, 1661, and 1673 cm−1). There was also a positive band assigned to aggregates (1608 cm−1). The CRB12 spectrum presented four negative bands connected with the lack (1668 and 1693 cm−1) and presence (1639 and 1682 cm−1) of intramolecular hydrogen bonded β-structures. The most intense positive bands observed on this spectrum (1612, 1622, and 1648 cm−1) could be connected with the formation of intermolecular hydrogen bonds, and their presence suggested aggregation of the gluten proteins. Comparison of the CRB12 and CRB18 spectra (see Figure 1f) showed that the most intense bands could be connected with pseudo-β-sheets (1613 cm−1) and hydrogen-bonded β-turns (1650 cm−1), which were regarded as signs of aggregation. Furthermore, there was observed a shift of the band from 1622 to 1628 cm−1, which indicated cleavage of the intermolecular H-bonds between β-sheets.18 The difference spectra of the carrot fiber are presented in Figure 1g-h. The CRR fiber caused changes in the region of aggregates (1605 cm−1), β-turns with intramolecular hydrogen bonds (1640 cm−1), and intermolecular hydrogen bonds (1654

cm−1). The band located at 1654 cm−1 could be also assigned to α-helix, but analysis of the structural changes indicated that this band is rather connected with β-turns. Changes in the αhelix region were observed in the studies of Nawrocka et al.10 The studies concerned direct interactions between gluten proteins and dietary fiber preparations used in three different concentrations without the presence of the starch. Results of these studies showed that α-helices from two protein complexes formed antiparallel-β-sheet structures, which was illustrated by the positive band at 1695 cm−1. Detailed analysis of the spectra showed that the band at 1622 cm−1 (CRR3) shifted to 1629 cm−1 (CRR6) as a result of increasing fiber content. This shift could be connected with the cleavage of the intermolecular hydrogen bonds in β-sheets.18 As seen in Figure 1g, there were other signs of aggregation, the increasing intensity of bands at 1606, 1611, 1640, and 1654 cm−1. Addition of 9% of CRR fiber caused the appearance of the band at 1684 cm−1, which could be assigned to β-sheets rich in Hbonds.20 The interactions between gluten proteins and cacao and carob preparations are presented in Figure 1m−o and i−l, respectively. The CAC fiber caused changes in the secondary structure of the gluten proteins mainly concerning β-sheet structures: pseudo-β-sheets (1616 cm−1), β-sheets (1632 cm−1), and antiparallel-β-sheets (1683 and 1693 cm−1). Detailed analysis of the CAC3 spectrum showed that additional bands appeared at 1621 and 1663 cm−1, which could be assigned to aggregated β-sheets and β-turns, respectively. Additions of 6 and 9% of CAC fiber caused the appearance of the bands connected with aggregates (1602 cm−1), aggregated β-sheets (1625 cm−1), H-bonded β-turns (1643, 1657 cm−1), and non-hydrogen bonded β-turns (1670 cm−1). In the CAC12 spectrum, bands connected with different types of aggregates (1611, 1655, 1665, and 1686 cm−1) were observed. In the case of the carob preparation, bands assigned to aggregated H-bonded β-sheets (1635 cm−1) were observed in all difference spectra. Detailed analysis of the CAR3 spectrum showed the presence of the bands connected with aggregated β-sheets (1619 and 1625 cm−1), β-turns (1673 cm−1), and interactions between glutamins and polysaccharide molecules (1650 cm−1).20 The spectrum of the CAR6 sample showed a negative band at 1618 cm−1 and a positive band at 1692 cm−1, simultaneously. This indicated that the 6% addition of carob fiber caused disaggregation of pseudo-β-sheets into antiparallel-β-sheets. Furthermore, bands connected with interactions of carbonyl groups in β-turns were observed (1641, 1655, and 1673 cm−1). A 9% addition of CAR fiber caused hydrogen bonding in β-turns (1637 and 1655 cm−1) and interaction between glutamins and polysaccharide molecules (1648 cm−1).20 A further increase of the fiber content also changed the structure of gluten proteins in a similar way. Bands assigned to aggregates (1607 cm−1), pseudo-β-sheets (1616 cm−1), aggregated β-sheets (1636 cm−1), H-bonded β-turns (1658 cm−1), and antiparallel-β-sheets (1675 and 1693 cm−1) were observed. The Raman spectra of oat and flax preparations were recorded only for two fiber contents (3 and 6%) because it was not possible to prepare dough samples with a higher fiber content. The changes in secondary structure caused by the flax preparation are shown in Figure 1p. Changes in the structure of gluten proteins concerned β-sheet-like structures (1614 cm−1, pseudo-β-sheets; and 1684 cm−1, antiparallel-β-sheets) and βturns (1643, 1657, and 1664 cm−1). Locations of these bands 2098


