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Studies on the granular structure of resistant starches (type 4) from normal, high amylose and waxy corn starch citrates
Food Research International 39 (2006) 332–341 www.elsevier.com/locate/foodres
Studies on the granular structure of resistant starches (type 4) from normal, high amylose and waxy corn starch citrates Xueju (Sherry) Xie, Qiang Liu *, Steve W. Cui Food Research Program, Agriculture and Agri-Food Canada, 93 Stone Road W., Guelph, Ont., Canada N1G 5C9 Received 20 June 2005; accepted 14 August 2005
Abstract Granular and crystalline structure of starch citrates from normal, high amylose and waxy corn starch were characterized using scanning electron microscopy (SEM), optical microscopy, X-ray diffraction and Fourier transform infrared spectroscopy (FT-IR) in this study. SEM showed that citric acid treatment induced changes in the morphology of starch granules. The granule structure of starch citrates was not collapsed or destroyed even after heating. Normal and high amylose corn starch citrates maintained birefringence but lost it upon heating at 100 C for 30 min. However, waxy corn starch citrate showed no birefringence, even before heating. Starch citrates showed different X-ray diffraction patterns before and after heating. A new peak at 1724 cm 1 (ester bond) was observed in FTIR for all starch citrates before and after heating, indicating starch citrates were heat-stable. After the deconvolution of spectra, the intensity ratio of 1016 cm 1/1045 cm 1 was used to calculate the ratio of amorphous to crystalline phase in the starch citrates. The ratio of 1016 cm 1/1045 cm 1 increased with an increase in the degree of substitution. Crown Copyright 2005 Published by Elsevier Ltd. All rights reserved. Keywords: Corn starch citrates; Enzyme resistant starch; X-ray diffraction; SEM; FT-IR; Optical microscopy
1. Introduction Most starches can be hydrolyzed by enzymes in the human gastrointestinal system. Some starch, however, escapes digestion in the small intestine and passes into the large bowel (Sievert & Pomeranz, 1989), and such starch is termed resistant starch (RS). Resistant starch in the diet is able to lower the glycemic response in humans and, hence, reduce the incidence of type II diabetes (Hoebler, Karinthi, Chiron, Champ, & Barry, 1999; Yue & Waring, 1998). Furthermore, resistant starch can be used as a food ingredient to improve cholesterol metabolism and reduce the risk of colon cancer (Yue & Waring, 1998). Resistant starch has been categorized into four classes: physically inaccessible starch (RS1), granular starch (RS2), retrograded amylose or high amylose starches
*
Corresponding author. Tel.: +1 519 780 8030; fax: +1 519 829 2600. E-mail address: [email protected] (Q. Liu).
(RS3), and chemically modified starches (RS4). It has been long known that chemical modification, such as esterification, etherification, and cross-linking of starch can build resistance to a-amylase (Hood & Arneson, 1976; Janzen, 1969; Leegwater & Luten, 1971). Chemical substitution of starch reduces its enzyme digestibility, probably because the bulky derivatizing groups sterically hinder formation of the enzyme-substrate complex (Conway & Hood, 1976). Woo (1999) obtained 5–70% RS4 starches by cross-linking wheat starch with mixtures of sodium trimetaphosphate and tripolyphosphates. It is well known that starch digestibility and rheological behavior are influenced by starch structure. Many techniques have been used to obtain information on starch structures. X-ray diffraction was employed to investigate different pattern of crystalline structure and crystallinity of resistant starch formed under different conditions (Eerlingen, Crombez, & Delcour, 1993). Fourier transform infrared spectroscopy (FT-IR) has been shown to be sensitive to changes in chain conformation, helicity, and double
0963-9969/$ - see front matter. Crown Copyright 2005 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2005.08.004
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helical structure (Goodfellow & Wilson, 1990). Chatel, Voirin, and Artaud (1997) have successfully identified starch acetates and hydroxypropyl starches by FT-IR. However, there is little information about the granular structure of RS4 starch in comparison with its native counterpart. Furthermore, the FT-IR technique of measuring degree of order of modified starch is not widely used. Studies on the granular structure of RS4 may provide insight into the relationship between starch structure and enzyme resistance. Our previous study showed that treatment of normal, waxy and high amylose corn starches with citric acid (140 C, 7 h) increased their resistant starch content to above 78% (Xie & Liu, 2004). The resistant starch content in those starch citrates was greater than 68% after heating for 30 min in boiling water, indicating they were thermally stable. The purpose of this study was to characterize the granular structure of type 4 resistant starch from different corn starch citrates. Our objectives were: (1) to study the changes in starch granule structure after modification by citric acid using polarized light microscopy and scanning electron microscopy and (2) to examine the effect of esterification with citric acid on the ratio of amorphous to crystalline phase of starch granules using X-ray diffractometry and FT-IR spectroscopy, respectively. 2. Materials and methods 2.1. Materials Corn starch citrates with different degrees of substitution (DS) and resistant starch contents were used in this study; they were obtained from our previous study by reacting starch with citric acid at 140 C for 3, 4, and 7 h (Xie & Liu, 2004). The starches used in the study were three normal corn starch citrates with RS and DS of 41.1% and 0.091 (3 h reaction time), 69.0% and 0.116 (5 h), and 78.8% and 0.120 (7 h), respectively, one waxy corn starch citrate with RS 87.5% and DS 0.160, and one high amylose corn starch citrate (70% amylose) with RS 86.4% and DS 0.140 (7 h reaction time). Controls contained only water (without citric acid) under the same reaction conditions. Starch citrates produced using a 7 h reaction time were selected for the heat-stability test, in which a 7% (w/w) slurry of starch citrate was heated at 100 C in a boiling water bath for 30 min with stirring, and then dried at room temperature to a moisture content 12% (Xie & Liu, 2004).
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2.2.2. Scanning electron microscopy SEM was performed on a Hitachi S-570 scanning electron microscope (Tokyo, Japan) equipped with Quartz PCI digital image acquisition software (Quartz Imaging Corp., Vancouver, BC, Canada). The starch samples were sprayed on a metal plate previously covered with doublesided adhesive, and gold-coated using an Emitech K550 sputter coater (Ashford, Kent, UK) under vacuum. The samples were examined at 10 kV accelerating voltage. Representative micrographs were taken for each sample at magnifications of 2000· and 6000·, respectively. 2.2.3. Wide angle X-ray diffraction A sealed tube X-ray diffraction instrument (Siemens/ Bruker, Madison, WI) with copper radiation (Cu Ka ˚ ), nickel filtered was used. The instrument k = 1.5418 A consisted of a Kristalloflex 760 generator, three-circle goniometer and Hi-Star area detector, equipped with GADDS software. Operation power was 40 kV and 40 mA. Collimator diameter was 0.8 mm. Sample to detector distance was 10 cm. The center of the detector was positioned at an angle of 25 from incident beam. The samples were prepared in thin-walled (0.01 mm) glass capillary tubes (1.0 mm in diameter). The results were given as X-ray diffraction spectra. 2.2.4. Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FT-IR) All spectra were obtained on a Digilab FTS 7000 spectrometer (Digilab USA, Randolph, MA, USA) at a resolution of 4 cm 1 by 32 scans. An attenuated total reflectance (ATR) accessory was used. The spectrometer was equipped with a thermoelectrically cooled deuterated tri-glycine sulfate (DTGS) detector. Granular samples were placed on the crystal in the sample compartment, which has a hinged cover to seal it from the environment. A spectrum of the empty cell was used as the background. All spectra were baseline-corrected, and then deconvoluted using Win-IR Pro software at absorbance range from 1200 to 800 cm 1. A half-band width of 15 cm 1 and a resolution enhancement factor of 1.5 with Bessel apodization were employed. Intensity measurements were performed on the deconvoluted spectra by recording the height of the absorbance bands from the baseline. Measurements were done in duplicate. 3. Results and discussion
2.2. Methods
3.1. Scanning electron microscopy
2.2.1. Light microscopy All samples were prepared by dispersing starch citrate in excess water on a microscope slide. Light microscopy was performed on an Olympus BX60 instrument (Olympus Optical Co., Ltd., Tokyo, Japan) equipped with a polarizer. A Sensys digital camera (Photometric Ltd., Tucson, Arizona, USA) was used for image capture.
