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Effect of acid hydrolysis on morphology, structure and digestion property of starch from Cynanchum auriculatum Royle ex Wight
Accepted Manuscript Title: Effect of acid hydrolysis on morphology, structure and digestion property of starch from Cynanchum auriculatum Royle ex Wight Author: Xingchi Wang Fanting Wen Shurong Zhang Ruru Shen Wei Jiang Jun Liu PII: DOI: Reference:
S0141-8130(16)32068-2 http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.01.002 BIOMAC 6903
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
19-10-2016 30-12-2016 1-1-2017
Please cite this article as: Xingchi Wang, Fanting Wen, Shurong Zhang, Ruru Shen, Wei Jiang, Jun Liu, Effect of acid hydrolysis on morphology, structure and digestion property of starch from Cynanchum auriculatum Royle ex Wight, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2017.01.002
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Effect of acid hydrolysis on morphology, structure and digestion property of starch from Cynanchum auriculatum Royle ex Wight
Xingchi Wang,1 Fanting Wen,1 Shurong Zhang, Ruru Shen, Wei Jiang, Jun Liu*
College of Food Science and Engineering, Yangzhou University, Yangzhou 225127, Jiangsu, China
Corresponding author. Tel: +86-514-87978158, E-mail: [email protected]. 1
The co-first author.
1
Highlights
A typical two-stage hydrolysis process was observed for C. auriculatum starch.
Amorphous regions were degraded rapidly at the early stage of hydrolysis.
The semi-crystalline growth rings were gradually hydrolyzed after 4 days.
The crystal type of acid hydrolyzed starch changed from CB-type to final A-type.
Acid hydrolysis caused RDS and SDS contents increased but RS content decreased.
Abstract: Effect of acid hydrolysis on the morphology, structure and digestion property of starch from Cynanchum auriculatum Royle ex Wight was investigated in this study. The hydrolysis degree of C. auriculatum starch rapidly increased to 63.69% after 4 days and reached 78.67% at the end of 9 days. Morphology observation showed that the starch granules remained intact during the first 4 days of hydrolysis. However, serious erosion phenomenon was observed after 5 days and starch granules completely fell into pieces after 7 days. During acid hydrolysis process, the crystal type of hydrolyzed starch changed from original CB-type to final A-type. Small-angle X-ray scattering patterns showed the semi-crystalline growth rings started to be hydrolyzed after 4 days. The proportions of single helix and amorphous 2
components as well as amylose content in starch gradually decreased, whereas the proportion of double helix components continuously increased during acid hydrolysis. However, the contents of rapidly digestible starch, slowly digestible starch and resistant starch were almost constant during acid hydrolysis process, indicating the in vitro digestion property of C. auriculatum starch was not affected by acid hydrolysis. Our results provided novel information on the inner structure of C. auriculatum starch granules.
Keywords: Cynanchum auriculatum Royle ex Wight; Morphology; Digestion property; Starch; Structure
1. Introduction Starch, the main carbohydrate source in human diet, is the most abundant storage polysaccharide in plants. Native starch is semi-crystalline in nature, which is composed of amylose and amylopectin. Amylose is a linear polymer composed of α-(1, 4)-linked glucopyranose units, while amylopectin is a highly branched polymer containing α-(1, 6)-linked branching points [1−2]. The functional properties of starch, 3
such as swelling, gelatinization, retrogradation, pasting and digestion properties depend on the molecular structure of starch granules (e.g. granule morphology, amylose content, amylopectin architecture, as well as crystalline and amorphous region arrangement) [3−4]. Thus, the functional properties of native starch could be greatly altered through different structural modification methods, including physical, chemical and enzymatic approaches [5−6]. Acid hydrolysis is one of the structural modification method widely used to produce thin boiling starches, which have wide applications in food, paper and textile industries [7]. Acid hydrolysis can change the morphology, crystalline type, amylose content, chain length distribution of amylopectin molecules, and crystalline organization of starch granules [8−10]. Beside, acid hydrolysis can also greatly change the swelling power, gelatinization, retrogradation, pasting, rheological and in vitro enzyme digestibility of starch granules [10−13]. To obtain starch with desired functional properties, it is essential to investigate the effect of acid hydrolysis on the structural and functional properties of starch. Moreover, acid hydrolysis is also very helpful to understand the inner structure of starch granules [14]. In general, the amorphous regions are more rapidly hydrolyzed compared to crystalline regions, and the residue after prolonged acid hydrolysis consists of acid-resistant crystalline parts of amylopectin [10]. Cynanchum auriculatum Royle ex Wight is a traditional Chinese medicine widely distributed in China. The root of C. auriculatum Royle ex Wight (also called ‘Baishouwu’ in Chinese) has been used as a famous tonic herbal drug as well as 4
healthy food for over 1000 years [15]. Nowadays, the root of C. auriculatum is usually processed into whole powder for human daily consumption in China. The dry root of C. auriculatum contains about 30% of starch, which is a potential raw material for food industry [16]. Till now, only few studies have been conducted on the structural and functional properties of C. auriculatum starch [16−17]. In our previous study, the structural characterizations of C. auriculatum starch were investigated by several instrumental methods. Results showed that the native C. auriculatum starch was both spherical and polygonal in shape with CB-type crystallinity. The proportions of single helix, double helix and amorphous components in C. auriculatum starch were 3.42%, 27.11% and 69.47%, respectively [16]. Miao et al. investigated the functional properties of starches isolated from two different cultivars of C. auriculatum, and found C. auriculatum starch had low digestibility [17]. However, the inner structure of C. auriculatum starch granule, which is the intrinsic character and key factor affecting the functional properties of C. auriculatum starch, is still unknown till now. In this study, acid hydrolysis was applied as a useful tool to reveal the inner structure of C. auriculatum starch granule. C. auriculatum starch was hydrolyzed with 2.2 M HCl at 35 °C for different times. Changes in the morphology and inner structure of C. auriculatum starch granule during acid hydrolysis were monitored by scanning electron microscope (SEM), X-ray powder diffraction (XRD), small-angle X-ray scattering (SAXS), attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy and solid-state 5
13
C cross polarization magic
angle spinning nuclear magnetic resonance (13C CP/MAS NMR) spectroscopy. Based on the results of instrumental analysis, an acid hydrolysis model was proposed to elucidate the inner structure of C. auriculatum starch granule for the first time. Moreover, the effect of acid hydrolysis on the digestion property of C. auriculatum starch was also investigated. 2. Materials and methods 2.1. Materials and reagents The dried root of C. auriculatum was kindly provided by the Yancheng Jinfang Shouwu Science and Technology Development Co., Ltd. (Jiangsu, China). Native starch was isolated from the root of C. auriculatum based on the method of Song et al. [18]. Amylose from potato, amylopectin from corn, porcine pancreatic α-amylase (EC 3.2.1.1) and amyloglucosidase from Aspergillus niger (EC 3.2.1.3) were all purchased from Sigma Chemical Co. (MO, USA). The glucose oxidase–peroxidase assay kits were purchased from Megazyme International Ireland Ltd. (Bray, Ireland). 2.2 Preparation of acid hydrolyzed starch Acid hydrolyzed C. auriculatum starch was prepared following the method of Cai et al. with some modifications [19]. Native C. auriculatum starch (2%, w/v) was treated with 2.2 M HCl at 35 °C for 1, 2, 3, 4, 5, 6, 7 and 9 days. The starch mixture was shaken three times daily to re-suspend the deposited granules. After hydrolysis, the granular residues were obtained by centrifugation at 5000 × g for 10 min. The hydrolysis degree of C. auriculatum starch was determined by measuring the content of solubilized sugars in the supernatant using the anthrone-H2SO4 method. The 6
residues obtained by centrifugation were further washed with deionized water for three times and dried at 35 °C to afford acid hydrolyzed starch samples. 2.3. Scanning electron microscopy (SEM) The morphology of acid hydrolyzed C. auriculatum starch was observed by S-4800 SEM (Hitachi Ltd., Japan). Sample was mounted on an aluminum stub and then sputter coated with gold. Clear images were taken at an accelerating voltage of 15 kV with magnification of 2500×. 2.4. X-ray powder diffractometry (XRD) XRD analysis of acid hydrolyzed C. auriculatum starch was performed on Bruker AXS D8 Advance X-ray diffractometer (Bruker AXS GmbH, Germany) operating at 40 kV and 40 mA with Ni-filtered Cu Kα radiation (λ = 0.154 nm). Sample was scanned at a rate of 1°/min with diffraction angle (2θ) of 3−40°. The relative crystallinity of starch was calculated as the percentage ratio of diffraction peak area to total diffraction area [20]. 2.5. Small-angle X-ray scattering (SAXS) SAXS analysis of acid hydrolyzed C. auriculatum starch was carried out on Bruker NanoStar SAXS system (Bruker AXS GmbH, Germany) equipped with Vantec 2000 area detector. Sample was first dispersed in distilled water to form slurries. Then, the optics and sample chamber were under vacuum to minimize air scattering. The obtained SAXS data were analyzed by Bruker NanoFit sofware. The relative position of SAXS peak (Smax) on the scattering vector axis was used to determine the Bragg spacing (2π/Smax) of starch. The intensity of scattering peak 7
(Imax) was calculated according to the method of Cai et al. [19]. 2.6. Fourier-transform infrared (FT-IR) spectroscopy FT-IR spectrum of acid hydrolyzed C. auriculatum starch was recorded on Varian 670 FT-IR spectrometer (Varian Inc., USA) equipped with a deuterated triglycine sulphate detector using horizontal germanium crystal ATR plate. The spectrum was recorded at 1200−800 cm−1, and then baseline corrected and deconvoluted by using Omnic software of version 8.0 (Thermo Scientific, MA). A half-width of 26 cm−1 and a resolution enhancement factor of 1.9 were employed. The ratio of absorbance height at 1045 cm−1 to the height at 1022 cm−1 was obtained from the deconvoluted spectrum. 2.7.
