Structure and Dynamics of Flour by Solid State NMR - American

Biomacromolecules 2004, 5, 1536-1544

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Structure and Dynamics of Flour by Solid State NMR: Effects of Hydration and Wheat Aging Lucia Calucci Istituto per i Processi Chimico-Fisici del CNR, Area della Ricerca di Pisa, via G. Moruzzi 1, 56124, Pisa, Italy

Luciano Galleschi Dipartimento di Scienze Botaniche dell’Universita` degli Studi di Pisa, via L. Ghini, 56126, Pisa, Italy

Marco Geppi* and Giulia Mollica Dipartimento di Chimica e Chimica Industriale dell’Universita` degli Studi di Pisa, via Risorgimento 35, 56126, Pisa, Italy Received February 10, 2004; Revised Manuscript Received April 1, 2004

The effects of accelerated aging of wheat seeds on structural and dynamic properties of dry and hydrated (ca 10 wt % H2O) flour at a molecular level were investigated by several high and low resolution solid-state NMR techniques. Identification and characterization of domains with different mobility was performed by 13 C direct excitation (DE) and cross-polarization (CP) magic angle spinning (MAS), as well as by 1H static and MAS experiments. 1H spin-lattice relaxation times (T1 and T1F) measurements were carried out to investigate molecular motions in different frequency ranges. Experimental data show that the main components of flour (starch and gluten proteins) are in a glassy phase, whereas the mobile fraction is constituted by lipids and, in hydrated samples, absorbed water. A lower proportion of rigid domains, as well as an increased dynamics of all flour components are observed after both seeds aging and flour hydration. Linear average dimensions between 20 and 200 Å are estimated for water domains in hydrated samples. Introduction Wheat flour is the most important ingredient in baked food products. Rheological properties of dough, which are in turn determined by flour quality and water-flour interactions, are recognized to be central to the successful manifacturing of bakery products. Flour characteristics are mainly determined by the quality of wheat seeds, that is by wheat texture and cultivar.1 Moreover, flour baking quality has been found to be affected by storage conditions of the precursor grain.2 Accelerated aging (incubation at high temperature and relative humidity) is a method employed to foresee seed storage potential.3-5 Wheat seeds subjected to accelerated aging lose their germinability,6-9 and degradation of their components (starch, proteins and lipids) has been observed.4-12 In particular, accelerated aging performed at 100% relative humidity and 35-45 °C results in an increase of soluble sugars and a decrease in starch and protein content.7-9,12 As a consequence, flour is obtained from aged seeds in low yield and with poor nutritive and baking quality. However, to the best of our knowledge, no studies are reported in the literature on the effects of accelerated aging of seeds on structural and dynamic properties of flour at a molecular level. Among the techniques allowing flour to be studied in the solid state, either dry or hydrated, solid state NMR (SS* To whom correspondence should be addressed. Tel: +39 0502219289. Fax: +39 0502219260. E-mail: [email protected].

NMR) spectroscopy has proved to be particularly successful in investigating local molecular structure, conformational order, and dynamics. In the last three decades, SS-NMR has been increasingly applied to characterize the main components of flour: carbohydrates (∼75 wt %), proteins (∼10 wt %), lipids (∼2 wt %), and water (∼12 wt %). Particular emphasis has been given to the investigation of amorphous and crystalline states of starch (see Tang et al.13 and references therein), properties of gluten proteins (see Calucci et al.14 and references therein), water organization and mobility in these systems, as well as influence of hydration on their structural and dynamic properties. 13C magic angle spinning (MAS) NMR spectra of flour have been reported and assigned to starch and protein components;15-18 the mobility of water in doughs and flour suspensions has been studied by proton relaxation.19,20 Nevertheless, NMR data on flour remain quite sparse in the literature. In the present work, we combine high resolution (13C direct-excitation (DE) and cross-polarization (CP) MAS and 1 H-MAS) and low resolution (free induction decay (FID) analysis and wide-line measurements of proton spin-lattice relaxation times in both the laboratory (T1) and rotating (T1F) frames) SS-NMR techniques to study the structural characteristics of flour components in different domains and dynamic states, the mobility of flour components and water, and the size of the domains they are located in. Measurements are performed on flour samples from bread wheat

10.1021/bm0499177 CCC: $27.50 © 2004 American Chemical Society Published on Web 05/04/2004