Figs. 1–3 show the SEM photographs of corn starch citrates (Figs. 1-B, 2-B and 3-B) and their controls (Figs. 1-A, 2-A and 3-A). The control starch granules (without citric acid treatment) showed a similar shape (round, angular) as their native counterparts (the SEM of native starches not shown in this paper). However, granule shapes changed when starches were treated with citric acid (Figs. 1-B, 2-B
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Fig. 1. Scanning electron micrographs (SEMs) of normal corn starch citrate and its control before and after heating (k = 1000· magnification). (A) Normal corn starch control, 2k; (B) normal corn starch citrate, 2k; (C) normal corn starch citrate, 6k; (A-h) normal corn starch control after heating, 2k; (B-h) normal corn starch citrate after heating, 2k; and (C-h) normal corn starch citrate after heating, 6k.
Fig. 2. Scanning electron micrographs (SEMs) of waxy corn starch citrate and its control before and after heating (k = 1000· magnification). (A) Waxy corn starch control, 2k; (B) waxy corn starch citrate, 2k; (C) waxy corn starch citrate, 6k; (B-h) waxy corn starch citrate after heating, 2k; and (C-h) waxy corn starch citrate after heating, 6k.
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Fig. 3. Scanning electron micrographs (SEMs) of high amylose corn starch (Hylon VII) citrate and its control before and after heating (k = 1000· magnification). (A) Hylon VII control, 2k; (B) hylon VII citrate, 2k; (C) hylon VII citrate, 6k; (B-h) hylon VII citrate after heating, 2k; and (C-h) hylon VII citrate after heating, 6k.
and 3-B). Some of the granules were doughnut-shaped with their outer sides drawn inwards (Figs. 1C and 2C). Waxy corn starch citrate contained more doughnut-shaped granules than the normal and high amylose corn starch citrates. These doughnut shapes were found more frequently in larger granules. The doughnut shaped granules could be due to granular swelling followed by collapse. The swelling could have occurred early in the reaction when the starch granules underwent swelling in a concentrated solution of citric acid. Upon cooling, the swollen granules collapsed into the doughnut shape. The area at the center of granule is the original growing point (hilum). This area is believed to be less organized than the rest of the granule since in corn starch, enzyme and acid hydrolysis and cavitation originate at the hilum (Chabot, Allen, & Hood, 1978; Fuwa, Sugimoto, Tanaka, & Glover, 1978; Whistler & Thornburg, 1957). Fannon, Hauber, and BeMiller (1992) reported that the surface pores on maize granules are openings to channels leading into the granule interior, mostly extending to the central cavity (Huber & BeMiller, 2000). SEM (Figs. 1–3) suggest that chemical reagents such as citric acid could directly access a loosely organized region in the center of starch by channels and cavities, which might lead to an alteration in the granule morphology. Figs. 1–3 also show that the granule surfaces of starch citrates were less smooth than those of their controls. Scanning electron micrographs of heated starch citrates (7% starch was heated at 100 C for 30 min) are shown in
Figs. 1B-h, 2B-h and 3B-h. Normal corn starch control (Fig. 1A-h) was gelatinized by heating and starch granules were swollen and disrupted or collapsed; a similar result was observed for waxy corn starch controls (not shown). In contrast, starch citrate granules did not swell or they exhibited much less swelling than control granules (Figs. 1B-h, 2B-h and 3B-h). This agrees with our previous study, which showed that citrate substitution prevented granular swelling and gelatinization (Xie & Liu, 2004). After heating at 100 C for 30 min, the doughnut shape of starch citrate granules remained virtually unchanged (Figs. 1C-h and 2Bh). It further confirms that the citrate substitution caused cross-linking of starch. Since when citric acid is heated, it will dehydrate to yield an anhydride. The citric anhydride can react with starch to give distarch citrate (Xie & Liu, 2004). The cross-links preserve granule structure during heating in water. However, the surface of some larger granules became rougher after heating. This was more pronounced in waxy corn starch citrate (Fig. 2C-h). 3.2. Optical microscopy Figs. 4 and 5 show polarized light photomicrographs of starch citrates and their controls before and after heating. For the clarity of pictures, photomicrographs of starches that lost birefringence were taken under normal light. Before heating, all controls (Figs. 4B and 5A and D), normal corn starch citrate (Fig. 4A) and high amylose corn starch
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Fig. 4. Polarized light micrographs of normal corn starch citrate and its control before and after heating. (A) Starch citrate before heating; (B) starch control before heating; (C) starch citrate after heating; and (D) starch control after heating.
citrate (Fig. 5E) exhibited birefringence. The loss of birefringence was observed only in waxy corn starch citrate (Fig. 5B). In general, the crystalline organization of starch can be altered by physical, chemical, and biochemical modification. Introducing the substituted groups of citric acid resulted in complete loss of birefringence in waxy corn starch citrate in comparison with normal corn and high amylose corn starch citrates, indicating that waxy corn starch has fragile granules which are easily disrupted during citric acid modification. When all starch citrates and their controls (7% solid in water) were heated at 100 C for 30 min, loss of birefringence was observed for all starches (Fig. 4D) (waxy and high amylose control not shown in the figure). Granules were swollen and disrupted in normal corn and waxy corn controls. However, undisrupted granules were found in high amylose corn control (image not shown). The granules of all starch citrates did not collapse or disrupt as their controls did, even though they lost the Maltese cross pattern. In general, heating starch in the presence of excess water results in the loss of birefringence because of the melting of starch crystallites, and is accompanied by rapid swelling of the granule, as seen in control starches in this study. However, modification of starch by citric acid restricted starch granules from further swelling and disruption during heating (Figs. 4C and 5C and F). Resistant starch contents were above 68% in these starch citrates after heating for 30 min at 100 C due to the formation of cross linkage on citration.
3.3. X-ray diffraction The X-ray diffractograms of starch citrates and their controls are presented in Fig. 6A. Normal corn and waxy corn starch controls showed an A-pattern with major reflections at 2h = 15.3 and 23.4, and an unresolved doublet at 17 and 18. High amylose corn starch control gave a B-type pattern with diffraction peaks at 2h = 17.6 and a few other peaks at 2h values of 20.4, 22.7 and 24, as well as an additional peak at 2h = 6. The X-ray patterns of control starches were modified by addition of citric acid (Fig. 6A). The crystalline peaks of all starch citrates became smaller or even disappeared in comparison with their controls. Waxy corn starch citrate showed the loss of all peaks in the X-ray diffraction pattern, which is in agreement with the result of birefringence loss (Fig. 5B). The diffraction peaks of normal corn starch centered at 2h = 15.3, 17–18 and 23.4 decreased in intensity on citration. A similar decrease in peak intensity (centered at 2h = 17.6 and 20.4) was also observed in high amylose starch citrate (Fig. 6A). However, the peaks centered at 2h = 6, 22.7 and 24.0 in high amylose corn starch disappeared on citration (Fig. 6A). When compared to their controls (Fig. 6A), the decrease in long-range order (based on X-ray diffraction pattern) followed the order: waxy citrate starch > normal corn starch citrate > high amylose corn starch citrate. This trend coincidentally correlated with the amylopectin content. Also, our previous study (Xie & Liu, 2004) showed that waxy corn starch had the highest
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Fig. 5. Polarized light micrographs of waxy corn starch citrate and high amylose corn starch citrate and their controls before and after heating. (A) Waxy corn starch control before heating; (B) waxy corn starch citrate before heating; (C) waxy corn starch citrate after heating; (D) high amylose corn starch control before heating; (E) high amylose corn starch citrate before heating; and (F) high amylose corn starch citrate after heating.