13
C solid-state cross polarization magic angle spinning nuclear magnetic
resonance (CP/MAS NMR) spectroscopy The solid-state NMR spectrum of acid hydrolyzed C. auriculatum starch was recorded on Bruker Avance III 400WB spectrometer (Bruker Biospin GmbH, Germany) equipped with a double resonance H/X CP-MAS 4 mm probe. Spectrum was obtained using the standard CP/MAS technique with a spinning rate of 6 kHz and the magic angle of 54.7°. The cross polarization contact time was set as 1.2 ms with a recycle delay of 3 s for an acquisition time of 15.7 ms. Quantitative analysis of NMR spectrum was carried out by decomposing the spectrum into respective amorphous and ordered subspectra according the method of Paris et al. [21]. Then, the ordered subspectrum was peak fitted by using PeakFit software of version 4.1.2 (Jandel Scientific Software, CA). Quantitative analyses of single helices, double 8
helices and amorphous conformational features within each sample were performed by the method of Tan et al. [22]. 2.8. Determination of amylose content Amylose content in acid hydrolyzed C. auriculatum starch was determined by the iodine colorimetric method [23]. Apparent amylose content was calculated from the standard curve of starch–iodine mixture at 620 nm, which was established by using amylose and amylopectin as standards. 2.9. In vitro digestibility In vitro digestion of acid hydrolyzed C. auriculatum starch was carried out following the method of Miao et al. with some modifications [17]. To prepare enzyme solution, 6 g of porcine pancreatic α-amylase was dispersed in 50 ml of deionized water and centrifuged at 3000 g for 15 min. The obtained supernatant (30 ml) was transferred into a beaker and mixed with 1000 U of amyloglucosidase. Afterwards, 20 mg of acid hydrolyzed C. auriculatum starch was suspended in 2 ml of 0.1 M sodium phosphate buffer (pH 6.0) and heated in a boiling water bath for 20 min with stirring. After cooling to room temperature, the cooked starch was hydrolyzed with previously prepared enzyme solution (0.5 ml) at 37 °C with shaking. Hydrolyzed solution (0.5 ml) was taken at 20 and 120 min, respectively; and immediately mixed with 4 ml of absolute ethanol to deactivate the enzymes. After centrifugation at 5000 g for 10 min, the amount of glucose in the supernatant was determined by glucose oxidase-peroxidase assay kits. The proportions of rapidly digestible starch (RDS, digested within 20 min), slowly digestible starch (SDS, 9
digested between 20 and 120 min) and resistant starch (RS, undigested after 120 min) were calculated using the following equations:
G 20 FG ) 0.9 100 TG G120 G 20 SDS(%) = ( ) 0.9 100 TG RDS(%) = (
(1) (2)
RS(%) = 100 − RDS(%)− SDS(%)
(3)
where G20 and G120 were glucose contents released after 20 and 120 min, respectively; FG and TG were the free glucose and total glucose contents in acid hydrolyzed C. auriculatum starch sample, respectively. 2.10. Statistical analysis Data were expressed as mean ± standard deviation (SD). Analysis of variance (ANOVA) using Tukey's test was performed by SPSS 13.0 software package. Difference was considered to be statistically significant if p < 0.05. 3. Results and discussion 3.1. Hydrolysis kinetics of C. auriculatum starch The hydrolysis kinetics of C. auriculatum starch granules was evaluated by the content of soluble sugar in the hydrolyzate. The hydrolysis degree of C. auriculatum starch by 2.2 M HCl at 35 °C for 9 days was presented in Fig. 1. The hydrolysis degree of C. auriculatum starch gradually increased with the increase of hydrolysis time. Moreover, a typical two-stage hydrolysis process could be observed: a relatively rapid hydrolysis rate at early stage (from 0 to 4 days) followed by a progressively decreased hydrolysis rate thereafter. As reported, the initial rapid hydrolysis stage is presumably due to the hydrolysis of the amorphous regions 10
within starch granules, whereas the subsequent slow hydrolysis stage is attributed to the hydrolysis of the crystalline regions of starch granules [24]. In general, the amorphous regions of starch granules are more susceptible to acid hydrolysis than the crystalline ones for two reasons. On one hand, the dense packing of double helixes within crystalline regions can prevent the penetration of H3O+ ions. On the other hand, the hydrolysis of glucosidic bonds within crystalline regions usually requires relatively higher activation energy [25]. Based on the model proposed by Biliaderis et al. [25], the hydrolysis dynamics of C. auriculatum starch at the first hydrolysis stage (from 0 to 4 days) can be fitted by the following equation:
100 Ln ( ) 0.296 t 0.18 100 x
(4)
where 0.296 was the calculated apparent rate constant (day−1) of the first hydrolysis stage, x was the starch hydrolysis rate (%) at specific time and t was the hydrolysis time (day). Biliaderis et al. compared the hydrolysis rate of different kinds of starches (from corn, pea, bean and lentil) and found apparent hydrolysis rate constant varied from 0.0453 to 0.192 day−1 for the tested starches [25]. By contrast, the apparent hydrolysis rate constant of C. auriculatum starch was much higher than the reported starches, indicating that C. auriculatum starch had relatively lower resistance to acid hydrolysis. The low resistance of C. auriculatum starch to acid hydrolysis could be also confirmed by comparing the hydrolysis progress of C. auriculatum starch with other C-type starches at the same hydrolysis conditions. As shown in Fig. 1, the hydrolysis degree of C. auriculatum starch increased to 11
78.67% at the end of 9 days. However, the hydrolysis degrees of rhizoma Dioscorea and pea starches remained over 30% after 25 days of hydrolysis [26−27]. Fig. 1 3.2. Morphology of acid hydrolyzed C. auriculatum starch granules The morphology of acid hydrolyzed C. auriculatum starch granules was examined by SEM and presented in Fig. 2. In our previous study, native C. auriculatum starch was found to exhibit both spherical and polygonal shapes with the granule size ranging from 2 to 12 μm [16]. As shown in Fig. 2a and Fig. 2b, C. auriculatum starch granules retained their original morphology during the first two days of hydrolysis. When the hydrolysis time reached 3 to 4 days, the outer surface of starch granules became rough due to the corrosion effect of HCl (Fig. 2c and Fig. 2d). Notably, the starch granules remained intact during the first four days of hydrolysis, although the hydrolysis rate at this stage was very fast. As reported, the amorphous regions of starch granules are preferentially degraded during the initial stage of acid hydrolysis [14, 24, 28, 29]. Our results further indicated that the amorphous regions of C. auriculatum starch were probably located at the inner districts of granules, while the crystalline regions mainly existed at the peripheral parts of granules. Similar phenomenon was also observed in the acid hydrolysis process of other C-type starches, such as rhizoma Dioscorea, rhizoma and pea starches [19, 26, 27, 30]. The prolonged acid treatment of C. auriculatum starch for 5 to 6 days had more serious erosion effect on starch, causing deformation and fracture on the surface of granules as well as adhesion between granules (Fig. 2e and Fig. 2f). 12
Thus, the crystalline regions at the peripheral parts of starch granules were gradually degraded at this stage. After 7 days of hydrolysis, the starch granules completely fell into pieces and formed lumps due to the more extensive acid erosion (Fig. 2g and Fig. 2h). Fig. 2 3.3. XRD spectra of acid hydrolyzed C. auriculatum starch Unbranched and singly branched amylopectin chains with more than 10 glucose units may form double helices and array into either A- or B- crystalline structures [10]. The semi-crystalline nature of acid hydrolyzed C. auriculatum starch granules was characterized by XRD and shown in Fig. 3. Our previous study suggested the native C. auriculatum starch exhibited strong diffraction peaks at 17.3° and 22.6° 2θ as well as and some small peaks at about 5.8° and 15.5° 2θ, suggesting C. auriculatum starch is CB-type crystallinity. During the first three days of hydrolysis, no significant difference was observed in the XRD patterns between native and acid hydrolyzed starches. Notably, the characteristic diffraction peak of B-type polymorphs at 5.8° 2θ significantly decreased after 4 days of hydrolysis and almost disappeared at 6 days of hydrolysis. By contrast, a new diffraction peak (at 18.2° 2θ) appeared in the neighborhood of 17.3° 2θ after 6 days of hydrolysis, which was attributed to the characteristic diffraction peak of A-type polymorphs. In addition, the broad diffraction peak at 22.6° 2θ in native starch gradually became sharp from 5 days of hydrolysis and thereafter. Our result suggested that the B-type polymorphs in the CB-type starch were preferentially degraded or faster hydrolyzed than A-type 13
polymorphs during the acid hydrolysis process. Moreover, the disappearance of characteristic B-type diffraction peak (at 5.8° 2θ) and the formation of new A-type diffraction peak (at 18.2° 2θ) further indicated that the crystal type of hydrolyzed C. auriculatum starch changed from original CB-type to final A-type. Similar results were also found by other investigators [19, 26, 27, 31]. As reported, B-type polymorphs are more susceptible to acid hydrolysis than A-type polymorphs, because B-type polymorphs have relatively looser packed structures [10]. By combining with the results of XRD and SEM, it could be concluded that B-type polymorphs in C. auriculatum starch were mainly degraded when the deformation and fracture of starch granules occurred. The crystalline degrees of acid hydrolyzed C. auriculatum starches were also calculated and summarized in Table 1. The crystalline degree of acid hydrolyzed C. auriculatum starches significantly increased during the first four days of hydrolysis and was almost constant thereafter, which further confirmed that the amorphous region of starch granules was preferentially hydrolyzed at early stage of acid hydrolysis. Fig. 3 Table 1 3.4. SAXS spectra of acid hydrolyzed C. auriculatum starch Native starch granules exhibit a layered organization with alternating amorphous and semi-crystalline radial growth rings. The amorphous growth rings are composed of amylose and amylopectin in disordered conformation, while the semi-crystalline