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Structure and Dynamics of Flour by SS-NMR

(Triticum aestiVum cv Centauro) seeds either unaged or subjected to accelerated aging for 4 and 10 days to highlight changes induced by the aging process in the microscopic properties of flour components. Both hydrated and dry samples were studied in order to discern the influence of water on intrinsic flour characteristics as well as on NMR spectral and relaxation behavior. Experimental Section Materials. Accelerated aging was performed according to Delouche and Baskin3 by incubation of wheat seeds (Triticum aestiVum cv Centauro) at 40 °C and 100% relative humidity for 4 and 10 days, as previously reported.9 Seeds were then air-dried under a laminar flow hood until they reached their original weight and ground in a break rollmill (Labormill 4RB, Italy); the so obtained flour was stored at -20 °C. Flour samples from seeds untreated and treated for 4 and 10 days contained 14, 11, and 8 ( 1 wt % water, respectively, as determined by calculating the weight loss after the samples were oven dried to constant weight (three replicates were considered for each treatment period). In the following, we will refer to these samples as “naturally hydrated” flours. Dry flour samples were prepared from naturally hydrated ones by pumping under vacuum (10-2 mbar) for 6 h and further dehydrating in a vacuum desiccator over P2O5 for at least 5 days. A sample containing about 12 wt % water was prepared by placing dry flour from untreated wheat over a saturated solution of K2CO3 at room temperature for 3 days (44% relative humidity).21 The quantity of absorbed water was determined gravimetrically. We will refer to this sample as “artificially hydrated” flour. NMR Measurements. High resolution 13C and 1H NMR experiments were performed at 20.0 ( 0.1 °C on a doublechannel Varian InfinityPlus 400 spectrometer, working at 399.89 MHz for proton and at 100.75 MHz for carbon-13, equipped with either a 7.5 mm or a 3.2 mm CP-MAS probehead. 1H and 13C 90° pulse lengths were 4.0 and 1.8 µs on 7.5 and 3.2 mm probeheads, respectively. 1H spectra were recorded with 16 scans, using a recycle delay of 5 s, at different MAS frequencies ranging from 0 to 7 kHz (7.5 mm rotors) and from 3 to 25 kHz (3.2 mm rotors). 13C CP-MAS experiments were carried out using a recycle delay of 5 s under decoupling conditions on both probeheads at different contact times, decoupling powers, and spinning frequencies; the standard CP-MAS spectra were recorded with a contact time of 500 µs, a 7 kHz MAS frequency, and 40 scans. Direct-excitation 13C spectra were recorded on the 7.5 mm probehead using a depth pulse sequence22 in order to suppress probe and rotor background signals, using a recycle delay of 2 s, a spinning rate of 7 kHz, and 1600 scans. Low resolution 1H experiments were carried out on a single-channel Varian XL-100 spectrometer interfaced with a Stelar DS-NMR acquisition system and equipped with a 5 mm probehead. These measurements were performed onresonance on static samples at a frequency of 20.00 MHz. For these experiments, flours were enclosed in standard 5

Figure 1. 13C DE-MAS (a) and CP-MAS (b) spectra of dry flour from unaged seeds recorded at 20 °C.

mm NMR glass tubes sealed on the top, with the exception of the artificially hydrated sample, which was encapsulated in a short glass tube in order to eliminate the empty space above the flour. Measurements were performed every 5 or 10° on heating from -10 to +80 °C, waiting 15 min for temperature equilibration of the sample. The temperature was controlled within 0.1 °C. The 90° pulse length was 2.4 µs and the recycle delay 5 s. Free induction decays were recorded using the quadrupolar echo pulse sequence with an echo delay of 12 µs, a dwell time of 1 µs, up to 4096 points and 512 scans. Proton spin-lattice relaxation times in the laboratory (T1) and rotating (T1F) frames were measured using the inversion-recovery and variable spin-lock time pulse sequences followed by quadrupolar echo, respectively. In both cases, 32 scans were accumulated, and the signal intensity was determined by the first point of the onresonance FID. At least 30 different delays and 32 different spin-lock times were used to measure T1 and T1F, respectively. The spin-lock field was 110 kHz. The 1H FID-T1F correlation experiment,23 which consists of recording 1H FIDs after the application of a variable spin-lock time wide-line pulse sequence, was carried out accumulating 32 scans, with the same acquisition parameters used to record the FID and to measure the T1F. Results and Discussion 13 C MAS NMR. Two kinds of 13C NMR spectra, namely C CP- and DE-MAS, were recorded on all of the flour samples at room temperature and at a spinning rate of 7 kHz; an example is shown in Figure 1. In the CP-MAS experiment,