DS (0.16) and RS content (87.5%) in comparison with those of normal corn and high amylose corn starches. In general, amylopectin molecules of A-type starches have shorter chains in both long- and short-chain fractions, and larger amounts of the short-chain fractions than those of the B-type starches. The ratio of short-chain/long-chain has been reported as 3.9 for waxy corn, 2.9 for normal corn, and 0.8 for high amylose corn, respectively (Hizukuri, 1985). The short chain fraction of amylopectin has been found to play an important role in starch crystallinity (Cheetham & Tao, 1998). As seen in Fig. 6A, introducing citric acid groups has an effect on both A-type starches (waxy corn and normal corn starch) and B-type starch (high amylose corn starch). When citric acid penetrates the starch granule through channels and cavities, it could disrupt the crystalline structure of granules due to a concentrated solution of citric acid. The reaction should occur both in the amorphous phase and crystalline phase. Substitution of citric acid groups on starch chains could form a highly cross-linked starch and thus limit starch chain mobility.
When starch citrates were heated at 100 C for 30 min, the decrease in resistant starch content was in the order of waxy corn starch citrate > normal corn starch citrate > high amylose corn starch citrate (Xie & Liu, 2004). Differences in X-ray diffraction patterns of three starch citrates before and after heating are presented in Fig. 6B. Normal corn starch citrate lost most crystalline peaks after heating except a peak at 2h = 20.2. A similar result was obtained for heated high amylose corn starch citrate, in which most peaks disappeared except a peak at 2h = 20.2. It indicates the long-range ordered structure of normal corn and high amylose corn starch citrates was almost completely disrupted. This is consistent with the previous observation that these starch granules lost birefringence after heating (Figs. 4C and 5F). The new X-ray diffraction pattern at 2h = 20.2 may reflect a new structure of amorphous regions in these starch citrates after heating. Also, the X-ray diffraction pattern of heated normal corn and high amylose corn starch citrates may indicate that retrograded amylose could contribute to the new single peak at 2h = 20.2. Both unheated and heated waxy corn starch
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Fig. 6. X-ray diffraction patterns of starch citrates and their controls: (A) before heating; (B) after heating. AM: high amylose corn starch control; AMC: high amylose corn starch citrate; NC: normal corn starch control; NCC: normal corn starch citrate; AP: waxy corn starch control; APC: waxy corn starch citrate; AMC-h: heated high amylose corn starch citrate; APC-h: heated waxy corn starch citrate; NCC-h: heated normal corn starch citrate.
citrate showed the same X-ray diffraction pattern. This is in agreement with results obtained by polarized light microscopy which showed that both heated and unheated waxy corn citrates lost birefringence (Fig. 5B and C). 3.4. FT-IR spectroscopy Fig. 7 presents the FT-IR spectra of starch citrates and their controls. The spectral patterns of all control starches were similar. This result is in agreement with Iizuka and Aishima (1999) who investigated spectral patterns of five starches (corn starch, potato starch, rice starch, amylose and amylopectin) by FT-IR/ATR, and found that all five starches exhibited a similar pattern. The band absorbance in starch has been assigned and matched with the vibrational modes of the chemical bonds by many researchers. In our study, all starch citrates showed 10 peaks in the regions of 3700–800 cm 1 (Fig. 7). From previous studies, Irudayaraj and Yang (2002) showed that the absorbance at 3381 and 2931 cm 1 can be attributed to O–H and C–H bond stretching, respectively. The absorbance at 1350 cm 1 has been attributed to the bended modes of
O–C–H, C–C–H, and C–O–H (Bellon-Maurel, Vallat, & Goffinet, 1995). Absorbance at 1150 and 1080 cm 1 are both assigned as the coupling of C–O, C–C and O–H bond stretching, bending and asymmetric stretching of the C–O–C glycosidic bridge (Goodfellow & Wilson, 1990; van Soest, Tournois, de Wit, & Vliegenthart, 1995). Absorbance at 1022 cm 1 is assigned to the vibration of C–O–H deformation (van Soest et al., 1995), and absorbance at 930 and 860 cm 1 are assigned both for C–H bending (Irudayaraj & Yang, 2002). A new peak at 1724 cm 1 was evident in all starch citrates but not found in their controls. This new absorption peak can be attributed to the characteristic ester group from citric acid in the starch citrate structure. Chatel et al. (1997) differentiated acetylated starch from other modified starches by FT-IR and found that the absorbance at 1724 cm 1 corresponded to the stretching vibration of the C@O bond from the acetyl group. Also, the absorption peak at 1746 cm 1 has been identified as the ester carbonyl group in bromoacetylated starch (Won, Chu, & Yu, 1996) and octanoated starch (Aburto et al., 1997).
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Fig. 7. FT-IR spectra of starch citrates and their controls. AP: waxy corn starch; AM: high amylose corn starch.
Native starch granule is partially crystalline with a degree of crystallinity of 20–40% (Hizukuri, 1996). X-ray diffractometry methods have been normally used for the determination of starch crystallinity. However, a method based on FT-IR was developed for the determination of starch crystallinity (van Soest et al., 1995). The IR absorbance band at 1047 cm 1 was sensitive to ordered or crystalline structure and the band at 1022 cm 1 was associated
with amorphous structure in starch. Thus, the ratio of intensity of 1022 cm 1/1047 cm 1 can express the degree of order in starch (van Soest, de Wit, Tournois, & Vliegenthart, 1994; van Soest et al., 1995). In our FT-IR study, when the original spectra of starch citrates (Fig. 8A) were deconvoluted (Fig. 8B) for better resolution of overlapping peaks, 10 peaks were evident in the spectra in the range of 1200–800 cm 1. The peak at 1016 cm 1 of starch citrates
Fig. 8. Original (A) and deconvoluted (B) FT-IR spectra of starch citrates (7 h reaction time). APC: waxy corn starch citrate; AP: waxy corn starch control; AMC: high amylose corn starch citrate; AM: high amylose corn starch control; NCC: normal corn starch citrate; NC: normal corn starch control.
