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Structure characterization of C-type starch granule by acid hydrolysis
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HYDROCOLLOIDS Food Hydrocolloids 22 (2008) 1283–1290 www.elsevier.com/locate/foodhyd
Structure characterization of C-type starch granule by acid hydrolysis Wang Shujuna,, Yu Jinglinb, Yu Jiugaoc, Pang Jipinga, Liu Hongyand a
College of Pharmaceutical Science and Technology, Tianjin University, Tianjin 300072, China College of Traditional Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin 300072, China c College of Science, Department of Chemistry, Tianjin University, Tianjin 300072, China d Henan Agricultural Academy of Sciences, Henan Province 450002, China
b
Received 26 March 2007; accepted 26 June 2007
Abstract The structural changes of C-type Dioscorea rhizoma starch are evaluated by scanning electron microscope (SEM), X-ray powder diffraction (XRD) and cross polarization/magic-angle spinning (CP/MAS) 13C nuclear magic resonance (NMR) during acid hydrolysis. SEM shows that the amorphous areas are mainly located in the core part of C-type starch granules or distribute alternately in the crystalline regions. XRD analysis reveals that the B-type polymorphs present in the C-type starch granules are preferentially degraded or degraded faster than A-type polymorphs. NMR spectra confirm that the amorphous regions in the starch granules are firstly hydrolyzed and could be hydrolyzed completely as long as the hydrolysis time is enough. After 40 days of hydrolysis, the acid-modified starch shows typical A-type characteristics whether for the X-ray diffraction pattern or for the 13C CP/MAS NMR spectra. The finding suggests that the B-type polymorphs consisting of the C-type starch granules are more unstable than the A-type polymorphs. This is not in agreement with the fact that B-type starches (potato starch) are more resistant to acid and enzyme than A-type cereal starches. r 2007 Elsevier Ltd. All rights reserved. Keywords: Starch; Acid hydrolysis; Amorphous region; Polymorphs
1. Introduction Starch is the predominant carbohydrate reserve found in plants. It is generally believed that starch granules are composed mainly of two types of glucose polymer–amylose, which is essentially a linear chain molecule, and amylopectin, which is branched. Amylose and amylopectin are packed into granules, which are part crystalline and part amorphous in structure. There is evidence that the crystallites consist of parallel, left-handed double helices formed from the short chains of amylopectin. Two types of crystallite, or polymorph structures, A and B, have been identified in starch granules, which can be distinguished by the packing density of the double helices. A-type polymorphs are more dense than B-type polymorphs (Gidley & Bociek, 1985; Hinrichs et al., 1987; Imberty & Perez, 1988; Sarko & Wu, 1978; Veregin, Fyfe, Marchessault, & Taylor, 1986; Zobel & Stephen, 1995). Starches from different Corresponding author.
E-mail address: [email protected] (W. Shujun). 0268-005X/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2007.06.012
botanical sources have either A (e.g., maize or rice), B (e.g., potato or canna) or a mixture of A and B types of polymorph (e.g., pea or yam) (Cairns, Bogracheva, Ring, Hedley, & Morris, 1997; Ratnayake, Hoover, Shahidi, Perera, & Jane, 2001; Wang, Gao, Liu, Chen, Yu, & Xiao, 2006; Wang, Liu, Gao, Chen, Yu, & Xiao, 2006; Wang, Yu, Gao, Liu, & Xiao, 2006). Dioscoreae (Chinese name Shanyao), the rhizome of various species of genus Dioscorea opposita Thunb. (Dioscoreaceae), has been used as an important invigorant in traditional Chinese medicine (TCM) for many years (Zuo & Tang, 2003). Total carbohydrates of D. opposita Thunb. vary from 20% to 60%. Starch is the most abundant carbohydrate in this rhizoma of D. opposita Thunb., making up about 40% content in the total biomass (Ni & Song, 2002). However, there are few investigations on the properties of starch present in D. opposita Thunb. In order to understand the properties of these starches, the physicochemical, thermal, morphological and crystalline properties were characterized by means of various methods. The starches from different D. opposita Thunb.
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cultivars showed the typical C-type X-ray diffraction patterns under the same testing conditions (Wang, Gao, Liu et al., 2006; Wang, Liu et al., 2006; Wang, Yu et al., 2006). As mentioned previously, native starches can be classified into three groups, the ‘‘A’’, ‘‘B’’ and ‘‘C’’ starches, on the basis of X-ray diffraction data. Although starch granular structure in general is not fully understood, there is even less understanding about the granular structure of C-type starches, even though they are relatively common. For example, it is not known if the A and B polymorphs are distributed in different granules, or if the two types occur within the same granules. If C-type starches consist of A- and B-type granules, then it is likely that their properties would be intermediate between those of A- and B-type starches. If, on the other hand, both polymorphs exist within all granules, then C-type starches would have unique properties dependent upon the arrangement of those two polymorphs in the granule. Bogracheva, Morris, Ring, and Hedley (1998) used a combination of techniques (including differential scanning calorimetry (DSC), X-ray powder diffraction (XRD), nuclear magnetic resonance (NMR)) to study the structure and properties of C-type starch from pea seeds. C-type starch granules contained both types of polymorph, the B-type polymorphs are in the center of the granule and surrounded by the A-type polymorphs. However, it is not known whether the amorphous area of C-type starch granule consists of the A- or B-type polymorphs or the mixture of A- and B-type polymorphs. Whether the amorphous regions are located in the center part or in the outer part of granules is not clear. Acid modification of starch could be very helpful to understand the inner structure of starch granules (Kang, Kim, Lee, & Kim, 1997; Kim & Ahn, 1996; Lawal, Adebowale, Ogunsanwo, Barba, & Ilo, 2005; Olayide, 2004; Shi & Seib, 1992). The purpose of the present work, as a continuation of a previous communication (Wang, Gao, Yu, & Xiao, 2006), is to further demonstrate the polymorph structure of C-type starch granules by SEM, XRD and 13C CP/MAS NMR during acid hydrolysis. This research can add to our understanding of polymorph structure of C-type starch. 2. Materials and methods 2.1. Materials D. JXXCM starch obtained from dried rhizoma of Dioscorea opposita Thunb. cv. Jiaxiangxichangmao in our laboratory was used throughout the study (Wang, Liu et al., 2006).