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Table 1. Percentage Distribution of Disulfide Bridge Conformation for Control Sample and Fiber-Modified (CHB, CRB, CRR, CAC, CAR, OAT, and FLX) Glutena

a

Standard deviations are given in parentheses. C, constant.

addition, postulated that the addition of wheat bran induced the formation of intermolecular β-sheets from β-turns. One of the reasons for the occurrence of this phenomenon may be redistribution of water in gluten dough after bran addition, which involves partial dehydration of the gluten network. However, previous studies,10 also carried out on gluten dough, showed that the addition of fiber preparations caused the formation of intermolecular antiparallel-β-sheets from α-helices rather than β-turns. Similar changes were observed by Zhou et al.8 in gluten proteins washed out from dough. There was observed a decrease in the intensity of the band connected with α-helix with a simultaneous increase in the intensity of the band assigned to intermolecular β-sheets (pseudo-β-sheets). The authors postulated that the long chains of HMW subunits of glutenins tended to form hydrogen bonds to each other, creating intermolecular β-sheets as a result of the decrease of water availability after the addition of fiber preparation. Thus, it can be claimed that the observed formation of hydrogen bonds in the β-structures is connected with interactions between sidechain amino acids or between side-chain amino acids and polysaccharide molecules rather than gluten proteins and water molecules. It has to be noted that the band located at 1658 cm−1 can be assigned to the helical structures and is regarded as the lower spectral limit for assignment of this structure. However, previous9 and present studies indicated that the band at 1658 cm−1 was connected with hydrogen-bonded carbonyl groups in β-turns rather than α-helix. When the carbonyl groups in βturns are not hydrogen-bonded, this band is located at ca. 1670 cm−1.6 A shift of this band toward lower wavenumbers increases with the number of new hydrogen bonds. Similar results were obtained by Nawrocka et al.,9 who observed the band shifts of different lengths depending on the type of dietary fiber. Similar

indicated aggregate formation connected by hydrogen bonds. In the case of oat fiber, it was impossible to calculate the second difference spectrum. For this reason, it was difficult to determine the nature of the changes in the secondary structure of gluten proteins by oat preparation. Previous studies9,10 have shown that oat fiber interacts differently with gluten proteins compared to other fibers. Analysis of the difference spectra indicated that all dietary fiber preparations apart from oat caused similar changes in the secondary structure of gluten proteins. The most noticeable changes induced by six of seven dietary fiber additives were observed in the regions connected with hydrogen-bonded βsheets (1614 and 1684 cm−1) and β-turns (1640 and 1657 cm−1). The results suggested that the observed changes concerned mainly glutenins. Structural analysis shows that αhelical structures are characteristic for a native gluten and gliadin fractions, whereas antiparallel-β-sheet structure dominated in glutenin.21 β-Turn structures might be also related to the β-spiral domains in glutenin polypeptides.22 An increase in content of β-sheets was observed in the studies of Correa et al.,7 who found this phenomenon to be connected with protein−water interactions and hence the increasing level of glutenin hydration. According to Belton et al.,23 β-sheets and other extended structures are related to the higher chain mobility produced by increasing hydration of glutenins. As protein is hydrated, protein−protein bonds are replaced by protein−water interactions, and this would allow sufficient movement of sections in the polypeptide chain to form βsheets. However, dietary fiber preparations are known for their ability to absorb considerable amounts of water,2,24 and gluten proteins and fibers compete with each other during the process of dough mixing. Bock and Damodaran,4 who studied changes in the secondary structure of gluten dough after wheat bran 2099


