Characterization of amylose and amylopectin fractions separated from potato, banana, corn, and cassava starches

International Journal of Biological Macromolecules 132 (2019) 32–42

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Characterization of amylose and amylopectin fractions separated from potato, banana, corn, and cassava starches Paulo Vitor França Lemos a,⁎, Leandro Santos Barbosa a, Ingrid Graça Ramos a, Rodrigo Estevam Coelho b, Janice Izabel Druzian a a b

Faculty of Pharmacy, Federal University of Bahia, Rua Barão de Jeremoabo, 147, Campus Universitário de Ondina, 40. 170-115, Salvador, BA, Brazil Federal Institute of Bahia, Rua Emídio Santos, SN, 40301-015, Barbalho, BA, Brazil

a r t i c l e

i n f o

Article history: Received 21 December 2018 Received in revised form 13 March 2019 Accepted 13 March 2019 Available online 14 March 2019 Keywords: Amylose-1-butanol Crystallinity Glass transition Thermogravimetry Deconvolution Polymodal distribution of molecular weight

a b s t r a c t Analytical techniques such HPSEC, DSC, and TGA have been employed for amylose determination in starch samples, though spectrophotometry by iodine binding is most commonly used. The vast majority of these techniques require an analytical curve, using amylose and amylopectin standards with physicochemical properties similar to those found in the original starch. The current study aimed to obtain the amylose and amylopectin fractions from potato, banana, corn, and cassava starches, characterize them, and evaluate their behavior via thermogravimetric curves. Blue amylose iodine complex and HPSEC-DRI methods have obtained high purity amylose and amylopectin fractions. All molecular weights of the obtained amylose and amylopectin fractions were similar to those presented in other reports. Different results were obtained by deconvolution of the amylopectin polymodal distribution. All amyloses presented as semi-crystalline V-type polymorphs, while all amylopectin fractions were amorphous. The Tg of all Vamyloses presented were directly proportional to their respective crystalline index. TGA evaluations have shown that selective precipitation of amylose with 1-butanol strongly changes its thermal behavior. Therefore, the separation procedure used was an ineffective pathway for obtaining standards for thermal studies. © 2019 Elsevier B.V. All rights reserved.

1. Introduction Amylose is a glucose polymer that comprises about 20% of normal starches. It is a relatively linear 1,4-α-D-glucan with a small number of long branches. Meanwhile, amylopectin is a 1,4-α-D-glucan containing high-density branches, and is about 5% α-1,6 glycosidic bonds [1,2]. The relative proportion of amylose:amylopectin varies considerably, not only between different types of plants, but also within a single species of plant or plant organ. The growth and development conditions of the plant also affect this relative proportion [2,3]. Several studies have shown that variation in this ratio implies changes in its physicochemical characteristics and interactions with other molecules, resulting in different swelling capacity [4], solubility in water [5], microscopic properties [4], texture and stability of starch products [6,7], and barrier/mechanical properties in starch films [8]. The amylose ratio affects some properties that are particularly useful in the food industry, such as gelatinization, solubility, pasting Abbreviations: S-P, separation procedure; S-A, Sigma-Aldrich; Mw, molecular weight. ⁎ Corresponding author. E-mail addresses: [email protected] (P.V.F. Lemos), [email protected] (J.I. Druzian).

https://doi.org/10.1016/j.ijbiomac.2019.03.086 0141-8130/© 2019 Elsevier B.V. All rights reserved.

characteristics, and texture. This therefore makes amylose content an important quality parameter for starch-based products [9]. Spectrophotometry is the method most frequently used to determine the amylose ratio of starches. This method is described in ISO (International Organization for Standardization) textbooks as being for the determination of amylose in milled, semi-milled, and parboiled rice (ISO 6647-1/2:2007 [10,11]). Some drawbacks are cited in the method described by ISO, such as amylose overestimation due to iodine's capability of also binding to amylopectin, even in small quantities; use of reagents; sample treatment requirement (scattering processes, gelatinization, and reaction); and being time consuming [12,13]. Moreover, experimental evidence using the iodine-binding method has shown that each different starch source requires the construction of an analytical curve. This means that measuring starches from different sources is usually difficult [9]. High purity amyloses are not widely available for use as standards, and commercially available amyloses are generally inconsistent in quality [14]. This is a recurring problem for those attempting to use these methods for amylose analysis. Enzymatic [2,15,16], chromatographic [12,13,17], and calorimetric [18–20] techniques have also been reported in the literature for determining the amylose ratio. However, this generally requires sample preparation or involves high costs.

