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Crystallinity changes in wheat starch during the bread-making process: Starch crystallinity in the bread crust
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Journal of Cereal Science 45 (2007) 219–226 www.elsevier.com/locate/yjcrs
Crystallinity changes in wheat starch during the bread-making process: Starch crystallinity in the bread crust C. Primo-Martı´ na,, N.H. van Nieuwenhuijzena,b, R.J. Hamera,c, T. van Vlieta,b a
Wageningen Center for Food Sciences, Wageningen, the Netherlands b Wageningen University, Wageningen, the Netherlands c TNO Quality of Life, Zeist, the Netherlands
Received 13 June 2006; received in revised form 23 August 2006; accepted 24 August 2006
Abstract The crystallinity of starch in crispy bread crust was quantified using several different techniques. Confocal scanning laser microscopy (CSLM) demonstrated the presence of granular starch in the crust and remnants of granules when moving towards the crumb. Differential scanning calorimetry (DSC) showed an endothermic transition at 70 1C associated with the melting of crystalline amylopectin. The relative starch crystallinity, as determined by X-ray and DSC, from different types of breads was found to lie between 36% and 41% (X-ray) and between 32% and 43% (DSC) for fresh bread crust. Storage of breads in a closed box (22 1C) for up to 20 days showed an increase in crust crystallinity due to amylopectin retrogradation both by X-ray and DSC. However, DSC thermograms of 1-day old bread crust showed no amylopectin retrogradation and after 2 days storage, amylopectin retrogradation in the crust was hardly detectable. 13C CP MAS NMR was used to characterize the physical state of starch in flour and bread crumb and crust. The intensity of the peaks showed a dependence on the degree of starch gelatinization. Comparison of the results for two different types of bread showed that the baking process influenced the extent of starch crystallinity in the bread crust. Amylopectin retrogradation, which is the main process responsible for the staling of bread crumb, cannot be responsible for crispness deterioration of the crust as amylopectin retrogradation upon storage of breads could only be measured in the crust after 2 days storage. Under the same conditions loss of bread crust crispness proceeds over shorter times. r 2006 Elsevier Ltd. All rights reserved. Keywords: Crust; Bread; Crystallinity; Starch; X-ray; DSC; CSLM; NMR
1. Introduction The structure and phase transitions of starch are topics of considerable interest, since they are related to the functionality of the starch (Biliaderis, 1992). Native starch is present as semi-crystalline granules. The semi-crystalline properties of native starch are related to the short-chain fraction of amylopectin arranged as double helices and Abbreviations: CSLM, confocal scanning laser microscopy; 13C CP/ MAS NMR, Solid-state 13C cross polarization magic angle spinning nuclear magnetic resonance; DRC, relative crystallinity determined using DSC; DSC, differential scanning calorimetry; PLM, polarized light microscopy; RH, relative humidity; XRC, relative crystallinity determined using X-ray Corresponding author. Tel.: +31 317 475120; fax: +31 317 475347. E-mail address: [email protected] (C. Primo-Martı´ n). 0733-5210/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jcs.2006.08.009
packed in small crystallites (Imberty et al., 1991; Sarko and Wu, 1978; Svensson and Eliasson, 1995). Linear amylose molecules are apparently present in an amorphous state in the granule (Zeleznak and Hoseney, 1987). The physical properties of wheat flour starch are subjected to changes during baking and subsequent storage of bread. These transformations largely determine the structure and texture of the solid matrix of the final product (Zobel and Kulp, 1996). When starch is suspended in water and heated it undergoes a series of processes called gelatinization. The changes occurring during gelatinization are (not in order of occurrence): swelling, loss of birefringence, melting, loss of crystallinity, increase of viscosity of the suspension and leaching of amylose from the granules. During the baking process, the starch in the bread crumb is gelatinized resulting in an amorphous
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structure. During storage of the bread, the crystalline structure of the starch is slowly recovered at the shortrange scale (double helices) (Keetels et al., 1996). This process is commonly known as retrogradation. Retrogradation is technologically important since it produces significant changes in the mechanical properties of the crumb affecting its sensory appreciation. The characterization of starch in bread crumb and its role during bread staling have been extensively studied (Hug-Iten et al., 2001, 2003; Jime´nez and Martı´ nez-Anaya, 2001; Morgan et al., 1992). This is however not the case for bread crust. In the crust the presence of intact, non-swollen starch granules has been described previously (Moss, 1975; Pomeranz et al., 1984; Primo-Martı´ n et al., 2006). But, little attention has been paid to the aging of the crust, its structural composition, the state of starch in the crust (Primo-Martı´ n et al., 2006) and its possible significance in the loss of crispness. In this work the crystallinity of the starch in the bread crust was studied as the extent of crystallinity most likely influences crust properties as well as subsequent changes during storage. The crystallinity of starch in the crust was determined using differential scanning calorimetry (DSC), wide angle X-ray diffraction, 13C cross polarization magic angle spinning nuclear magnetic resonance (13C CP MAS NMR) and polarization microscopy which provides complementary information about the state of the starch. In addition, the state of the starch in the crust, in the crumb and in the flour were compared. Confocal scanning microscopy (CSLM) was used to gain an insight into the organisation of the bread crumb and crust. 2. Materials and methods 2.1. Materials Breads, rusk rolls and crispy rolls, were prepared using flour of the variety Spring (16.5% protein, 74% starch, 6.7% damaged starch, 0.57% ash). Spring flour was purchased from Meneba (Meneba Meel BV, Rotterdam, The Netherlands). 2.2. Methods 2.2.1. Bread-baking Rusk rolls: Part-baked rusk rolls (8 cm diameter, 4 cm height) were prepared at the TNO baking laboratory (TNO Quality of life, Zeist, The Netherlands). Wheat flour (3000 g), water (1845 ml), dry yeast (50 g), salt (70 g) and ascorbic acid (20 ppm) were used in the formulation as described previously (Primo-Martı´ n et al., 2006). Crispy rolls: Crispy rolls were prepared at the TNO baking laboratory (TNO Food and Nutrition, Zeist, The Netherlands). Wheat flour (3000 g), water (1782 ml), dry yeast (50 g), salt (70 g) and ascorbic acid (20 ppm) were mixed in a mixer (Kemper SP 15, Kemper, The Netherlands). All ingredients (at 5 1C), except water, were blended
for 1 min at low speed (140 rpm). Next, water (at 10 1C) was added and mixed at low speed for 2 min. Then, the dough was kneaded at high speed (280 rpm) until a final dough temperature of 26 1C. After mixing, the dough was allowed to rest for 15 min and was then divided and rounded. Proofing was performed at 30 1C and 80% RH until a fixed volume of gas was produced (500 ml) in a SJA Fermentograph (Franken, Goes, The Netherlands). The breads were part-baked at 180 1C during 10 min and 75 1C dew point in a Becker oven (Becker BV, Nederweert, The Netherlands). The part-baked breads were allowed to cool to room temperature for 30 min, frozen at 30 1C and stored at 18 1C until use. The temperature profile during breadmaking was recorded at the centre of the dough using a Microlink 3200 probe (Manchester, UK) and at the top of the dough using an IR-sensor Heimann KT 15 (Mera Benelux B.V, BerkelEnschot, The Netherlands). Breads (rusk and crispy rolls) were baked off in a Bakermat Mastermind oven (Leventi, Gilze, The Netherlands) pre-heated at 250 1C under the following conditions: 5 s steam and baking during 5 min at 235 1C. After baking the products were allowed to cool at ambient temperature (22–23 1C) for 30 min (fresh bread). The fresh bread was packed in a sealed plastic box and stored for 1, 2 and 20 d in a plastic bag (22 1C). The crust was separated from the crumb using a knife. Any remaining crumb was carefully removed from under the crust. Crust and crumb samples were freeze dried and ground (0.25 mm sieve) for analysis. 2.2.2. Confocal scanning laser microscopy Two fluorescent probes (fluorescein 5-isothiocyanate (FITC, 0.85% in water) and Rhodamine B (0.15% in water) were used to stain the bread samples (crumb and crust). Fluorescent probes were added as aqueous solutions (1.67 ml of each solution per 10 g of flour) to the mixing water during bread making. At the concentrations used, FITC stains starch (green), while Rhodamine B reacts more specifically with protein (red). The samples were studied using a Leica TCS SP (Leica Microsystems, Heidelberg, Germany) CSLM with an Ar/Kr laser. The excitation wavelengths used were 488 and 568 nm for FITC and Rhodamine B, respectively, and the emission maxima were at 518 and 625 nm. A 3D image was obtained by taking an average of two images every 12 mm in the z-direction for a total of 60 steps. Images from the separated channels were overlain to allow a simultaneous imaging of starch and protein. 2.2.3. Polarized light microscopy (PLM) Flour, bread crumb and crust were observed using a polarized light microscope (Zeiss Axioplan MC 100) with a quarter wave-plate. Samples were prepared by mixing 2–3 mg of finely powdered, freeze-dried sample with a droplet of water.
