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Effect of defatting on acid hydrolysis rate of maize starch with different amylose contents
International Journal of Biological Macromolecules 62 (2013) 652–656
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Effect of defatting on acid hydrolysis rate of maize starch with different amylose contents Benxi Wei a,b , Xiuting Hu a,b , Bao Zhang a,b , Hongyan Li a,b , Xueming Xu a,b , Zhengyu Jin a,b,∗ , Yaoqi Tian a,b,∗∗ a b
The State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, China Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi 214122, China
a r t i c l e
i n f o
Article history: Received 5 September 2013 Received in revised form 27 September 2013 Accepted 5 October 2013 Available online 12 October 2013 Keywords: Maize starch Defatting Physiochemical Acid hydrolysis rate
a b s t r a c t The effect of defatting on the physiochemical properties and the acid hydrolysis rate of maize starch with different amylose contents was evaluated in this study. The increase in the number of pores and the stripping of starch surface layers were observed after defatting by scanning electron microscopy. X-ray diffraction spectrum showed that the peaks attributing to the amylose–lipid complex disappeared. The relative crystallinity increased by 19% for high-amylose maize starch (HMS) on defatting, while the other tested starches virtually unchanged. Differential scanning calorimetry study indicated an increase in the thermal stability for the defatted starches. Compared with native waxy maize starch, the acid hydrolysis rate of the defatted one increased by 6% after 10 days. For normal maize starch (NMS) and HMS, the higher rate of hydrolysis was observed during the first 5 days. Thereafter, the hydrolysis rate was lower than that of their native counterpart. The increase in susceptibility to acid hydrolysis (in the first 5 days) was mainly attributed to the defective and porous structures formed during defatting process, while the decrease of hydrolysis rate for NMS and HMS samples (after the first 5 days) probably resulted from the increase in the relative crystallinity. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved.
1. Introduction Acid hydrolysis has been widely used to modify starch granule structure and produce thin boiling starch, which was extensively used in food, textile and paper industries [1]. In these fields, acidmodified starch is prepared with dilute sulfuric acid (H2 SO4 ) or hydrochloric acid (HCl) at 25–55 ◦ C for different periods. Two distinct stages were observed in the course of acid hydrolysis of starch as a function of time. The first stage (i) was attributed to a relatively fast hydrolysis of starch, mainly hydrolyzing the amorphous lamellae, while a slow hydrolysis of the crystalline lamellae occurred during the second stage (ii) [2,3]. Due to the two-stage hydrolysis pattern, acid-modified process of starch mainly took 5–15 days [4]. The effects of different factors on the hydrolysis were investigated. The major results showed that the first stage (i) could be affected by granule size, pores on the surface, amylose content,
∗ Corresponding author at: The State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, China. Tel.: +86 510 85913299; fax: +86 510 85913299. ∗∗ Corresponding author at: The State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, China. Tel.: +86 510 85328571; fax: +86 510 85328571. E-mail addresses: [email protected] (Z. Jin), [email protected] (Y. Tian).
amylopectin content, the mode of distribution of ␣ (1–6) branches between the amorphous and the crystalline regions, and degree of packing of the double helices within the crystallites [4]. However, little attention was attracted to the natural lipids presented in starch granules. Starch granules usually contained lipids. The content and the composition of these lipids varied in different kinds of plants. It was generally known that the amount of total starch lipids was 0.01–1.46% in cereals [5,6], 0.01–0.87% in legumes [7], and 0.08–0.19% in tuber and roots [6,8,9]. The lipids presented in starch could be classified into three categories, including nonstarch lipids, starch surface lipids, and internal starch lipids [10]. Starch lipids presented in free state were connected with starch components, either linking via ionic and hydrogen bonds to hydroxyl groups (non-starch lipids and starch surface lipids) or in the form of amylose–inclusion complexes (starch surface lipids and internal starch lipids) [11,12]. Therefore, it could be deduced that surface lipids and non-starch lipids might prevent the starch granule hydrolyzing by acid at the first hydrolysis stage (i). Lipid-complexd amylose chains showed great resistance to the acid hydrolysis [13,14]. The complexes, formed by internal starch lipids and amylose, hindered the attack of acid on glucosidic bonds, since the glucose unit should change its conformation from chair to half-chair in order to be hydrolyzed [15]. Therefore, it could be hypothesized that defatting treatment could increase the
0141-8130/$ – see front matter. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2013.10.008
B. Wei et al. / International Journal of Biological Macromolecules 62 (2013) 652–656
653
Table 1 Removal ratio of starch lipids by the methanol/water extracted method. Sample
WMS NMS HMS a b c
Lipids contents (mg per 100 g starch) Acid hydrolyzeda (total lipids)
Solvent extractedb
Removal ratio by solvent extracted method (%)
134 ± 19c 523 ± 31 1042 ± 39
125 ± 26 508 ± 23 1006 ± 31
93.3 97.1 96.5
Lipids obtained by extraction after acid hydrolysis (24% HCl) of native starches at 70 ◦ C for 30 min. Lipids obtained by methanol–water (85%, v/v) extraction at 75 ◦ C for 24 h. Mean ± SD of at least triplicate.
accessibility of acid to the starch granules and increase the degree of acid hydrolysis of starches. The present study, thus, was designed to determine the effect of defatting on the susceptibility of granular starches to acid hydrolysis. Maize starches with different amylose contents were studied, since the lipids content was related to amylose content or long chain (1 → 4)-␣-d-glucan content [16]. This would provide a deeper insight into the effect of defatting on acid hydrolysis rate of starches.