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signals arising from carbons strongly dipolarly coupled to protons (typically 13C nuclei directly bound to protons and in a rigid environment) are remarkably enhanced. The CPMAS spectrum of flour is dominated by the peaks of the most abundant component, i.e., starch (60-110 ppm), showing only very weak peaks from aliphatic (10-40 ppm) and aromatic (∼130 ppm) side chain carbons and from main chain peptide carbonyl carbons (∼175 ppm) of gluten proteins, in agreement with spectra previously reported in the literature.15-18 Possible contributions in the 40-65 ppm spectral region from protein main-chain carbons cannot be excluded. Starch signals show very broad lines, allowing only the C-1 (∼102 ppm) and C-6 (∼62 ppm) resonances of R-Dglucose to be resolved, whereas C-2, C-3, and C-5 all resonate within the broad peak at ∼73 ppm and C-4 shows up as a high-frequency shoulder of the same peak. No signal multiplicity characteristic of starch crystalline forms13,24-27 is distinguished for the C-1 carbon signal. Since line broadening due to experimental conditions (for instance insufficient decoupling power or matching between some molecular motional frequencies and the spinning or decoupling frequencies)28 could be excluded by performing experiments at different decoupling powers and spinning frequencies (results not shown), broad signals could be unambiguously assigned to chemical shift distributions associated to conformational distributions of glucose moieties in amorphous starch.24-27 The CP dynamics of the starch peaks is typical of very rigid phases, with an optimal CP time of about 500 µs, suggesting that starch is in a glassy phase, in agreement with its phase diagram.29 Significant differences in the CP dynamics were not observed among the different starch carbons in each flour sample, nor among the different flour samples. In the DE-MAS spectra, recorded with a very short recycle delay (2 s), only fast-relaxing carbon nuclei, usually located in mobile environments, are revealed. As shown in Figure 1, in these spectra, the olefin and alkyl carbon signals from lipids, resonating at about 130 ppm and in the range 10-40 ppm, respectively,14,17 are much more intense than expected on the basis of flour composition, due to the very mobile nature of lipids in cereal seeds. It must be noticed that the carboxyl carbon signal at about 175 ppm does not show this behavior because, despite the high mobility of the lipid phase, these carbons have long spin-lattice relaxation times owing to the absence of directly bound protons. Signals from starch are also present in the DE-MAS spectra, even though characterized by a much smaller signal-to-noise ratio with respect to the CP spectra, thus confirming that starch carbons are essentially in rigid environments. However, the ratio between the intensity of the C-6 peak and that of the other starch peaks is much higher in the DE spectra with respect to the corresponding CP ones; this feature is ascribable to the shorter 13C T1 of C-6, due to conformational motions of the CH2OH groups.13 Very weak signals from mobile carbons in gluten proteins could also be present in the DE-MAS spectra, hidden by the much stronger peaks of both starch and lipids. No remarkable differences in both the 13C CP- and DEMAS spectral features were revealed among the investigated

Calucci et al.