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Table 2. Analysis of Aromatic Amino Acids (Tyrosine and Tryptophan) Environment for Control Sample and Fiber-Modified (CHB, CRB, CRR, CAC, CAR, OAT, and FLX) Glutena

a


conformation in comparison with control sample. Simultaneously, the other fibers except for OAT fiber decreased the number of S−S bonds. The OAT preparation did not influence S−S bonds in this conformation. In the case of the t-g-g conformation, the amount of disulfide bridges increased for CAC and OAT fibers, decreased for CRB, CRR, CAR, and FLX fibers, and remained unchanged for CHB fiber compared to control sample. The number of S−S bonds in the t-g-t conformation increased for three fibers (CRR, CAC, CAR) and remained the same for the others. Analysis of the percentage distribution for disulfide bridges conformation showed that the number of S−S bonds in the g-g-g conformation increased with increasing fiber content only for the CAC preparation, decreased for CHB, CRB, CRR, CAR and FLX fibers, and remained unchanged for the OAT preparation. The amount of SSt‑g‑g increased for CRB, CRR, and CAR fibers, remaining constant only for flax. In this case, a correlation was not established for chokeberry and cacao preparations. The number of S−S bonds in t-g-t conformation increased for two fibers, OAT and FLX, and slightly decreased only for CRR. For other preparations, a relationship was not established. Results from a previous study, in which interactions between gluten proteins and fiber preparations in gluten dough have been studied,10 showed that the same correlation was observed for the g-g-g conformation in the case of three fibers, cranberry, cacao, and flax, and for the t-g-t conformation in the case of two fibers, carob and flax. The reason for the differences in the disulfide bridges conformation can be connected with the source of the gluten proteins; that is, they derived from the model flour dough in the present studies and gluten dough in the previous studies. According to Zhou et al.,8 the increase in the number of trans−gauche−gauche and trans−gauche−trans S−S bonds led to the displacement of polypeptide chain that appeared in abnormal protein folding and subunits aggregation.

changes in this band connected with hydrogen-bonded carbonyl groups of β-turns were observed by Linlaud et al.,6 who studied interactions between gluten proteins and pectins. According to Nawrocka et al.,10 the used dietary fibers were characterized by different contents of pectin. Changes in helical structures were observed in previous studies10 concerning interactions between gluten proteins and dietary fibers in gluten dough (without starch). All difference spectra showed a negative band located in the α-helix region (1650−1658 cm−1) and a positive band assigned to antiparallel-β-sheets (ca. 1695 cm−1). These results indicated that a fiber component induced a connection of α-helices from two protein complexes to form antiparallel-β-sheet structures. Furthermore, it was suggested that starch protects the gluten proteins against undesirable changes concerning α-helical structures. Changes in Disulfide Bridge (S−S) Region. Gluten proteins contain ca. 2% of cysteine, which participates in the formation of intra- and intermolecular disulfide bridges (S−S). These bonds are extremely important for the structure and functionality of gluten proteins because they are the main target for most redox reactions that occur during dough preparation.25 Analysis of the disulfide region of the control sample showed five bands located at 503 cm−1 (SSg‑g‑g), 515 and 521sh cm−1 (SSt‑g‑g), and 530sh and 536 cm−1 (SSt‑g‑t).15 Table 1 presents the results from the deconvolution of the S−S band of the control sample and gluten proteins modified by dietary fibers. Deconvoluted spectra of the region depicting disulfide bridges for all samples are presented in Figure S1 in the Supporting Information. The control sample contained 41, 41, and 18% of disulfide bridges in the g-g-g, t-g-g, and t-g-t conformations, respectively. Similar results were obtained in a previous study9 and for gluten washed out from commercial flour.26 As shown in Table 1, 3% addition of the CRB and FLX fibers caused increases in the number of disulfide bridges in the g-g-g 2100