14
growth rings are composed of alternating lamellar structures of crystalline and amorphous regions with repeat distance ranging from 9 to 10 nm [32]. Change in the lamellar structure of C. auriculatum starch during acid hydrolysis was determined by SAXS and shown in Fig. 4. The main scattering peak (Smax) corresponded to lamellar repeat distance or Bragg spacing (2π/Smax) of semi-crystalline growth rings. In our previous study, the scattering peak of native C. auriculatum starch was observed at the scattering vector of 0.682 nm, corresponding to lamellar repeat distance of 9.21 nm. Notably, the scattering peak positions were unchanged during the first four days of hydrolysis, indicating that the semi-crystalline growth rings remained compact and were not hydrolyzed by acid at this stage. By combining with the results of hydrolysis kinetics, morphology change, XRD and SAXS patterns, it could be predicted that the amorphous regions including bulk amorphous core parts and amorphous growth rings were rapidly hydrolyzed at the early stage of acid hydrolysis, which caused significant increase in crystalline degree of starch granules. However, the scattering peaks of starch disappeared after 4 days of hydrolysis, suggesting the crystalline and amorphous lamellaes in semi-crystalline growth rings were concomitantly hydrolyzed [14, 19]. Therefore, little change was observed in the relative crystalline degree in XRD patterns. Moreover, the intensity of scattering peak in starch usually depends on the degree of ordered semi-crystalline structures and/or on the differences in electron density between crystalline and amorphous lamellae with respect to the amorphous background [33−34]. The intensity of scattering peak in native C. auriculatum starch was 33.52. As summarized in Table 1, 15
the intensity of scattering peak of C. auriculatum starch significantly increased to 77.91 at the first day of hydrolysis, and subsequently decreased to 8.85 until the end of 4 days of hydrolysis, and disappeared thereafter. This result was in consistent with that of others [14, 19, 35]. The initial rapid increase in peak intensity was probably caused by the hydrolysis of amorphous regions at the core parts, and subsequent decrease in peak intensity could be attributed to the hydrolysis of amorphous growth rings. The final disappearance of peak intensity was due to hydrolysis of semi-crystalline growth rings. Fig. 4 3.5. FT-IR spectra of acid hydrolyzed C. auriculatum starch The amylopectin branch chains can form two types of helices. One type of helices is packed in short-range order and defined as the double helical order, and another type of helices is packed in long-range order which is related to the packing of double helices forming crystallinity [8]. Change in the short-range ordered structure of starch during acid hydrolysis was monitored by ATR FT-IR and presented in Fig. 5. In FT-IR spectra, the bands at 1045 and 1022 cm−1 are corresponded to the ordered/crystalline and amorphous regions in starch, respectively [36]. Thus, the ratio of absorbance at 1045/1022 cm−1 can be used to quantify the degree of short-range order. As shown in Table 1, the ratios of absorbance at 1045/1022 cm−1 for acid hydrolyzed C. auriculatum starch varied in the range of 0.557−0.578. This indicated that no significant changes in short-range molecular order occurred during the whole hydrolysis period. This result was in 16
agreement of that of acid hydrolyzed lotus rhizome C-type starch [19]. Cai et al. suggested the unchanged ratio of absorbance at 1045/1022 cm−1 was due to the fact that the particle size of C-type lotus rhizome starch was much larger penetration depth of ATR FT-IR technique (about 2 μm thick in external regions) [19]. However, a significant increase in the ratio of absorbance at 1045/1022 cm−1 was observed in acid hydrolyzed A-type waxy maize starches with similar particle size [20]. This suggested that the change in the short-range molecular order might be closely related to the crystalline type of starch. Fig. 5 3.6. 13C CP/MAS NMR spectra of acid hydrolyzed C. auriculatum starch Change in the molecular structure of C. auriculatum starch granules during acid hydrolysis were also monitored by
13
C CP/MAS NMR technique. As presented in
Fig. 6A, signals at 94–105 ppm were assigned to C1, while the broad signal at about 82 ppm was due to amorphous domain of C4. The overlapping signals at 68–78 ppm were attributed to C2, C3 and C5, and the signal at around 62 ppm was assigned to C6 [37]. In the
13
C NMR spectra, the multiplicity of C1 domain (94–105 ppm) is
usually related with the crystalline type of starch granules as characterized by XRD. In general, A-type starch exhibits a triplet C-1 resonance at about 102, 101 and 100 ppm, whereas B-type starch shows a doublet C-1 resonance at around 101 and 100 ppm. The C-1 resonance type of C-type starch is mainly dependent on the relative proportion of A- and B-type polymorphs [38]. As shown in Fig. 6B, acid hydrolyzed starch for 1−4 days showed inconspicuous doublet peaks at 101.2 and 100.2 ppm, 17
which was much similar to the C-1 resonance of native C. auriculatum starch [16]. This indicated that acid hydrolyzed starch remained CB-type crystalline at the first four days of hydrolysis, which was in agreement with the result of XRD spectra. Notably, C1 resonance of acid hydrolyzed starch for 5−9 days gradually split into triplet at about 99.7, 100.6 and 101.8 ppm, corresponding to the characteristics of A-type starch. This suggested that the crystalline type of acid hydrolyzed starch granules gradually changed from CB-type to A-type after 4 days of hydrolysis. Similar phenomenon was also observed in the acid hydrolysis process of other C-type starches [19, 27, 31]. The ordered subspectra of acid hydrolyzed C. auriculatum starch were obtained by subtracting the amorphous starch spectrum from the acid hydrolyzed starch spectrum until there was no residual intensity at 84 ppm. As shown in Fig. 6C, the ordered subspectra of acid hydrolyzed starch showed clear crystalline peaks at C1 resonances. The subspectra further indicated the change in the crystalline type of acid hydrolyzed starch after 4 days of hydrolysis. The ordered subspectra of acid hydrolyzed starches were subsequently fitted to individual peaks by PeakFit Software. The fitted peak at about 103 ppm is attributed to the C1 of single-helical lipid-amylose complexes (V-type amylase), and peaks at 100–102 ppm corresponded to double-helix conformation [22]. The proportions of single helix, double helix and amorphous components in acid hydrolyzed starch were calculated and summarized in Table 1. The proportions of single helix and amorphous components gradually decreased, whereas the proportion of double helix components continuously 18
increased during acid hydrolysis. Our result was consistent with that of other C-type starches [19, 29]. Fig. 6 3.7. Amylose content of acid hydrolyzed C. auriculatum starch Native starch granules usually contain two forms of amylose: the free amylose and lipid-complexed amylose [2]. Wang et al. suggested that amylose was predominantly located at the core area of starch granules and formed the bulk amorphous region with disordered amylopectin chains [14]. Some amylose chains exhibited a spoke-like pattern, which transversely penetrated the lamellar stacks of growth rings and radiated from the core to outer surface of starch granules [10]. The original amylose content in native C. auriculatum starch was measured as 28.04% in our previous study [16]. As shown in Table 1, about one half of amylose (52.46%) was rapidly hydrolyzed at the first three days of hydrolysis, which accompanied the hydrolysis of amorphous region of starch. Thus, the fast hydrolyzed amylose was more likely to be located at the amorphous regions (core area) of starch granules. However, only 9.09% of amylose was further slowly hydrolyzed from day 4 to day 9, which was closely related to the deformation of starch granule morphology and the hydrolysis of growth rings as well as transformation of crystalline type. This suggested that the slowly hydrolyzed amylose probably transversely penetrated the lamellar stacks of growth rings, and was hydrolyzed along with the amorphous and semi-crystalline growth rings. At the end of 9 days of hydrolysis, 38.45% of amylose remained in the acid hydrolyzed starch. The remaining un-hydrolyzed amylose was 19
possibly located at the semi-crystalline growth rings, which was more resistant to acid hydrolysis than amorphous growth rings [34]. The gradual decrease of amylose content in C. auriculatum starch during acid hydrolysis was in agreement with previous reports [14, 19]. Cai et al. reported that all the amylose in lotus rhizome starch was hydrolyzed after 36 h of hydrolysis [19]. Wang et al. reported the amylose content in pea starch decreased to zero after 6 days of hydrolysis [14]. These further suggested the acid hydrolysis characteristics of amylose from different starches were closely related with the source and distribution of amylose in starches. 3.8. Prediction of the acid hydrolysis process of C. auriculatum starch Combining all the information obtained above, the whole acid hydrolysis process of C. auriculatum starch could be divided into three stages according to the growth ring model as proposed by Wang et al. [14]. As shown in Fig. 7, the amorphous core is mainly composed of amylose and disordered amylopectin and is surrounded by alternated amorphous and semicrystalline growth rings. The amorphous growth rings are mainly composed of extended chains of amylopectin interconnecting the crystalline regions containing interspersed amylose molecules, while the semi-crystalline growth rings are composed of crystalline amylopectin interspersed with amylose molecules. At the first stage of acid hydrolysis (during the first four days), the amorphous regions containing amylose were located at the inner of C. auriculatum starch granules and were rapidly degraded, which caused significant increase in the crystalline degree of starch granules. Although the outer surface was slightly eroded when the hydrolysis time reached 3 to 4 days, the 20