2006). Two grams (dry basis) of native D. JXXCM starch was hydrolyzed by suspending it in 80 ml of 2.2 mol/L HCl solution at 35 1C for 2, 4, 8, 16 and 32 days without stirring. After hydrolysis, the suspension was filtered by a G4-type anti-acid filler (Tian-Ma Company, Tianjin, China) under low pressure. The filter cake was washed several times with distilled water until the pH value of the filtrate was 7. The resulting filter cake was washed 2–4 times with acetone again. The resulting acid-modified starch was dried at room temperature (25 1C) overnight (air stream) and utilized throughout the whole experiment. 2.3. Morphological properties Scanning electron micrographs were obtained with an environmental scanning electron microscope (ESEM, Philips XL-3, Eindhoven, Holland). Acid-thinned starch samples were suspended in acetone to obtain a 1% suspension. One drop of the starch–acetone suspension was applied on an aluminum stub using double-sided adhesive tape and the starch was coated with gold powder to avoid charging under the electron beam after the acetone volatilized. An accelerating potential of 30 kV was used during micrography. 2.4. X-ray diffractometry X-ray powder diffraction measurements were done using a Panalytical X’Pert Pro diffractometer (PANalytical, Holand). The procedure is followed by the method reported previously (Wang, Liu et al., 2006). 2.5.
13
C CP/MAS solid-state NMR
Solid-state 13C CP/MAS NMR spectra were recorded at 75 MHz using a Bruker MSL 300 NMR spectrometer (Great Britain Bruker Spectrospin Ltd., Conventry, UK) operating at room temperature. Samples were spun at the magic angle (54.51 or 54.71). A magic-angle spinning (m.a.s.) rate of 2.5 KHz and a decouping field of 61 KHz were used. The 901 pulse width was 4 ms with a recycle time of 5 s. A contact time of 1 ms was used for all the samples; spectral width was 20 kHz; acquisition time, 27 ms. Each sample was packed into a 7 mm m.a.s. sample rotor with tight push-fitting caps. The rotor held 200–240 mg of starch sample. Spectra are referenced to external Me4Si via the low field resonance of adamantane (38.6 ppm). About 10,000 scans were accumulated for each spectrum to obtain a satisfactory signal–noise ratio. A polynomial baseline was manually corrected where necessary after Fourier transformation and phasing. 2.6. Particle size analysis
2.2. Preparation of acid-modified starch The acid-modified starch was prepared according to the methods in our previous study (Wang, Gao, Yu et al.,
Particle size analysis of acid-modified starches was done using a laser light-scattering particle size analyzer (Mastersizer S, version 2.15, Malvern Instruments Ltd.,
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Malvern, UK). The focal length was 100 mm. Starch (0.1 g, db) was dissolved in 150 ml distilled water and mixed at a very slow speed using a magnetic stirrer for 1 h at room temperature prior to measurement. An obscuration level of 20% was maintained during measurements on the Mastersizer. 3. Results and discussion 3.1. Scanning electron microscopy (SEM) SEM photographs of native starch and acid-modified starches are displayed in Fig. 1. SEM of native starch shows the presence of large elliptic and oval or small spherical granules. The surface of the granule appears to be smooth, with no evidence of any fissures. During 2–4 days of the hydrolysis, no obvious changes occur on the shape and size of the starch granules from the SEM photographs (Fig. 1—a1, a2, b1, b2, c1, c2). After 8 days of hydrolysis,
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some fissures could be observed on the surface of some starch granules. The fissures are formed due to the erosion of acid on the surface of the starch granule. The length and width of the planform of starch granules change little compared with native starch granules after 16 days of hydrolysis. However, the three-dimensional space of starch granules becomes smaller. That is, the volume of starch granules (after acid hydrolysis) occupied becomes smaller than the native starch granule. When the starch granules are subjected to 32 days of acid hydrolysis, they become still smaller and shriveled (Fig. 1f) due to the degradation of amorphous areas located in the interior of starch granules. However, the surface of most starch granules still remains smooth and intact. After 40 days of hydrolysis, most starch granules fall to pieces completely due to the heavy acid erosion. From SEM of the fragmentized starch granules, it could be deduced that starch granules are hollow or some tunnels develop at the interior of starch granules before they are fractured. As we all know, the
Fig. 1. SEM photographs of native and modified starches. a1, a2: Native starch; b1, b2: 2 days; c1, c2: 4 days; d1, d2: 8 days; e1, e2: 16 days; f1, f2: 32days; and g1, g2, g3, g4: 40 days.
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Fig. 1. (Continued)
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amorphous regions of starch granules are preferentially degraded during the process of acid hydrolysis. This suggests that the amorphous regions are mainly locates in the core parts of starch granules or distribute alternately in the crystalline regions. Further information could be obtained by TEM or AFM. 13
3.2.