2. Materials and methods
2.4. Differential scanning calorimetry (DSC) Thermal analysis of native and defatted maize starches was performed by a DSC7000 instrument (Seiko Instruments Inc., Chiba, Japan). Water (11 L) was added with a microsyringe to native and defatted starches (3.0 mg) in an aluminum DSC pan. The mixtures were sealed and equilibrate at 4 ◦ C for 24 h. The thermal behaviors of the starch samples were studied by heating samples at a heating rate of 5 ◦ C/min from 20 ◦ C to 120 ◦ C under ultrahigh-purity nitrogen atmosphere. An empty pan was used as a reference. The onset temperature (To ), peak temperature (Tp ), and conclusion temperature (Tc ) together with enthalpy change (H) were quantified by the Muse software.
2.1. Materials 2.5. Field emission scanning electron microscopy (FE-SEM) Waxy maize starch (WMS) was kindly donated by Tianjin Tingfung Starch Development Co., Ltd. (Tianjin, China). Normal maize starch (NMS) and high-amylose maize starch (amylomaize V, HMS) were purchased from Puluoxing Starch Co., Ltd. (Hangzhou, China). All other chemicals and reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. (Suzhou, China) and of analytical grade unless otherwise stated. The amylose contents for WMS, NMS, and HMS were 0.5%, 26%, and 62%, respectively.
2.2. Starch defatting Defatted starches were prepared according to the method of Schoch [17]. Maize starches with different amylose contents were Soxhlet extracted with 85% aqueous methanol at 75 ◦ C for 24 h. The solvent was removed by vacuum evaporation and the starch was air-dried to a constant moisture of ∼10%. Lipids content was determined using the method of Jitngarmkusol et al. [18] and calculated on a dry starch basis.
2.3. X-ray diffraction (XRD) pattern and relative crystallinity (RC) Prior to XRD test, native and defatted starch samples were milled to powers (200 mesh) and hydrated at 75% relative humidity (RH) in a sealed vessel using saturated sodium chloride. Basically, the samples (0.8 g) were pressed into a pellet (10 mm × 25 mm) with hydraulic press. XRD pattern was obtained using a Bruker D8Advance XRD instrument (Bruker AXS Inc., Karlsruhe, Germany). The diffractograms were collected under the conditions of 40 kV ˚ radiand 30 mA with nickel-filtered Cu-K␣ (wavelength 1.5405 A) ation. Powdered samples were scanned from 4◦ to 30◦ (2) with a scanning rate of 4◦ /min. Each sample was scanned at least in triplicate. The RC (ratio of the crystalline portion to the sum of crystalline and amorphous portions) of the starches and diffraction angle (2) of X-ray pattern were analyzed by Jade 5.0 software (Materials Data Inc., CA, USA) and quantitatively estimated using the method of Nara et al. [19].
FE-SEM was performed using a Hitachi S-4800 (Hitachi, Japan) at an acceleration voltage of 1 kV. The starch samples were placed on aluminum specimen stubs with double-sided adhesive tape and coated with gold for observation. 2.6. Acid hydrolysis of the native and defatted starches The starches were hydrolyzed with 3.16 M H2 SO4 at 40 ◦ C (10 g starch per 100 mL acid) for 10 days with constant stirring at the speed of 200 rpm. At different time intervals, aliquots of the reaction mixtures were neutralized and centrifuged (3773 × g). The supernatant liquid was assayed for total carbohydrates [20]. The acid hydrolysis rate was determined by expressing the solubilized carbohydrates as a percentage of the initial dry starch. 2.7. Statistical analysis Statistical analysis was performed using ORIGIN 7.5 (OriginLab Inc., Hampton, USA). Data were expressed as means ± standard deviations and analyzed by a one-way analysis of variance (ANOVA). A probability P < 0.05 was considered significant throughout the study. 3. Results and discussion 3.1. Comparison of lipids content between starches Starches derived from different origins usually contained quantities of lipids either on the surface of starches or the inner starch granules. In this study, the lipids contents for WMS, NMS, and HMS were 0.13%, 0.52%, and 1.04%, respectively. It can be seen from Table 1, methanol–water (85%, v/v) was a good solvent for defatting, as it resulted in almost complete removal of the starch lipids after 24 h. The lipid contents for maize starches were within the range as reported in the literature [5], and was positively correlated with amylose content. Since amylose dispersed among amylopectin molecules and might be located primarily in the amorphous zones
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B. Wei et al. / International Journal of Biological Macromolecules 62 (2013) 652–656
Fig. 1. SEM micrographs of native maize starches with different amylose contents and the corresponding defatted samples: (a) waxy maize starch (WMS); (b) defatted waxy maize starch (WMS-d); (c) normal maize starch (NMS); (d) defatted normal maize starch (NMS-d); (e) high-amylose maize starch (HMS); and (f) defatted high-amylose maize starch (HMS-d).
of the growth rings [21], this suggested that NMS and HMS could include more internal lipids than surface lipids.
3.2. SEM Fig. 1 illustrates SEM pictures of native and defatted maize starches with different amylose contents. Defatted maize starches showed no obvious granule disruption comparing with native maize samples (Fig. 1a). However, a variety of surface changes were detected after defatting in SEM pictures. For the defatted waxy maize starch (WMS-d), many small pores appeared in the starch granule and the smooth starch surface became coarse. The surface layer was peeled off after treating in hot methanol–water solvent (Fig. 1b), indicating that surface lipids were removed. Comparing with NMS (Fig. 1d), the pores in the granule became larger and deeper and the surface layer was also stripped for the defatted normal maize starch (NMS-d). Similarly, many small pores appeared in defatted high-amylose maize starch (HMS-d) and the surface became rough by defatting (Fig. 1f). It could be concluded that the defatting treatment caused serious changes in maize starch, since defatting could remove the starch lipids and induce the disruption of hydrogen bonds between polymer chains. This might loosen starch granules and the acid could penetrate and degrade the starch granules easily [22].