morphology of starch granules was still intact. Moreover, the semi-crystalline growth rings remained compact and were not hydrolyzed by acid at this stage. At the second stage of acid hydrolysis (from 4 to 6 days), the semi-crystalline growth rings containing small proportion of amylose began to be hydrolyzed by acid, which caused deformation and fracture on the surface of granules as well as adhesion between granules. The crystalline and amorphous lamellaes in semi-crystalline growth rings were concomitantly hydrolyzed at this stage. However, B-type polymorphs in C. auriculatum starch were preferentially degraded or faster hydrolyzed than A-type polymorphs. At the third stage of acid hydrolysis (from 6 to 9 days), the starch granules completely fell into pieces and formed lumps. The crystal type of acid hydrolyzed starch changed from CB-type to final A-type with crystalline degree almost unchanged. Fig. 7 3.9. In vitro digestibility of acid hydrolyzed C. auriculatum starch For nutritional purposes, starch can be classified into RDS, SDS and RS based on their digestion properties. RDS represents starch that is rapidly and thoroughly hydrolyzed in small intestine, and SDS represents starch that is slowly and completely digested in small intestine. However, RS is starch which resists digestion in small intestine and functions as a substrate for bacterial fermentation in large intestine [39]. In this study, the acid hydrolyzed C. auriculatum starch was cooked in a boiling water bath for 20 min before enzyme hydrolysis. The RDS, SDS and RS contents in acid hydrolyzed C. auriculatum starch were determined and shown in 21
Table 2. Notably, no statistical significance was observed between different acid hydrolyzed starch samples in their RDS, SDS and RS contents (p > 0.05). This was due to the cooking process had completely destroyed the initial structure of acid hydrolyzed starch granules and finally turned the starch into amorphous state [40]. Therefore, different acid hydrolyzed starch samples showed the similar tendency of enzyme hydrolysis. Results indicated that the in vitro digestion property of C. auriculatum starch was not significantly affected by acid hydrolysis. Similar results were also obtained by other investigators [40−41]. Table 2 4. Conclusion Acid hydrolysis had a significant impact on both structure and digestion property of C. auriculatum starch. At the early stage of hydrolysis (from 0 to 4 days), the amorphous regions located at the inner of starch granules were degraded rapidly, causing significant increases in the crystalline degree of starch granules. The morphology of starch granules and the semi-crystalline growth rings were still intact at this stage. After 4 days of hydrolysis, the surface of starch granules and the semi-crystalline growth rings were gradually hydrolyzed, causing the starch granules finally fell into pieces and formed lumps. B-type polymorphs in C. auriculatum starch were preferentially degraded or faster hydrolyzed than A-type polymorphs, resulting in the crystal type of acid hydrolyzed starch changed from original CB-type to final A-type. Thus, our results provided novel information on the inner structure of C. auriculatum starch granules. In addition, acid hydrolysis did not significantly alter 22
the in vitro digestibility of C. auriculatum starch. Effects of acid concentration and hydrolysis temperature on the inner structure and functional properties of C. auriculatum starch can be further investigated in future. Acknowledgements This work was supported by Grants-in-Aid for scientific research from the National Natural Science Foundation of China (No. 31571788 and 31101216), Natural Science Foundation of Jiangsu Province (No. BK20151310), Innovation and Entrepreneurship Training Program for College Students in Jiangsu Province (No. 201611117045Z), Qing Lan Project of Jiangsu Province, Jiangsu Provincial Government Scholarship for Overseas Studies, and High Level Talent Support Program of Yangzhou University.
23
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[34] S.S. Kozlov, A. Blennow, A.V. Krivandin, V.P. Yuryev, Structural and thermodynamic properties of starches extracted from GBSS and GWD suppressed potato lines, Int. J. Biol. Macromol. 40 (2007) 449−460. [35] P.J. Jenkins, A.M. Donald, The effect of acid hydrolyis on native starch granule structure, Starch-Stärke 49 (1997) 262−267. [36] J. Huang, L. Zhao, J. Man, J. Wang, W. Zhou, H. Huai, C. Wei, Comparison of physicochemical properties of B-type nontraditional starches from different sources, Int. J. Biol. Macromol. 78 (2015) 165−172. [37] Y. Su, H. Du, Y. Huo, Y. Xu, J. Wang, L. Wang, S. Zhao, S. Xiong, Characterization of cationic starch flocculants synthesized by dry process with ball milling activating method, Int. J. Biol. Macromol. 87 (2016) 34−40. [38] C. Mutungi, L. Passauer, C. Onyango, D. Jaros, H. Rohm, Debranched cassava starch crystallinity determination by Raman spectroscopy: Correlation of features in Raman spectra with X-ray diffraction and
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spectroscopy, Carbohyd. Polym. 87 (2012) 598−606. [39] H.N. Englyst, S.M. Kingman, J.H. Cummings, Classification and measurement of nutritionally important starch fractions, Eur. J. Clin. Nutr. 46 (1992) S33−S50. [40] G. Zhang, M. Venkatachalam, B.R. Hamaker, Structural basis for the slow digestion property of native cereal starches, Biomacromolecules 7 (2006) 3259−3266.W. [41] Song, S. Janaswamy, Y. Yao, Structure and in vitro digestibility of normal corn starch: effect of acid treatment, autoclaving, and β-amylolysis, J Agr. Food Chem. 58 (2010) 9753−9758.
28
Figure Captions Fig. 1. The hydrolysis kinetics of C. auriculatum starch during 9 days. Experimental data were expressed as mean ± SD of triplicates. Fig. 2. SEM images (2500 ×) of acid hydrolyzed C. auriculatum starch particles at different hydrolysis times: (a) 1 day, (b) 2 days, (c) 3 days, (d) 4 days, (e) 5 days, (f) 6 days, (g) 7 days and (h) 9 days. Fig. 3. XRD patterns of acid hydrolyzed C. auriculatum starch at different hydrolysis times: (a) 1 day, (b) 2 days, (c) 3 days, (d) 4 days, (e) 5 days, (f) 6 days, (g) 7 days and (h) 9 days. Fig. 4. SAXS patterns of acid hydrolyzed C. auriculatum starch at different hydrolysis times: (a) 1 day, (b) 2 days, (c) 3 days, (d) 4 days, (e) 5 days, (f) 6 days, (g) 7 days and (h) 9 days. Fig. 5. ATR-FTIR deconvoluted spectra of acid hydrolyzed C. auriculatum starch at different hydrolysis times: (a) 1 day, (b) 2 days, (c) 3 days, (d) 4 days, (e) 5 days, (f) 6 days, (g) 7 days and (h) 9 days. Fig. 6.
13
C CP/MAS NMR original spectra (A) with enlarged C1 region (B) and
ordered subspectra (C) of acid hydrolyzed C. auriculatum starch at different hydrolysis times: (a) 1 day, (b) 2 days, (c) 3 days, (d) 4 days, (e) 5 days, (f) 6 days, (g) 7 days and (h) 9 days. Fig. 7. The predicted acid hydrolysis process of C. auriculatum starch.
29
Hydrolysis degree (%)
100 80 60 40 20 0 0
3
6
Hydrolysis time (days) Fig. 1.
30
9
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
Fig. 2.
31
Relative intensity
a b c d e f g h 0
5
10
15
20
25
Diffraction angel (2q )
Fig. 3.
32
30
35
40
h
Relative intensity
g f e d c b a 0.02
0.04
0.06
0.08
0.1
Scattering vector (Å–1)
Fig. 4.
33
0.12
0.14
0.16
1022
995
1045
Relative intensity
a b c d e f g h 1200
1100
1000
900 -1
Wavenumber (cm )
Fig. 5.
34
800
C2, 3, 5
(A)
C6
C1 C4
a b c d e f g h 120
110
100
90
80
70
60
50
Chemical shift (ppm) 101.2
100.2
103.3
(B)
94.7
a b c d e f g 101.8
100.6
99.7
h 110
105
100
Chemical shift (ppm)
35
95
90
C2, 3, 5
(C)
C6 C1 C4
a b c d e f g h 120
110
100
90 80 Chemical shift (ppm)
Fig. 6.
36
70
60
50
Bulk amorphous region
Amorphous growth ring
(1) Amorphous regions were rapidly hydrolyzed Semi-crystalline growth ring
1−4 days Native C. auriculatum starch
Semi-crystalline growth rings were slowly hydrolyzed
(2)
(3) Granules fell into pieces and formed lumps
7−9 days
5−6 days
Fig. 7.
37
Table 1 Effect of acid hydrolysis on the long-range order (relative crystallinity and SAXS parameters), short-range order (ratio of 1045/1022 cm−1 and relative conformation proportions) and amylose content of C. auriculatum starch. Hydrolysis time
Relative
SAXS parametersa
Ratio of
Relative conformation proportion (%)b
Amylose
(day)
crystallinity (%)
Smax (nm–1)
d (nm)
Imax
1045/1022 cm−1
Single helix
Double helix
Amorphous
content (%)
1
26.59
0.682
9.21
77.91
0.563
3.05
30.78
66.17
20.73
2
27.84
0.682
9.21
60.09
0.569
2.42
34.75
62.83
15.48
3
29.47
0.682
9.21
18.17
0.561
1.95
37.04
61.01
13.33
4
31.18
0.682
9.21
8.85
0.569
1.50
40.42
58.08
12.84
5
31.57
−c
−
−
0.575
1.28
41.76
56.96
11.34
6
31.76
−
−
−
0.557
0.96
43.02
56.02
11.09
7
32.06
−
−
−
0.578
0.75
44.31
54.94
10.57
9
32.23
−
−
−
0.569
0.61
44.80
54.59
10.08
a
SAXS parameters of Smax, peak position; d, Bragg spacing (2/Smax); Imax, peak intensity. The relative conformation proportion was calculated from 13C CP/MAS NMR spectrum. c Data were not detected. b
37
Table 2 Effect of acid hydrolysis on the RDS, SDS and RS contents in C. auriculatum starch.a Hydrolysis time (day)
RDS (%)
SDS (%)
RS (%)
1
76.20 ± 0.35
8.35 ± 0.21
15.45 ± 0.15
2
76.29 ± 1.42
8.40 ± 0.18
15.31 ± 1.24
3
76.14 ± 3.45
8.44 ± 0.05
15.42 ± 3.41
4
77.94 ± 2.23
8.27 ± 0.08
13.79 ± 2.14
5
77.03 ± 0.61
8.36 ± 0.18
14.61 ± 0.80
6
76.72 ± 1.13
8.49 ± 0.05
14.79 ± 1.08
7
77.48 ± 2.18
8.35 ± 0.11
14.17 ± 2.07
9
76.60 ± 0.54
8.33 ± 0.03
15.07 ± 0.57
a
Data (Mean ± SD) in the same column with different letters were significantly different (p < 0.05).