C CP/MAS solid-state NMR
The effect of acid hydrolysis on the 13C CP/MAS spectra of starches is shown in Fig. 2. Substantial similarities are observed in all spectra with resolved resonances in the ranges 60–64 and 94–105 ppm. These two peaks are assigned to C-1 and C-6 sites in hexapyranoses, respectively. The peak at 94–105 ppm becomes more and more narrower with increasing hydrolysis time. The major signal intensity in all spectra is in the range 68–78 ppm and is associated with C2, C3 and C5. The weak signal at about 82 ppm in the spectra of native and acid-modified starches subjected to short hydrolysis time is due to the C-4 site (Atichokudomchai, Varavinit, & Chinachoti, 2004; Gidley, & Bociek, 1985; Marchessault & Taylor, 1985; Veregin et al., 1986). The broad shoulder that appears at 95 ppm could arise from the amorphous areas for C1 and the broad resonance at 82 ppm from the amorphous domains for C4. Such an assignment was based on the fact that these broad resonances are absent in the A- and B-type spherulitic crystal spectra, but are present dominantly in that from amorphous samples. As for C-type native and amorphous
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pea starches, these resonances from amorphous areas for C1 and C4 peaks are also present in the spectra of 13C CP/ MAS NMR (Bogracheva et al., 1998). Acid-modified starches (except for starch subjected to 40 days of hydrolysis) showed C1 spectra similar to that of native starch, indicating that acid modification did not have an effect on molecular packing of the double helices in the crystalline regions. After 40 days of hydrolysis, three resonances for C1 could be seen from the 13C NMR spectra of starch (Fig. 2g), which is a typical A-type characteristic. The intensity of C1 and C4 amorphous resonances (95 and 82 ppm) was found to decrease gradually with increasing hydrolysis time and disappeared when the hydrolysis time reached 40 days. In general, amorphous compounds give broad resonances as the distribution of the local molecular environment gives rise to a broad distribution of chemical shifts for each carbon. Ordered materials show narrower resonances due to more regularity of the environment (Gidley & Bociek, 1985; Veregin et al., 1986), reflecting the stricter polymer configurations in the ordered parts of the starch (Paris, Bizot, Buzare, & Buleon, 1999). Then the disappearance of these broad resonances on hydrolysis is due to a decrease in the amount of noncrystalline material in the starch, indicating that the amorphous regions in the starch granules were preferentially degraded and would be completely decomposed as long as the hydrolysis time is enough. As for the resonance for C2, C3 and C5, only one strong and sharp peak (72.5 ppm) is present in the 13C CP/MAS NMR spectra of C-type native starch (Fig. 2a). This result is in agreement with that of the 13C CP/MAS spectra of C-type pea starch (Bogracheva et al., 1998). The peak
72.5ppm 75.9ppm
70.9ppm 4000
g
3500
g
3000
f
2500
e
2000
d
1500
c
1000
b
500
a
Relative diffraction intensity / cps
f
e
d
c C2,3,5 b C1
95 ppm C4
a
C6
0 120
100
80
60
40
Chemical Shift (ppm) Fig. 2. 13C CP/MAS NMR spectra of native and acid-modified starches at various hydrolysis times. (a) Native starch, (b) 2 days, (c) 4 days, (d) 8 days, (e) 16 days, (f) 32 days and (g) 40 days.
5
10
15 20 25 Diffraction angle (2θ)
30
35
Fig. 3. X-ray powder diffraction spectra of native and acid-modified starches at various hydrolysis times. (a) Native starch, (b) 2 days, (c) 4 days, (d) 8 days, (e) 16 days, (f) 32 days and (g) 40 days.
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becomes more and more wide and splits into three peaks (75.9, 72.5 and 70.9 ppm) up to 40 days of hydrolysis. The large differences observed between 13C CP/MAS NMR
spectra of native starch and acid-modified starch suggest substantial conformational differences between the two types of structures (C- and A-type polymorphs).
Table 1 Effect of hydrolysis time on the average particle size Hydrolysis time (days) Average particle diameter (mm)
0 25.7
2 25.2
4 24.6
8 23.8
16 21.2
32 17.6
40 10.2
Crystalline areas Amorphous areas
Crystalline (A-polymorphs)
Amorphous (B-polymorphs)
Native C-type starch granule Amorphous areas are degraded firstly
Hydrolysis for the amorphous area
Hydrolysis for the crystalline area
Fragments after hydrolysis Fig. 4. Structure pattern of C-type starch.