3.3. XRD patterns and relative crystallinity (RC) The XRD patterns and the RC of native and defatted maize starches were shown in Fig. 2. WMS, NMS, WMS-d, and NMS-d exhibited the typical A-type pattern of cereal starches, with strong peaks at 2 = 15◦ and 23◦ and a dual peak at 17◦ and 18◦ . Both of the WMS and NMS showed no significant changes in RC after defatting. However, the peaks at 20◦ for the WMS-d and NMS-d samples disappeared after the treatment of Soxhlet extraction. That could be ascribed to the loss of starch lipids after defatting, since the peaks at 20◦ was a V-characteristic of amylose–lipid complex [23,24]. These results indicated that defatting could remove the starch lipids but induce no obvious crystalline changes for WMS and NMS. HMS showed the presence of a typical B-type X-ray pattern with strong reflections at 2 = 6◦ and 17◦ , and an unresolved doublet peaks at 2 = 22◦ and 23◦ (Fig. 2). Defatting resulted in an increase in RC (19%), the disappearance of the peak at 2 = 15◦ , and weakening of the peaks at 2 = 6◦ and 20◦ . This indicated that the X-ray pattern of HMS was transformed to a combination of A + B-type pattern after the removal of starch lipids. 3.4. Thermal properties The transition temperatures named To , Tp , Tc and the enthalpy change (H), were presented in Table 2. Defatted starches showed
B. Wei et al. / International Journal of Biological Macromolecules 62 (2013) 652–656
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Table 2 Gelatinization parametersa of native and defatted maize starches with different amylose contents. Samplesb
Gelatinization parameters To (◦ C)
Tp (◦ C)
Tc (◦ C)
Tc –To
H (J/g)
WMS WMS-d
62.8 ± 0.2ac 61.5 ± 0.2a
69.2 ± 0.1b 68.5 ± 0.1bc
77.4 ± 0.2c 77.8 ± 0.4c
14.6c 16.3b
18.1 ± 0.4a 16.2 ± 0.3b
NMS NMS-d
63.3 ± 0.5b 60.9 ± 0.1c
67.9 ± 0.2c 65.9 ± 0.3d
73.3 ± 0.2d 70.3 ± 0.1e
10.0d 9.4d
14.1 ± 0.2c 12.3 ± 0.2d
HMS HMS-d
62.9 ± 0.3a 62.5 ± 0.2a
76.7 ± 0.1a 77.2 ± 0.3a
97.6 ± 0.2a 95.8 ± 0.3b
34.7a 33.3a
13.9 ± 0.2c 12.5 ± 0.3d
a To , onset temperature of gelatinization; Tp , peak temperature of gelatinization; Tc , conclusion temperature of gelatinization; Tc –To , the gelatinization temperatures range; and H, the enthalpy change of gelatinization. b WMS, waxy maize starch; WMS-d, defatted waxy maize starch; NMS, normal maize starch; NMS-d, defatted normal maize starch; HMS, high-amylose maize starch; and HMS-d, defatted high-amylose maize starch. c Mean ± SD of triplicate; values followed by the same letter within the same column are not significantly different (P > 0.05) by Tukey’s HSD test.
20 °
Relative crystallinity
Diffraction Intensity
WMS
47.1%
WMS-d 44.3%
NMS
35.4%
NMS-d 37.0%
HMS
30.5%
HMS-d 36.2% 5
10
15
20
25
30
35
Diffraction Angle (2θ) Fig. 2. XRD patterns of native and defatted maize starches with different amylose contents: WMS, waxy maize starch; WMS-d, defatted waxy maize starch; NMS, normal maize starch; NMS-d, defatted normal maize starch; HMS, high-amylose maize starch; and HMS-d, defatted high-amylose maize starch.
no significant changes in gelatinization temperature (To , Tp and Tc ) for WMS-d and HMS-d compared to their native counterparts. However, for NMS, To , Tp and Tc significantly decreased after the process of defatting, suggesting that double helices presented in crystalline and non-crystalline arrays may have been disrupted during the defatting process. The results were
similar to the study by Uthumporn et al., [22] who reported that defatting induced the shifting of gelatinization parameters to lower temperatures compared to un-defatted starches. The gelatinization temperature range (Tc –To ) showed significant increase for WMS-d compared to WMS, while no significant differences were detected between NMS and NMS-d or HMS and HMS-d. Tc –To reflected variations in crystalline shape, crystalline size, degree of crystal perfection, and the type of starch chain intertwining [amylose–amylose, (AM–AM), amylose–amylopectin (AM–AMP), amylopectin–amylopectin (AMP–AMP)] that generated the double helical chains of starch crystallites [2]. Therefore, in starches with very high amylose content (HMS and HMS-d), crystallinites could originate not only from AMP–AMP, but also from the helices between AM–AM and AM–AMP [25]. Consequently, Tc –To for HMS/HMS-d was much higher than WMS/WMS-d and NMS/NMS-d. Compared to WMS, the broader temperature range of WMS-d might result from disruption of the granule surface and further prompted the more imperfection of crystallinity by defatting. Gelatinization enthalpy (H) has been shown to reflect loss of molecular (double helical) order [26]. Lower enthalpy values were observed in the defatted starches for all treated samples, which was in agreement with the report of Uthumporn et al. [22]. Variety of factors could result in the differences in H among native starches, such as long branched chain lengths of amylopectin and interaction of lipids with amylose. In this study, two factors might lead to the decrease of H for defatted samples: (1) removal of the amylose–lipids complex by defatting, and (2) the slightly depolymerization of starch helixes within the defatted starch granules. 100
100 NMS-d NMS
Hydrolysis Rate (%)
WMS-d WMS
HMS-d HMS
80
80
60
60
40
40
20
20
(a) 0
0
2
4
6
8
10 0
(b) 2
4 6 8 Time (days)
10 0
(c) 2
4
6
8
10
0
Fig. 3. Acid hydrolysis (3.16 M H2 SO4 , 40 ◦ C) profiles (solubilized carbohydrate as a function of time) of defatted and native maize starch: (a) defatted and native waxy maize starch (WMS-d/WMS); (b) defatted and native normal maize starch (NMS-d/NMS); and (c) defatted and native high-amylose maize starch (HMS-d/HMS).