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2.00
1.60E-02
1.95
1.20E-02 3h
1.85
5h
1.80 1.75
0h
8.00E-03
1.70
Intensity of 1724
Intensity Ratio of 1016/1045
1.90
1.65 4.00E-03
1.60 intensity ratio of 1016/1045
1.55 1.50 0.00
intensity of 1724 0.00E+00
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0.12
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Degree of Substitution
Fig. 9. The relationship between the degree of substitution and intensity of ester bond in normal corn starch citrate and the amorphous/crystalline ratio in starch.
became sharper than their controls, whereas the peak at 1045 cm 1 appeared more flat than their controls. The ratio of 1016 cm 1/1045 cm 1 was calculated to represent the amount of amorphous to crystalline phase in starch citrate. The ratio for waxy, high amylose, and normal corn starch citrate (in comparison with their controls) was 2.1 (1.6), 2.0 (1.6) and 1.9 (1.7), respectively. The higher value indicates that more amorphous phase existed in the starch granule. Similar results were obtained from X-ray diffraction, in which starch citrates (waxy, normal and high amylose corn starch citrate) exhibited greater amorphous content than their controls (Fig. 6A). Fig. 9 shows the relationship between intensity ratio of 1016 cm 1/1045 cm 1 and the intensity of 1724 cm 1 (ester group) and the degree of substitution of normal corn starch citrates produced by different reaction times (3–7 h). As expected, with an increase in the degree of substitution from reaction time 0 to 7 h, the intensity of 1724 cm 1 increased, indicating that more ester groups were present in starch by covalent bond linkage between citric acid and starch molecules. The ratio of 1016 cm 1/1045 cm 1 increased when the degree of substitution increased from 0 (0 h reaction time) to 0.116 (5 h reaction time), which indicated that the citrate substituent altered chain packing and generated more amorphous structures in starch. However, starch citrate with 5 h reaction time had the same ratio of 1016 cm 1/1045 cm 1 as 7 h. DS of these two starch citrates (0.116 for 5 h vs. 0.120 for 7 h) may be too close to cause the difference in the ratio of intensity at 1016 cm 1/1045 cm 1. As DS of normal corn starch citrate increased from 0.09 for 3 h to 0.12 for 7 h, peak intensities at 1150, 1124, 1079, 922 and 860 cm 1 increased gradually (data not shown). It indicates that introduction of citric acid groups affects the vibrational modes of some chemical bonds in starch. When starch citrates were heated at 100 C for 30 min, the ratio of 1016 cm 1/1045 cm 1 increased from 1.9 to 2.2 for normal corn starch citrate and from 2.0 to 2.3 for high amylose corn starch citrate, indicating that gran-
ular structure was further altered by heating. However, the ratio of 1016 cm 1/1045 cm 1 for heated waxy corn starch citrate was the same as unheated sample (2.1). This is in agreement with the result of X-ray diffraction pattern that both heated and unheated waxy corn starch citrates exhibited the same pattern with no crystalline peak shown in the spectra (Fig. 6B). Absorbance at 1724 cm 1 (Fig. 7) still appeared on the FT-IR spectra for all heated starch citrates (not shown). In our previous study, the DS of starch citrate was slightly decreased after heating (Xie & Liu, 2004). The resistant starch contents of starch citrates were above 68% after heating. This indicates that the ester bonds with cross-links in starch citrates are strong enough to survive the heating process. The citric acid can still affect molecular structure in a way that protects starch from enzyme attack. 4. Summary The granular structure of three types of corn starch citrates have been evaluated using scanning electron microscopy and polarized light microscopy. By reacting with citric acid, the granule structure of corn starches changed, especially forming the doughnut-like shapes with their outside drawn inwards. The surface of corn starch citrates were rough compared with their controls. The effects were more pronounced in larger granules. Polarized light microscopy showed that all starch citrates and their controls (except waxy corn starch citrate) exhibited birefringence. All starch citrates lost birefringence after heating at 100 C for 30 min. However, they maintained their granular form and resisted swelling. X-ray diffraction patterns showed the crystalline peaks of all starch citrates became smaller or even disappeared for waxy corn starch citrate as compared to their controls. After heating, normal corn starch citrate and high amylose corn starch citrate produced a new X-ray diffraction pattern with the loss of most crystalline peaks except a peak at 2h = 20.2. From
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FT-IR results, the absorbance at 1724 cm 1, which corresponded to the ester bond in starch, was apparent in all starch citrates both before and after heating. It indicates that these starch citrates were heat-stable. As the degree of substitution increased, the intensity of 1724 cm 1 (ester bond) increased, as well as the amount of amorphous to crystalline phase (1016 cm 1/1045 cm 1) in starch citrate. Acknowledgements This study was carried out with the financial support of the Ontario Corn ProducersÕ Association (OCPA) and Agriculture and Agri-Food Canada. The authors thank Elizabeth Donner for technical assistance. The authors also thank Dr. Paul A. Seib for helpful discussions. References Aburto, J., Thiebaud, S., Alric, I., Borredon, E., Bikiaris, D., Prions, J., et al. (1997). Properties of octanoated starch and its blends with polyethylene. Carbohydrate Polymers, 34, 101–112. Bellon-Maurel, V., Vallat, C., & Goffinet, D. (1995). Quantitative analysis of individual sugars during starch hydrolysis by FT-IR/ATR spectrometry. Part I. Multivariate calibration study-repeatability and reproducibility. Applied Spectroscopy, 49, 556–562. Chabot, J. F., Allen, J. E., & Hood, L. F. (1978). Freeze-etch ultra structure of waxy maize and acid hydrolyzed waxy maize starch granules. Journal of Food Science, 43, 727–730. Chatel, S., Voirin, A., & Artaud, J. (1997). Starch identification and determination in sweetened fruit preparations. 2. Optimization of dialysis and gelatinization steps, infrared identification of starch chemical modifications. Journal of Agricultural and Food Chemistry, 45, 425–430. Cheetham, N. W. H., & Tao, L. (1998). Variation in crystalline type with amylose content in maize starch granules: an X-ray powder diffraction study. Carbohydrate Polymers, 36, 277–284. Conway, R. L., & Hood, L. F. (1976). Pancreatic alpha-amylase hydrolysis products of modified and unmodified tapioca starches. Die Starke, 28, 342. Eerlingen, R. C., Crombez, M., & Delcour, J. A. (1993). Enzyme-resistant starch. I. Quantitative and qualitative influence of incubation time and temperature of autoclaved starch on resistant starch formation. Cereal Chemistry, 70(3), 339–344. Fannon, J. E., Hauber, R. J., & BeMiller, J. N. (1992). Surface pores of starch granules. Cereal Chemistry, 69, 284–288. Fuwa, H., Sugimoto, Y., Tanaka, M., & Glover, D. V. (1978). Susceptibility of various starch granules to amylase as seen by scanning electron microscope. Starch, 30, 186–191.