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2.2.4. Wide angle X-ray powder diffraction Wide angle X-ray diffraction (XRD) measurements of freeze-dried and ground samples were performed with a PHILIPS PW 3020 diffractometer (Philips, The Netherlands) in the reflection geometry. The X-ray generator was equipped with a copper tube operating at 40 kV and 50 mA and irradiating the sample with monochromatic Cu ka radiation with a wave length of 0.154 nm. XRD diffractograms were acquired at room temperature over a 2y range of 7–301 with a measurement time of 5 s per 2y intervals. The step size was 0.051 and the number of steps 461. Crystallinity was quantified by integrating the area under the crystalline peaks (15, 17, 18 and 231). Relative crystallinity (XRC) was expressed as X RC ¼
Is 100, If
where Is is the integrated area of crystalline peaks in the sample and If is the integrated area of crystalline peaks in flour (native starch). The integrated areas were obtained after subtracting the background using the program DRXWin 2.2 (Primo-Martı´ n, 1999). 2.2.5. Differential scanning calorimetry Freeze-dried crust and crumb samples were weighed and distilled water added at a 3:1 (v/w) water to sample ratio in stainless steel pans (TA Instruments Inc., USA). DSC measurements were performed with a Perkin-Elmer DSC 7 calorimeter (Perkin-Elmer Corp., USA). Indium was used to calibrate the system. The samples were heated from 10 to 130 1C at 10 1C/ min. An empty stainless steel pan was used as a reference during the DSC measurement. The enthalpy was expressed in J/g of sample (db). Relative crystallinity (DRC) was calculated as: DRC ¼
DH s 100, DH f
where DHs represents the melting enthalpy (J/g) of starch crystallites in the sample (peak 70 1C) that did not gelatinize during baking and DHf represents the enthalpy (J/g) of starch gelatinization in the flour. DRC of stored samples was calculated as DRC ¼
221
pulse was 5 ms, which corresponds to the spin lock frequency of 50 kHz. Samples of fresh bread crust or freeze-dried bread crumb were equilibrated to reach a water activity of 0.48 before measurement. 3. Results 3.1. Temperature profile during bread baking During baking of the crispy rolls the temperature of the dough was recorded at two positions: at the surface, the crust, at the centre of the dough, and the crumb (Fig. 1). The temperature during baking of rusk rolls was not recorded because the breads are baked in a tin which prevents the possibility of measuring the temperature at the surface with the IR sensor. The temperature near the surface of the dough of the crispy rolls increased very fast during the first minutes of baking. The sudden transient decrease of the temperature at the top of the dough (crust) at different intervals is caused by vapour injections in the oven. The surface temperature of 100 1C was reached after approximately one minute of baking. In the centre of the bread the temperature increased slowly reaching 100 1C after 8 min baking. 3.2. Confocal scanning laser microscopy of bread In the CSLM image of the stained bread (Fig. 2) the left side corresponds to the crust and the right side to the crumb. In the crust the existence of a continuous protein network (stained in red) and a discontinuous starch network (stained in green) (Primo-Martı´ n et al., 2006) can be observed. Starch granules are not fully gelatinized as they conserve their granular shape. When moving from the outer crust towards the crumb an increased extent of starch gelatinization is observed. The granular shape of the starch starts to disappear and only remnants of granules are recognized.
DH sr 100, DH f
where DHsr ¼ DHs+DHr and DHr represents the enthalpy (J/g) of amylopectin retrogradation (peak 55 1C). 2.2.6. Solid-state 13C cross polarization magic angle spinning nuclear magnetic resonance 13 C CP MAS NMR was performed on a Bruker AMX300 spectrometer (Bruker, Rheinstetten, Germany) operating at 20 1C. Spectra were referenced to external glycine (C ¼ O at 175.7 ppm). Around 3600 scans were averaged for each spectrum. The contact time was 1 ms. A spectral width of 125 kHz and line broadening of 50 Hz was used. The spinning rate was 5 kHz. The duration of the 90 degree
Fig. 1. Temperature profile in the crumb and crust of crispy bread recorded during part-baking.
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Fig. 2. 3D CSLM image of a transverse section of bread. The left side of the image corresponds to the crust and the right side to the crumb. Starch is stained (green) using FITC and proteins (red) using Rhodamine B. Size of the image is 1.0 2.4 0.7 mm. The arrow shows roughly the gradual transition between crust and crumb.
Fig. 3. Polarized light microscopy images of flour (a), bread crumb (b), and bread crust (c) showing the birefringency of flour and bread crust and the loss of birefringency of the bread crumb.
3.3. Polarized light microscopy The semi-crystalline starch granules in flour show Maltese cross patterns under polarized light, resulting from a high degree of molecular orientation in the starch granule (Fig. 3a). Starch undergoes gelatinization when heated in the presence of water. The level of gelatinization after baking will depend on the temperature and the water available in the system. The temperature/water process during breadmaking (Fig. 1) gave rise to different results in respect to the birefringence of starch in crumb and crust. A polarized image of the crumb (Fig. 3b) shows loss of the Maltese cross patterns for most granules because the molecular orientation has been disrupted during baking. Nevertheless some granules in the crust still show molecular orientation (Fig. 3c). 3.4. Wide angle X-ray powder diffraction X-ray diffraction was used to investigate changes in crystallinity of starch due to processing and storage of bread. Four peaks were identified in the flour at 2y angles of 15, 17, 18 and 231 (equivalent to d spacings of 5.8, 5.1, 4.9 and 3.8 A˚, respectively) corresponding to the A-type
pattern of cereal starch (Fig. 4). The X-ray difraction pattern of the bread crust also showed the existence of crystallinity but the peaks had a lower intensity. Peaks were found at 15, 17, 18, 20 and 231 while the X-ray diffractogram of the fresh crumb showed only one peak at 201 (Fig. 5). This peak at 201 found in both bread crumb and crust corresponds to the presence of crystalline V-type amylose–lipid complexes formed during processing, which depends on the kind and amount of the complexing agent, water content and heating temperature (Zobel and Kulp, 1996). The degree of starch crystallinity in the bread crust was determined for a fresh and a 20-day stored bread crust (Fig. 4). The relative crystallinity (Table 1) of the bread crust was significantly increased during bread staling from 36% and 41% (fresh bread crust of crispy rolls and rusk rolls, respectively) to 52% and 68% crystallinity (crispy rolls and rusk rolls 20-day old bread crust, respectively). The amylopectin retrogradation in the crumb led to the formation of a crystalline B-type pattern during staling of bread (Fig. 5). After 20 d the crystallinity of starch in the crumb increased from 0 (fresh crumb) to 3474% and 2673% for rusk rolls and crispy rolls, respectively. The development of A- or B-type diffraction patterns during
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Table 1 Relative crystallinity (XRC) in bread crust samples as determined using Xray diffraction XRC %a
Sample Rusk roll
Fresh crust 20 days crust 20 days crumb
4172 6976 3474
Crispy roll
Fresh crust 20 days crust 20 days crumb
3673 5271 2673
a XRC, the relative crystallinity, is calculatetd using: XRC ¼ (Is/If)100, where Is is the integrated area of crystalline peaks in the sample (15, 17, 18 and 231) and If is the integrated area of crystalline peaks in flour (15, 17, 18 and 23 1C). Crystallinity of fresh crumb is zero.