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B. Wei et al. / International Journal of Biological Macromolecules 62 (2013) 652–656
3.5. Acid hydrolysis of native and defatted maize starches with different amylose contents Fig. 3 shows the trend of acid hydrolysis rate for native and defatted starches at different time intervals. All starches exhibited a two-stage hydrolysis pattern. A relatively faster hydrolysis rate was observed during the first 5 days and followed by a slower rate from 5 days to 10 days for WMS/WMS-d and NMS/NMS-d. However, for HMS/HMS-d, the turning point of the hydrolysis rate was at 7 days. The same hydrolysis patterns have been also reported in literature [4], while the final hydrolysis rate in this study was significantly higher than the results previously reported [2,3], which might result from the higher acid concentration used (3.16 M, H2 SO4 ) in this study compared to the literature (2.2 M, HCl). Removal of the starch lipids led to a significant increase in acid hydrolysis rate (6%) for WMS-d after 10 days (Fig. 3a). This might be ascribed to the appearance of pores in starch granules and the stripping of starch surface layers, which increased the granule susceptibility to acid hydrolysis. For NMS/NMS-d and HMS/HMS-d, defatting caused quite different effects on acid hydrolysis rate compared to WMS/WMS-d (Fig. 3b/c). The higher rate of hydrolysis was evident for the first 5 days. Thereafter, the rate of hydrolysis was lower than that of their native counterparts. In the first 5 days, the higher acid hydrolysis rate could be contributed to the rapid hydrolysis of leaching amylose derived from the defatting process, as amylose was partially dissolved out of the granule after defatting [27]. Once the released amylose chains were hydrolyzed, the effect of the increased RC on acid hydrolysis became dominant.
rate of WMS increased after defatting due to the increase of the pores in starch granule. However, for NMS and HMS, the hydrolysis rate was higher at first 5 days and became lower than that of native starches in the following hydrolysis process. This would provide a deeper insight into the effect of defatting on acid hydrolysis rate of starches and improve the efficiency in producing thin boiling starch. Acknowledgements This study was financially supported by Natural Science Foundation of China (Nos. 31230057 and 31201288) and Natural Science Foundation of Jiangsu Province (No. BK2012115). References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14]
4. Conclusions
[15] [16] [17] [18]
Defatting affected physicochemical properties and acid hydrolysis rate of maize starches with different amylose contents. Lipids were almost completely removed by Soxhlet extraction with methanol/water solvent. It induced not only the increase of pores in the starch granule and peeling of the starch surface, but also the growing of the relative crystallinity. However, the XRD patterns did not change for all the starch samples. Defatting also caused high susceptibility of maize starches to acid hydrolysis. Acid hydrolysis
[19] [20] [21] [22] [23] [24] [25] [26] [27]
V. Singh, S. Ali, Carbohydr. Polym. 41 (2000) 191–195. L. Jayakody, R. Hoover, Food Res. Int. 35 (2002) 665–680. W. Li, H. Corke, T. Beta, Carbohydr. Polym. 69 (2007) 398–405. D. Le Corre, J. Bras, A. Dufresne, Biomacromolecules 11 (2010) 1139–1153. W.R. Morrison, J. Cereal Sci. 8 (1988) 1–15. T. Vasanthan, R. Hoover, Food Chem. 43 (1992) 19–27. R. Hoover, S.C. Rorke, A.M. Martin, J. Food Biochem. 15 (1991) 117–136. L. Emiola, L. Delarosa, J. Food Biochem. 5 (1981) 115–130. G. Goshima, M. Abe, N. Sato, K. Ohashi, H. Tsuge, Starch – Stärke 37 (1985) 10–14. W. Morrison, Starch – Stärke 33 (1981) 408–410. F. Mikus, R. Hixon, R. Rundle, J. Am. Chem. Soc. 68 (1946) 1115–1123. M.N. Azudin, W.R. Morrison, J. Cereal Sci. 4 (1986) 23–31. W.R. Morrison, R.F. Tester, M.J. Gidley, J. Karkalas, Carbohydr. Res. 245 (1993) 289–302. G.G. Gelders, J.P. Duyck, H. Goesaert, J.A. Delcour, Carbohydr. Polym. 60 (2005) 379–389. Y. Nakazawa, Y.-J. Wang, Carbohydr. Res. 338 (2003) 2871–2882. J. South, W. Morrison, O. Nelson, J. Cereal Sci. 14 (1991) 267–278. T.J. Schoch, J. Am. Chem. Soc. 64 (1942) 2954–2956. S. Jitngarmkusol, J. Hongsuwankul, K. Tananuwong, Food Chem. 110 (2008) 23–30. S. Nara, T. Komiya, Starch – Stärke 35 (1983) 407–410. G.L. Miller, Anal. Chem. 31 (1959) 426–428. A.M. Smith, Biomacromolecules 2 (2001) 335–341. U. Uthumporn, A. Karim, A. Fazilah, Ind. Crops Prod. 43 (2013) 441–449. H. Tang, K. Watanabe, T. Mitsunaga, Carbohydr. Polym. 49 (2002) 217–224. N.W. Cheetham, L. Tao, Carbohydr. Polym. 36 (1998) 277–284. P. Jenkins, A. Donald, Int. J. Biol. Macromol. 17 (1995) 315–321. D. Cooke, M.J. Gidley, Carbohydr. Res. 227 (1992) 103–112. R.F. Tester, W.R. Morrison, Cereal Chem. 67 (1990) 551–557.