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Goodfellow, B. J., & Wilson, R. H. (1990). A Fourier transform IR study of the gelation of amylose and amylopectin. Biopolymers, 30, 1183–1189. Hizukuri, S. (1985). Relationship between the distribution of the chain length of amylopectin and the crystalline structure of starch granules. Carbohydrate Research, 141, 295–306. Hizukuri, S. (1996). Starch: Analytical aspects. Carbohydrates in food (pp. 147–160). New York: Marcel Dekker. Hoebler, C., Karinthi, A., Chiron, H., Champ, M., & Barry, J. L. (1999). Bioavailability of starch in bread rich in amylose: metabolic responses in healthy subjects and starch structure. European Journal of Clinical Nutrition, 53, 360–366. Hood, L. F., & Arneson, V. G. (1976). In vitro digestibility of hydroxypropyl distarch phosphate and unmodified tapioca starch. Cereal Chemistry, 53, 282–290. Huber, K. C., & BeMiller, J. N. (2000). Channels of maize and sorghum starch granules. Carbohydrate Polymers, 41, 269–276. Iizuka, K., & Aishima, T. (1999). Starch gelation process observed by FTIR/ATR spectrometry with multivariate data analysis. Journal of Food Science, 64, 653–658. Irudayaraj, J., & Yang, H. (2002). Depth profiling of a heterogeneous food-packing model using step-scan Fourier transform infrared photoacoustic spectroscopy. Journal of Food Engineering, 55, 25–33. Janzen, G. L. (1969). Digestibility of starches and phosphatized starches by means of pancreatin. Starch, 38, 231–237. Leegwater, D. C., & Luten, J. B. (1971). A study on the in vitro digestibility of hydroxypropyl starches by pancreatin. Starch, 23, 430–432. Sievert, D., & Pomeranz, Y. (1989). Enzyme-resistant starch. I. Characterization and evaluation by enzymic, thermoanalytical, and microscopic methods. Cereal Chemistry, 66(4), 342–347. van Soest, J. J. G., de Wit, D., Tournois, H., & Vliegenthart, J. F. G. (1994). Retrogradation of potato starch as studied by Fourier transform infrared spectroscopy. Starch, 46, 453–457. van Soest, J. J. G., Tournois, H., de Wit, D., & Vliegenthart, J. F. G. (1995). Short-range structure in (partially) crystalline potato starch determined with attenuated total reflectance Fourier-transform IR spectroscopy. Carbohydrate Research, 279, 201–214. Whistler, R. L., & Thornburg, W. L. (1957). Development of starch granules in corn endosperm. Journal of Agricultural and Food Chemistry, 5, 203–207. Won, C. Y., Chu, C. C., & Yu, T. J. (1996). Synthesis of starch-based drug carrier for the control/release of estrone hormone. Carbohydrate Polymers, 32, 239–244. Woo, K. S. (1999). Cross-linked, RS4 type resistant starch: preparation and properties. Dissertation, Department of Grain Science and Industry. Kansas State University, KS, USA. Xie, X. J., & Liu, Q. (2004). Development of new resistant starch-citrate starch as a functional food ingredient. Starch, 56, 364–370. Yue, P., & Waring, S. (1998). Resistant starch in food applications. Cereal Foods World, 43, 691–695.
Studies on the granular structure of resistant starches (type 4) from normal, high amylose and waxy corn starch citrates Xueju (Sherry) Xie, Qiang Liu *, Steve W. Cui Food Research Program, Agriculture and Agri-Food Canada, 93 Stone Road W., Guelph, Ont., Canada N1G 5C9 Received 20 June 2005; accepted 14 August 2005
Abstract Granular and crystalline structure of starch citrates from normal, high amylose and waxy corn starch were characterized using scanning electron microscopy (SEM), optical microscopy, X-ray diffraction and Fourier transform infrared spectroscopy (FT-IR) in this study. SEM showed that citric acid treatment induced changes in the morphology of starch granules. The granule structure of starch citrates was not collapsed or destroyed even after heating. Normal and high amylose corn starch citrates maintained birefringence but lost it upon heating at 100 C for 30 min. However, waxy corn starch citrate showed no birefringence, even before heating. Starch citrates showed different X-ray diffraction patterns before and after heating. A new peak at 1724 cm 1 (ester bond) was observed in FTIR for all starch citrates before and after heating, indicating starch citrates were heat-stable. After the deconvolution of spectra, the intensity ratio of 1016 cm 1/1045 cm 1 was used to calculate the ratio of amorphous to crystalline phase in the starch citrates. The ratio of 1016 cm 1/1045 cm 1 increased with an increase in the degree of substitution. Crown Copyright 2005 Published by Elsevier Ltd. All rights reserved. Keywords: Corn starch citrates; Enzyme resistant starch; X-ray diffraction; SEM; FT-IR; Optical microscopy
1. Introduction Most starches can be hydrolyzed by enzymes in the human gastrointestinal system. Some starch, however, escapes digestion in the small intestine and passes into the large bowel (Sievert & Pomeranz, 1989), and such starch is termed resistant starch (RS). Resistant starch in the diet is able to lower the glycemic response in humans and, hence, reduce the incidence of type II diabetes (Hoebler, Karinthi, Chiron, Champ, & Barry, 1999; Yue & Waring, 1998). Furthermore, resistant starch can be used as a food ingredient to improve cholesterol metabolism and reduce the risk of colon cancer (Yue & Waring, 1998). Resistant starch has been categorized into four classes: physically inaccessible starch (RS1), granular starch (RS2), retrograded amylose or high amylose starches
*
Corresponding author. Tel.: +1 519 780 8030; fax: +1 519 829 2600. E-mail address: [email protected] (Q. Liu).
(RS3), and chemically modified starches (RS4). It has been long known that chemical modification, such as esterification, etherification, and cross-linking of starch can build resistance to a-amylase (Hood & Arneson, 1976; Janzen, 1969; Leegwater & Luten, 1971). Chemical substitution of starch reduces its enzyme digestibility, probably because the bulky derivatizing groups sterically hinder formation of the enzyme-substrate complex (Conway & Hood, 1976). Woo (1999) obtained 5–70% RS4 starches by cross-linking wheat starch with mixtures of sodium trimetaphosphate and tripolyphosphates. It is well known that starch digestibility and rheological behavior are influenced by starch structure. Many techniques have been used to obtain information on starch structures. X-ray diffraction was employed to investigate different pattern of crystalline structure and crystallinity of resistant starch formed under different conditions (Eerlingen, Crombez, & Delcour, 1993). Fourier transform infrared spectroscopy (FT-IR) has been shown to be sensitive to changes in chain conformation, helicity, and double
0963-9969/$ - see front matter. Crown Copyright 2005 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2005.08.004
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helical structure (Goodfellow & Wilson, 1990). Chatel, Voirin, and Artaud (1997) have successfully identified starch acetates and hydroxypropyl starches by FT-IR. However, there is little information about the granular structure of RS4 starch in comparison with its native counterpart. Furthermore, the FT-IR technique of measuring degree of order of modified starch is not widely used. Studies on the granular structure of RS4 may provide insight into the relationship between starch structure and enzyme resistance. Our previous study showed that treatment of normal, waxy and high amylose corn starches with citric acid (140 C, 7 h) increased their resistant starch content to above 78% (Xie & Liu, 2004). The resistant starch content in those starch citrates was greater than 68% after heating for 30 min in boiling water, indicating they were thermally stable. The purpose of this study was to characterize the granular structure of type 4 resistant starch from different corn starch citrates. Our objectives were: (1) to study the changes in starch granule structure after modification by citric acid using polarized light microscopy and scanning electron microscopy and (2) to examine the effect of esterification with citric acid on the ratio of amorphous to crystalline phase of starch granules using X-ray diffractometry and FT-IR spectroscopy, respectively. 2. Materials and methods 2.1. Materials Corn starch citrates with different degrees of substitution (DS) and resistant starch contents were used in this study; they were obtained from our previous study by reacting starch with citric acid at 140 C for 3, 4, and 7 h (Xie & Liu, 2004). The starches used in the study were three normal corn starch citrates with RS and DS of 41.1% and 0.091 (3 h reaction time), 69.0% and 0.116 (5 h), and 78.8% and 0.120 (7 h), respectively, one waxy corn starch citrate with RS 87.5% and DS 0.160, and one high amylose corn starch citrate (70% amylose) with RS 86.4% and DS 0.140 (7 h reaction time). Controls contained only water (without citric acid) under the same reaction conditions. Starch citrates produced using a 7 h reaction time were selected for the heat-stability test, in which a 7% (w/w) slurry of starch citrate was heated at 100 C in a boiling water bath for 30 min with stirring, and then dried at room temperature to a moisture content 12% (Xie & Liu, 2004).