3.5. DSC Fig. 4. X-ray diffractograms of flour and fresh and 20 days aged bread crust of rusk rolls.
Fig. 5. X-ray diffractograms of fresh bread crumb and 20 days aged bread crumb of rusk rolls and crispy rolls.
aging is dependent on the amount of water present. A water content higher than 43% leads to development of a B- type pattern while a water content lower than 29% leads to an A-type pattern (Osella et al., 2005). This confirms the development during storage of a B-type pattern in the crumb and a probable A-type pattern development in the crust.
The extent of gelatinization of starch is governed by moisture content and temperature history (Fukuoka et al., 2002). When a starch/water mixture, with a limited amount of water, is heated, three endothermic peaks can be observed (Fukuoka et al., 2002). The first peak (55–60 1C) corresponds to moisture-mediated disorganization of starch crystallites and the second peak (70 1C) is the melting of the remaining crystallites (Donovan, 1979). The third peak (100 1C) has been attributed to dissociation of amylose–lipid complexes formed at lower temperatures during the process. Typical DSC thermograms for crumb and crust samples are shown in Fig. 6. Fresh crumb thermograms showed a single peak at 100 1C corresponding to dissociation of amylose–lipid complexes. The absence of the first and second peak indicated that starch in the crumb had completely gelatinized during baking. However, thermograms of the fresh crust showed, in addition to the peak corresponding to the amylose–lipid complex, a peak at 70 1C. This peak corresponded to the starch crystals that did not gelatinize during baking. The gelatinization temperature of these remaining granules is increased due to the heat-moisture treatment during baking. Heatmoisture treatment of starches has been shown to produce an increase in the gelatinization temperature (Hoover et al., 1994; Jacobs and Delcour, 1998) and reflects a decrease in the destabilization effect of the amorphous regions on the melting of starch crystallites during gelatinization (Hoover and Vasanthan, 1994). Storage of rusk and crispy rolls for 1 day did not show any retrogradation peak in the crust samples. After 2 days storage, the crust showed a retrogradation peak at 5171 1C (DH ¼ 0.2170.04 J/g for rusk rolls and DH ¼ 0.144 J/g for crispy rolls) although it was not always detectable (data not shown). Crust stored for 20 days showed a small retrogradation peak at 5071 1C (DH ¼ 0.8570.20 J/g for rusk rolls and DH ¼ 1.1770.29 J/g for crispy rolls).
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Fig. 6. DSC thermograms of fresh bread crumb (a), 20 days aged bread crumb (b), fresh bread crust (c) and 20 days aged bread crust (d) of rusk rolls. The samples were heated from 10 to 130 1C at 10 1C/min.
Table 2 Enthalpy of melting of crystals and relative crystallinity (DRC) in bread crust samples as determined using DSC DH (J/g)
DRC %b
Fresh crust 1 day crust 2 days crust 20 days crust 20 days crumb
3.170.3 3.270.3 2.970.1 3.370.1 5.370.6a
4374 4474 4073 5774c 7377c
Fresh crust 1 day crust 2 days crust 20 days crust 20 days crumb
2.370.3 1.970.2 1.870.1 1.970.2 5.070.1a
3274 2773 2572 4473c 7073c
Sample
NMR spectra of wheat flour showed the peak of anomeric C atoms (C-1) as a triplet with peaks at 101.6 and 100.7 ppm and a shoulder at 99.4 ppm. This is characteristic of A-type starch (Veregin et al., 1986). Spectra from bread crumb showed a narrowing of the C-1 peak, a displacement to a larger chemical shift (102.1 ppm) and a loss of the triplet characteristics. Bread crust showed the same trend with a peak at 102.3 and 101.9 ppm for rusk rolls and crispy rolls, respectively (Fig. 7). This is indicative of the presence of V-type starch (Veregin et al., 1987) although amorphous amylopectin, as well as amorphous amylose could also contribute to this peak. When starch gelatinizes an increase in relative intensity of the peaks at 82 and 103.3 ppm occurs (Morgan et al., 1992). An increase in relative peak intensity of the peak at 82 and 102 ppm was also found for bread crumb and crust compared to flour. This indicates an increase in the amount of amorphous starch (Gidley and Bociek, 1985). A higher intensity was found for bread crumb than for bread crust (rusk roll and crisp roll). This suggests that the starch in the bread crust gelatinized to a lesser extent than the starch in bread crumb. A decrease in relative peak intensity of the C atom in the CH2OH side group (C-6, 61 ppm) was observed in bread crumb when compared with native flour. 4. Discussion and conclusion
Rusk roll
Crispy roll
a
Enthalpy of amylopectin retrogradation (peak 55 1C). DRC was calculated using the formula DRC ¼ (DHs/DHf)100. c DRC was calculated using the formula DRC ¼ (DHsr/DHf)100, where DHs: melting enthalpy (J/g) of starch crystallites (peak 70 1C), DHf: enthalpy (J/g) of starch gelatinization in the flour, DHsr ¼ DHs+DHr and DHr: enthalpy (J/g) of amylopectin retrogradation (peak 55 1C). Entalphy of fresh crumb is zero. b
The relative crystallinity of starch in the bread crust is shown in Table 2. DSC also showed a somewhat lower relative starch crystallinity for bread crust from rusk rolls than for crispy rolls, as observed previously using X-ray diffraction. Also with this technique, bread crust staled for 20 days showed an increase in starch crystallinity. Retrogradation of starch in bread crumb staled for 20 days led to crystallinities of 7377% and 7073% for rusk rolls and crispy rolls, respectively. 3.6. Solid-state
13
C CP/MAS NMR
Fig. 7 shows the 13C CP MAS NMR spectra for bread crust, crumb and flour equilibrated at aw ¼ 0.48. The
The aim of this work was to study the crystallinity of the starch in the bread crust as well as its subsequent changes during storage. Studying the baking process gives an insight into the processes that occur and that result in changes in the physical state of the starch. The temperature/water content history of bread crumb and crust during bread baking is widely different (Fig. 1) and in our view is responsible for the different state of the starch in crumb and crust. During the first minutes of baking, water evaporates very fast from the outside contours of the dough and the morphology of the crust is set. A temperature higher than 100 1C is rapidly reached in the crust (Fig. 1). The fast evaporation of water from the crust due to the high temperature of the surface of the crust impairs the full gelatinization of the starch in the crust. The fact that the starch is not fully gelatinized in the bread crust has important consequences for its material properties (e.g. glass transition, water sorption) both directly after baking and upon storage. Bread crust deterioration has been described previously as a loss of crust crispness accompanied by an increase in toughness (Luyten et al., 2004; Primo-Martı´ n et al., 2006; Zobel and Kulp, 1996). The major cause of crust staling has been reported to be due to an increase of the content of water (that acts as a plasticizer). There is a redistribution of water due to migration from the crumb to the crust or due to water absorbed from the air onto the crust (Primo-Martı´ n et al., 2006). In this study the water activity of the crust increased from 0.53 (fresh bread crust) to 0.92 (bread crust staled for 20 days).
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Fig. 7. Solid state NMR spectrum of flour, bread crust (rusk roll and crispy roll) and crumb equilibrated at 0.48 water activity.