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Effect of defatting on acid hydrolysis rate of maize starch with different amylose contents Benxi Wei a,b , Xiuting Hu a,b , Bao Zhang a,b , Hongyan Li a,b , Xueming Xu a,b , Zhengyu Jin a,b,∗ , Yaoqi Tian a,b,∗∗ a b
The State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, China Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi 214122, China
a r t i c l e
i n f o
Article history: Received 5 September 2013 Received in revised form 27 September 2013 Accepted 5 October 2013 Available online 12 October 2013 Keywords: Maize starch Defatting Physiochemical Acid hydrolysis rate
a b s t r a c t The effect of defatting on the physiochemical properties and the acid hydrolysis rate of maize starch with different amylose contents was evaluated in this study. The increase in the number of pores and the stripping of starch surface layers were observed after defatting by scanning electron microscopy. X-ray diffraction spectrum showed that the peaks attributing to the amylose–lipid complex disappeared. The relative crystallinity increased by 19% for high-amylose maize starch (HMS) on defatting, while the other tested starches virtually unchanged. Differential scanning calorimetry study indicated an increase in the thermal stability for the defatted starches. Compared with native waxy maize starch, the acid hydrolysis rate of the defatted one increased by 6% after 10 days. For normal maize starch (NMS) and HMS, the higher rate of hydrolysis was observed during the first 5 days. Thereafter, the hydrolysis rate was lower than that of their native counterpart. The increase in susceptibility to acid hydrolysis (in the first 5 days) was mainly attributed to the defective and porous structures formed during defatting process, while the decrease of hydrolysis rate for NMS and HMS samples (after the first 5 days) probably resulted from the increase in the relative crystallinity. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved.
1. Introduction Acid hydrolysis has been widely used to modify starch granule structure and produce thin boiling starch, which was extensively used in food, textile and paper industries [1]. In these fields, acidmodified starch is prepared with dilute sulfuric acid (H2 SO4 ) or hydrochloric acid (HCl) at 25–55 ◦ C for different periods. Two distinct stages were observed in the course of acid hydrolysis of starch as a function of time. The first stage (i) was attributed to a relatively fast hydrolysis of starch, mainly hydrolyzing the amorphous lamellae, while a slow hydrolysis of the crystalline lamellae occurred during the second stage (ii) [2,3]. Due to the two-stage hydrolysis pattern, acid-modified process of starch mainly took 5–15 days [4]. The effects of different factors on the hydrolysis were investigated. The major results showed that the first stage (i) could be affected by granule size, pores on the surface, amylose content,
∗ Corresponding author at: The State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, China. Tel.: +86 510 85913299; fax: +86 510 85913299. ∗∗ Corresponding author at: The State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122, China. Tel.: +86 510 85328571; fax: +86 510 85328571. E-mail addresses: [email protected] (Z. Jin), [email protected] (Y. Tian).
amylopectin content, the mode of distribution of ␣ (1–6) branches between the amorphous and the crystalline regions, and degree of packing of the double helices within the crystallites [4]. However, little attention was attracted to the natural lipids presented in starch granules. Starch granules usually contained lipids. The content and the composition of these lipids varied in different kinds of plants. It was generally known that the amount of total starch lipids was 0.01–1.46% in cereals [5,6], 0.01–0.87% in legumes [7], and 0.08–0.19% in tuber and roots [6,8,9]. The lipids presented in starch could be classified into three categories, including nonstarch lipids, starch surface lipids, and internal starch lipids [10]. Starch lipids presented in free state were connected with starch components, either linking via ionic and hydrogen bonds to hydroxyl groups (non-starch lipids and starch surface lipids) or in the form of amylose–inclusion complexes (starch surface lipids and internal starch lipids) [11,12]. Therefore, it could be deduced that surface lipids and non-starch lipids might prevent the starch granule hydrolyzing by acid at the first hydrolysis stage (i). Lipid-complexd amylose chains showed great resistance to the acid hydrolysis [13,14]. The complexes, formed by internal starch lipids and amylose, hindered the attack of acid on glucosidic bonds, since the glucose unit should change its conformation from chair to half-chair in order to be hydrolyzed [15]. Therefore, it could be hypothesized that defatting treatment could increase the
0141-8130/$ – see front matter. Crown Copyright © 2013 Published by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2013.10.008
B. Wei et al. / International Journal of Biological Macromolecules 62 (2013) 652–656
653
Table 1 Removal ratio of starch lipids by the methanol/water extracted method. Sample
WMS NMS HMS a b c
Lipids contents (mg per 100 g starch) Acid hydrolyzeda (total lipids)
Solvent extractedb
Removal ratio by solvent extracted method (%)
134 ± 19c 523 ± 31 1042 ± 39
125 ± 26 508 ± 23 1006 ± 31
93.3 97.1 96.5
Lipids obtained by extraction after acid hydrolysis (24% HCl) of native starches at 70 ◦ C for 30 min. Lipids obtained by methanol–water (85%, v/v) extraction at 75 ◦ C for 24 h. Mean ± SD of at least triplicate.
accessibility of acid to the starch granules and increase the degree of acid hydrolysis of starches. The present study, thus, was designed to determine the effect of defatting on the susceptibility of granular starches to acid hydrolysis. Maize starches with different amylose contents were studied, since the lipids content was related to amylose content or long chain (1 → 4)-␣-d-glucan content [16]. This would provide a deeper insight into the effect of defatting on acid hydrolysis rate of starches.