Fragments after hydrolysis
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3.3. XRD diffraction The XRD patterns of acid-modified starches and their native counterparts are shown in Fig. 3. The intensity of the peak at 6.512y is found to decrease with increasing hydrolysis time and disappeared after 16 days of hydrolysis, indicating that the B-type crystal structure is completely degraded. Another striking difference is observed for the peak at around the 2y value of 20.11 in the XRD spectra. Native C-type starch shows only one broad peak at 20.112y. The peak splits into two broad peaks at 19.91 and 20.91 after 40 days of hydrolysis, which is the typical A-type characteristic. The disappearance of the characteristic B-type diffraction peak and the development of the typical A-type diffraction peak show that the crystal type of native starch changes from typical C- to Atype pattern. This result is much different from that of other acid-modified starches which exhibited the same crystalline type in comparison with the unmodified starch (Atichokudomchai & Varavinit, 2003; Lawal et al., 2005; Olayide, 2004; Wang, Truong, & Wang, 2003). This result reveals that B-type polymorphs in the C-type starch granule is first degraded or degraded faster than A-type polymorphs in the process of acid hydrolysis. Generally, amorphous areas were first hydrolyzed, followed by the crystalline regions (Wang & Wang, 2001). That is, B-type polymorphs present in C-type polymorphs mainly constitute the amorphous regions, while the crystalline areas are primarily composed of A-type polymorphs. 3.4. Particle size analysis The average particle size of native starch and acidmodified starches is listed in Table 1. Native starch and acid-modified starches showed the same bimodal distribution (figure not given). The average particle size of unmodified starch granules was 25.7 mm. During 2–8 days of acid hydrolysis, the particle size of acidmodified starches changes little compared with native starch. However, the average particle size reduces from 25.7 mm for native starch to 21.2 mm for acid-modified starch for 16 days. The average particle size is found to decrease further with increasing hydrolysis time. When the starch is subjected to 40 days of hydrolysis, the average particle diameter decreases to 10.2 mm. The reduction in granule size of acid-modified starch could be due to the cleavage of starch chains during the hydrolysis. 4. Conclusions Structural changes during acid hydrolysis could provide information on the granular structure of starch granules. Acid hydrolysis starts from the hilum of the starch granule (amorphous areas) and then the outer crystalline parts. The amorphous regions are mainly located in the core of the starch granules or distribute alternately in the crystalline areas. The amorphous areas could be degraded completely
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as long as the hydrolysis time is enough. XRD and NMR data reveal that B-type polymorphs present in the C-type starch are preferentially attacked by the acid or it is degraded faster than A-type polymorphs. B-type polymorphs in C-type starch granules mainly constitute the amorphous regions, while the crystalline areas are primarily composed of A-type polymorphs. B-type polymorphs exist in the core of C-type starch granules, which is surrounded by A-type polymorphs. The structure pattern of C-type starch is shown in Fig. 4. Acknowledgments We acknowledge the financial assistance of the New Century Excellent Scholar Foundation (N.C.E.S.F., China). We also acknowledge very helpful discussions regarding the 13C CP/NMR data and X-ray diffraction data with Dr. Y. Chen and Dr. H.X. Chen. We also acknowledge the technical assistance of K.Y. Zhu, T. Xue and F.M. Jin for the 13C CP/NMR, SEM and XRD measurements. References Atichokudomchai, N., & Varavinit, S. (2003). Characterization and utilization of acid-modified cross-linked tapioca starch in pharmaceutical tablets. Carbohydrate Polymers, 53, 263–270. Atichokudomchai, N., Varavinit, S., & Chinachoti, P. (2004). A study of ordered structure in acid-modified starch by 13C CP/MAS solid-state NMR. Carbohydrate Polymers, 58, 383–389. Bogracheva, T. Y., Morris, V. J., Ring, S. G., & Hedley, C. L. (1998). The granular structure of C-type pea starch and its role in gelatinization. Biopolymers, 45, 323–332. Cairns, P., Bogracheva, T. Y., Ring, S. G., Hedley, C. L., & Morris, V. J. (1997). Determination of the polymorphic composition of smooth pea starch. Carbohydrate Polymers, 32, 275–282. Gidley, M. J., & Bociek, S. M. (1985). Molecular organization in starches: A 13C CP/MAS NMR study. Journal of the American Chemical Society, 107, 7040–7044. Hinrichs, W., Butiner, G., Steifa, M., Betzei, C., Zabei, V., Pfannemuller, B., et al. (1987). An amylose antiparallel double helix at atomic resolution. Science, 238, 205–208. Imberty, A., & Perez, S. (1988). A revisit to the three-dimensional structure of B-type starch. Biopolymers, 27, 1205–1221. Kang, K. J., Kim, S., Lee, S. K., & Kim, S. K. (1997). Relationship between molecular structure of acid-hydrolyzed rice starch and retrogradation. Korean Journal of Food Science and Technology, 29, 876–881. Kim, R. E., & Ahn, S. Y. (1996). Gelling properties of acid-modified red bean starch gels. Agriculture Chemistry and Biotechnology, 39, 49–53. Lawal, O. S., Adebowale, K. O., Ogunsanwo, B. M., Barba, L. L., & Ilo, N. S. (2005). Oxidized and acid thinned starch derivatives of hybrid maize: Functional characteristics, wide-angle X-ray diffractometry and thermal properties. International Journal of Biological Macromolecules, 35, 71–79. Marchessault, R. H., & Taylor, M. G. (1985). Solid-state 13C-C.P.-M.A.S. N.M.R. of starches. Carbohydrate Research, 144, C1–C5. Ni, S. Y., & Song, X. H. (2002). Nutritional components analysis of Chinese yam. Jiangsu Pharmaceutical and Clinical Research, 10, 26–27. Olayide, S. L. (2004). Composition, physicochemical properties and retrogradation characteristics of native, oxidised, acetylated and acidthinned new cocoyam (Xanthosoma sagittifolium) starch. Food Chemistry, 87, 205–218.