samples, thus suggesting that structural or dynamic changes induced by seeds aging or flour hydration are not dramatic or affect only a very small fraction of flour. 1 H MAS NMR. At 20 °C, the static 1H spectra of dry flour samples consist of two unresolved signals with halfheight widths of about 2 and 50 kHz (Figure 2a), corresponding to the exponential and Pake components of the FID (see below), respectively. A bigger proportion of the narrower signal is present in the spectra of hydrated flours, where also a slight narrowing of the lines can be observed with respect to the spectra of dry flours. Signals with line widths of the order of a few Hz are never revealed, thus indicating the absence of liquidlike water. Similar findings were reported for the 1H static spectra of starch30,31 and gluten14 with low moisture content. In the spectra recorded under MAS conditions on both dry and hydrated samples (Figure 2b), some fine structure is observed even at very low spinning rates (500 Hz), arising from the resolution of the narrower line, that does not experience any significant change increasing the spinning rate above 1.5 kHz. For the dry samples, the sharp signals can be ascribed to lipids (0.9, 1.3, 1.6, 2.1, 2.2, 2.8, and 5.3 ppm), which therefore represent the most mobile fraction of flour, in agreement with what was observed in 13C DE-MAS spectra, and, to a lesser extent, to proteins (weak signals at 2.5, 4.0-4.5 ppm) (Figure 2d).14,32 In the hydrated samples, a broad signal due to bound water is also present, centered at 4.6 ppm (Figure 2c), which is indeed roughly proportional to the estimated water content. At a spinning rate of 7 kHz a partial split of the broader signal into spinning sidebands is observed (Figure 2b), ascribable to the inhomogeneous component of the homonuclear dipolar interactions;33 similar spectral features are observed at higher spinning rates (up to 25 kHz), except for the position of the sidebands. 1 H FID Analysis. 1H FIDs have been recorded on resonance on dry and hydrated flour samples between -10 and +80 °C every 5 or 10 °C on increasing the temperature; a representative FID is shown in Figure 3. At least two components with quite different decay rates can be distinguished in each FID. To reproduce the experimental decays, nonlinear least-squares fittings of acquired data were carried out using a linear combination of suitable analytical functions chosen among exponential, Weibullian, Gaussian, Abragamian, and Pake functions.34 The best results were obtained by using a weighted sum, S(t), of an exponential, E(t), and a Pake, P(t), function, describing slow and fast magnetization decays and associated with protons in mobile and very rigid domains, respectively. With this choice, a good reproduction of the experimental FID was obtained at each temperature and for each sample: an example is reported in Figure 3. In particular, the Pake function revealed to be the only function able to correctly reproduce the small hole observable in the FIDs at about 30 µs.35 The addition of a third function in the linear combination did not bring any significant improvement in the fit quality. The analytical function used in the FID analysis can thus be written as S(t) ) AE(t) + BP(t)

(1)



Figure 2. (a)1H static and (b) 1H MAS (7 kHz spinning rate) spectra of dry flour from unaged seeds recorded at 20 °C; (c) expansion of the 1H MAS (7 kHz spinning rate) spectrum of naturally hydrated flour from unaged seeds recorded at 20 °C; (d) expansion of (b).

where A and B are the weights, in arbitrary units, of the exponential and Pake functions, respectively. The exponential function E(t) ) e-(t/T2)

(2)

is characterized by the spin-spin relaxation time T2, whereas the Pake function, obtained by inverse Fourier transform of the original expression in the frequency domain,36 can be written as35 P(t) )

xπ6e

[

-β2t2/2

(x ) (x ) ]

cos Rt C xRt

6Rt +S π

6Rt sin Rt (3) π xRt

where C and S are the Fresnell functions that can be approximated to37 π 1 + 0.926x 1 sin x2 C(x) ≈ + 2 2 + 1.792x + 3.104x2 2 1 π cos x2 (4) 2 3 2 2 + 4.142x + 3.492x + 6.670x

( )

( )

π 1 1 + 0.926x S(x) ≈ cos x2 2 2 + 1.792x + 3.104x2 2 1 π sin x2 (5) 2 3 2 2 + 4.142x + 3.492x + 6.670x

( )

( )

and R ) 3γ2p/4R3HH, γ being the proton gyromagnetic ratio. The Pake function is therefore characterized by the parameters RHH and β, which, in the original formulation, represent respectively the distance between two nearest neighbor protons and the width of the Gaussian line due to the dipolar

interactions between not nearest neighbor protons. In a complex system such as flour, RHH must be intended as an average parameter. Fittings performed considering A, B, T2, RHH, and β as parameters showed a strong correlation between β and RHH; therefore, FID analyses were performed fixing RHH to the value of 1.9 Å and optimizing the other parameters. This value of RHH, which is physically meaningful for the systems investigated, gave indeed the best results in fittings performed on a large set of experimental data (FIDs recorded on different samples at different temperatures). The best fit values of the Pake weight percentage (100*B/(A + B)), β, and T2 are reported in Figure 4. At room temperature, dry samples are mainly constituted by rigid starch and gluten components (90-95% of protons contribute to the Pake function), whereas the mobile component, characterized by T2 values of about 200 µs, is essentially associated to lipids, as previously observed by 1 H MAS. A regular decreasing trend of the Pake weight percentage with increasing the temperature is observed for all the dry samples; at the same time, β values decrease. This behavior is due to an expected increase in the mobility of the system, resulting from both a higher mobility within the rigid domains and a bigger proportion of flour components in mobile environments (up to about 15% protons contribute to the exponential component). A peculiar trend of the β values as a function of temperature is observed for the flour obtained from unaged seeds, where a sudden decrease, corresponding to a remarkable increase in the mobility of the rigid domains, takes place between 20 and 30 °C, suggesting the occurrence of a solid-solid phase