2. Materials and methods
2.4. Differential scanning calorimetry (DSC) Thermal analysis of native and defatted maize starches was performed by a DSC7000 instrument (Seiko Instruments Inc., Chiba, Japan). Water (11 L) was added with a microsyringe to native and defatted starches (3.0 mg) in an aluminum DSC pan. The mixtures were sealed and equilibrate at 4 ◦ C for 24 h. The thermal behaviors of the starch samples were studied by heating samples at a heating rate of 5 ◦ C/min from 20 ◦ C to 120 ◦ C under ultrahigh-purity nitrogen atmosphere. An empty pan was used as a reference. The onset temperature (To ), peak temperature (Tp ), and conclusion temperature (Tc ) together with enthalpy change (H) were quantified by the Muse software.
2.1. Materials 2.5. Field emission scanning electron microscopy (FE-SEM) Waxy maize starch (WMS) was kindly donated by Tianjin Tingfung Starch Development Co., Ltd. (Tianjin, China). Normal maize starch (NMS) and high-amylose maize starch (amylomaize V, HMS) were purchased from Puluoxing Starch Co., Ltd. (Hangzhou, China). All other chemicals and reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. (Suzhou, China) and of analytical grade unless otherwise stated. The amylose contents for WMS, NMS, and HMS were 0.5%, 26%, and 62%, respectively.
2.2. Starch defatting Defatted starches were prepared according to the method of Schoch [17]. Maize starches with different amylose contents were Soxhlet extracted with 85% aqueous methanol at 75 ◦ C for 24 h. The solvent was removed by vacuum evaporation and the starch was air-dried to a constant moisture of ∼10%. Lipids content was determined using the method of Jitngarmkusol et al. [18] and calculated on a dry starch basis.
2.3. X-ray diffraction (XRD) pattern and relative crystallinity (RC) Prior to XRD test, native and defatted starch samples were milled to powers (200 mesh) and hydrated at 75% relative humidity (RH) in a sealed vessel using saturated sodium chloride. Basically, the samples (0.8 g) were pressed into a pellet (10 mm × 25 mm) with hydraulic press. XRD pattern was obtained using a Bruker D8Advance XRD instrument (Bruker AXS Inc., Karlsruhe, Germany). The diffractograms were collected under the conditions of 40 kV ˚ radiand 30 mA with nickel-filtered Cu-K␣ (wavelength 1.5405 A) ation. Powdered samples were scanned from 4◦ to 30◦ (2) with a scanning rate of 4◦ /min. Each sample was scanned at least in triplicate. The RC (ratio of the crystalline portion to the sum of crystalline and amorphous portions) of the starches and diffraction angle (2) of X-ray pattern were analyzed by Jade 5.0 software (Materials Data Inc., CA, USA) and quantitatively estimated using the method of Nara et al. [19].
FE-SEM was performed using a Hitachi S-4800 (Hitachi, Japan) at an acceleration voltage of 1 kV. The starch samples were placed on aluminum specimen stubs with double-sided adhesive tape and coated with gold for observation. 2.6. Acid hydrolysis of the native and defatted starches The starches were hydrolyzed with 3.16 M H2 SO4 at 40 ◦ C (10 g starch per 100 mL acid) for 10 days with constant stirring at the speed of 200 rpm. At different time intervals, aliquots of the reaction mixtures were neutralized and centrifuged (3773 × g). The supernatant liquid was assayed for total carbohydrates [20]. The acid hydrolysis rate was determined by expressing the solubilized carbohydrates as a percentage of the initial dry starch. 2.7. Statistical analysis Statistical analysis was performed using ORIGIN 7.5 (OriginLab Inc., Hampton, USA). Data were expressed as means ± standard deviations and analyzed by a one-way analysis of variance (ANOVA). A probability P < 0.05 was considered significant throughout the study. 3. Results and discussion 3.1. Comparison of lipids content between starches Starches derived from different origins usually contained quantities of lipids either on the surface of starches or the inner starch granules. In this study, the lipids contents for WMS, NMS, and HMS were 0.13%, 0.52%, and 1.04%, respectively. It can be seen from Table 1, methanol–water (85%, v/v) was a good solvent for defatting, as it resulted in almost complete removal of the starch lipids after 24 h. The lipid contents for maize starches were within the range as reported in the literature [5], and was positively correlated with amylose content. Since amylose dispersed among amylopectin molecules and might be located primarily in the amorphous zones
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Fig. 1. SEM micrographs of native maize starches with different amylose contents and the corresponding defatted samples: (a) waxy maize starch (WMS); (b) defatted waxy maize starch (WMS-d); (c) normal maize starch (NMS); (d) defatted normal maize starch (NMS-d); (e) high-amylose maize starch (HMS); and (f) defatted high-amylose maize starch (HMS-d).
of the growth rings [21], this suggested that NMS and HMS could include more internal lipids than surface lipids.
3.2. SEM Fig. 1 illustrates SEM pictures of native and defatted maize starches with different amylose contents. Defatted maize starches showed no obvious granule disruption comparing with native maize samples (Fig. 1a). However, a variety of surface changes were detected after defatting in SEM pictures. For the defatted waxy maize starch (WMS-d), many small pores appeared in the starch granule and the smooth starch surface became coarse. The surface layer was peeled off after treating in hot methanol–water solvent (Fig. 1b), indicating that surface lipids were removed. Comparing with NMS (Fig. 1d), the pores in the granule became larger and deeper and the surface layer was also stripped for the defatted normal maize starch (NMS-d). Similarly, many small pores appeared in defatted high-amylose maize starch (HMS-d) and the surface became rough by defatting (Fig. 1f). It could be concluded that the defatting treatment caused serious changes in maize starch, since defatting could remove the starch lipids and induce the disruption of hydrogen bonds between polymer chains. This might loosen starch granules and the acid could penetrate and degrade the starch granules easily [22].