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charge acceptor.29 The I(850)/I(830) value increased to 2.35 for 12% CAR fiber addition but halved to 1.27 for the highest fiber content. The biggest decline in the value of the ratio, for example, the formation of the largest number of new hydrogen bonds, was observed for CAC fiber, whereas the biggest increase in the ratio value was obtained for CAR fiber. The lowest values of the ratio did not exceed the value of 0.9, which indicated the formation of moderate-to-weak hydrogen bonds.30 The ratio increase observed for the CHB and CAR fibers indicated that the OH groups can be strongly bound to a negatively charged acceptor such as a carboxylate group.31 An increase in the ratio value was observed after the addition of pectin and locust bean gum to the dough by Linlaud et al.6 According to Nawrocka et al.,10 the pectin content in the fiber preparation did not affect the value of the tyrosine doublet. However, comparison of the fiber chemical composition and values of the tyrosine doublet indicated a relationship between the content of polyphenols other than anthocyanins and the ratio value for the CHB and CAR preparations.9 Taddei et al.13 studied interactions between anthocyanins and gliadins, and the tyrosine doublet ratio decreased for gliadins−anthocyanins mixtures. A decrease in the I(850)/I(830) ratio was also observed after the addition of the konjac glucomannan (watersoluble polysaccharide) to the dough characterized by 55% water absorption by Zhou et al.8 Dough samples enriched by dietary fiber preparations in the present study were characterized by water absorption of ca. 60%. Thus, it can be said that compounds rich in hydroxyl groups, for example, polysaccharides, compete for water with gluten proteins. The tryptophan (TRP) band at 760 cm−1 has been proposed as an indicator of the strength of H-bonding and hydrophobicity of the indole ring.6 A decrease in the band intensity suggests that TRP residues come from a buried hydrophobic environment and contribute to the formation of a more disordered structure. In contrast, an increase in the band intensity is connected with the opposite phenomenon. The addition of dietary fiber preparations caused strong changes in the intensity of the TRP band for all samples except for CRB fiber as seen in Table 2. It can be said that the intensity of the TRP band remained constant for the CRB fiber. Similar results were obtained by Nawrocka et al.,10 who studied interactions between gluten proteins and fiber preparations in the gluten dough. Generally, the addition of dietary fibers caused a 2−3fold increase in the intensity of the TRP band. These results suggested an increment in the burriedness of the TRP residues that contributed to the more ordered structure. Similar results were obtained for gluten proteins after the addition of sodium stearoyl lactylate by Ferrer et al.27 and of locust bean gum by Linlaud et al.6 Thermogravimetric Analysis. The thermogravimetric parameters, weight loss and degradation temperature (Td), are shown in Table 3. The thermal degradation profiles and their first derivatives are presented in Figure S2 in the Supporting Information. Initial weight loss in the gluten samples at 60−150 °C (see Figure S2 in the Supporting Information) is connected with the loss of free and bound water with increasing temperature. Further increase in the temperature caused breakage of the covalent peptide bonds, disulfide bridges, and O−N and O−O linkages, leading to decomposition of the gluten proteins.16 The degradation temperature (Td) and weight loss for control sample were 322 °C and 75.4%, respectively. Similar results were obtained by Wang et al.,21 who studied the thermal properties of gluten