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Figure 3. (a) Experimental and best-fit calculated proton FIDs recorded at 60 °C on dry flour from seeds aged for 4 days. (b) Best fit Exponential and Pake functions. (c) Residues of the fit procedure. Only the first 400 FID points are shown.

transition. A decrease of β, even though less dramatic, is also observed in the same temperature range for the corresponding naturally and artificially hydrated samples. No abrupt changes of β are found for flour samples from aged seeds, either dry or naturally hydrated. Moreover, accelerated aging of wheat results in a small decrease of the Pake component in the FIDs and in a slight increase of exponential T2. Naturally hydrated flours show lower β values with respect to the corresponding dry samples, in agreement with the plasticizing role of water in dough, as well as higher T2 values. Moreover, the percentages of protons in the exponential and Pake components agree with the known water contents in the different samples (moisture level decreases with aging time), strongly suggesting that the slowly decaying FID component (corresponding to the narrower component of the static 1H spectrum) is dominated by H2O protons. T2 values are compatible with absorbed water molecules undergoing relatively fast reorientational motions but not with the presence of free water (T2 ≈1 s). For naturally hydrated

Figure 4. Best fit parameters obtained by proton FID analysis. (a) Weight percentage of the Pake function. (b) Pake β values. (c) Exponential T2 values. Full and open symbols represent dry and hydrated samples, respectively. Circles, squares and triangles refer to flour from seeds unaged and aged for 4 and 10 days, respectively. Diamonds refer to the artificially hydrated sample. Lines are drawn to guide the eyes. Maximum estimated errors are 1% for Pake weights, 2% for β, 10 and 30% for T2 of hydrated and dry samples, respectively.



Figure 5. (a) 1H T1; (b) 1H T1F 1/PWRA (see definition of PWRA in eq 7). Full and open symbols represent dry and hydrated samples, respectively. Circles, squares, and triangles refer to flour from seeds unaged and aged for 4 and 10 days, respectively. Diamonds refer to the artificially hydrated sample. Lines are drawn to guide the eyes. Maximum errors are 2% for T1 and 10% for T1F 1/PWRA.

flours, Pake weight percentage and β values show decreasing trends with increasing the temperature, similar to those observed for the corresponding dry samples, in the temperature ranges from -10 °C to +30, +50, and +70 °C for samples with 0, 4 and 10 days aging times, respectively. At higher temperatures, an inversion of the trends occurs, that can be attributed to a progressive water loss due to evaporation. This interpretation is supported by the fact that regularly decreasing trends of Pake weight percentage and β values with increasing the temperature are observed for the artificially hydrated encapsulated sample up to 80 °C. A further evidence is represented by the decrease of the intensity of the FID first point, taking place by increasing the temperature only in naturally hydrated samples. Regular increasing trends of T2 are observed for hydrated samples on heating, even though partially contrasted at higher temperatures by water evaporation, as a consequence of the fastening of water dynamics. 1H T and T . The proton spin-lattice relaxation times 1 1G in both laboratory (T1) and rotating (T1F) frames have been measured for all of the samples in the temperature range between -10 and +80 °C in order to obtain information on dynamic processes occurring in the MHz (T1) and kHz (T1F) ranges, as well as on heterodomain dimensions on a spatial scale of tens to hundreds of angstroms. However, the extraction of dynamic information from relaxation data is not straightforward. In particular, the measured relaxation times can be remarkably different from the intrinsic ones due to the mixing effects of proton spin diffusion, efficient over a spatial scale of few hundreds of angstroms for T1 and of few tens of angstroms for T1F.38 The interpretation of relaxation data in terms of specific motional processes of flour components and, eventually, water molecules is even more complex owing to the structural and dynamic heterogeneity of the samples. In all cases, the magnetization recovery in T1 measurements was well fitted by a monoexponential function, whereas in T1F measurements, the magnetization decay was monoexponential for dry samples and biexponential for

hydrated ones. This implies that the averaging effect of spin diffusion is complete in both the T1 and T1F time scales for dry samples, whereas it is complete in the T1 time scale, but only partial in the T1F one, for hydrated samples. As mentioned above, the T1 and T1F values permit an estimation of the heterodomains average size, considering the following equation for diffusion in three dimensions:38 L ≈ x6Dst