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2.2.2. Scanning electron microscopy SEM was performed on a Hitachi S-570 scanning electron microscope (Tokyo, Japan) equipped with Quartz PCI digital image acquisition software (Quartz Imaging Corp., Vancouver, BC, Canada). The starch samples were sprayed on a metal plate previously covered with doublesided adhesive, and gold-coated using an Emitech K550 sputter coater (Ashford, Kent, UK) under vacuum. The samples were examined at 10 kV accelerating voltage. Representative micrographs were taken for each sample at magnifications of 2000· and 6000·, respectively. 2.2.3. Wide angle X-ray diffraction A sealed tube X-ray diffraction instrument (Siemens/ Bruker, Madison, WI) with copper radiation (Cu Ka ˚ ), nickel filtered was used. The instrument k = 1.5418 A consisted of a Kristalloflex 760 generator, three-circle goniometer and Hi-Star area detector, equipped with GADDS software. Operation power was 40 kV and 40 mA. Collimator diameter was 0.8 mm. Sample to detector distance was 10 cm. The center of the detector was positioned at an angle of 25 from incident beam. The samples were prepared in thin-walled (0.01 mm) glass capillary tubes (1.0 mm in diameter). The results were given as X-ray diffraction spectra. 2.2.4. Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FT-IR) All spectra were obtained on a Digilab FTS 7000 spectrometer (Digilab USA, Randolph, MA, USA) at a resolution of 4 cm 1 by 32 scans. An attenuated total reflectance (ATR) accessory was used. The spectrometer was equipped with a thermoelectrically cooled deuterated tri-glycine sulfate (DTGS) detector. Granular samples were placed on the crystal in the sample compartment, which has a hinged cover to seal it from the environment. A spectrum of the empty cell was used as the background. All spectra were baseline-corrected, and then deconvoluted using Win-IR Pro software at absorbance range from 1200 to 800 cm 1. A half-band width of 15 cm 1 and a resolution enhancement factor of 1.5 with Bessel apodization were employed. Intensity measurements were performed on the deconvoluted spectra by recording the height of the absorbance bands from the baseline. Measurements were done in duplicate. 3. Results and discussion
2.2. Methods
3.1. Scanning electron microscopy
2.2.1. Light microscopy All samples were prepared by dispersing starch citrate in excess water on a microscope slide. Light microscopy was performed on an Olympus BX60 instrument (Olympus Optical Co., Ltd., Tokyo, Japan) equipped with a polarizer. A Sensys digital camera (Photometric Ltd., Tucson, Arizona, USA) was used for image capture.
Figs. 1–3 show the SEM photographs of corn starch citrates (Figs. 1-B, 2-B and 3-B) and their controls (Figs. 1-A, 2-A and 3-A). The control starch granules (without citric acid treatment) showed a similar shape (round, angular) as their native counterparts (the SEM of native starches not shown in this paper). However, granule shapes changed when starches were treated with citric acid (Figs. 1-B, 2-B
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Fig. 1. Scanning electron micrographs (SEMs) of normal corn starch citrate and its control before and after heating (k = 1000· magnification). (A) Normal corn starch control, 2k; (B) normal corn starch citrate, 2k; (C) normal corn starch citrate, 6k; (A-h) normal corn starch control after heating, 2k; (B-h) normal corn starch citrate after heating, 2k; and (C-h) normal corn starch citrate after heating, 6k.
Fig. 2. Scanning electron micrographs (SEMs) of waxy corn starch citrate and its control before and after heating (k = 1000· magnification). (A) Waxy corn starch control, 2k; (B) waxy corn starch citrate, 2k; (C) waxy corn starch citrate, 6k; (B-h) waxy corn starch citrate after heating, 2k; and (C-h) waxy corn starch citrate after heating, 6k.
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Fig. 3. Scanning electron micrographs (SEMs) of high amylose corn starch (Hylon VII) citrate and its control before and after heating (k = 1000· magnification). (A) Hylon VII control, 2k; (B) hylon VII citrate, 2k; (C) hylon VII citrate, 6k; (B-h) hylon VII citrate after heating, 2k; and (C-h) hylon VII citrate after heating, 6k.
and 3-B). Some of the granules were doughnut-shaped with their outer sides drawn inwards (Figs. 1C and 2C). Waxy corn starch citrate contained more doughnut-shaped granules than the normal and high amylose corn starch citrates. These doughnut shapes were found more frequently in larger granules. The doughnut shaped granules could be due to granular swelling followed by collapse. The swelling could have occurred early in the reaction when the starch granules underwent swelling in a concentrated solution of citric acid. Upon cooling, the swollen granules collapsed into the doughnut shape. The area at the center of granule is the original growing point (hilum). This area is believed to be less organized than the rest of the granule since in corn starch, enzyme and acid hydrolysis and cavitation originate at the hilum (Chabot, Allen, & Hood, 1978; Fuwa, Sugimoto, Tanaka, & Glover, 1978; Whistler & Thornburg, 1957). Fannon, Hauber, and BeMiller (1992) reported that the surface pores on maize granules are openings to channels leading into the granule interior, mostly extending to the central cavity (Huber & BeMiller, 2000). SEM (Figs. 1–3) suggest that chemical reagents such as citric acid could directly access a loosely organized region in the center of starch by channels and cavities, which might lead to an alteration in the granule morphology. Figs. 1–3 also show that the granule surfaces of starch citrates were less smooth than those of their controls. Scanning electron micrographs of heated starch citrates (7% starch was heated at 100 C for 30 min) are shown in
Figs. 1B-h, 2B-h and 3B-h. Normal corn starch control (Fig. 1A-h) was gelatinized by heating and starch granules were swollen and disrupted or collapsed; a similar result was observed for waxy corn starch controls (not shown). In contrast, starch citrate granules did not swell or they exhibited much less swelling than control granules (Figs. 1B-h, 2B-h and 3B-h). This agrees with our previous study, which showed that citrate substitution prevented granular swelling and gelatinization (Xie & Liu, 2004). After heating at 100 C for 30 min, the doughnut shape of starch citrate granules remained virtually unchanged (Figs. 1C-h and 2Bh). It further confirms that the citrate substitution caused cross-linking of starch. Since when citric acid is heated, it will dehydrate to yield an anhydride. The citric anhydride can react with starch to give distarch citrate (Xie & Liu, 2004). The cross-links preserve granule structure during heating in water. However, the surface of some larger granules became rougher after heating. This was more pronounced in waxy corn starch citrate (Fig. 2C-h). 3.2. Optical microscopy Figs. 4 and 5 show polarized light photomicrographs of starch citrates and their controls before and after heating. For the clarity of pictures, photomicrographs of starches that lost birefringence were taken under normal light. Before heating, all controls (Figs. 4B and 5A and D), normal corn starch citrate (Fig. 4A) and high amylose corn starch
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Fig. 4. Polarized light micrographs of normal corn starch citrate and its control before and after heating. (A) Starch citrate before heating; (B) starch control before heating; (C) starch citrate after heating; and (D) starch control after heating.
citrate (Fig. 5E) exhibited birefringence. The loss of birefringence was observed only in waxy corn starch citrate (Fig. 5B). In general, the crystalline organization of starch can be altered by physical, chemical, and biochemical modification. Introducing the substituted groups of citric acid resulted in complete loss of birefringence in waxy corn starch citrate in comparison with normal corn and high amylose corn starch citrates, indicating that waxy corn starch has fragile granules which are easily disrupted during citric acid modification. When all starch citrates and their controls (7% solid in water) were heated at 100 C for 30 min, loss of birefringence was observed for all starches (Fig. 4D) (waxy and high amylose control not shown in the figure). Granules were swollen and disrupted in normal corn and waxy corn controls. However, undisrupted granules were found in high amylose corn control (image not shown). The granules of all starch citrates did not collapse or disrupt as their controls did, even though they lost the Maltese cross pattern. In general, heating starch in the presence of excess water results in the loss of birefringence because of the melting of starch crystallites, and is accompanied by rapid swelling of the granule, as seen in control starches in this study. However, modification of starch by citric acid restricted starch granules from further swelling and disruption during heating (Figs. 4C and 5C and F). Resistant starch contents were above 68% in these starch citrates after heating for 30 min at 100 C due to the formation of cross linkage on citration.