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Paris, M., Bizot, H., Buzare, J. Y., & Buleon, A. (1999). Crystallinity and structuring role of water in native and recrystallized starches by 13C CP-MAS NMR spectroscopy : Spectral decomposition. Carbohydrate Polymers, 39, 327–339. Ratnayake, W. S., Hoover, R., Shahidi, F., Perera, C., & Jane, J. (2001). Composition, molecular structure, and physicochemical properties of starches from four field pea (Pisum sativum L.) cultivars. Food Chemistry, 74, 189–202. Sarko, A., & Wu, H. C. H. (1978). The crystal structures of A-, B- and Cpolymorphs of amylose and starch. Starch/Sta¨rke, 30, 73–78. Shi, Y.-C., & Seib, P. A. (1992). The structure of four waxy starches related to gelatinization and retrogradation. Carbohydrate Research, 227, 131–145. 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. Wang, L. F., & Wang, Y. J. (2001). Structures and physicochemical properties of acid-thinned corn, potato and rice starches. Starch/ Stearke, 53, 570–576. Wang, S. J., Gao, W. Y., Liu, H. Y., Chen, H. X., Yu, J. G., & Xiao, P. G. (2006). Studies on the physicochemical, morphological, thermal and
crystalline properties of starches separated from different Dioscorea opposita cultivars. Food Chemistry, 99, 38–44. Wang, S. J., Gao, W. Y., Yu, J. L., & Xiao, P. G. (2006). The crystalline changes of starch from Dioscorea rhizoma by acid hydrolysis. Chinese Chemical Letter, 17, 1255–1258. Wang, S. J., Liu, H. Y., Gao, W. Y., Chen, H. X., Yu, J. G., & Xiao, P. G. (2006). Characterization of new starches separated from different Chinese yam (Dioscorea opposita Thunb.) cultivars. Food Chemistry, 99, 30–37. Wang, S. J., Yu, J. L., Gao, W. Y., Liu, H. Y., & Xiao, P. G. (2006). New starches from traditional Chinese medicine (TCM)—Chinese yam (Dioscorea opposita Thunb.) cultivars. Carbohydrate Research, 341, 289–293. Wang, Y. J., Truong, V. D., & Wang, L. F. (2003). Structures and rheological properties of corn starch as affected by acid hydrolysis. Carbohydrate Polymers, 52, 327–333. Zobel, H. H., & Stephen, A. M. (1995). In A. M. Stephen (Ed.), Food polysaccharides and their application (p. 19). New York: Marcel Dekker. Zuo, Y. F., & Tang, D. C. (2003). Science of Chinese materia medica. Shanghai, China: Shanghai University of Traditional Chinese Medicine Press pp. 301–303.
FOOD
HYDROCOLLOIDS Food Hydrocolloids 22 (2008) 1283–1290 www.elsevier.com/locate/foodhyd
Structure characterization of C-type starch granule by acid hydrolysis Wang Shujuna,, Yu Jinglinb, Yu Jiugaoc, Pang Jipinga, Liu Hongyand a
College of Pharmaceutical Science and Technology, Tianjin University, Tianjin 300072, China College of Traditional Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin 300072, China c College of Science, Department of Chemistry, Tianjin University, Tianjin 300072, China d Henan Agricultural Academy of Sciences, Henan Province 450002, China
b
Received 26 March 2007; accepted 26 June 2007
Abstract The structural changes of C-type Dioscorea rhizoma starch are evaluated by scanning electron microscope (SEM), X-ray powder diffraction (XRD) and cross polarization/magic-angle spinning (CP/MAS) 13C nuclear magic resonance (NMR) during acid hydrolysis. SEM shows that the amorphous areas are mainly located in the core part of C-type starch granules or distribute alternately in the crystalline regions. XRD analysis reveals that the B-type polymorphs present in the C-type starch granules are preferentially degraded or degraded faster than A-type polymorphs. NMR spectra confirm that the amorphous regions in the starch granules are firstly hydrolyzed and could be hydrolyzed completely as long as the hydrolysis time is enough. After 40 days of hydrolysis, the acid-modified starch shows typical A-type characteristics whether for the X-ray diffraction pattern or for the 13C CP/MAS NMR spectra. The finding suggests that the B-type polymorphs consisting of the C-type starch granules are more unstable than the A-type polymorphs. This is not in agreement with the fact that B-type starches (potato starch) are more resistant to acid and enzyme than A-type cereal starches. r 2007 Elsevier Ltd. All rights reserved. Keywords: Starch; Acid hydrolysis; Amorphous region; Polymorphs
1. Introduction Starch is the predominant carbohydrate reserve found in plants. It is generally believed that starch granules are composed mainly of two types of glucose polymer–amylose, which is essentially a linear chain molecule, and amylopectin, which is branched. Amylose and amylopectin are packed into granules, which are part crystalline and part amorphous in structure. There is evidence that the crystallites consist of parallel, left-handed double helices formed from the short chains of amylopectin. Two types of crystallite, or polymorph structures, A and B, have been identified in starch granules, which can be distinguished by the packing density of the double helices. A-type polymorphs are more dense than B-type polymorphs (Gidley & Bociek, 1985; Hinrichs et al., 1987; Imberty & Perez, 1988; Sarko & Wu, 1978; Veregin, Fyfe, Marchessault, & Taylor, 1986; Zobel & Stephen, 1995). Starches from different Corresponding author.
E-mail address: [email protected] (W. Shujun). 0268-005X/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodhyd.2007.06.012
botanical sources have either A (e.g., maize or rice), B (e.g., potato or canna) or a mixture of A and B types of polymorph (e.g., pea or yam) (Cairns, Bogracheva, Ring, Hedley, & Morris, 1997; Ratnayake, Hoover, Shahidi, Perera, & Jane, 2001; Wang, Gao, Liu, Chen, Yu, & Xiao, 2006; Wang, Liu, Gao, Chen, Yu, & Xiao, 2006; Wang, Yu, Gao, Liu, & Xiao, 2006). Dioscoreae (Chinese name Shanyao), the rhizome of various species of genus Dioscorea opposita Thunb. (Dioscoreaceae), has been used as an important invigorant in traditional Chinese medicine (TCM) for many years (Zuo & Tang, 2003). Total carbohydrates of D. opposita Thunb. vary from 20% to 60%. Starch is the most abundant carbohydrate in this rhizoma of D. opposita Thunb., making up about 40% content in the total biomass (Ni & Song, 2002). However, there are few investigations on the properties of starch present in D. opposita Thunb. In order to understand the properties of these starches, the physicochemical, thermal, morphological and crystalline properties were characterized by means of various methods. The starches from different D. opposita Thunb.