(6)

where L is the average diffusion path length occurring in a time t and Ds is the diffusion coefficient. When a heterogeneous system consisting of domains characterized by different intrinsic relaxation times is considered, and a single, spin diffusion averaged, relaxation time is measured for all protons, t must be replaced with the experimental relaxation time value and L represents the maximum linear average domain dimension. Here, using a value of 6 × 10-16 m2/s for Ds, typical of solid polymers,39 we derive L values of 200-300 and 20-35 Å from T1 and T1F experimental values, respectively (it must be noticed that for T1F a halved value of the diffusion coefficient must be considered38). Taking into account the effects of spin diffusion above-described, the estimated average linear domain dimensions are less than about 30 Å in dry flours and in the range of 20-200 Å in hydrated ones. T1’s of 150-300 and 80-150 ms have been determined for dry and hydrated samples, respectively (Figure 5a). T1F’s range between 5 and 8 ms for dry samples (Figure 5b), whereas for hydrated samples the two T1F components have values in the ranges 1-4 and 5-15 ms. In particular, below room temperature, one of the two T1F components is of the same order of magnitude of the T1F measured in the dry samples and has a relative weight similar to that determined for the Pake function in FID analyses, suggesting that it could be roughly associated to protons in rigid domains. However, T1F values and weights do not show regular trends as a function of temperature, essentially because of the correlation among the fitting parameters in the analysis of the mag-

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netization decay. Such correlation tends to increase with increasing the temperature, as the ratio between the two T1F components decreases down to less than 3. To clarify these aspects, in the case of biexponential T1F decay, two recently developed procedures have been used: one correlates the different exponential components of the T1F decay to the different FID components23 and the other allows the proton relaxation to be completely interpreted in terms of dynamics, by discarding the multiexponential behavior and thus eliminating the spin diffusion effects.40 In the first case, FID-T1F correlation experiments were recorded on the hydrated flour sample from unaged wheat and analyzed following a suitable procedure to obtain three matrices (a, b, and c) whose i,j elements correspond to the ith FID and jth T1F components. a represents the percentage of proton nuclei corresponding to the given FID and T1F components; b, ranging from -1 to 1, gives the correlation between the two components (-1, 0, and 1 indicate no, average, and complete correlation, respectively); c is a measure of the weighted relaxation rate corresponding to the selected couple of components, allowing the proton T1F relaxation sinks to be identified.23 As an example, the results obtained for the naturally hydrated flour from unaged seeds at 0 °C are reported in Figure 6. It is possible to observe that almost 70% of protons are associated to both the Pake function and the longest T1F component and that a very strong correlation (b ) 0.70) is found between these two components, as well as between the exponential FID function and the shortest T1F component. Therefore, the two T1F components, apart from some mixing effect due to spin diffusion, can be individually associated to the rigid (mainly starch) and mobile (lipids and water) fractions of the sample. The c elements clearly indicate that two comparably important relaxation sinks are present in the rigid and mobile fractions of the sample, with the less efficient relaxation of protons in rigid domains being compensated by their larger number. Therefore, a quantitative description of spin-lattice relaxation in the rotating frame in terms of dynamics should mandatorily take into account motional processes in both rigid and mobile environments. To eliminate the effects of spin diffusion and obtain a purely “dynamic” spin-diffusion free quantity, thus simplifying the comparison between T1F values from dry and naturally hydrated flours, the inverse of the population weighted rate average (PWRA) quantity,40 defined as wi

PWRA )

∑i T

1Fi

∑i wi

(7)

where wi is the weight of the component with relaxation time T1Fi and i runs over the number of components, was used instead of the values of the individual T1F components (it must be noticed that 1/PWRA is equal to T1F in the case of a monoexponential decay). The obtained 1/PWRA values are shown in Figure 5b. For the dry samples, T1 regularly decreases by increasing temperature as expected for solid samples in the rigid-lattice