The increase in hydration (plasticisation) of the components will induce, at room temperature, a glass to rubber transition of the macromolecules (proteins and polysaccharides) present in the amorphous regions. The uptake of water by the starch will depend on its state. Water sorption isotherms have shown that native starches absorb less water than gelatinized starch at the same water activity (Charles et al., 2003; Nieuwenhuijzen, N.H. van, Tromp H., Hamer, R.J., van Vliet, T., unpublished observations). Also an effect of the degree of starch crystallinity on the glass transition (Tg) has been previously described (Zeleznak and Hoseney, 1987). These authors found an increase of Tg of crystalline starch compared to amorphous starch at the same water content. So, the extent of starch crystallinity after baking will most likely influence the kinetics of crust staling and with that the loss of crispness. The different techniques used in this study showed that only part of the starch in the crust gelatinizes. 13C NMR showed the presence of V-type starch in crust and crumb compared to the flour where starch is only present in the A pattern. The formation of V-type starch in crumb and crust follows from the displacement of the chemical shift of C-1. The changes observed in the NMR spectra between crust, crumb and flour (Section 3.5) supported the changes in starch crystallinity as observed with DSC. X-ray diffraction also showed the presence of an A-type pattern in crust samples in addition to V-type. A-type crystallinity is probably not seen in the NMR spectra because it may be hidden within the first peak (C-1) (Baik et al., 2003). Thus solid-state NMR and X-ray provide complementary information about the molecular organization of starch. 13 C NMR showed a decrease in relative intensity in the C6 position compared to the other C positions in crust and crumb samples, with a larger decrease for the crumb. This is probably due to the higher rotational mobility of this side group in gelatinized starch. This higher mobility makes the cross-polarisation from the 13C to the protons
connected to it, less efficient. The lower intensity of the C-6 peak therefore supports the notion that bread crumb contains a large fraction of gelatinised starch. However, polarized light microscopy, DSC and X-ray analyses indicated the presence of crystalline starch in the crust. The crystalline pattern of the crust obtained by X-ray diffraction was the same as the pattern of the flour. Nonetheless, both DSC and 13C NMR showed differences between flour and crust crystallinity. DSC thermograms of the flour showed a gelatinization peak at 65 1C (data not shown) while DSC thermograms of bread crust showed the melting of the remaining crystals at 70 1C. This difference in melting temperatures could be explained by the occurrence of different crystalline granules that differ in stability. During baking the amount of water in the crust will not be sufficient to gelatinize all the starch, thus only the less stable crystals will gelatinize. The crystals remaining after the baking process will be more stable as reflected in a higher melting temperature in the DSC thermogram. The quantification of starch crystallinity using DSC and Xray was similar for fresh bread samples. However, after retrogradation the crystallinity of the staled samples differed considerably for the crumb samples depending on the methodology employed. This is explained by the different sensitivity of these techniques to amylopectin chain organization at different length scales (e.g. double helices, crystalline domains). Molecular ordering over short length scales does not appear as crystalline order as determined by X-ray diffraction (Baik et al., 2003). The study of starch crystallinity (DSC, X-ray) was performed using freeze-dried samples. Starch crystallinity is influenced by the water content of the sample, requiring that all the samples used had the same water content. To achieve the same water content two options were possible: a) freeze-drying the samples (achieving a final water content of E6%) or b) equilibrating the samples to certain water content. The last option involved storage of the
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sample at certain relative humidity up to equilibrium of the samples. This meant a long storage time (41 day) in the presence of water, that could result in starch retrogradation which would interfere with the quantification of crystallinity. Therefore, the first option was chosen. A possible effect of the freeze-drying process on the absolute crystallinity values was minimized by using relative crystallinity values, as all samples (crumb, crust and flour) were subjected to the freeze-drying process. It may be concluded that during baking ca. 40% of the starch in the crust does not gelatinize due to a lack of water. The fraction that loses its crystallinity regains it by retrogradation but only after a long time. By comparison, the gelatinized starch in the crumb regains its crystallinity much faster. It is not clear if this is due to the lower water content in the crust as compared to the crumb, as this would affect the kinetics of recrystallization. This aspect is highly relevant since this means that amylopectin retrogradation, which is the main process responsible for the staling of bread crumb, cannot be responsible for crispness deterioration of the crust. Amylopectin retrogradation was measurable in bread crust after two days of storage with a low enthalpy that was not always detectable. Under the same conditions loss of bread crust crispness proceeds over much shorter times (shorter than a day) (Primo-Martı´ n et al., 2006). Acknowledgments We thank Dr. Vicent Primo Martı´ n (University of Valencia, Spain) for providing the program DRX-Win 2.2 for analysis of the X-ray diffractograms and for helpful discussion and assistance in performing X-ray measurements. We also thank the Wageningen NMR Centre and Peng Rong for their assistance with the NMR experiments. References Baik, M.-Y., Dickinson, L.C., Chinachoti, P., 2003. Solid-state 13C CP/ MAS NMR studies on aging of starch in white bread. Journal of Agricultural and Food Chemistry 51, 1242–1248. Biliaderis, C.G., 1992. Structures and phase transitions of starch in food systems. Food Technology 46, 98–109. Charles, A.L., Hsien-Ming, K., Huang, T.C., 2003. Physical investigations of surface membrane–water relationship of intact and gelatizined wheat–starch systems. Carbohydrate Research 338, 2403–2408. Donovan, J.W., 1979. Phase transition of the starch-water system. Biopolymers 18, 263–275. Fukuoka, M., Ohta, K.I., Watanbe, H., 2002. Determination of the terminal extent of starch gelatinization in a limited water system by DSC. Journal of Food Engineering 53, 39–42. Gidley, M.J., Bociek, S.M., 1985. Molecular organization in starches: a 13 C CP/MAS NMR study. Journal of American Chemical Society 107, 7040–7044.