3.3. XRD patterns and relative crystallinity (RC) The XRD patterns and the RC of native and defatted maize starches were shown in Fig. 2. WMS, NMS, WMS-d, and NMS-d exhibited the typical A-type pattern of cereal starches, with strong peaks at 2 = 15◦ and 23◦ and a dual peak at 17◦ and 18◦ . Both of the WMS and NMS showed no significant changes in RC after defatting. However, the peaks at 20◦ for the WMS-d and NMS-d samples disappeared after the treatment of Soxhlet extraction. That could be ascribed to the loss of starch lipids after defatting, since the peaks at 20◦ was a V-characteristic of amylose–lipid complex [23,24]. These results indicated that defatting could remove the starch lipids but induce no obvious crystalline changes for WMS and NMS. HMS showed the presence of a typical B-type X-ray pattern with strong reflections at 2 = 6◦ and 17◦ , and an unresolved doublet peaks at 2 = 22◦ and 23◦ (Fig. 2). Defatting resulted in an increase in RC (19%), the disappearance of the peak at 2 = 15◦ , and weakening of the peaks at 2 = 6◦ and 20◦ . This indicated that the X-ray pattern of HMS was transformed to a combination of A + B-type pattern after the removal of starch lipids. 3.4. Thermal properties The transition temperatures named To , Tp , Tc and the enthalpy change (H), were presented in Table 2. Defatted starches showed
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Table 2 Gelatinization parametersa of native and defatted maize starches with different amylose contents. Samplesb
Gelatinization parameters To (◦ C)
Tp (◦ C)
Tc (◦ C)
Tc –To
H (J/g)
WMS WMS-d
62.8 ± 0.2ac 61.5 ± 0.2a
69.2 ± 0.1b 68.5 ± 0.1bc
77.4 ± 0.2c 77.8 ± 0.4c
14.6c 16.3b
18.1 ± 0.4a 16.2 ± 0.3b
NMS NMS-d
63.3 ± 0.5b 60.9 ± 0.1c
67.9 ± 0.2c 65.9 ± 0.3d
73.3 ± 0.2d 70.3 ± 0.1e
10.0d 9.4d
14.1 ± 0.2c 12.3 ± 0.2d
HMS HMS-d
62.9 ± 0.3a 62.5 ± 0.2a
76.7 ± 0.1a 77.2 ± 0.3a
97.6 ± 0.2a 95.8 ± 0.3b
34.7a 33.3a
13.9 ± 0.2c 12.5 ± 0.3d
a To , onset temperature of gelatinization; Tp , peak temperature of gelatinization; Tc , conclusion temperature of gelatinization; Tc –To , the gelatinization temperatures range; and H, the enthalpy change of gelatinization. b WMS, waxy maize starch; WMS-d, defatted waxy maize starch; NMS, normal maize starch; NMS-d, defatted normal maize starch; HMS, high-amylose maize starch; and HMS-d, defatted high-amylose maize starch. c Mean ± SD of triplicate; values followed by the same letter within the same column are not significantly different (P > 0.05) by Tukey’s HSD test.
20 °
Relative crystallinity
Diffraction Intensity
WMS
47.1%
WMS-d 44.3%
NMS
35.4%
NMS-d 37.0%
HMS
30.5%
HMS-d 36.2% 5
10
15
20
25
30
35
Diffraction Angle (2θ) Fig. 2. XRD patterns of native and defatted maize starches with different amylose contents: WMS, waxy maize starch; WMS-d, defatted waxy maize starch; NMS, normal maize starch; NMS-d, defatted normal maize starch; HMS, high-amylose maize starch; and HMS-d, defatted high-amylose maize starch.
no significant changes in gelatinization temperature (To , Tp and Tc ) for WMS-d and HMS-d compared to their native counterparts. However, for NMS, To , Tp and Tc significantly decreased after the process of defatting, suggesting that double helices presented in crystalline and non-crystalline arrays may have been disrupted during the defatting process. The results were
similar to the study by Uthumporn et al., [22] who reported that defatting induced the shifting of gelatinization parameters to lower temperatures compared to un-defatted starches. The gelatinization temperature range (Tc –To ) showed significant increase for WMS-d compared to WMS, while no significant differences were detected between NMS and NMS-d or HMS and HMS-d. Tc –To reflected variations in crystalline shape, crystalline size, degree of crystal perfection, and the type of starch chain intertwining [amylose–amylose, (AM–AM), amylose–amylopectin (AM–AMP), amylopectin–amylopectin (AMP–AMP)] that generated the double helical chains of starch crystallites [2]. Therefore, in starches with very high amylose content (HMS and HMS-d), crystallinites could originate not only from AMP–AMP, but also from the helices between AM–AM and AM–AMP [25]. Consequently, Tc –To for HMS/HMS-d was much higher than WMS/WMS-d and NMS/NMS-d. Compared to WMS, the broader temperature range of WMS-d might result from disruption of the granule surface and further prompted the more imperfection of crystallinity by defatting. Gelatinization enthalpy (H) has been shown to reflect loss of molecular (double helical) order [26]. Lower enthalpy values were observed in the defatted starches for all treated samples, which was in agreement with the report of Uthumporn et al. [22]. Variety of factors could result in the differences in H among native starches, such as long branched chain lengths of amylopectin and interaction of lipids with amylose. In this study, two factors might lead to the decrease of H for defatted samples: (1) removal of the amylose–lipids complex by defatting, and (2) the slightly depolymerization of starch helixes within the defatted starch granules. 100
100 NMS-d NMS
Hydrolysis Rate (%)
WMS-d WMS
HMS-d HMS
80
80
60
60
40
40
20
20
(a) 0
0
2
4
6
8
10 0
(b) 2
4 6 8 Time (days)
10 0
(c) 2
4
6
8
10
0
Fig. 3. Acid hydrolysis (3.16 M H2 SO4 , 40 ◦ C) profiles (solubilized carbohydrate as a function of time) of defatted and native maize starch: (a) defatted and native waxy maize starch (WMS-d/WMS); (b) defatted and native normal maize starch (NMS-d/NMS); and (c) defatted and native high-amylose maize starch (HMS-d/HMS).