Analysis of the S−S bond location (see Table 1 and Figure S1 in the Supporting Information) showed that the band corresponding to the t-g-t conformation was successfully reconvoluted with one Gaussian component with the maximum at 520 cm−1 after the addition of CHB and CAC fibers for all fiber contents. In the case of the other fibers and control sample, deconvolution of the spectrum in the t-g-g region required application of two Gaussians located at 515 and 521 cm−1. The band at 515 cm−1 is regarded as indicative for the formation of intrachain disulfide bonds.27 Disappearance of this band after the addition of some fiber preparations might be connected with cleavage of the intrachain disulfide bonds. In the case of CRR and CAR preparations, it could be assumed that the increasing content of dietary fiber affected the cleavage of the S−S bonds. The opposite tendencythese bonds appear after the addition of the higher amount of fiberwas observed for CRB and FLX fibers, whereas OAT fiber did not influence the intrachain S−S bridges. Generally, it can be said that the observed transformation of the disulfide bridges from stable conformation (g-g-g) to less stable conformations (t-g-g and t-g-t) led to weakening of the gluten network. Weakening of the protein structure could be caused by the limited access of the gluten proteins to water28 because the dietary fiber preparations absorb considerable amounts of water. Rhazi et al.28 have shown that during the dehydration process, SH groups disappeared because they participated in the formation of disulfide bridges. The studies of Zhou et al.8 also confirmed that S−S bridges adopted a more stable conformation (g-g-g) in conditions of high water absorption (81%). Changes in Aromatic Amino Acid Environment. Raman bands corresponding to the oscillation of two amino acids, tyrosine (TYR) and tryptophan (TRP), provided information about H-bonding (tyrosine doublet) and hydrophobic environment (TRP band at 760 cm−1). Although disulfide bridges are assumed to be the main bonds responsible for gluten network formation during dough mixing, hydrogen bonds, formed by tyrosine residues, also participate in this process.25 The tyrosine doublet (I(850)/I(830)) is regarded as a good indicator of hydrogen bonding by the phenolic hydroxyl group. A decrease in the tyrosine doublet value reflected an increase in burriedness, suggesting possible involvement of TYR residues in intermolecular or intramolecular interactions. I(850)/I(830) for the control sample is 1.72. This value is slightly higher in comparison with those obtained by Nawrocka et al.9 and Ferrer et al.27 As shown in Table 2, the I(850)/I(830) values decreased after 3% addition of dietary fiber for almost all preparations other than CRB, for which this ratio remained constant. Further increase in content of dietary fiber preparations caused an increase of the ratio value for only two fibers, CHB and CAR, and decreases of the ratio for CRR, CAC, OAT, and FLX. Correlation between the value of the tyrosine doublet and content of the fiber preparation was not established for the CRB fiber. In the case of the CRB fiber, the value of the tyrosine doublet decreased for the 3, 6, and 9% fiber contents and increased for the highest contents (12 and 18%). However, an opposite relationship was observed for the CAR fiber. Similar results were obtained by Ferrer et al.,27 who studied interactions between gluten proteins and emulsifiers in dough. The authors suggested that an increase in the tyrosine doublet was connected with the fact that the tyrosine residues are extremely strongly hydrogen bonded and act as a positive 2101


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the more open and weak gluten structure, whereas a decrease can be attributed to the more compact and stronger gluten structure. Addition of three fibers (CHB, CAC, and CAR) caused decreases in weight loss; CRB, CRR, and OAT did not change the parameter. In the cases of CAC, CAR, and OAT fibers, the TGA results are in good agreement with the results of rheological studies obtained previously.11 Extensional tests showed that CAC and CAR contributed to the considerable increase in the dough resistance to extension, which was >2fold higher on average compared with the control sample, whereas OAT did not influence the parameter value. The observed increase in the resistance to extension indicated the formation of a more compact and stronger gluten network in dough. The increase of the FLX fiber addition from 3 to 6% caused a slight increase in the weight loss. It could also be confirmed by the extensional studies of Koca and Anil.32 Using 5% addition of the flaxseed flour to the wheat dough, they observed an increase in the resistance to extension. However, further increase in the flaxseed content decreased this parameter value. Furthermore, the slope coefficients of the simple linear regression of the weight loss depending on the content of dietary fiber for five fibers (CHB, CRB, CRR, CAC, and CAR) were determined. The regression was not established for the OAT and FLX fibers due to the small number of data. Values of the slope were −0.44, −0.37, −0.27, −0.13, and −0.06 for CHB, CAR, CAC, CRB, and CRR, respectively. The last two values are regarded as insignificant. It can be said that the slope illustrates the power of the fiber influence on the gluten protein folding/aggregation. Hence, the fibers in terms of folding/ aggregation power can be sorted in the descending order CHB > CAR > CAC > CRR > CRB. Table 3 and Figure S2 in the Supporting Information additionally present thermal properties of the dietary fiber preparations. The degradation temperature took a few values for the fiber preparations and was probably connected with the chemical composition of the fiber. The degradation temperature is observed in the ranges 327−450 °C for cellulose,33 200−327 °C for hemicellulose,33 200−500 °C for lignin,34 and 180−270 °C for pectins.35 Conclusions. FT-Raman spectroscopy and thermogravimetry revealed that the addition of dietary fiber preparations caused changes in the structure of the gluten proteins, leading to a more complex and stronger network (possibility of protein aggregation or abnormal folding). Analysis of the Raman difference spectra indicated that all dietary fibers apart from oat caused similar changes in the secondary structure of gluten proteins concerning hydrogen-bonded β-sheets and β-turns. The results suggested that the observed formation of hydrogen bonds in the β-structures can be connected with interactions between side-chain amino acids or between side-chain amino acids and polysaccharide molecules rather than gluten proteins and water molecules. Aggregation or abnormal folding of gluten proteins was confirmed by the thermogravimetric analysis in the case of chokeberry, cacao, and carob fibers. The mechanism of interactions between dietary fibers and gluten proteins is complex and probably depends on the chemical composition of the dietary fiber preparations. For this reason, further studies in this field are needed.