38
S0141-8130(16)32068-2 http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.01.002 BIOMAC 6903
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
19-10-2016 30-12-2016 1-1-2017
Please cite this article as: Xingchi Wang, Fanting Wen, Shurong Zhang, Ruru Shen, Wei Jiang, Jun Liu, Effect of acid hydrolysis on morphology, structure and digestion property of starch from Cynanchum auriculatum Royle ex Wight, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2017.01.002
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Effect of acid hydrolysis on morphology, structure and digestion property of starch from Cynanchum auriculatum Royle ex Wight
Xingchi Wang,1 Fanting Wen,1 Shurong Zhang, Ruru Shen, Wei Jiang, Jun Liu*
College of Food Science and Engineering, Yangzhou University, Yangzhou 225127, Jiangsu, China
Corresponding author. Tel: +86-514-87978158, E-mail: [email protected]. 1
The co-first author.
1
Highlights
A typical two-stage hydrolysis process was observed for C. auriculatum starch.
Amorphous regions were degraded rapidly at the early stage of hydrolysis.
The semi-crystalline growth rings were gradually hydrolyzed after 4 days.
The crystal type of acid hydrolyzed starch changed from CB-type to final A-type.
Acid hydrolysis caused RDS and SDS contents increased but RS content decreased.
Abstract: Effect of acid hydrolysis on the morphology, structure and digestion property of starch from Cynanchum auriculatum Royle ex Wight was investigated in this study. The hydrolysis degree of C. auriculatum starch rapidly increased to 63.69% after 4 days and reached 78.67% at the end of 9 days. Morphology observation showed that the starch granules remained intact during the first 4 days of hydrolysis. However, serious erosion phenomenon was observed after 5 days and starch granules completely fell into pieces after 7 days. During acid hydrolysis process, the crystal type of hydrolyzed starch changed from original CB-type to final A-type. Small-angle X-ray scattering patterns showed the semi-crystalline growth rings started to be hydrolyzed after 4 days. The proportions of single helix and amorphous 2
components as well as amylose content in starch gradually decreased, whereas the proportion of double helix components continuously increased during acid hydrolysis. However, the contents of rapidly digestible starch, slowly digestible starch and resistant starch were almost constant during acid hydrolysis process, indicating the in vitro digestion property of C. auriculatum starch was not affected by acid hydrolysis. Our results provided novel information on the inner structure of C. auriculatum starch granules.
Keywords: Cynanchum auriculatum Royle ex Wight; Morphology; Digestion property; Starch; Structure
1. Introduction Starch, the main carbohydrate source in human diet, is the most abundant storage polysaccharide in plants. Native starch is semi-crystalline in nature, which is composed of amylose and amylopectin. Amylose is a linear polymer composed of α-(1, 4)-linked glucopyranose units, while amylopectin is a highly branched polymer containing α-(1, 6)-linked branching points [1−2]. The functional properties of starch, 3
such as swelling, gelatinization, retrogradation, pasting and digestion properties depend on the molecular structure of starch granules (e.g. granule morphology, amylose content, amylopectin architecture, as well as crystalline and amorphous region arrangement) [3−4]. Thus, the functional properties of native starch could be greatly altered through different structural modification methods, including physical, chemical and enzymatic approaches [5−6]. Acid hydrolysis is one of the structural modification method widely used to produce thin boiling starches, which have wide applications in food, paper and textile industries [7]. Acid hydrolysis can change the morphology, crystalline type, amylose content, chain length distribution of amylopectin molecules, and crystalline organization of starch granules [8−10]. Beside, acid hydrolysis can also greatly change the swelling power, gelatinization, retrogradation, pasting, rheological and in vitro enzyme digestibility of starch granules [10−13]. To obtain starch with desired functional properties, it is essential to investigate the effect of acid hydrolysis on the structural and functional properties of starch. Moreover, acid hydrolysis is also very helpful to understand the inner structure of starch granules [14]. In general, the amorphous regions are more rapidly hydrolyzed compared to crystalline regions, and the residue after prolonged acid hydrolysis consists of acid-resistant crystalline parts of amylopectin [10]. Cynanchum auriculatum Royle ex Wight is a traditional Chinese medicine widely distributed in China. The root of C. auriculatum Royle ex Wight (also called ‘Baishouwu’ in Chinese) has been used as a famous tonic herbal drug as well as 4
healthy food for over 1000 years [15]. Nowadays, the root of C. auriculatum is usually processed into whole powder for human daily consumption in China. The dry root of C. auriculatum contains about 30% of starch, which is a potential raw material for food industry [16]. Till now, only few studies have been conducted on the structural and functional properties of C. auriculatum starch [16−17]. In our previous study, the structural characterizations of C. auriculatum starch were investigated by several instrumental methods. Results showed that the native C. auriculatum starch was both spherical and polygonal in shape with CB-type crystallinity. The proportions of single helix, double helix and amorphous components in C. auriculatum starch were 3.42%, 27.11% and 69.47%, respectively [16]. Miao et al. investigated the functional properties of starches isolated from two different cultivars of C. auriculatum, and found C. auriculatum starch had low digestibility [17]. However, the inner structure of C. auriculatum starch granule, which is the intrinsic character and key factor affecting the functional properties of C. auriculatum starch, is still unknown till now. In this study, acid hydrolysis was applied as a useful tool to reveal the inner structure of C. auriculatum starch granule. C. auriculatum starch was hydrolyzed with 2.2 M HCl at 35 °C for different times. Changes in the morphology and inner structure of C. auriculatum starch granule during acid hydrolysis were monitored by scanning electron microscope (SEM), X-ray powder diffraction (XRD), small-angle X-ray scattering (SAXS), attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy and solid-state 5
13
C cross polarization magic
angle spinning nuclear magnetic resonance (13C CP/MAS NMR) spectroscopy. Based on the results of instrumental analysis, an acid hydrolysis model was proposed to elucidate the inner structure of C. auriculatum starch granule for the first time. Moreover, the effect of acid hydrolysis on the digestion property of C. auriculatum starch was also investigated. 2. Materials and methods 2.1. Materials and reagents The dried root of C. auriculatum was kindly provided by the Yancheng Jinfang Shouwu Science and Technology Development Co., Ltd. (Jiangsu, China). Native starch was isolated from the root of C. auriculatum based on the method of Song et al. [18]. Amylose from potato, amylopectin from corn, porcine pancreatic α-amylase (EC 3.2.1.1) and amyloglucosidase from Aspergillus niger (EC 3.2.1.3) were all purchased from Sigma Chemical Co. (MO, USA). The glucose oxidase–peroxidase assay kits were purchased from Megazyme International Ireland Ltd. (Bray, Ireland). 2.2 Preparation of acid hydrolyzed starch Acid hydrolyzed C. auriculatum starch was prepared following the method of Cai et al. with some modifications [19]. Native C. auriculatum starch (2%, w/v) was treated with 2.2 M HCl at 35 °C for 1, 2, 3, 4, 5, 6, 7 and 9 days. The starch mixture was shaken three times daily to re-suspend the deposited granules. After hydrolysis, the granular residues were obtained by centrifugation at 5000 × g for 10 min. The hydrolysis degree of C. auriculatum starch was determined by measuring the content of solubilized sugars in the supernatant using the anthrone-H2SO4 method. The 6
residues obtained by centrifugation were further washed with deionized water for three times and dried at 35 °C to afford acid hydrolyzed starch samples. 2.3. Scanning electron microscopy (SEM) The morphology of acid hydrolyzed C. auriculatum starch was observed by S-4800 SEM (Hitachi Ltd., Japan). Sample was mounted on an aluminum stub and then sputter coated with gold. Clear images were taken at an accelerating voltage of 15 kV with magnification of 2500×. 2.4. X-ray powder diffractometry (XRD) XRD analysis of acid hydrolyzed C. auriculatum starch was performed on Bruker AXS D8 Advance X-ray diffractometer (Bruker AXS GmbH, Germany) operating at 40 kV and 40 mA with Ni-filtered Cu Kα radiation (λ = 0.154 nm). Sample was scanned at a rate of 1°/min with diffraction angle (2θ) of 3−40°. The relative crystallinity of starch was calculated as the percentage ratio of diffraction peak area to total diffraction area [20]. 2.5. Small-angle X-ray scattering (SAXS) SAXS analysis of acid hydrolyzed C. auriculatum starch was carried out on Bruker NanoStar SAXS system (Bruker AXS GmbH, Germany) equipped with Vantec 2000 area detector. Sample was first dispersed in distilled water to form slurries. Then, the optics and sample chamber were under vacuum to minimize air scattering. The obtained SAXS data were analyzed by Bruker NanoFit sofware. The relative position of SAXS peak (Smax) on the scattering vector axis was used to determine the Bragg spacing (2π/Smax) of starch. The intensity of scattering peak 7
(Imax) was calculated according to the method of Cai et al. [19]. 2.6. Fourier-transform infrared (FT-IR) spectroscopy FT-IR spectrum of acid hydrolyzed C. auriculatum starch was recorded on Varian 670 FT-IR spectrometer (Varian Inc., USA) equipped with a deuterated triglycine sulphate detector using horizontal germanium crystal ATR plate. The spectrum was recorded at 1200−800 cm−1, and then baseline corrected and deconvoluted by using Omnic software of version 8.0 (Thermo Scientific, MA). A half-width of 26 cm−1 and a resolution enhancement factor of 1.9 were employed. The ratio of absorbance height at 1045 cm−1 to the height at 1022 cm−1 was obtained from the deconvoluted spectrum. 2.7.