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3.5. Acid hydrolysis of native and defatted maize starches with different amylose contents Fig. 3 shows the trend of acid hydrolysis rate for native and defatted starches at different time intervals. All starches exhibited a two-stage hydrolysis pattern. A relatively faster hydrolysis rate was observed during the first 5 days and followed by a slower rate from 5 days to 10 days for WMS/WMS-d and NMS/NMS-d. However, for HMS/HMS-d, the turning point of the hydrolysis rate was at 7 days. The same hydrolysis patterns have been also reported in literature [4], while the final hydrolysis rate in this study was significantly higher than the results previously reported [2,3], which might result from the higher acid concentration used (3.16 M, H2 SO4 ) in this study compared to the literature (2.2 M, HCl). Removal of the starch lipids led to a significant increase in acid hydrolysis rate (6%) for WMS-d after 10 days (Fig. 3a). This might be ascribed to the appearance of pores in starch granules and the stripping of starch surface layers, which increased the granule susceptibility to acid hydrolysis. For NMS/NMS-d and HMS/HMS-d, defatting caused quite different effects on acid hydrolysis rate compared to WMS/WMS-d (Fig. 3b/c). The higher rate of hydrolysis was evident for the first 5 days. Thereafter, the rate of hydrolysis was lower than that of their native counterparts. In the first 5 days, the higher acid hydrolysis rate could be contributed to the rapid hydrolysis of leaching amylose derived from the defatting process, as amylose was partially dissolved out of the granule after defatting [27]. Once the released amylose chains were hydrolyzed, the effect of the increased RC on acid hydrolysis became dominant.
rate of WMS increased after defatting due to the increase of the pores in starch granule. However, for NMS and HMS, the hydrolysis rate was higher at first 5 days and became lower than that of native starches in the following hydrolysis process. This would provide a deeper insight into the effect of defatting on acid hydrolysis rate of starches and improve the efficiency in producing thin boiling starch. Acknowledgements This study was financially supported by Natural Science Foundation of China (Nos. 31230057 and 31201288) and Natural Science Foundation of Jiangsu Province (No. BK2012115). References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14]
4. Conclusions
[15] [16] [17] [18]
Defatting affected physicochemical properties and acid hydrolysis rate of maize starches with different amylose contents. Lipids were almost completely removed by Soxhlet extraction with methanol/water solvent. It induced not only the increase of pores in the starch granule and peeling of the starch surface, but also the growing of the relative crystallinity. However, the XRD patterns did not change for all the starch samples. Defatting also caused high susceptibility of maize starches to acid hydrolysis. Acid hydrolysis
[19] [20] [21] [22] [23] [24] [25] [26] [27]
V. Singh, S. Ali, Carbohydr. Polym. 41 (2000) 191–195. L. Jayakody, R. Hoover, Food Res. Int. 35 (2002) 665–680. W. Li, H. Corke, T. Beta, Carbohydr. Polym. 69 (2007) 398–405. D. Le Corre, J. Bras, A. Dufresne, Biomacromolecules 11 (2010) 1139–1153. W.R. Morrison, J. Cereal Sci. 8 (1988) 1–15. T. Vasanthan, R. Hoover, Food Chem. 43 (1992) 19–27. R. Hoover, S.C. Rorke, A.M. Martin, J. Food Biochem. 15 (1991) 117–136. L. Emiola, L. Delarosa, J. Food Biochem. 5 (1981) 115–130. G. Goshima, M. Abe, N. Sato, K. Ohashi, H. Tsuge, Starch – Stärke 37 (1985) 10–14. W. Morrison, Starch – Stärke 33 (1981) 408–410. F. Mikus, R. Hixon, R. Rundle, J. Am. Chem. Soc. 68 (1946) 1115–1123. M.N. Azudin, W.R. Morrison, J. Cereal Sci. 4 (1986) 23–31. W.R. Morrison, R.F. Tester, M.J. Gidley, J. Karkalas, Carbohydr. Res. 245 (1993) 289–302. G.G. Gelders, J.P. Duyck, H. Goesaert, J.A. Delcour, Carbohydr. Polym. 60 (2005) 379–389. Y. Nakazawa, Y.-J. Wang, Carbohydr. Res. 338 (2003) 2871–2882. J. South, W. Morrison, O. Nelson, J. Cereal Sci. 14 (1991) 267–278. T.J. Schoch, J. Am. Chem. Soc. 64 (1942) 2954–2956. S. Jitngarmkusol, J. Hongsuwankul, K. Tananuwong, Food Chem. 110 (2008) 23–30. S. Nara, T. Komiya, Starch – Stärke 35 (1983) 407–410. G.L. Miller, Anal. Chem. 31 (1959) 426–428. A.M. Smith, Biomacromolecules 2 (2001) 335–341. U. Uthumporn, A. Karim, A. Fazilah, Ind. Crops Prod. 43 (2013) 441–449. H. Tang, K. Watanabe, T. Mitsunaga, Carbohydr. Polym. 49 (2002) 217–224. N.W. Cheetham, L. Tao, Carbohydr. Polym. 36 (1998) 277–284. P. Jenkins, A. Donald, Int. J. Biol. Macromol. 17 (1995) 315–321. D. Cooke, M.J. Gidley, Carbohydr. Res. 227 (1992) 103–112. R.F. Tester, W.R. Morrison, Cereal Chem. 67 (1990) 551–557.