Table 3. Thermogravimetric Parameters, Weight Loss at 600 °C and Degradation Temperature (Td), for Control Sample and Fiber-Modified (CHB, CRB, CRR, CAC, CAR, OAT, and FLX) Glutena

a


proteins during frozen storage. As seen from Table 3, addition of the fiber preparation caused changes in weight loss, whereas the degradation temperature remained unchanged. The changes in the weight loss probably depend on the weight loss of the dietary fiber preparation as suggested by the results shown in Table 3. If the value of the weight loss was lower in comparison with the control sample, the parameter value decreased. If the weight loss was higher than or comparable to that of the control sample, the parameter remained constant. The constant value of the degradation temperature indicated that the gluten proteins modified by dietary fibers remain thermally stable. According to Khatkar et al.,16 changes in the weight loss provide information about the structure of the gluten network. An increase in the parameter value suggested the formation of 2102


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(10) Nawrocka, A.; Szymańska-Chargot, M.; Miś, A.; Kowalski, R.; Gruszecki, W. I. Raman studies of gluten proteins aggregation induced by dietary fibres. Food Chem. 2016, 194, 86−94. (11) Nawrocka, A.; Miś, A.; Szymańska-Chargot, M. Characteristics of relationships between structure of gluten proteins and dough rheology − influence of dietary fibres studied by FT-Raman spectroscopy. Food Biophys. 2016, 11, 81−90. (12) Sivam, A. S.; Sun-Waterhouse, D.; Perera, C. O.; Waterhouse, G. I. N. Application of FT-IR and Raman spectroscopy for the study of biopolymers in breads fortified with fibre and polyphenols. Food Res. Int. 2013, 50, 574−585. (13) Taddei, P.; Zanna, N.; Tozzi, S. Raman characterization of the interactions between gliadins and anthocyanins. J. Raman Spectrosc. 2013, 44, 1435−1439. (14) Herrero, A. M.; Carmona, P.; Cofrades, S.; Jimenez-Colmenero, F. Raman spectroscopic determination of structural changes in meat batters upon soy protein addition and heat treatment. Food Res. Int. 2008, 41, 765−772. (15) Sugeta, H. Normal vibrations and molecular conformations of dialkyl disulphides. Spectrochim. Acta, Part A 1975, 31, 1729−1737. (16) Khatkar, B. S.; Barak, S.; Mudgil, D. Effects of gliadin addition on the rheological, microscopic and thermal characteristics of wheat gluten. Int. J. Biol. Macromol. 2013, 53, 38−41. (17) Popineau, Y.; Bonenfant, S.; Cornec, M.; Pezolet, M. A study by infrared spectroscopy of the conformations of gluten proteins differing in their gliadin and glutenin composition. J. Cereal Sci. 1994, 20, 15− 22. (18) Mangavel, C.; Barbot, J.; Popineau, Y.; Gueguen, J. Evolution of wheat gliadins conformation during film formation: A Fourier transform infrared study. J. Agric. Food Chem. 2001, 49, 867−872. (19) Juszczyk, P.; Kołodziejczyk, A. S.; Grzonka, Z. FTIR spectroscopic studies on aggregation process of the β-amyloid 11− 28 fragment and its variants. J. Pept. Sci. 2009, 15, 23−29. (20) Secundo, F.; Guerrieri, N. ATR-FT/IR study on the interactions between gliadins and dextrin and their effect on protein secondary structure. J. Agric. Food Chem. 2005, 53, 1757−1764. (21) Wang, P.; Xu, L.; Nikoo, M.; Ocen, D.; Wu, F.; Yang, N.; Jin, Z.; Xu, X. Effect