13
C solid-state cross polarization magic angle spinning nuclear magnetic
resonance (CP/MAS NMR) spectroscopy The solid-state NMR spectrum of acid hydrolyzed C. auriculatum starch was recorded on Bruker Avance III 400WB spectrometer (Bruker Biospin GmbH, Germany) equipped with a double resonance H/X CP-MAS 4 mm probe. Spectrum was obtained using the standard CP/MAS technique with a spinning rate of 6 kHz and the magic angle of 54.7°. The cross polarization contact time was set as 1.2 ms with a recycle delay of 3 s for an acquisition time of 15.7 ms. Quantitative analysis of NMR spectrum was carried out by decomposing the spectrum into respective amorphous and ordered subspectra according the method of Paris et al. [21]. Then, the ordered subspectrum was peak fitted by using PeakFit software of version 4.1.2 (Jandel Scientific Software, CA). Quantitative analyses of single helices, double 8
helices and amorphous conformational features within each sample were performed by the method of Tan et al. [22]. 2.8. Determination of amylose content Amylose content in acid hydrolyzed C. auriculatum starch was determined by the iodine colorimetric method [23]. Apparent amylose content was calculated from the standard curve of starch–iodine mixture at 620 nm, which was established by using amylose and amylopectin as standards. 2.9. In vitro digestibility In vitro digestion of acid hydrolyzed C. auriculatum starch was carried out following the method of Miao et al. with some modifications [17]. To prepare enzyme solution, 6 g of porcine pancreatic α-amylase was dispersed in 50 ml of deionized water and centrifuged at 3000 g for 15 min. The obtained supernatant (30 ml) was transferred into a beaker and mixed with 1000 U of amyloglucosidase. Afterwards, 20 mg of acid hydrolyzed C. auriculatum starch was suspended in 2 ml of 0.1 M sodium phosphate buffer (pH 6.0) and heated in a boiling water bath for 20 min with stirring. After cooling to room temperature, the cooked starch was hydrolyzed with previously prepared enzyme solution (0.5 ml) at 37 °C with shaking. Hydrolyzed solution (0.5 ml) was taken at 20 and 120 min, respectively; and immediately mixed with 4 ml of absolute ethanol to deactivate the enzymes. After centrifugation at 5000 g for 10 min, the amount of glucose in the supernatant was determined by glucose oxidase-peroxidase assay kits. The proportions of rapidly digestible starch (RDS, digested within 20 min), slowly digestible starch (SDS, 9
digested between 20 and 120 min) and resistant starch (RS, undigested after 120 min) were calculated using the following equations:
G 20 FG ) 0.9 100 TG G120 G 20 SDS(%) = ( ) 0.9 100 TG RDS(%) = (
(1) (2)
RS(%) = 100 − RDS(%)− SDS(%)
(3)
where G20 and G120 were glucose contents released after 20 and 120 min, respectively; FG and TG were the free glucose and total glucose contents in acid hydrolyzed C. auriculatum starch sample, respectively. 2.10. Statistical analysis Data were expressed as mean ± standard deviation (SD). Analysis of variance (ANOVA) using Tukey's test was performed by SPSS 13.0 software package. Difference was considered to be statistically significant if p < 0.05. 3. Results and discussion 3.1. Hydrolysis kinetics of C. auriculatum starch The hydrolysis kinetics of C. auriculatum starch granules was evaluated by the content of soluble sugar in the hydrolyzate. The hydrolysis degree of C. auriculatum starch by 2.2 M HCl at 35 °C for 9 days was presented in Fig. 1. The hydrolysis degree of C. auriculatum starch gradually increased with the increase of hydrolysis time. Moreover, a typical two-stage hydrolysis process could be observed: a relatively rapid hydrolysis rate at early stage (from 0 to 4 days) followed by a progressively decreased hydrolysis rate thereafter. As reported, the initial rapid hydrolysis stage is presumably due to the hydrolysis of the amorphous regions 10
within starch granules, whereas the subsequent slow hydrolysis stage is attributed to the hydrolysis of the crystalline regions of starch granules [24]. In general, the amorphous regions of starch granules are more susceptible to acid hydrolysis than the crystalline ones for two reasons. On one hand, the dense packing of double helixes within crystalline regions can prevent the penetration of H3O+ ions. On the other hand, the hydrolysis of glucosidic bonds within crystalline regions usually requires relatively higher activation energy [25]. Based on the model proposed by Biliaderis et al. [25], the hydrolysis dynamics of C. auriculatum starch at the first hydrolysis stage (from 0 to 4 days) can be fitted by the following equation:
100 Ln ( ) 0.296 t 0.18 100 x
(4)
where 0.296 was the calculated apparent rate constant (day−1) of the first hydrolysis stage, x was the starch hydrolysis rate (%) at specific time and t was the hydrolysis time (day). Biliaderis et al. compared the hydrolysis rate of different kinds of starches (from corn, pea, bean and lentil) and found apparent hydrolysis rate constant varied from 0.0453 to 0.192 day−1 for the tested starches [25]. By contrast, the apparent hydrolysis rate constant of C. auriculatum starch was much higher than the reported starches, indicating that C. auriculatum starch had relatively lower resistance to acid hydrolysis. The low resistance of C. auriculatum starch to acid hydrolysis could be also confirmed by comparing the hydrolysis progress of C. auriculatum starch with other C-type starches at the same hydrolysis conditions. As shown in Fig. 1, the hydrolysis degree of C. auriculatum starch increased to 11
78.67% at the end of 9 days. However, the hydrolysis degrees of rhizoma Dioscorea and pea starches remained over 30% after 25 days of hydrolysis [26−27]. Fig. 1 3.2. Morphology of acid hydrolyzed C. auriculatum starch granules The morphology of acid hydrolyzed C. auriculatum starch granules was examined by SEM and presented in Fig. 2. In our previous study, native C. auriculatum starch was found to exhibit both spherical and polygonal shapes with the granule size ranging from 2 to 12 μm [16]. As shown in Fig. 2a and Fig. 2b, C. auriculatum starch granules retained their original morphology during the first two days of hydrolysis. When the hydrolysis time reached 3 to 4 days, the outer surface of starch granules became rough due to the corrosion effect of HCl (Fig. 2c and Fig. 2d). Notably, the starch granules remained intact during the first four days of hydrolysis, although the hydrolysis rate at this stage was very fast. As reported, the amorphous regions of starch granules are preferentially degraded during the initial stage of acid hydrolysis [14, 24, 28, 29]. Our results further indicated that the amorphous regions of C. auriculatum starch were probably located at the inner districts of granules, while the crystalline regions mainly existed at the peripheral parts of granules. Similar phenomenon was also observed in the acid hydrolysis process of other C-type starches, such as rhizoma Dioscorea, rhizoma and pea starches [19, 26, 27, 30]. The prolonged acid treatment of C. auriculatum starch for 5 to 6 days had more serious erosion effect on starch, causing deformation and fracture on the surface of granules as well as adhesion between granules (Fig. 2e and Fig. 2f). 12
Thus, the crystalline regions at the peripheral parts of starch granules were gradually degraded at this stage. After 7 days of hydrolysis, the starch granules completely fell into pieces and formed lumps due to the more extensive acid erosion (Fig. 2g and Fig. 2h). Fig. 2 3.3. XRD spectra of acid hydrolyzed C. auriculatum starch Unbranched and singly branched amylopectin chains with more than 10 glucose units may form double helices and array into either A- or B- crystalline structures [10]. The semi-crystalline nature of acid hydrolyzed C. auriculatum starch granules was characterized by XRD and shown in Fig. 3. Our previous study suggested the native C. auriculatum starch exhibited strong diffraction peaks at 17.3° and 22.6° 2θ as well as and some small peaks at about 5.8° and 15.5° 2θ, suggesting C. auriculatum starch is CB-type crystallinity. During the first three days of hydrolysis, no significant difference was observed in the XRD patterns between native and acid hydrolyzed starches. Notably, the characteristic diffraction peak of B-type polymorphs at 5.8° 2θ significantly decreased after 4 days of hydrolysis and almost disappeared at 6 days of hydrolysis. By contrast, a new diffraction peak (at 18.2° 2θ) appeared in the neighborhood of 17.3° 2θ after 6 days of hydrolysis, which was attributed to the characteristic diffraction peak of A-type polymorphs. In addition, the broad diffraction peak at 22.6° 2θ in native starch gradually became sharp from 5 days of hydrolysis and thereafter. Our result suggested that the B-type polymorphs in the CB-type starch were preferentially degraded or faster hydrolyzed than A-type 13