Hoover, R., Vasanthan, T., 1994. Effect of heat-moisture treatment on the structure and physicochemical properties of cereal, legume, and tuber starches. Carbohydrate Research 252, 33–53. Hoover, R., Vasanthan, T., Senanayake, N.J., Martin, A.M., 1994. The effects of defatting and heat-moisture treatment on the retrogradation of starch gels from wheat, oat, potato, and lentil. Carbohydrate Research 261, 13–24. Hug-Iten, S., Escher, F., Conde-Petit, B., 2001. Structural properties of starch in bread and bread model systems: influence of an antistaling aamylase. Cereal Chemistry 78, 421–428. Hug-Iten, S., Escher, F., Conde-Petit, B., 2003. Staling of bread: role of amylose and amylopectin and influence of starch-degrading enzymes. Cereal Chemistry 80, 654–661. Imberty, A., Buleon, A., Tran, V., Perez, S., 1991. Recent advances in knowledge of starch structure. Staerke 10, 375–384. Jacobs, H., Delcour, J.A., 1998. Hydrothermal modifications of granular starch, with retention of the granular structure: a review. Journal of Agricultural and Food Chemistry 46, 2895–2905. Jime´nez, T., Martı´ nez-Anaya, M.A., 2001. Amylases and hemicellulases in: breadmaking. degradation by-products and potential relationship with functionality. Food Science and Technology International 7, 5–14. Keetels, C., Oostergetel, G.T., van Vliet, T., 1996. Retrogradation of amylopectin in concentrated starch gels. Carbohydrate Polymers 30, 61–64. Luyten, H., Plijter, J.J., van Vliet, T., 2004. Crispy/crunchy crusts of cellular solid foods: a literature review with discussion. Journal of Texture Studies 35, 445–492. Morgan, K.R., Furneaux, R.H., Stanley, R.A., 1992. Observation by solid-state 13C CP MAS NMR spectroscopy of the transformations of wheat starch associated with the making and staling of bread. Carbohydrate Research 235, 15–22. Moss, R., 1975. Bread microstructure as affected by cysteine, potassium bromate and ascorbic acid. Cereal Foods World 20, 289–292. Osella, C.A., Sa´nchez, H.D., Carrara, C.R., de la Torre, M.A., Buera, M.P., 2005. Water redistribution and structural changes of starch during storage of a gluten-free bread. Starch 57, 208–216. Pomeranz, Y., Meyer, D., Seibel, W., 1984. Wheat, wheat-rye and rye dough and bread studied by scanning electron microscopy. Cereal Chemistry 61, 53–59. Primo-Martı´ n, C., Pijpekamp, A., van de Vliet, T., van de Jongh, H.H.J., Plijter, J.J., Hamer, R.J., 2006. The role of the gluten network in the crispness of bread crust. Journal of Cereal Science 43, 342–352. Primo-Martı´ n, V., 1999. DRXWin & CreaFit 2.0: graphical and analytical tools for powder XRD patterns. Powder Diffraction 14, 70–73. Sarko, A., Wu, H.C.H., 1978. The crystal structures of A-, B- and Cpolymorphs of amylose and starch. Starch 30, 73–78. Svensson, E., Eliasson, A.C., 1995. Crystalline changes in native wheat and potato starches at intermediate water levels during gelatinization. Carbohydrate Polymers 26, 171–176. Veregin, R.P., Fyfe, C.A., Marchessault, R.H., Taylor, M.G., 1986. Characterization of the crystalline A and B starch polymorphs and investigation of starch crystallization by high-resolution 13C/CP MAS NMR. Macromolecules 19, 1030–1034. Veregin, R.P., Fyfe, C.A., Marchessault, R.H., 1987. Investigation of the crystalline ‘‘V’’ amylose complexes by high-resolution 13C/CP MAS NMR. Macromolecules 20, 3007–3012. Zeleznak, K.J., Hoseney, R.C., 1987. The glass transition in starch. Cereal Chemistry 64, 121–124. Zobel, H.F., Kulp, K., 1996. The staling mechanism. In: Hebeda, R.E., Zobel, H.F. (Eds.), Baked Goods Freshness: Technology, Evaluation, and Inhibition of Staling. Marcel Dekker, New York, pp. 1–64.
Journal of Cereal Science 45 (2007) 219–226 www.elsevier.com/locate/yjcrs
Crystallinity changes in wheat starch during the bread-making process: Starch crystallinity in the bread crust C. Primo-Martı´ na,, N.H. van Nieuwenhuijzena,b, R.J. Hamera,c, T. van Vlieta,b a
Wageningen Center for Food Sciences, Wageningen, the Netherlands b Wageningen University, Wageningen, the Netherlands c TNO Quality of Life, Zeist, the Netherlands
Received 13 June 2006; received in revised form 23 August 2006; accepted 24 August 2006
Abstract The crystallinity of starch in crispy bread crust was quantified using several different techniques. Confocal scanning laser microscopy (CSLM) demonstrated the presence of granular starch in the crust and remnants of granules when moving towards the crumb. Differential scanning calorimetry (DSC) showed an endothermic transition at 70 1C associated with the melting of crystalline amylopectin. The relative starch crystallinity, as determined by X-ray and DSC, from different types of breads was found to lie between 36% and 41% (X-ray) and between 32% and 43% (DSC) for fresh bread crust. Storage of breads in a closed box (22 1C) for up to 20 days showed an increase in crust crystallinity due to amylopectin retrogradation both by X-ray and DSC. However, DSC thermograms of 1-day old bread crust showed no amylopectin retrogradation and after 2 days storage, amylopectin retrogradation in the crust was hardly detectable. 13C CP MAS NMR was used to characterize the physical state of starch in flour and bread crumb and crust. The intensity of the peaks showed a dependence on the degree of starch gelatinization. Comparison of the results for two different types of bread showed that the baking process influenced the extent of starch crystallinity in the bread crust. Amylopectin retrogradation, which is the main process responsible for the staling of bread crumb, cannot be responsible for crispness deterioration of the crust as amylopectin retrogradation upon storage of breads could only be measured in the crust after 2 days storage. Under the same conditions loss of bread crust crispness proceeds over shorter times. r 2006 Elsevier Ltd. All rights reserved. Keywords: Crust; Bread; Crystallinity; Starch; X-ray; DSC; CSLM; NMR
1. Introduction The structure and phase transitions of starch are topics of considerable interest, since they are related to the functionality of the starch (Biliaderis, 1992). Native starch is present as semi-crystalline granules. The semi-crystalline properties of native starch are related to the short-chain fraction of amylopectin arranged as double helices and Abbreviations: CSLM, confocal scanning laser microscopy; 13C CP/ MAS NMR, Solid-state 13C cross polarization magic angle spinning nuclear magnetic resonance; DRC, relative crystallinity determined using DSC; DSC, differential scanning calorimetry; PLM, polarized light microscopy; RH, relative humidity; XRC, relative crystallinity determined using X-ray Corresponding author. Tel.: +31 317 475120; fax: +31 317 475347. E-mail address: [email protected] (C. Primo-Martı´ n). 0733-5210/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jcs.2006.08.009
packed in small crystallites (Imberty et al., 1991; Sarko and Wu, 1978; Svensson and Eliasson, 1995). Linear amylose molecules are apparently present in an amorphous state in the granule (Zeleznak and Hoseney, 1987). The physical properties of wheat flour starch are subjected to changes during baking and subsequent storage of bread. These transformations largely determine the structure and texture of the solid matrix of the final product (Zobel and Kulp, 1996). When starch is suspended in water and heated it undergoes a series of processes called gelatinization. The changes occurring during gelatinization are (not in order of occurrence): swelling, loss of birefringence, melting, loss of crystallinity, increase of viscosity of the suspension and leaching of amylose from the granules. During the baking process, the starch in the bread crumb is gelatinized resulting in an amorphous