of frozen storage on the conformational, thermal and microscopic properties of gluten: Comparative studies on gluten-, glutenin- and gliadin-rich fractions. Food Hydrocolloids 2014, 35, 238− 246. (22) Wellner, R.; Mills, E. N. C.; Brownsey, G.; Wilson, R. H.; Brown, N.; Freeman, J.; Halford, N. G.; Shewry, P. R.; Belton, P. S. Changes in protein secondary structure during gluten deformation studied by dynamic Fourier transform infrared spectroscopy. Biomacromolecules 2005, 6, 255−261. (23) Belton, P. S.; Colquhoun, I. J.; Grant, A.; Wellner, N.; Field, J. M.; Shewry, P. R.; Tatham, A. S. FTIR and NMR studies on the hydration of a high-Mr subunit of glutenin. Int. J. Biol. Macromol. 1995, 17, 74−80. (24) Skendi, A.; Papageorgiou, M.; Biliaderis, C. G. Effect of barley bglucan molecular size and level on wheat dough rheological properties. J. Food Eng. 2009, 91, 594−601. (25) Wieser, H. Chemistry of gluten proteins. Food Microbiol. 2007, 24, 115−119. (26) Gomez, A. V.; Ferrer, E. G.; Anon, M. C.; Puppo, M. C. Changes in secondary structure of gluten proteins due to emulsifiers. J. Mol. Struct. 2013, 1033, 51−58. (27) Ferrer, E. G.; Gomez, A. V.; Anon, M. C.; Puppo, M. C. Structural changes in gluten protein structure after addition of emulsifier. A Raman spectroscopy study. Spectrochim. Acta, Part A 2011, 79, 278−281. (28) Rhazi, L.; Cazalis, R.; Aussenac, T. Sulfhydryl-disulphide changes in storage proteins of developing wheat grain: influence on the SDS-unextractable glutenin polymer formation. J. Cereal Sci. 2003, 38, 3−13. (29) Honzatko, R. B.; Williams, R. W. Raman spectroscopy of avidin: secondary structure, disulphide conformation, and the environment of tyrosine. Biochemistry 1982, 21, 6201−6205.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b05712. Band profile (band position, bandwidth, band amplitude, and fwhm) of Gaussian components of S−S region for all samples; deconvoluted disulfide bridge region for modified samples by CHB, CRB, CRR, CAC, CAR, FLX, and OAT fibers; thermogravimetric profiles and derivative thermogravimetric profiles of gluten proteins (control sample), gluten proteins modified by dietary fiber preparations, and dietary fiber preparations (PDF)

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AUTHOR INFORMATION

Corresponding Author

*(A.N.) E-mail: [email protected]. Phone: +48 81 744 50 61. Fax: +48 81 744 50 67. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED AGR, aggregates; aβS, antiparallel-β-sheet; βS, β-sheet; βT, βturns; CAC, cacao fiber; CAR, carob fiber; CHB, chokeberry fiber; CMC, carboxymethylcellulose; CRB, cranberry fiber; CRR, carrot fiber; FLX, flax fiber; FT-Raman, Fourier transform Raman spectroscopy; HMP, pectin of high methylation degree; HPMC, hydroxypropylmethylcellulose; LMP, pectin of low methylation degree; MCC, microcrystalline cellulose; OAT, oat fiber; pβS, pseudo-β-sheet; SSg‑g‑g, disulfide bridges in gauche− gauche−gauche conformation; SSt‑g‑g, disulfide bridges in trans−gauche−gauche conformation; SSt‑g‑t, disulfide bridges in trans−gauche−trans conformation; TGA, thermogravimetry; TRP, tryptophan; TYR, tyrosine

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REFERENCES

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