3.3. X-ray diffraction The X-ray diffractograms of starch citrates and their controls are presented in Fig. 6A. Normal corn and waxy corn starch controls showed an A-pattern with major reflections at 2h = 15.3 and 23.4, and an unresolved doublet at 17 and 18. High amylose corn starch control gave a B-type pattern with diffraction peaks at 2h = 17.6 and a few other peaks at 2h values of 20.4, 22.7 and 24, as well as an additional peak at 2h = 6. The X-ray patterns of control starches were modified by addition of citric acid (Fig. 6A). The crystalline peaks of all starch citrates became smaller or even disappeared in comparison with their controls. Waxy corn starch citrate showed the loss of all peaks in the X-ray diffraction pattern, which is in agreement with the result of birefringence loss (Fig. 5B). The diffraction peaks of normal corn starch centered at 2h = 15.3, 17–18 and 23.4 decreased in intensity on citration. A similar decrease in peak intensity (centered at 2h = 17.6 and 20.4) was also observed in high amylose starch citrate (Fig. 6A). However, the peaks centered at 2h = 6, 22.7 and 24.0 in high amylose corn starch disappeared on citration (Fig. 6A). When compared to their controls (Fig. 6A), the decrease in long-range order (based on X-ray diffraction pattern) followed the order: waxy citrate starch > normal corn starch citrate > high amylose corn starch citrate. This trend coincidentally correlated with the amylopectin content. Also, our previous study (Xie & Liu, 2004) showed that waxy corn starch had the highest
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Fig. 5. Polarized light micrographs of waxy corn starch citrate and high amylose corn starch citrate and their controls before and after heating. (A) Waxy corn starch control before heating; (B) waxy corn starch citrate before heating; (C) waxy corn starch citrate after heating; (D) high amylose corn starch control before heating; (E) high amylose corn starch citrate before heating; and (F) high amylose corn starch citrate after heating.
DS (0.16) and RS content (87.5%) in comparison with those of normal corn and high amylose corn starches. In general, amylopectin molecules of A-type starches have shorter chains in both long- and short-chain fractions, and larger amounts of the short-chain fractions than those of the B-type starches. The ratio of short-chain/long-chain has been reported as 3.9 for waxy corn, 2.9 for normal corn, and 0.8 for high amylose corn, respectively (Hizukuri, 1985). The short chain fraction of amylopectin has been found to play an important role in starch crystallinity (Cheetham & Tao, 1998). As seen in Fig. 6A, introducing citric acid groups has an effect on both A-type starches (waxy corn and normal corn starch) and B-type starch (high amylose corn starch). When citric acid penetrates the starch granule through channels and cavities, it could disrupt the crystalline structure of granules due to a concentrated solution of citric acid. The reaction should occur both in the amorphous phase and crystalline phase. Substitution of citric acid groups on starch chains could form a highly cross-linked starch and thus limit starch chain mobility.
When starch citrates were heated at 100 C for 30 min, the decrease in resistant starch content was in the order of waxy corn starch citrate > normal corn starch citrate > high amylose corn starch citrate (Xie & Liu, 2004). Differences in X-ray diffraction patterns of three starch citrates before and after heating are presented in Fig. 6B. Normal corn starch citrate lost most crystalline peaks after heating except a peak at 2h = 20.2. A similar result was obtained for heated high amylose corn starch citrate, in which most peaks disappeared except a peak at 2h = 20.2. It indicates the long-range ordered structure of normal corn and high amylose corn starch citrates was almost completely disrupted. This is consistent with the previous observation that these starch granules lost birefringence after heating (Figs. 4C and 5F). The new X-ray diffraction pattern at 2h = 20.2 may reflect a new structure of amorphous regions in these starch citrates after heating. Also, the X-ray diffraction pattern of heated normal corn and high amylose corn starch citrates may indicate that retrograded amylose could contribute to the new single peak at 2h = 20.2. Both unheated and heated waxy corn starch
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Fig. 6. X-ray diffraction patterns of starch citrates and their controls: (A) before heating; (B) after heating. AM: high amylose corn starch control; AMC: high amylose corn starch citrate; NC: normal corn starch control; NCC: normal corn starch citrate; AP: waxy corn starch control; APC: waxy corn starch citrate; AMC-h: heated high amylose corn starch citrate; APC-h: heated waxy corn starch citrate; NCC-h: heated normal corn starch citrate.
citrate showed the same X-ray diffraction pattern. This is in agreement with results obtained by polarized light microscopy which showed that both heated and unheated waxy corn citrates lost birefringence (Fig. 5B and C). 3.4. FT-IR spectroscopy Fig. 7 presents the FT-IR spectra of starch citrates and their controls. The spectral patterns of all control starches were similar. This result is in agreement with Iizuka and Aishima (1999) who investigated spectral patterns of five starches (corn starch, potato starch, rice starch, amylose and amylopectin) by FT-IR/ATR, and found that all five starches exhibited a similar pattern. The band absorbance in starch has been assigned and matched with the vibrational modes of the chemical bonds by many researchers. In our study, all starch citrates showed 10 peaks in the regions of 3700–800 cm 1 (Fig. 7). From previous studies, Irudayaraj and Yang (2002) showed that the absorbance at 3381 and 2931 cm 1 can be attributed to O–H and C–H bond stretching, respectively. The absorbance at 1350 cm 1 has been attributed to the bended modes of
O–C–H, C–C–H, and C–O–H (Bellon-Maurel, Vallat, & Goffinet, 1995). Absorbance at 1150 and 1080 cm 1 are both assigned as the coupling of C–O, C–C and O–H bond stretching, bending and asymmetric stretching of the C–O–C glycosidic bridge (Goodfellow & Wilson, 1990; van Soest, Tournois, de Wit, & Vliegenthart, 1995). Absorbance at 1022 cm 1 is assigned to the vibration of C–O–H deformation (van Soest et al., 1995), and absorbance at 930 and 860 cm 1 are assigned both for C–H bending (Irudayaraj & Yang, 2002). A new peak at 1724 cm 1 was evident in all starch citrates but not found in their controls. This new absorption peak can be attributed to the characteristic ester group from citric acid in the starch citrate structure. Chatel et al. (1997) differentiated acetylated starch from other modified starches by FT-IR and found that the absorbance at 1724 cm 1 corresponded to the stretching vibration of the C@O bond from the acetyl group. Also, the absorption peak at 1746 cm 1 has been identified as the ester carbonyl group in bromoacetylated starch (Won, Chu, & Yu, 1996) and octanoated starch (Aburto et al., 1997).
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Fig. 7. FT-IR spectra of starch citrates and their controls. AP: waxy corn starch; AM: high amylose corn starch.