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structure. During storage of the bread, the crystalline structure of the starch is slowly recovered at the shortrange scale (double helices) (Keetels et al., 1996). This process is commonly known as retrogradation. Retrogradation is technologically important since it produces significant changes in the mechanical properties of the crumb affecting its sensory appreciation. The characterization of starch in bread crumb and its role during bread staling have been extensively studied (Hug-Iten et al., 2001, 2003; Jime´nez and Martı´ nez-Anaya, 2001; Morgan et al., 1992). This is however not the case for bread crust. In the crust the presence of intact, non-swollen starch granules has been described previously (Moss, 1975; Pomeranz et al., 1984; Primo-Martı´ n et al., 2006). But, little attention has been paid to the aging of the crust, its structural composition, the state of starch in the crust (Primo-Martı´ n et al., 2006) and its possible significance in the loss of crispness. In this work the crystallinity of the starch in the bread crust was studied as the extent of crystallinity most likely influences crust properties as well as subsequent changes during storage. The crystallinity of starch in the crust was determined using differential scanning calorimetry (DSC), wide angle X-ray diffraction, 13C cross polarization magic angle spinning nuclear magnetic resonance (13C CP MAS NMR) and polarization microscopy which provides complementary information about the state of the starch. In addition, the state of the starch in the crust, in the crumb and in the flour were compared. Confocal scanning microscopy (CSLM) was used to gain an insight into the organisation of the bread crumb and crust. 2. Materials and methods 2.1. Materials Breads, rusk rolls and crispy rolls, were prepared using flour of the variety Spring (16.5% protein, 74% starch, 6.7% damaged starch, 0.57% ash). Spring flour was purchased from Meneba (Meneba Meel BV, Rotterdam, The Netherlands). 2.2. Methods 2.2.1. Bread-baking Rusk rolls: Part-baked rusk rolls (8 cm diameter, 4 cm height) were prepared at the TNO baking laboratory (TNO Quality of life, Zeist, The Netherlands). Wheat flour (3000 g), water (1845 ml), dry yeast (50 g), salt (70 g) and ascorbic acid (20 ppm) were used in the formulation as described previously (Primo-Martı´ n et al., 2006). Crispy rolls: Crispy rolls were prepared at the TNO baking laboratory (TNO Food and Nutrition, Zeist, The Netherlands). Wheat flour (3000 g), water (1782 ml), dry yeast (50 g), salt (70 g) and ascorbic acid (20 ppm) were mixed in a mixer (Kemper SP 15, Kemper, The Netherlands). All ingredients (at 5 1C), except water, were blended
for 1 min at low speed (140 rpm). Next, water (at 10 1C) was added and mixed at low speed for 2 min. Then, the dough was kneaded at high speed (280 rpm) until a final dough temperature of 26 1C. After mixing, the dough was allowed to rest for 15 min and was then divided and rounded. Proofing was performed at 30 1C and 80% RH until a fixed volume of gas was produced (500 ml) in a SJA Fermentograph (Franken, Goes, The Netherlands). The breads were part-baked at 180 1C during 10 min and 75 1C dew point in a Becker oven (Becker BV, Nederweert, The Netherlands). The part-baked breads were allowed to cool to room temperature for 30 min, frozen at 30 1C and stored at 18 1C until use. The temperature profile during breadmaking was recorded at the centre of the dough using a Microlink 3200 probe (Manchester, UK) and at the top of the dough using an IR-sensor Heimann KT 15 (Mera Benelux B.V, BerkelEnschot, The Netherlands). Breads (rusk and crispy rolls) were baked off in a Bakermat Mastermind oven (Leventi, Gilze, The Netherlands) pre-heated at 250 1C under the following conditions: 5 s steam and baking during 5 min at 235 1C. After baking the products were allowed to cool at ambient temperature (22–23 1C) for 30 min (fresh bread). The fresh bread was packed in a sealed plastic box and stored for 1, 2 and 20 d in a plastic bag (22 1C). The crust was separated from the crumb using a knife. Any remaining crumb was carefully removed from under the crust. Crust and crumb samples were freeze dried and ground (0.25 mm sieve) for analysis. 2.2.2. Confocal scanning laser microscopy Two fluorescent probes (fluorescein 5-isothiocyanate (FITC, 0.85% in water) and Rhodamine B (0.15% in water) were used to stain the bread samples (crumb and crust). Fluorescent probes were added as aqueous solutions (1.67 ml of each solution per 10 g of flour) to the mixing water during bread making. At the concentrations used, FITC stains starch (green), while Rhodamine B reacts more specifically with protein (red). The samples were studied using a Leica TCS SP (Leica Microsystems, Heidelberg, Germany) CSLM with an Ar/Kr laser. The excitation wavelengths used were 488 and 568 nm for FITC and Rhodamine B, respectively, and the emission maxima were at 518 and 625 nm. A 3D image was obtained by taking an average of two images every 12 mm in the z-direction for a total of 60 steps. Images from the separated channels were overlain to allow a simultaneous imaging of starch and protein. 2.2.3. Polarized light microscopy (PLM) Flour, bread crumb and crust were observed using a polarized light microscope (Zeiss Axioplan MC 100) with a quarter wave-plate. Samples were prepared by mixing 2–3 mg of finely powdered, freeze-dried sample with a droplet of water.
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2.2.4. Wide angle X-ray powder diffraction Wide angle X-ray diffraction (XRD) measurements of freeze-dried and ground samples were performed with a PHILIPS PW 3020 diffractometer (Philips, The Netherlands) in the reflection geometry. The X-ray generator was equipped with a copper tube operating at 40 kV and 50 mA and irradiating the sample with monochromatic Cu ka radiation with a wave length of 0.154 nm. XRD diffractograms were acquired at room temperature over a 2y range of 7–301 with a measurement time of 5 s per 2y intervals. The step size was 0.051 and the number of steps 461. Crystallinity was quantified by integrating the area under the crystalline peaks (15, 17, 18 and 231). Relative crystallinity (XRC) was expressed as X RC ¼
Is 100, If
where Is is the integrated area of crystalline peaks in the sample and If is the integrated area of crystalline peaks in flour (native starch). The integrated areas were obtained after subtracting the background using the program DRXWin 2.2 (Primo-Martı´ n, 1999). 2.2.5. Differential scanning calorimetry Freeze-dried crust and crumb samples were weighed and distilled water added at a 3:1 (v/w) water to sample ratio in stainless steel pans (TA Instruments Inc., USA). DSC measurements were performed with a Perkin-Elmer DSC 7 calorimeter (Perkin-Elmer Corp., USA). Indium was used to calibrate the system. The samples were heated from 10 to 130 1C at 10 1C/ min. An empty stainless steel pan was used as a reference during the DSC measurement. The enthalpy was expressed in J/g of sample (db). Relative crystallinity (DRC) was calculated as: DRC ¼
DH s 100, DH f
where DHs represents the melting enthalpy (J/g) of starch crystallites in the sample (peak 70 1C) that did not gelatinize during baking and DHf represents the enthalpy (J/g) of starch gelatinization in the flour. DRC of stored samples was calculated as DRC ¼
221
pulse was 5 ms, which corresponds to the spin lock frequency of 50 kHz. Samples of fresh bread crust or freeze-dried bread crumb were equilibrated to reach a water activity of 0.48 before measurement. 3. Results 3.1. Temperature profile during bread baking During baking of the crispy rolls the temperature of the dough was recorded at two positions: at the surface, the crust, at the centre of the dough, and the crumb (Fig. 1). The temperature during baking of rusk rolls was not recorded because the breads are baked in a tin which prevents the possibility of measuring the temperature at the surface with the IR sensor. The temperature near the surface of the dough of the crispy rolls increased very fast during the first minutes of baking. The sudden transient decrease of the temperature at the top of the dough (crust) at different intervals is caused by vapour injections in the oven. The surface temperature of 100 1C was reached after approximately one minute of baking. In the centre of the bread the temperature increased slowly reaching 100 1C after 8 min baking. 3.2. Confocal scanning laser microscopy of bread In the CSLM image of the stained bread (Fig. 2) the left side corresponds to the crust and the right side to the crumb. In the crust the existence of a continuous protein network (stained in red) and a discontinuous starch network (stained in green) (Primo-Martı´ n et al., 2006) can be observed. Starch granules are not fully gelatinized as they conserve their granular shape. When moving from the outer crust towards the crumb an increased extent of starch gelatinization is observed. The granular shape of the starch starts to disappear and only remnants of granules are recognized.
DH sr 100, DH f
where DHsr ¼ DHs+DHr and DHr represents the enthalpy (J/g) of amylopectin retrogradation (peak 55 1C). 2.2.6. Solid-state 13C cross polarization magic angle spinning nuclear magnetic resonance 13 C CP MAS NMR was performed on a Bruker AMX300 spectrometer (Bruker, Rheinstetten, Germany) operating at 20 1C. Spectra were referenced to external glycine (C ¼ O at 175.7 ppm). Around 3600 scans were averaged for each spectrum. The contact time was 1 ms. A spectral width of 125 kHz and line broadening of 50 Hz was used. The spinning rate was 5 kHz. The duration of the 90 degree
Fig. 1. Temperature profile in the crumb and crust of crispy bread recorded during part-baking.
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Fig. 2. 3D CSLM image of a transverse section of bread. The left side of the image corresponds to the crust and the right side to the crumb. Starch is stained (green) using FITC and proteins (red) using Rhodamine B. Size of the image is 1.0 2.4 0.7 mm. The arrow shows roughly the gradual transition between crust and crumb.
Fig. 3. Polarized light microscopy images of flour (a), bread crumb (b), and bread crust (c) showing the birefringency of flour and bread crust and the loss of birefringency of the bread crumb.