polymorphs during the acid hydrolysis process. Moreover, the disappearance of characteristic B-type diffraction peak (at 5.8° 2θ) and the formation of new A-type diffraction peak (at 18.2° 2θ) further indicated that the crystal type of hydrolyzed C. auriculatum starch changed from original CB-type to final A-type. Similar results were also found by other investigators [19, 26, 27, 31]. As reported, B-type polymorphs are more susceptible to acid hydrolysis than A-type polymorphs, because B-type polymorphs have relatively looser packed structures [10]. By combining with the results of XRD and SEM, it could be concluded that B-type polymorphs in C. auriculatum starch were mainly degraded when the deformation and fracture of starch granules occurred. The crystalline degrees of acid hydrolyzed C. auriculatum starches were also calculated and summarized in Table 1. The crystalline degree of acid hydrolyzed C. auriculatum starches significantly increased during the first four days of hydrolysis and was almost constant thereafter, which further confirmed that the amorphous region of starch granules was preferentially hydrolyzed at early stage of acid hydrolysis. Fig. 3 Table 1 3.4. SAXS spectra of acid hydrolyzed C. auriculatum starch Native starch granules exhibit a layered organization with alternating amorphous and semi-crystalline radial growth rings. The amorphous growth rings are composed of amylose and amylopectin in disordered conformation, while the semi-crystalline
14
growth rings are composed of alternating lamellar structures of crystalline and amorphous regions with repeat distance ranging from 9 to 10 nm [32]. Change in the lamellar structure of C. auriculatum starch during acid hydrolysis was determined by SAXS and shown in Fig. 4. The main scattering peak (Smax) corresponded to lamellar repeat distance or Bragg spacing (2π/Smax) of semi-crystalline growth rings. In our previous study, the scattering peak of native C. auriculatum starch was observed at the scattering vector of 0.682 nm, corresponding to lamellar repeat distance of 9.21 nm. Notably, the scattering peak positions were unchanged during the first four days of hydrolysis, indicating that the semi-crystalline growth rings remained compact and were not hydrolyzed by acid at this stage. By combining with the results of hydrolysis kinetics, morphology change, XRD and SAXS patterns, it could be predicted that the amorphous regions including bulk amorphous core parts and amorphous growth rings were rapidly hydrolyzed at the early stage of acid hydrolysis, which caused significant increase in crystalline degree of starch granules. However, the scattering peaks of starch disappeared after 4 days of hydrolysis, suggesting the crystalline and amorphous lamellaes in semi-crystalline growth rings were concomitantly hydrolyzed [14, 19]. Therefore, little change was observed in the relative crystalline degree in XRD patterns. Moreover, the intensity of scattering peak in starch usually depends on the degree of ordered semi-crystalline structures and/or on the differences in electron density between crystalline and amorphous lamellae with respect to the amorphous background [33−34]. The intensity of scattering peak in native C. auriculatum starch was 33.52. As summarized in Table 1, 15
the intensity of scattering peak of C. auriculatum starch significantly increased to 77.91 at the first day of hydrolysis, and subsequently decreased to 8.85 until the end of 4 days of hydrolysis, and disappeared thereafter. This result was in consistent with that of others [14, 19, 35]. The initial rapid increase in peak intensity was probably caused by the hydrolysis of amorphous regions at the core parts, and subsequent decrease in peak intensity could be attributed to the hydrolysis of amorphous growth rings. The final disappearance of peak intensity was due to hydrolysis of semi-crystalline growth rings. Fig. 4 3.5. FT-IR spectra of acid hydrolyzed C. auriculatum starch The amylopectin branch chains can form two types of helices. One type of helices is packed in short-range order and defined as the double helical order, and another type of helices is packed in long-range order which is related to the packing of double helices forming crystallinity [8]. Change in the short-range ordered structure of starch during acid hydrolysis was monitored by ATR FT-IR and presented in Fig. 5. In FT-IR spectra, the bands at 1045 and 1022 cm−1 are corresponded to the ordered/crystalline and amorphous regions in starch, respectively [36]. Thus, the ratio of absorbance at 1045/1022 cm−1 can be used to quantify the degree of short-range order. As shown in Table 1, the ratios of absorbance at 1045/1022 cm−1 for acid hydrolyzed C. auriculatum starch varied in the range of 0.557−0.578. This indicated that no significant changes in short-range molecular order occurred during the whole hydrolysis period. This result was in 16
agreement of that of acid hydrolyzed lotus rhizome C-type starch [19]. Cai et al. suggested the unchanged ratio of absorbance at 1045/1022 cm−1 was due to the fact that the particle size of C-type lotus rhizome starch was much larger penetration depth of ATR FT-IR technique (about 2 μm thick in external regions) [19]. However, a significant increase in the ratio of absorbance at 1045/1022 cm−1 was observed in acid hydrolyzed A-type waxy maize starches with similar particle size [20]. This suggested that the change in the short-range molecular order might be closely related to the crystalline type of starch. Fig. 5 3.6. 13C CP/MAS NMR spectra of acid hydrolyzed C. auriculatum starch Change in the molecular structure of C. auriculatum starch granules during acid hydrolysis were also monitored by
13
C CP/MAS NMR technique. As presented in
Fig. 6A, signals at 94–105 ppm were assigned to C1, while the broad signal at about 82 ppm was due to amorphous domain of C4. The overlapping signals at 68–78 ppm were attributed to C2, C3 and C5, and the signal at around 62 ppm was assigned to C6 [37]. In the
13
C NMR spectra, the multiplicity of C1 domain (94–105 ppm) is
usually related with the crystalline type of starch granules as characterized by XRD. In general, A-type starch exhibits a triplet C-1 resonance at about 102, 101 and 100 ppm, whereas B-type starch shows a doublet C-1 resonance at around 101 and 100 ppm. The C-1 resonance type of C-type starch is mainly dependent on the relative proportion of A- and B-type polymorphs [38]. As shown in Fig. 6B, acid hydrolyzed starch for 1−4 days showed inconspicuous doublet peaks at 101.2 and 100.2 ppm, 17
which was much similar to the C-1 resonance of native C. auriculatum starch [16]. This indicated that acid hydrolyzed starch remained CB-type crystalline at the first four days of hydrolysis, which was in agreement with the result of XRD spectra. Notably, C1 resonance of acid hydrolyzed starch for 5−9 days gradually split into triplet at about 99.7, 100.6 and 101.8 ppm, corresponding to the characteristics of A-type starch. This suggested that the crystalline type of acid hydrolyzed starch granules gradually changed from CB-type to A-type after 4 days of hydrolysis. Similar phenomenon was also observed in the acid hydrolysis process of other C-type starches [19, 27, 31]. The ordered subspectra of acid hydrolyzed C. auriculatum starch were obtained by subtracting the amorphous starch spectrum from the acid hydrolyzed starch spectrum until there was no residual intensity at 84 ppm. As shown in Fig. 6C, the ordered subspectra of acid hydrolyzed starch showed clear crystalline peaks at C1 resonances. The subspectra further indicated the change in the crystalline type of acid hydrolyzed starch after 4 days of hydrolysis. The ordered subspectra of acid hydrolyzed starches were subsequently fitted to individual peaks by PeakFit Software. The fitted peak at about 103 ppm is attributed to the C1 of single-helical lipid-amylose complexes (V-type amylase), and peaks at 100–102 ppm corresponded to double-helix conformation [22]. The proportions of single helix, double helix and amorphous components in acid hydrolyzed starch were calculated and summarized in Table 1. The proportions of single helix and amorphous components gradually decreased, whereas the proportion of double helix components continuously 18
increased during acid hydrolysis. Our result was consistent with that of other C-type starches [19, 29]. Fig. 6 3.7. Amylose content of acid hydrolyzed C. auriculatum starch Native starch granules usually contain two forms of amylose: the free amylose and lipid-complexed amylose [2]. Wang et al. suggested that amylose was predominantly located at the core area of starch granules and formed the bulk amorphous region with disordered amylopectin chains [14]. Some amylose chains exhibited a spoke-like pattern, which transversely penetrated the lamellar stacks of growth rings and radiated from the core to outer surface of starch granules [10]. The original amylose content in native C. auriculatum starch was measured as 28.04% in our previous study [16]. As shown in Table 1, about one half of amylose (52.46%) was rapidly hydrolyzed at the first three days of hydrolysis, which accompanied the hydrolysis of amorphous region of starch. Thus, the fast hydrolyzed amylose was more likely to be located at the amorphous regions (core area) of starch granules. However, only 9.09% of amylose was further slowly hydrolyzed from day 4 to day 9, which was closely related to the deformation of starch granule morphology and the hydrolysis of growth rings as well as transformation of crystalline type. This suggested that the slowly hydrolyzed amylose probably transversely penetrated the lamellar stacks of growth rings, and was hydrolyzed along with the amorphous and semi-crystalline growth rings. At the end of 9 days of hydrolysis, 38.45% of amylose remained in the acid hydrolyzed starch. The remaining un-hydrolyzed amylose was 19