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cultivars showed the typical C-type X-ray diffraction patterns under the same testing conditions (Wang, Gao, Liu et al., 2006; Wang, Liu et al., 2006; Wang, Yu et al., 2006). As mentioned previously, native starches can be classified into three groups, the ‘‘A’’, ‘‘B’’ and ‘‘C’’ starches, on the basis of X-ray diffraction data. Although starch granular structure in general is not fully understood, there is even less understanding about the granular structure of C-type starches, even though they are relatively common. For example, it is not known if the A and B polymorphs are distributed in different granules, or if the two types occur within the same granules. If C-type starches consist of A- and B-type granules, then it is likely that their properties would be intermediate between those of A- and B-type starches. If, on the other hand, both polymorphs exist within all granules, then C-type starches would have unique properties dependent upon the arrangement of those two polymorphs in the granule. Bogracheva, Morris, Ring, and Hedley (1998) used a combination of techniques (including differential scanning calorimetry (DSC), X-ray powder diffraction (XRD), nuclear magnetic resonance (NMR)) to study the structure and properties of C-type starch from pea seeds. C-type starch granules contained both types of polymorph, the B-type polymorphs are in the center of the granule and surrounded by the A-type polymorphs. However, it is not known whether the amorphous area of C-type starch granule consists of the A- or B-type polymorphs or the mixture of A- and B-type polymorphs. Whether the amorphous regions are located in the center part or in the outer part of granules is not clear. Acid modification of starch could be very helpful to understand the inner structure of starch granules (Kang, Kim, Lee, & Kim, 1997; Kim & Ahn, 1996; Lawal, Adebowale, Ogunsanwo, Barba, & Ilo, 2005; Olayide, 2004; Shi & Seib, 1992). The purpose of the present work, as a continuation of a previous communication (Wang, Gao, Yu, & Xiao, 2006), is to further demonstrate the polymorph structure of C-type starch granules by SEM, XRD and 13C CP/MAS NMR during acid hydrolysis. This research can add to our understanding of polymorph structure of C-type starch. 2. Materials and methods 2.1. Materials D. JXXCM starch obtained from dried rhizoma of Dioscorea opposita Thunb. cv. Jiaxiangxichangmao in our laboratory was used throughout the study (Wang, Liu et al., 2006).
2006). Two grams (dry basis) of native D. JXXCM starch was hydrolyzed by suspending it in 80 ml of 2.2 mol/L HCl solution at 35 1C for 2, 4, 8, 16 and 32 days without stirring. After hydrolysis, the suspension was filtered by a G4-type anti-acid filler (Tian-Ma Company, Tianjin, China) under low pressure. The filter cake was washed several times with distilled water until the pH value of the filtrate was 7. The resulting filter cake was washed 2–4 times with acetone again. The resulting acid-modified starch was dried at room temperature (25 1C) overnight (air stream) and utilized throughout the whole experiment. 2.3. Morphological properties Scanning electron micrographs were obtained with an environmental scanning electron microscope (ESEM, Philips XL-3, Eindhoven, Holland). Acid-thinned starch samples were suspended in acetone to obtain a 1% suspension. One drop of the starch–acetone suspension was applied on an aluminum stub using double-sided adhesive tape and the starch was coated with gold powder to avoid charging under the electron beam after the acetone volatilized. An accelerating potential of 30 kV was used during micrography. 2.4. X-ray diffractometry X-ray powder diffraction measurements were done using a Panalytical X’Pert Pro diffractometer (PANalytical, Holand). The procedure is followed by the method reported previously (Wang, Liu et al., 2006). 2.5.
13
C CP/MAS solid-state NMR
Solid-state 13C CP/MAS NMR spectra were recorded at 75 MHz using a Bruker MSL 300 NMR spectrometer (Great Britain Bruker Spectrospin Ltd., Conventry, UK) operating at room temperature. Samples were spun at the magic angle (54.51 or 54.71). A magic-angle spinning (m.a.s.) rate of 2.5 KHz and a decouping field of 61 KHz were used. The 901 pulse width was 4 ms with a recycle time of 5 s. A contact time of 1 ms was used for all the samples; spectral width was 20 kHz; acquisition time, 27 ms. Each sample was packed into a 7 mm m.a.s. sample rotor with tight push-fitting caps. The rotor held 200–240 mg of starch sample. Spectra are referenced to external Me4Si via the low field resonance of adamantane (38.6 ppm). About 10,000 scans were accumulated for each spectrum to obtain a satisfactory signal–noise ratio. A polynomial baseline was manually corrected where necessary after Fourier transformation and phasing. 2.6. Particle size analysis
2.2. Preparation of acid-modified starch The acid-modified starch was prepared according to the methods in our previous study (Wang, Gao, Yu et al.,
Particle size analysis of acid-modified starches was done using a laser light-scattering particle size analyzer (Mastersizer S, version 2.15, Malvern Instruments Ltd.,
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Malvern, UK). The focal length was 100 mm. Starch (0.1 g, db) was dissolved in 150 ml distilled water and mixed at a very slow speed using a magnetic stirrer for 1 h at room temperature prior to measurement. An obscuration level of 20% was maintained during measurements on the Mastersizer. 3. Results and discussion 3.1. Scanning electron microscopy (SEM) SEM photographs of native starch and acid-modified starches are displayed in Fig. 1. SEM of native starch shows the presence of large elliptic and oval or small spherical granules. The surface of the granule appears to be smooth, with no evidence of any fissures. During 2–4 days of the hydrolysis, no obvious changes occur on the shape and size of the starch granules from the SEM photographs (Fig. 1—a1, a2, b1, b2, c1, c2). After 8 days of hydrolysis,
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some fissures could be observed on the surface of some starch granules. The fissures are formed due to the erosion of acid on the surface of the starch granule. The length and width of the planform of starch granules change little compared with native starch granules after 16 days of hydrolysis. However, the three-dimensional space of starch granules becomes smaller. That is, the volume of starch granules (after acid hydrolysis) occupied becomes smaller than the native starch granule. When the starch granules are subjected to 32 days of acid hydrolysis, they become still smaller and shriveled (Fig. 1f) due to the degradation of amorphous areas located in the interior of starch granules. However, the surface of most starch granules still remains smooth and intact. After 40 days of hydrolysis, most starch granules fall to pieces completely due to the heavy acid erosion. From SEM of the fragmentized starch granules, it could be deduced that starch granules are hollow or some tunnels develop at the interior of starch granules before they are fractured. As we all know, the
Fig. 1. SEM photographs of native and modified starches. a1, a2: Native starch; b1, b2: 2 days; c1, c2: 4 days; d1, d2: 8 days; e1, e2: 16 days; f1, f2: 32days; and g1, g2, g3, g4: 40 days.