3.3. Polarized light microscopy The semi-crystalline starch granules in flour show Maltese cross patterns under polarized light, resulting from a high degree of molecular orientation in the starch granule (Fig. 3a). Starch undergoes gelatinization when heated in the presence of water. The level of gelatinization after baking will depend on the temperature and the water available in the system. The temperature/water process during breadmaking (Fig. 1) gave rise to different results in respect to the birefringence of starch in crumb and crust. A polarized image of the crumb (Fig. 3b) shows loss of the Maltese cross patterns for most granules because the molecular orientation has been disrupted during baking. Nevertheless some granules in the crust still show molecular orientation (Fig. 3c). 3.4. Wide angle X-ray powder diffraction X-ray diffraction was used to investigate changes in crystallinity of starch due to processing and storage of bread. Four peaks were identified in the flour at 2y angles of 15, 17, 18 and 231 (equivalent to d spacings of 5.8, 5.1, 4.9 and 3.8 A˚, respectively) corresponding to the A-type
pattern of cereal starch (Fig. 4). The X-ray difraction pattern of the bread crust also showed the existence of crystallinity but the peaks had a lower intensity. Peaks were found at 15, 17, 18, 20 and 231 while the X-ray diffractogram of the fresh crumb showed only one peak at 201 (Fig. 5). This peak at 201 found in both bread crumb and crust corresponds to the presence of crystalline V-type amylose–lipid complexes formed during processing, which depends on the kind and amount of the complexing agent, water content and heating temperature (Zobel and Kulp, 1996). The degree of starch crystallinity in the bread crust was determined for a fresh and a 20-day stored bread crust (Fig. 4). The relative crystallinity (Table 1) of the bread crust was significantly increased during bread staling from 36% and 41% (fresh bread crust of crispy rolls and rusk rolls, respectively) to 52% and 68% crystallinity (crispy rolls and rusk rolls 20-day old bread crust, respectively). The amylopectin retrogradation in the crumb led to the formation of a crystalline B-type pattern during staling of bread (Fig. 5). After 20 d the crystallinity of starch in the crumb increased from 0 (fresh crumb) to 3474% and 2673% for rusk rolls and crispy rolls, respectively. The development of A- or B-type diffraction patterns during
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Table 1 Relative crystallinity (XRC) in bread crust samples as determined using Xray diffraction XRC %a
Sample Rusk roll
Fresh crust 20 days crust 20 days crumb
4172 6976 3474
Crispy roll
Fresh crust 20 days crust 20 days crumb
3673 5271 2673
a XRC, the relative crystallinity, is calculatetd using: XRC ¼ (Is/If)100, where Is is the integrated area of crystalline peaks in the sample (15, 17, 18 and 231) and If is the integrated area of crystalline peaks in flour (15, 17, 18 and 23 1C). Crystallinity of fresh crumb is zero.
3.5. DSC Fig. 4. X-ray diffractograms of flour and fresh and 20 days aged bread crust of rusk rolls.
Fig. 5. X-ray diffractograms of fresh bread crumb and 20 days aged bread crumb of rusk rolls and crispy rolls.
aging is dependent on the amount of water present. A water content higher than 43% leads to development of a B- type pattern while a water content lower than 29% leads to an A-type pattern (Osella et al., 2005). This confirms the development during storage of a B-type pattern in the crumb and a probable A-type pattern development in the crust.
The extent of gelatinization of starch is governed by moisture content and temperature history (Fukuoka et al., 2002). When a starch/water mixture, with a limited amount of water, is heated, three endothermic peaks can be observed (Fukuoka et al., 2002). The first peak (55–60 1C) corresponds to moisture-mediated disorganization of starch crystallites and the second peak (70 1C) is the melting of the remaining crystallites (Donovan, 1979). The third peak (100 1C) has been attributed to dissociation of amylose–lipid complexes formed at lower temperatures during the process. Typical DSC thermograms for crumb and crust samples are shown in Fig. 6. Fresh crumb thermograms showed a single peak at 100 1C corresponding to dissociation of amylose–lipid complexes. The absence of the first and second peak indicated that starch in the crumb had completely gelatinized during baking. However, thermograms of the fresh crust showed, in addition to the peak corresponding to the amylose–lipid complex, a peak at 70 1C. This peak corresponded to the starch crystals that did not gelatinize during baking. The gelatinization temperature of these remaining granules is increased due to the heat-moisture treatment during baking. Heatmoisture treatment of starches has been shown to produce an increase in the gelatinization temperature (Hoover et al., 1994; Jacobs and Delcour, 1998) and reflects a decrease in the destabilization effect of the amorphous regions on the melting of starch crystallites during gelatinization (Hoover and Vasanthan, 1994). Storage of rusk and crispy rolls for 1 day did not show any retrogradation peak in the crust samples. After 2 days storage, the crust showed a retrogradation peak at 5171 1C (DH ¼ 0.2170.04 J/g for rusk rolls and DH ¼ 0.144 J/g for crispy rolls) although it was not always detectable (data not shown). Crust stored for 20 days showed a small retrogradation peak at 5071 1C (DH ¼ 0.8570.20 J/g for rusk rolls and DH ¼ 1.1770.29 J/g for crispy rolls).
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Fig. 6. DSC thermograms of fresh bread crumb (a), 20 days aged bread crumb (b), fresh bread crust (c) and 20 days aged bread crust (d) of rusk rolls. The samples were heated from 10 to 130 1C at 10 1C/min.
Table 2 Enthalpy of melting of crystals and relative crystallinity (DRC) in bread crust samples as determined using DSC DH (J/g)
DRC %b
Fresh crust 1 day crust 2 days crust 20 days crust 20 days crumb
3.170.3 3.270.3 2.970.1 3.370.1 5.370.6a
4374 4474 4073 5774c 7377c
Fresh crust 1 day crust 2 days crust 20 days crust 20 days crumb
2.370.3 1.970.2 1.870.1 1.970.2 5.070.1a
3274 2773 2572 4473c 7073c
Sample
NMR spectra of wheat flour showed the peak of anomeric C atoms (C-1) as a triplet with peaks at 101.6 and 100.7 ppm and a shoulder at 99.4 ppm. This is characteristic of A-type starch (Veregin et al., 1986). Spectra from bread crumb showed a narrowing of the C-1 peak, a displacement to a larger chemical shift (102.1 ppm) and a loss of the triplet characteristics. Bread crust showed the same trend with a peak at 102.3 and 101.9 ppm for rusk rolls and crispy rolls, respectively (Fig. 7). This is indicative of the presence of V-type starch (Veregin et al., 1987) although amorphous amylopectin, as well as amorphous amylose could also contribute to this peak. When starch gelatinizes an increase in relative intensity of the peaks at 82 and 103.3 ppm occurs (Morgan et al., 1992). An increase in relative peak intensity of the peak at 82 and 102 ppm was also found for bread crumb and crust compared to flour. This indicates an increase in the amount of amorphous starch (Gidley and Bociek, 1985). A higher intensity was found for bread crumb than for bread crust (rusk roll and crisp roll). This suggests that the starch in the bread crust gelatinized to a lesser extent than the starch in bread crumb. A decrease in relative peak intensity of the C atom in the CH2OH side group (C-6, 61 ppm) was observed in bread crumb when compared with native flour. 4. Discussion and conclusion
Rusk roll
Crispy roll
a
Enthalpy of amylopectin retrogradation (peak 55 1C). DRC was calculated using the formula DRC ¼ (DHs/DHf)100. c DRC was calculated using the formula DRC ¼ (DHsr/DHf)100, where DHs: melting enthalpy (J/g) of starch crystallites (peak 70 1C), DHf: enthalpy (J/g) of starch gelatinization in the flour, DHsr ¼ DHs+DHr and DHr: enthalpy (J/g) of amylopectin retrogradation (peak 55 1C). Entalphy of fresh crumb is zero. b
The relative crystallinity of starch in the bread crust is shown in Table 2. DSC also showed a somewhat lower relative starch crystallinity for bread crust from rusk rolls than for crispy rolls, as observed previously using X-ray diffraction. Also with this technique, bread crust staled for 20 days showed an increase in starch crystallinity. Retrogradation of starch in bread crumb staled for 20 days led to crystallinities of 7377% and 7073% for rusk rolls and crispy rolls, respectively. 3.6. Solid-state
13
C CP/MAS NMR
Fig. 7 shows the 13C CP MAS NMR spectra for bread crust, crumb and flour equilibrated at aw ¼ 0.48. The
The aim of this work was to study the crystallinity of the starch in the bread crust as well as its subsequent changes during storage. Studying the baking process gives an insight into the processes that occur and that result in changes in the physical state of the starch. The temperature/water content history of bread crumb and crust during bread baking is widely different (Fig. 1) and in our view is responsible for the different state of the starch in crumb and crust. During the first minutes of baking, water evaporates very fast from the outside contours of the dough and the morphology of the crust is set. A temperature higher than 100 1C is rapidly reached in the crust (Fig. 1). The fast evaporation of water from the crust due to the high temperature of the surface of the crust impairs the full gelatinization of the starch in the crust. The fact that the starch is not fully gelatinized in the bread crust has important consequences for its material properties (e.g. glass transition, water sorption) both directly after baking and upon storage. Bread crust deterioration has been described previously as a loss of crust crispness accompanied by an increase in toughness (Luyten et al., 2004; Primo-Martı´ n et al., 2006; Zobel and Kulp, 1996). The major cause of crust staling has been reported to be due to an increase of the content of water (that acts as a plasticizer). There is a redistribution of water due to migration from the crumb to the crust or due to water absorbed from the air onto the crust (Primo-Martı´ n et al., 2006). In this study the water activity of the crust increased from 0.53 (fresh bread crust) to 0.92 (bread crust staled for 20 days).