possibly located at the semi-crystalline growth rings, which was more resistant to acid hydrolysis than amorphous growth rings [34]. The gradual decrease of amylose content in C. auriculatum starch during acid hydrolysis was in agreement with previous reports [14, 19]. Cai et al. reported that all the amylose in lotus rhizome starch was hydrolyzed after 36 h of hydrolysis [19]. Wang et al. reported the amylose content in pea starch decreased to zero after 6 days of hydrolysis [14]. These further suggested the acid hydrolysis characteristics of amylose from different starches were closely related with the source and distribution of amylose in starches. 3.8. Prediction of the acid hydrolysis process of C. auriculatum starch Combining all the information obtained above, the whole acid hydrolysis process of C. auriculatum starch could be divided into three stages according to the growth ring model as proposed by Wang et al. [14]. As shown in Fig. 7, the amorphous core is mainly composed of amylose and disordered amylopectin and is surrounded by alternated amorphous and semicrystalline growth rings. The amorphous growth rings are mainly composed of extended chains of amylopectin interconnecting the crystalline regions containing interspersed amylose molecules, while the semi-crystalline growth rings are composed of crystalline amylopectin interspersed with amylose molecules. At the first stage of acid hydrolysis (during the first four days), the amorphous regions containing amylose were located at the inner of C. auriculatum starch granules and were rapidly degraded, which caused significant increase in the crystalline degree of starch granules. Although the outer surface was slightly eroded when the hydrolysis time reached 3 to 4 days, the 20
morphology of starch granules was still intact. Moreover, the semi-crystalline growth rings remained compact and were not hydrolyzed by acid at this stage. At the second stage of acid hydrolysis (from 4 to 6 days), the semi-crystalline growth rings containing small proportion of amylose began to be hydrolyzed by acid, which caused deformation and fracture on the surface of granules as well as adhesion between granules. The crystalline and amorphous lamellaes in semi-crystalline growth rings were concomitantly hydrolyzed at this stage. However, B-type polymorphs in C. auriculatum starch were preferentially degraded or faster hydrolyzed than A-type polymorphs. At the third stage of acid hydrolysis (from 6 to 9 days), the starch granules completely fell into pieces and formed lumps. The crystal type of acid hydrolyzed starch changed from CB-type to final A-type with crystalline degree almost unchanged. Fig. 7 3.9. In vitro digestibility of acid hydrolyzed C. auriculatum starch For nutritional purposes, starch can be classified into RDS, SDS and RS based on their digestion properties. RDS represents starch that is rapidly and thoroughly hydrolyzed in small intestine, and SDS represents starch that is slowly and completely digested in small intestine. However, RS is starch which resists digestion in small intestine and functions as a substrate for bacterial fermentation in large intestine [39]. In this study, the acid hydrolyzed C. auriculatum starch was cooked in a boiling water bath for 20 min before enzyme hydrolysis. The RDS, SDS and RS contents in acid hydrolyzed C. auriculatum starch were determined and shown in 21
Table 2. Notably, no statistical significance was observed between different acid hydrolyzed starch samples in their RDS, SDS and RS contents (p > 0.05). This was due to the cooking process had completely destroyed the initial structure of acid hydrolyzed starch granules and finally turned the starch into amorphous state [40]. Therefore, different acid hydrolyzed starch samples showed the similar tendency of enzyme hydrolysis. Results indicated that the in vitro digestion property of C. auriculatum starch was not significantly affected by acid hydrolysis. Similar results were also obtained by other investigators [40−41]. Table 2 4. Conclusion Acid hydrolysis had a significant impact on both structure and digestion property of C. auriculatum starch. At the early stage of hydrolysis (from 0 to 4 days), the amorphous regions located at the inner of starch granules were degraded rapidly, causing significant increases in the crystalline degree of starch granules. The morphology of starch granules and the semi-crystalline growth rings were still intact at this stage. After 4 days of hydrolysis, the surface of starch granules and the semi-crystalline growth rings were gradually hydrolyzed, causing the starch granules finally fell into pieces and formed lumps. B-type polymorphs in C. auriculatum starch were preferentially degraded or faster hydrolyzed than A-type polymorphs, resulting in the crystal type of acid hydrolyzed starch changed from original CB-type to final A-type. Thus, our results provided novel information on the inner structure of C. auriculatum starch granules. In addition, acid hydrolysis did not significantly alter 22
the in vitro digestibility of C. auriculatum starch. Effects of acid concentration and hydrolysis temperature on the inner structure and functional properties of C. auriculatum starch can be further investigated in future. Acknowledgements This work was supported by Grants-in-Aid for scientific research from the National Natural Science Foundation of China (No. 31571788 and 31101216), Natural Science Foundation of Jiangsu Province (No. BK20151310), Innovation and Entrepreneurship Training Program for College Students in Jiangsu Province (No. 201611117045Z), Qing Lan Project of Jiangsu Province, Jiangsu Provincial Government Scholarship for Overseas Studies, and High Level Talent Support Program of Yangzhou University.
23
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Figure Captions Fig. 1. The hydrolysis kinetics of C. auriculatum starch during 9 days. Experimental data were expressed as mean ± SD of triplicates. Fig. 2. SEM images (2500 ×) of acid hydrolyzed C. auriculatum starch particles at different hydrolysis times: (a) 1 day, (b) 2 days, (c) 3 days, (d) 4 days, (e) 5 days, (f) 6 days, (g) 7 days and (h) 9 days. Fig. 3. XRD patterns of acid hydrolyzed C. auriculatum starch at different hydrolysis times: (a) 1 day, (b) 2 days, (c) 3 days, (d) 4 days, (e) 5 days, (f) 6 days, (g) 7 days and (h) 9 days. Fig. 4. SAXS patterns of acid hydrolyzed C. auriculatum starch at different hydrolysis times: (a) 1 day, (b) 2 days, (c) 3 days, (d) 4 days, (e) 5 days, (f) 6 days, (g) 7 days and (h) 9 days. Fig. 5. ATR-FTIR deconvoluted spectra of acid hydrolyzed C. auriculatum starch at different hydrolysis times: (a) 1 day, (b) 2 days, (c) 3 days, (d) 4 days, (e) 5 days, (f) 6 days, (g) 7 days and (h) 9 days. Fig. 6.
13
C CP/MAS NMR original spectra (A) with enlarged C1 region (B) and
ordered subspectra (C) of acid hydrolyzed C. auriculatum starch at different hydrolysis times: (a) 1 day, (b) 2 days, (c) 3 days, (d) 4 days, (e) 5 days, (f) 6 days, (g) 7 days and (h) 9 days. Fig. 7. The predicted acid hydrolysis process of C. auriculatum starch.
29
Hydrolysis degree (%)
100 80 60 40 20 0 0
3
6
Hydrolysis time (days) Fig. 1.
30
9
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)
Fig. 2.
31
Relative intensity
a b c d e f g h 0
5
10
15
20
25
Diffraction angel (2q )
Fig. 3.
32
30
35
40
h
Relative intensity
g f e d c b a 0.02
0.04
0.06
0.08
0.1
Scattering vector (Å–1)
Fig. 4.
33
0.12
0.14
0.16
1022
995
1045
Relative intensity
a b c d e f g h 1200
1100
1000
900 -1
Wavenumber (cm )
Fig. 5.
34
800
C2, 3, 5
(A)
C6
C1 C4
a b c d e f g h 120
110
100
90
80
70
60
50
Chemical shift (ppm) 101.2
100.2
103.3
(B)
94.7
a b c d e f g 101.8
100.6
99.7
h 110
105
100
Chemical shift (ppm)
35
95
90
C2, 3, 5
(C)
C6 C1 C4
a b c d e f g h 120
110
100
90 80 Chemical shift (ppm)
Fig. 6.
36
70
60
50
Bulk amorphous region
Amorphous growth ring
(1) Amorphous regions were rapidly hydrolyzed Semi-crystalline growth ring
1−4 days Native C. auriculatum starch
Semi-crystalline growth rings were slowly hydrolyzed
(2)
(3) Granules fell into pieces and formed lumps
7−9 days
5−6 days
Fig. 7.
37
Table 1 Effect of acid hydrolysis on the long-range order (relative crystallinity and SAXS parameters), short-range order (ratio of 1045/1022 cm−1 and relative conformation proportions) and amylose content of C. auriculatum starch. Hydrolysis time
Relative
SAXS parametersa
Ratio of
Relative conformation proportion (%)b
Amylose
(day)
crystallinity (%)
Smax (nm–1)
d (nm)
Imax
1045/1022 cm−1
Single helix
Double helix
Amorphous
content (%)
1
26.59
0.682
9.21
77.91
0.563
3.05
30.78
66.17
20.73
2
27.84
0.682
9.21
60.09
0.569
2.42
34.75
62.83
15.48
3
29.47
0.682
9.21
18.17
0.561
1.95
37.04
61.01
13.33
4
31.18
0.682
9.21
8.85
0.569
1.50
40.42
58.08
12.84
5
31.57
−c
−
−
0.575
1.28
41.76
56.96
11.34
6
31.76
−
−
−
0.557
0.96
43.02
56.02
11.09
7
32.06
−
−
−
0.578
0.75
44.31
54.94
10.57
9
32.23
−
−
−
0.569
0.61
44.80
54.59
10.08
a
SAXS parameters of Smax, peak position; d, Bragg spacing (2/Smax); Imax, peak intensity. The relative conformation proportion was calculated from 13C CP/MAS NMR spectrum. c Data were not detected. b
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Table 2 Effect of acid hydrolysis on the RDS, SDS and RS contents in C. auriculatum starch.a Hydrolysis time (day)
RDS (%)
SDS (%)
RS (%)
1
76.20 ± 0.35
8.35 ± 0.21
15.45 ± 0.15
2
76.29 ± 1.42
8.40 ± 0.18
15.31 ± 1.24
3
76.14 ± 3.45
8.44 ± 0.05
15.42 ± 3.41
4
77.94 ± 2.23
8.27 ± 0.08
13.79 ± 2.14
5
77.03 ± 0.61
8.36 ± 0.18
14.61 ± 0.80
6
76.72 ± 1.13
8.49 ± 0.05
14.79 ± 1.08
7
77.48 ± 2.18
8.35 ± 0.11
14.17 ± 2.07
9
76.60 ± 0.54
8.33 ± 0.03
15.07 ± 0.57
a
Data (Mean ± SD) in the same column with different letters were significantly different (p < 0.05).
38