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Fig. 1. (Continued)
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amorphous regions of starch granules are preferentially degraded during the process of acid hydrolysis. This suggests that the amorphous regions are mainly locates in the core parts of starch granules or distribute alternately in the crystalline regions. Further information could be obtained by TEM or AFM. 13
3.2.
C CP/MAS solid-state NMR
The effect of acid hydrolysis on the 13C CP/MAS spectra of starches is shown in Fig. 2. Substantial similarities are observed in all spectra with resolved resonances in the ranges 60–64 and 94–105 ppm. These two peaks are assigned to C-1 and C-6 sites in hexapyranoses, respectively. The peak at 94–105 ppm becomes more and more narrower with increasing hydrolysis time. The major signal intensity in all spectra is in the range 68–78 ppm and is associated with C2, C3 and C5. The weak signal at about 82 ppm in the spectra of native and acid-modified starches subjected to short hydrolysis time is due to the C-4 site (Atichokudomchai, Varavinit, & Chinachoti, 2004; Gidley, & Bociek, 1985; Marchessault & Taylor, 1985; Veregin et al., 1986). The broad shoulder that appears at 95 ppm could arise from the amorphous areas for C1 and the broad resonance at 82 ppm from the amorphous domains for C4. Such an assignment was based on the fact that these broad resonances are absent in the A- and B-type spherulitic crystal spectra, but are present dominantly in that from amorphous samples. As for C-type native and amorphous
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pea starches, these resonances from amorphous areas for C1 and C4 peaks are also present in the spectra of 13C CP/ MAS NMR (Bogracheva et al., 1998). Acid-modified starches (except for starch subjected to 40 days of hydrolysis) showed C1 spectra similar to that of native starch, indicating that acid modification did not have an effect on molecular packing of the double helices in the crystalline regions. After 40 days of hydrolysis, three resonances for C1 could be seen from the 13C NMR spectra of starch (Fig. 2g), which is a typical A-type characteristic. The intensity of C1 and C4 amorphous resonances (95 and 82 ppm) was found to decrease gradually with increasing hydrolysis time and disappeared when the hydrolysis time reached 40 days. In general, amorphous compounds give broad resonances as the distribution of the local molecular environment gives rise to a broad distribution of chemical shifts for each carbon. Ordered materials show narrower resonances due to more regularity of the environment (Gidley & Bociek, 1985; Veregin et al., 1986), reflecting the stricter polymer configurations in the ordered parts of the starch (Paris, Bizot, Buzare, & Buleon, 1999). Then the disappearance of these broad resonances on hydrolysis is due to a decrease in the amount of noncrystalline material in the starch, indicating that the amorphous regions in the starch granules were preferentially degraded and would be completely decomposed as long as the hydrolysis time is enough. As for the resonance for C2, C3 and C5, only one strong and sharp peak (72.5 ppm) is present in the 13C CP/MAS NMR spectra of C-type native starch (Fig. 2a). This result is in agreement with that of the 13C CP/MAS spectra of C-type pea starch (Bogracheva et al., 1998). The peak
72.5ppm 75.9ppm
70.9ppm 4000
g
3500
g
3000
f
2500
e
2000
d
1500
c
1000
b
500
a
Relative diffraction intensity / cps
f
e
d
c C2,3,5 b C1
95 ppm C4
a
C6
0 120
100
80
60
40
Chemical Shift (ppm) Fig. 2. 13C CP/MAS NMR spectra of native and acid-modified starches at various hydrolysis times. (a) Native starch, (b) 2 days, (c) 4 days, (d) 8 days, (e) 16 days, (f) 32 days and (g) 40 days.
5
10
15 20 25 Diffraction angle (2θ)
30
35
Fig. 3. X-ray powder diffraction spectra of native and acid-modified starches at various hydrolysis times. (a) Native starch, (b) 2 days, (c) 4 days, (d) 8 days, (e) 16 days, (f) 32 days and (g) 40 days.
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becomes more and more wide and splits into three peaks (75.9, 72.5 and 70.9 ppm) up to 40 days of hydrolysis. The large differences observed between 13C CP/MAS NMR
spectra of native starch and acid-modified starch suggest substantial conformational differences between the two types of structures (C- and A-type polymorphs).
Table 1 Effect of hydrolysis time on the average particle size Hydrolysis time (days) Average particle diameter (mm)
0 25.7
2 25.2
4 24.6
8 23.8
16 21.2
32 17.6
40 10.2
Crystalline areas Amorphous areas
Crystalline (A-polymorphs)
Amorphous (B-polymorphs)
Native C-type starch granule Amorphous areas are degraded firstly
Hydrolysis for the amorphous area
Hydrolysis for the crystalline area
Fragments after hydrolysis Fig. 4. Structure pattern of C-type starch.