Native starch granule is partially crystalline with a degree of crystallinity of 20–40% (Hizukuri, 1996). X-ray diffractometry methods have been normally used for the determination of starch crystallinity. However, a method based on FT-IR was developed for the determination of starch crystallinity (van Soest et al., 1995). The IR absorbance band at 1047 cm 1 was sensitive to ordered or crystalline structure and the band at 1022 cm 1 was associated
with amorphous structure in starch. Thus, the ratio of intensity of 1022 cm 1/1047 cm 1 can express the degree of order in starch (van Soest, de Wit, Tournois, & Vliegenthart, 1994; van Soest et al., 1995). In our FT-IR study, when the original spectra of starch citrates (Fig. 8A) were deconvoluted (Fig. 8B) for better resolution of overlapping peaks, 10 peaks were evident in the spectra in the range of 1200–800 cm 1. The peak at 1016 cm 1 of starch citrates
Fig. 8. Original (A) and deconvoluted (B) FT-IR spectra of starch citrates (7 h reaction time). APC: waxy corn starch citrate; AP: waxy corn starch control; AMC: high amylose corn starch citrate; AM: high amylose corn starch control; NCC: normal corn starch citrate; NC: normal corn starch control.
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2.00
1.60E-02
1.95
1.20E-02 3h
1.85
5h
1.80 1.75
0h
8.00E-03
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Intensity Ratio of 1016/1045
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1.65 4.00E-03
1.60 intensity ratio of 1016/1045
1.55 1.50 0.00
intensity of 1724 0.00E+00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
Degree of Substitution
Fig. 9. The relationship between the degree of substitution and intensity of ester bond in normal corn starch citrate and the amorphous/crystalline ratio in starch.
became sharper than their controls, whereas the peak at 1045 cm 1 appeared more flat than their controls. The ratio of 1016 cm 1/1045 cm 1 was calculated to represent the amount of amorphous to crystalline phase in starch citrate. The ratio for waxy, high amylose, and normal corn starch citrate (in comparison with their controls) was 2.1 (1.6), 2.0 (1.6) and 1.9 (1.7), respectively. The higher value indicates that more amorphous phase existed in the starch granule. Similar results were obtained from X-ray diffraction, in which starch citrates (waxy, normal and high amylose corn starch citrate) exhibited greater amorphous content than their controls (Fig. 6A). Fig. 9 shows the relationship between intensity ratio of 1016 cm 1/1045 cm 1 and the intensity of 1724 cm 1 (ester group) and the degree of substitution of normal corn starch citrates produced by different reaction times (3–7 h). As expected, with an increase in the degree of substitution from reaction time 0 to 7 h, the intensity of 1724 cm 1 increased, indicating that more ester groups were present in starch by covalent bond linkage between citric acid and starch molecules. The ratio of 1016 cm 1/1045 cm 1 increased when the degree of substitution increased from 0 (0 h reaction time) to 0.116 (5 h reaction time), which indicated that the citrate substituent altered chain packing and generated more amorphous structures in starch. However, starch citrate with 5 h reaction time had the same ratio of 1016 cm 1/1045 cm 1 as 7 h. DS of these two starch citrates (0.116 for 5 h vs. 0.120 for 7 h) may be too close to cause the difference in the ratio of intensity at 1016 cm 1/1045 cm 1. As DS of normal corn starch citrate increased from 0.09 for 3 h to 0.12 for 7 h, peak intensities at 1150, 1124, 1079, 922 and 860 cm 1 increased gradually (data not shown). It indicates that introduction of citric acid groups affects the vibrational modes of some chemical bonds in starch. When starch citrates were heated at 100 C for 30 min, the ratio of 1016 cm 1/1045 cm 1 increased from 1.9 to 2.2 for normal corn starch citrate and from 2.0 to 2.3 for high amylose corn starch citrate, indicating that gran-
ular structure was further altered by heating. However, the ratio of 1016 cm 1/1045 cm 1 for heated waxy corn starch citrate was the same as unheated sample (2.1). This is in agreement with the result of X-ray diffraction pattern that both heated and unheated waxy corn starch citrates exhibited the same pattern with no crystalline peak shown in the spectra (Fig. 6B). Absorbance at 1724 cm 1 (Fig. 7) still appeared on the FT-IR spectra for all heated starch citrates (not shown). In our previous study, the DS of starch citrate was slightly decreased after heating (Xie & Liu, 2004). The resistant starch contents of starch citrates were above 68% after heating. This indicates that the ester bonds with cross-links in starch citrates are strong enough to survive the heating process. The citric acid can still affect molecular structure in a way that protects starch from enzyme attack. 4. Summary The granular structure of three types of corn starch citrates have been evaluated using scanning electron microscopy and polarized light microscopy. By reacting with citric acid, the granule structure of corn starches changed, especially forming the doughnut-like shapes with their outside drawn inwards. The surface of corn starch citrates were rough compared with their controls. The effects were more pronounced in larger granules. Polarized light microscopy showed that all starch citrates and their controls (except waxy corn starch citrate) exhibited birefringence. All starch citrates lost birefringence after heating at 100 C for 30 min. However, they maintained their granular form and resisted swelling. X-ray diffraction patterns showed the crystalline peaks of all starch citrates became smaller or even disappeared for waxy corn starch citrate as compared to their controls. After heating, normal corn starch citrate and high amylose corn starch citrate produced a new X-ray diffraction pattern with the loss of most crystalline peaks except a peak at 2h = 20.2. From
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FT-IR results, the absorbance at 1724 cm 1, which corresponded to the ester bond in starch, was apparent in all starch citrates both before and after heating. It indicates that these starch citrates were heat-stable. As the degree of substitution increased, the intensity of 1724 cm 1 (ester bond) increased, as well as the amount of amorphous to crystalline phase (1016 cm 1/1045 cm 1) in starch citrate. Acknowledgements This study was carried out with the financial support of the Ontario Corn ProducersÕ Association (OCPA) and Agriculture and Agri-Food Canada. The authors thank Elizabeth Donner for technical assistance. The authors also thank Dr. Paul A. Seib for helpful discussions. References Aburto, J., Thiebaud, S., Alric, I., Borredon, E., Bikiaris, D., Prions, J., et al. (1997). Properties of octanoated starch and its blends with polyethylene. Carbohydrate Polymers, 34, 101–112. Bellon-Maurel, V., Vallat, C., & Goffinet, D. (1995). Quantitative analysis of individual sugars during starch hydrolysis by FT-IR/ATR spectrometry. Part I. Multivariate calibration study-repeatability and reproducibility. Applied Spectroscopy, 49, 556–562. Chabot, J. F., Allen, J. E., & Hood, L. F. (1978). Freeze-etch ultra structure of waxy maize and acid hydrolyzed waxy maize starch granules. Journal of Food Science, 43, 727–730. Chatel, S., Voirin, A., & Artaud, J. (1997). Starch identification and determination in sweetened fruit preparations. 2. Optimization of dialysis and gelatinization steps, infrared identification of starch chemical modifications. Journal of Agricultural and Food Chemistry, 45, 425–430. Cheetham, N. W. H., & Tao, L. (1998). Variation in crystalline type with amylose content in maize starch granules: an X-ray powder diffraction study. Carbohydrate Polymers, 36, 277–284. Conway, R. L., & Hood, L. F. (1976). Pancreatic alpha-amylase hydrolysis products of modified and unmodified tapioca starches. Die Starke, 28, 342. Eerlingen, R. C., Crombez, M., & Delcour, J. A. (1993). Enzyme-resistant starch. I. Quantitative and qualitative influence of incubation time and temperature of autoclaved starch on resistant starch formation. Cereal Chemistry, 70(3), 339–344. Fannon, J. E., Hauber, R. J., & BeMiller, J. N. (1992). Surface pores of starch granules. Cereal Chemistry, 69, 284–288. Fuwa, H., Sugimoto, Y., Tanaka, M., & Glover, D. V. (1978). Susceptibility of various starch granules to amylase as seen by scanning electron microscope. Starch, 30, 186–191.
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