Figure 6. Graphic representation of a, b, and c matrix elements obtained from the analysis of a correlation experiment between 1H FID and T1F components, performed at 0 °C on naturally hydrated flour from unaged seeds.

regime. On the other hand, 1/PWRA is barely affected by temperature, indicating that the motions mainly contributing to proton relaxation in the rotating frame are in an intermediate motional regime. However, the quite high 1/PWRA values suggest that these motions are relative to a small


fraction of the sample only; following Tanner et al.30 these motions could be ascribed to the starch CH2OH groups. The smaller T1 and 1/PWRA determined for flours from aged seeds reflect an increased molecular mobility in both the MHz and kHz ranges. Hydration of flours results in a decrease in the T1 values, especially for samples with higher moisture level (Figure 5a), in agreement with the results shown in ref 41. A minimum in the T1 vs temperature curve is clearly observed for hydrated flour from seeds aged for 10 days (8 wt % H2O); this minimum shifts at lower temperatures for samples with higher water content (flour from seeds aged for 4 days and unaged), similarly to what was observed on starch samples.30 Furthermore, hydration causes a decrease of 1/PWRA for all of the samples below 30 °C; at higher temperatures, similar values are found for dry and hydrated samples (Figure 5b). By comparing the T1 and 1/PWRA trends for dry and artificially hydrated flour from unaged seeds, it is possible to argue that water dynamics represents an important relaxation path at low temperatures, and starch motions become more effective at higher temperatures. Conclusions Several high and low resolution SS-NMR techniques have been employed to investigate structure and dynamics of flour components, focusing on the effects of both flour hydration and wheat accelerated aging. The combination of suitable complementary experiments revealed that it is necessary to avoid possible mis-interpretations of data from single experiments, especially in the analysis of relaxation times. 13C MAS experiments showed that dry flour is essentially constituted by starch in a rigid amorphous phase; only a very small fraction of mobile components is observed, mainly ascribable to lipids. 1H MAS experiments confirmed that lipids constitute a very mobile phase and that, in hydrated flours, water is located in relatively mobile domains, even though no free water was detected. However, high-resolution MAS experiments resulted scarcely sensitive in revealing structural and dynamic changes caused by flour hydration and/or seeds accelerated aging. To this regard, low-resolution 1H FID analysis and spin-lattice relaxation times measurements revealed much more information and allowed slight changes in the dynamics of flour components to be highlighted. In particular, it was found that wheat aging results in a slightly higher amount of flour mobile fractions, as well as in a fastening of the dynamics in both the rigid and mobile components. Moreover, flour from unaged wheat seeds showed a solid-solid phase transition at about room temperature, not present in samples from aged seeds. The observed microscopic behavior might be roughly related to the deterioration of flour biopolymers induced by wheat aging (i.e., decrease of soluble protein content and deterioration of storage proteins of wheat); 9,12 however, further efforts are required to draw strict correlations between NMR findings and changes in flour composition. Dry flours were found to be homogeneous on a 30 Å spatial scale. Hydration of flour at a very low moisture level (8-14 wt % water) causes a minor plasticization of the rigid components. In