Fragments after hydrolysis
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3.3. XRD diffraction The XRD patterns of acid-modified starches and their native counterparts are shown in Fig. 3. The intensity of the peak at 6.512y is found to decrease with increasing hydrolysis time and disappeared after 16 days of hydrolysis, indicating that the B-type crystal structure is completely degraded. Another striking difference is observed for the peak at around the 2y value of 20.11 in the XRD spectra. Native C-type starch shows only one broad peak at 20.112y. The peak splits into two broad peaks at 19.91 and 20.91 after 40 days of hydrolysis, which is the typical A-type characteristic. The disappearance of the characteristic B-type diffraction peak and the development of the typical A-type diffraction peak show that the crystal type of native starch changes from typical C- to Atype pattern. This result is much different from that of other acid-modified starches which exhibited the same crystalline type in comparison with the unmodified starch (Atichokudomchai & Varavinit, 2003; Lawal et al., 2005; Olayide, 2004; Wang, Truong, & Wang, 2003). This result reveals that B-type polymorphs in the C-type starch granule is first degraded or degraded faster than A-type polymorphs in the process of acid hydrolysis. Generally, amorphous areas were first hydrolyzed, followed by the crystalline regions (Wang & Wang, 2001). That is, B-type polymorphs present in C-type polymorphs mainly constitute the amorphous regions, while the crystalline areas are primarily composed of A-type polymorphs. 3.4. Particle size analysis The average particle size of native starch and acidmodified starches is listed in Table 1. Native starch and acid-modified starches showed the same bimodal distribution (figure not given). The average particle size of unmodified starch granules was 25.7 mm. During 2–8 days of acid hydrolysis, the particle size of acidmodified starches changes little compared with native starch. However, the average particle size reduces from 25.7 mm for native starch to 21.2 mm for acid-modified starch for 16 days. The average particle size is found to decrease further with increasing hydrolysis time. When the starch is subjected to 40 days of hydrolysis, the average particle diameter decreases to 10.2 mm. The reduction in granule size of acid-modified starch could be due to the cleavage of starch chains during the hydrolysis. 4. Conclusions Structural changes during acid hydrolysis could provide information on the granular structure of starch granules. Acid hydrolysis starts from the hilum of the starch granule (amorphous areas) and then the outer crystalline parts. The amorphous regions are mainly located in the core of the starch granules or distribute alternately in the crystalline areas. The amorphous areas could be degraded completely
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as long as the hydrolysis time is enough. XRD and NMR data reveal that B-type polymorphs present in the C-type starch are preferentially attacked by the acid or it is degraded faster than A-type polymorphs. B-type polymorphs in C-type starch granules mainly constitute the amorphous regions, while the crystalline areas are primarily composed of A-type polymorphs. B-type polymorphs exist in the core of C-type starch granules, which is surrounded by A-type polymorphs. The structure pattern of C-type starch is shown in Fig. 4. Acknowledgments We acknowledge the financial assistance of the New Century Excellent Scholar Foundation (N.C.E.S.F., China). We also acknowledge very helpful discussions regarding the 13C CP/NMR data and X-ray diffraction data with Dr. Y. Chen and Dr. H.X. Chen. We also acknowledge the technical assistance of K.Y. Zhu, T. Xue and F.M. Jin for the 13C CP/NMR, SEM and XRD measurements. References Atichokudomchai, N., & Varavinit, S. (2003). Characterization and utilization of acid-modified cross-linked tapioca starch in pharmaceutical tablets. Carbohydrate Polymers, 53, 263–270. Atichokudomchai, N., Varavinit, S., & Chinachoti, P. (2004). A study of ordered structure in acid-modified starch by 13C CP/MAS solid-state NMR. Carbohydrate Polymers, 58, 383–389. Bogracheva, T. Y., Morris, V. J., Ring, S. G., & Hedley, C. L. (1998). The granular structure of C-type pea starch and its role in gelatinization. Biopolymers, 45, 323–332. Cairns, P., Bogracheva, T. Y., Ring, S. G., Hedley, C. L., & Morris, V. J. (1997). Determination of the polymorphic composition of smooth pea starch. Carbohydrate Polymers, 32, 275–282. Gidley, M. J., & Bociek, S. M. (1985). Molecular organization in starches: A 13C CP/MAS NMR study. Journal of the American Chemical Society, 107, 7040–7044. Hinrichs, W., Butiner, G., Steifa, M., Betzei, C., Zabei, V., Pfannemuller, B., et al. (1987). An amylose antiparallel double helix at atomic resolution. Science, 238, 205–208. Imberty, A., & Perez, S. (1988). A revisit to the three-dimensional structure of B-type starch. Biopolymers, 27, 1205–1221. Kang, K. J., Kim, S., Lee, S. K., & Kim, S. K. (1997). Relationship between molecular structure of acid-hydrolyzed rice starch and retrogradation. Korean Journal of Food Science and Technology, 29, 876–881. Kim, R. E., & Ahn, S. Y. (1996). Gelling properties of acid-modified red bean starch gels. Agriculture Chemistry and Biotechnology, 39, 49–53. Lawal, O. S., Adebowale, K. O., Ogunsanwo, B. M., Barba, L. L., & Ilo, N. S. (2005). Oxidized and acid thinned starch derivatives of hybrid maize: Functional characteristics, wide-angle X-ray diffractometry and thermal properties. International Journal of Biological Macromolecules, 35, 71–79. Marchessault, R. H., & Taylor, M. G. (1985). Solid-state 13C-C.P.-M.A.S. N.M.R. of starches. Carbohydrate Research, 144, C1–C5. Ni, S. Y., & Song, X. H. (2002). Nutritional components analysis of Chinese yam. Jiangsu Pharmaceutical and Clinical Research, 10, 26–27. Olayide, S. L. (2004). Composition, physicochemical properties and retrogradation characteristics of native, oxidised, acetylated and acidthinned new cocoyam (Xanthosoma sagittifolium) starch. Food Chemistry, 87, 205–218.
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