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Fig. 7. Solid state NMR spectrum of flour, bread crust (rusk roll and crispy roll) and crumb equilibrated at 0.48 water activity.
The increase in hydration (plasticisation) of the components will induce, at room temperature, a glass to rubber transition of the macromolecules (proteins and polysaccharides) present in the amorphous regions. The uptake of water by the starch will depend on its state. Water sorption isotherms have shown that native starches absorb less water than gelatinized starch at the same water activity (Charles et al., 2003; Nieuwenhuijzen, N.H. van, Tromp H., Hamer, R.J., van Vliet, T., unpublished observations). Also an effect of the degree of starch crystallinity on the glass transition (Tg) has been previously described (Zeleznak and Hoseney, 1987). These authors found an increase of Tg of crystalline starch compared to amorphous starch at the same water content. So, the extent of starch crystallinity after baking will most likely influence the kinetics of crust staling and with that the loss of crispness. The different techniques used in this study showed that only part of the starch in the crust gelatinizes. 13C NMR showed the presence of V-type starch in crust and crumb compared to the flour where starch is only present in the A pattern. The formation of V-type starch in crumb and crust follows from the displacement of the chemical shift of C-1. The changes observed in the NMR spectra between crust, crumb and flour (Section 3.5) supported the changes in starch crystallinity as observed with DSC. X-ray diffraction also showed the presence of an A-type pattern in crust samples in addition to V-type. A-type crystallinity is probably not seen in the NMR spectra because it may be hidden within the first peak (C-1) (Baik et al., 2003). Thus solid-state NMR and X-ray provide complementary information about the molecular organization of starch. 13 C NMR showed a decrease in relative intensity in the C6 position compared to the other C positions in crust and crumb samples, with a larger decrease for the crumb. This is probably due to the higher rotational mobility of this side group in gelatinized starch. This higher mobility makes the cross-polarisation from the 13C to the protons
connected to it, less efficient. The lower intensity of the C-6 peak therefore supports the notion that bread crumb contains a large fraction of gelatinised starch. However, polarized light microscopy, DSC and X-ray analyses indicated the presence of crystalline starch in the crust. The crystalline pattern of the crust obtained by X-ray diffraction was the same as the pattern of the flour. Nonetheless, both DSC and 13C NMR showed differences between flour and crust crystallinity. DSC thermograms of the flour showed a gelatinization peak at 65 1C (data not shown) while DSC thermograms of bread crust showed the melting of the remaining crystals at 70 1C. This difference in melting temperatures could be explained by the occurrence of different crystalline granules that differ in stability. During baking the amount of water in the crust will not be sufficient to gelatinize all the starch, thus only the less stable crystals will gelatinize. The crystals remaining after the baking process will be more stable as reflected in a higher melting temperature in the DSC thermogram. The quantification of starch crystallinity using DSC and Xray was similar for fresh bread samples. However, after retrogradation the crystallinity of the staled samples differed considerably for the crumb samples depending on the methodology employed. This is explained by the different sensitivity of these techniques to amylopectin chain organization at different length scales (e.g. double helices, crystalline domains). Molecular ordering over short length scales does not appear as crystalline order as determined by X-ray diffraction (Baik et al., 2003). The study of starch crystallinity (DSC, X-ray) was performed using freeze-dried samples. Starch crystallinity is influenced by the water content of the sample, requiring that all the samples used had the same water content. To achieve the same water content two options were possible: a) freeze-drying the samples (achieving a final water content of E6%) or b) equilibrating the samples to certain water content. The last option involved storage of the
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sample at certain relative humidity up to equilibrium of the samples. This meant a long storage time (41 day) in the presence of water, that could result in starch retrogradation which would interfere with the quantification of crystallinity. Therefore, the first option was chosen. A possible effect of the freeze-drying process on the absolute crystallinity values was minimized by using relative crystallinity values, as all samples (crumb, crust and flour) were subjected to the freeze-drying process. It may be concluded that during baking ca. 40% of the starch in the crust does not gelatinize due to a lack of water. The fraction that loses its crystallinity regains it by retrogradation but only after a long time. By comparison, the gelatinized starch in the crumb regains its crystallinity much faster. It is not clear if this is due to the lower water content in the crust as compared to the crumb, as this would affect the kinetics of recrystallization. This aspect is highly relevant since this means that amylopectin retrogradation, which is the main process responsible for the staling of bread crumb, cannot be responsible for crispness deterioration of the crust. Amylopectin retrogradation was measurable in bread crust after two days of storage with a low enthalpy that was not always detectable. Under the same conditions loss of bread crust crispness proceeds over much shorter times (shorter than a day) (Primo-Martı´ n et al., 2006). Acknowledgments We thank Dr. Vicent Primo Martı´ n (University of Valencia, Spain) for providing the program DRX-Win 2.2 for analysis of the X-ray diffractograms and for helpful discussion and assistance in performing X-ray measurements. We also thank the Wageningen NMR Centre and Peng Rong for their assistance with the NMR experiments. References Baik, M.-Y., Dickinson, L.C., Chinachoti, P., 2003. Solid-state 13C CP/ MAS NMR studies on aging of starch in white bread. Journal of Agricultural and Food Chemistry 51, 1242–1248. Biliaderis, C.G., 1992. Structures and phase transitions of starch in food systems. Food Technology 46, 98–109. Charles, A.L., Hsien-Ming, K., Huang, T.C., 2003. Physical investigations of surface membrane–water relationship of intact and gelatizined wheat–starch systems. Carbohydrate Research 338, 2403–2408. Donovan, J.W., 1979. Phase transition of the starch-water system. Biopolymers 18, 263–275. Fukuoka, M., Ohta, K.I., Watanbe, H., 2002. Determination of the terminal extent of starch gelatinization in a limited water system by DSC. Journal of Food Engineering 53, 39–42. Gidley, M.J., Bociek, S.M., 1985. Molecular organization in starches: a 13 C CP/MAS NMR study. Journal of American Chemical Society 107, 7040–7044.
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