hydrated samples, water is located in domains with average linear dimensions between 20 and 200 Å. Acknowledgment. We thank Dr. Silvia Ghiringhelli for technical assistance in the preparation of flour samples and Prof. Carlo Alberto Veracini for helpful discussions. This work was financially supported by CNR-Agenzia 2000. References and Notes (1) Cornell, H. J., Hoveling, A. W. Wheat Chemistry and Utilization; Technomic Publishing Co. Inc.: Basel, Switzerland, 1998. (2) Gras, P. W.; O’Riordan, B. Australian PostharVest Technical Conference, 1998. (3) Delouche, J. C.; Baskin, C. C. Seed Sci. Technol. 1973, 1, 427452. (4) McDonald, M. B. Seed Sci. Technol. 1999, 27, 177-237. (5) McDonald, M. B. Seed Sci. Res. 1998, 8, 265-275. (6) Ellis R. H.; Roberts E. H. Seed Sci. Technol. 1982, 9, 373-409. Roberts, E. H. Physiology of Seed Deterioration. Crop Science Society of America: Madison, WI, 1986; Vol. 11, pp 101-123. Stoyanova, S. D. Seed Sci. Technol. 1991, 19, 363-371. (7) Galleschi, L.; Capocchi, A.; Ghiringhelli, S.; Saviozzi, F.; Calucci, L.; Pinzino, C.; Zandomeneghi, M. J. Agric. Food. Chem. 2002, 50, 5450-5457. (8) Krishnan P.; Nagarajan, S.; Dadlani, M.; Moharir, A. V. Seed Sci. Technol. 2003, 31, 541-550. (9) Calucci, L.; Capocchi, A.; Galleschi, L.; Ghiringhelli, S.; Pinzino, C.; Saviozzi, F.; Zandomeneghi, M. J. Agric. Food. Chem. in press. (10) Dell’Aquila, A. Seed Sci. Res. 1994, 4, 293-298. (11) Rehman, Z. U.; Shah, W. H. Plant Food Hum. Nutr. 1999, 54, 109117. (12) Ghiringhelli, S. Ph.D. Thesis, Universita` degli Studi di Pisa, 2003. (13) Tang, H.; Hills, B. P. Biomacromolecules 2003, 4, 1269-1276. (14) Calucci, L.; Forte, C.; Galleschi, L.; Geppi, M.; Ghiringhelli, S. Int. J. Biol. Macromol. 2003, 32, 179-189. (15) O’Donnell, D. J.; Ackerman, J. J. H.; Maciel, G. E. J. Agric. Food Chem. 1981, 29, 514-518. (16) Garbow, J. R.; Schaefer, J. J. Agric. Food Chem. 1991, 39, 877880. (17) Gil A. M.; Alberti E.; Santos D. In Magnetic Resonance in Food Science-A View to the Next Century; Webb, G. A., Belton, P. S., Gil, A. M., Delgadillo, I., Eds.; The Royal Society of Chemistry: Cambridge, U.K., 2001; pp 43-53. (18) Baik, M.-Y.; Dickinson, L. C.; Chinachoti, P. J. Agric. Food Chem. 2003, 51, 1242-1248. (19) Richardson, S. J.; Baianu, I. C.; Steinberg, M. P. J. Agric. Food Chem. 1986, 34, 17-23. (20) Ruan, R. R.; Wang, X.; Chen, P. L.; Fulcher, R. G.; Pesheck, P.; Chakrabarti, S. Cereal Chem. 1999, 76, 231-235. (21) Hartley, L.; Chevance, F.; Hill, S. E.; Mitchel, J. R.; Blanshard, J. M. V. Carbohydr. Polym. 1995, 28, 83-89. (22) Cory, D. G.; Ritchey, W. M. J. Magn. Reson. 1988, 80, 128-132. (23) Geppi, M.; Kenwright, A. M.; Say, B. J. Solid State Nucl. Magn. Reson. 2000, 15, 195-199. (24) Gidley, M. J.; Bociek, S. M. J. Am. Chem. Soc. 1985, 107, 70407044. (25) Veregin, R. P.; Fyfe, C. A.; Marchessault, R. H.; Taylor, M. G. Macromolecules. 1986, 19, 1030-1034. (26) Morgan, K. R.; Furneaux, R. H.; Larsen, N. G. Carbohydr. Res. 1995, 276, 387-399. (27) Bogracheva, T. Ya.; Wang, Y. L.; Hedley, C. L. Biopolymers 2000, 58, 247-259. (28) Rothwell, W. P.; Waugh, J. S. J. Chem. Phys. 1981, 74, 2721-2732. (29) Cuq, B.; Abecassis, J.; Guilbert, S. Int. J. Food Sci. Technol. 2003, 38, 759-766. (30) Tanner, S. F.; Hills, B. P.; Parker, R. J. Chem. Soc. Farday Trans. 1991, 87, 2613-2621. (31) Kou, Y.; Dickinson, L. C.; Chinachoti, P. J. Agric. Food Chem. 2000, 48, 5489-5495. (32) Sacco, A.; Neri Bolsi, I.; Massini, R.; Spraul, M.; Humpfer, E.; Ghelli, S. J. Agric. Food Chem. 1998, 46, 4242-4249. (33) Schnell, I.; Spiess, H. W. J. Magn. Reson. 2001, 151, 153-227. (34) Hansen, E. W.; Kristiansen, P. E.; Pedersen, N. J. Phys. Chem. B 1998, 102, 5444-5450.

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Structure and Dynamics of Flour by Solid State NMR - American

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