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Thermogravimetry (TGA) was successfully applied for amylose ratio determination. Developed by Stawski (2008) [2], this method was successfully implemented in rice, potato, and wheat starch samples. This technique had the advantage of not including sample-processing steps, being fast, and being readily adaptable to routine analysis. However, as demonstrated in our previous work, the thermal behavior of native starches is related to the amylose ratio (major component), the structural organization of granules, and the presence of minor compounds, such as organic and inorganic phosphorous and lipids [21]. This, therefore, confirms that when using the TG method, standards of amylose and amylopectin from the same source of the analyzed starch must be used. Schoch, 1942 [22], described starch separation for the first time. Some authors [23–25] provided alternative paths and optimized this method, considering the purity of the amylose and amylopectin fractions obtained. High purity starch fractions are obtained by precipitating the amylose fraction of amylopectin with guest molecules. As only the former is able to make inclusion complexes, its solubility is reduced in an aqueous dispersion. Therefore, the insoluble amylose-guest molecule is recovered as a precipitate. Some guest molecules, such as methanol, 1-butanol, and pentasol, have been employed to precipitate free amyloses from aqueous dispersions. However, the thermal features of these specimens were not well characterized for thermoanalytical purposes, such as for the amylose determination method by TGA. Therefore, the present study aimed to obtain the amylose and amylopectin fractions from potato, banana, corn, and cassava starches, characterize these fractions, and evaluate their behavior via thermogravimetric curves.

2. Material and methods 2.1. Starch sample Commercial samples of potato (Solanum tuberosum) and corn (Zea mays) starches were employed. The cassava (Manihot esculenta) and banana (musa spp.) starches were provided by Embrapa - Cassava and Fruits, Cruz das Almas, BA. For comparison, potato amylose, maize, and potato amylopectin obtained from Sigma-Aldrich (S-A) were used. The other reagents used were analytical grade.

2.2. Starch separation method Based on the Montgomery & Senti (1958) [24] starch separation method, starch was initially pretreated for the extraction of fat and to facilitate starch extraction using only water. For this, the starch (5%) was added to a solution of 70% glycerol. The resulting suspension was stirred slowly while heating to 89 °C at a rate of 0.8 °C min−1, with N2 bubbling. The temperature was maintained for 1 h. Following this, the suspension was cooled, filtered, and washed with ethanol until the pretreatment solvent was fully removed. The pretreated starch aqueous solution (2%) was adjusted to pH 6.0–6.3 using phosphate buffer, stirred at 200 rpm, and heated to 98 °C. The solution was then cooled using an ice bath (0–2 °C), and subjected to centrifugation (42.200 ×g) for 1 h. The resulting supernatant, consisting of amylose produced by the separation procedure, was centrifuged until there was no precipitate. This supernatant was named AmL-SP. The resulting solid fractions were named Amp-SP. These were collected, mixed, lyophilized, and consisted of amylopectin produced by the separation procedure. Supernatants were mixed, combined with 1-butanol (1:3 v/v), and maintained at rest for 8 h. Then, the solution was centrifuged (4.689 ×g) for 10 min. The obtained solid fraction was lyophilized. This sample consisted of amylose.

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2.3. Scanning electron micrography (SEM) The micrographs of dried separated amylose and amylopectin fractions were obtained using a Tescan – Vega Lmu field emission scanning electron microscope (Oxford Instruments X-Act). A total of images of gold-coated samples were obtained at 15 kV, and the morphologies were evaluated at 1200 × magnification. 2.4. X-ray diffraction (XRD) The XRD patterns of the starch fractions were obtained using an Xray diffractometer Shimadzu XRD-6000 model, with a graphite monochromator operating with CuK radiation (λ = 1.5405 Å) at 40 kV, 30 mA, 0.02 (slit). The crystalline indices related to alpha quartz (%CI) of the samples were taken using the software OriginPro 8.1. This was done by determining the area under the peaks in the range of 3 and 30 2ϴ, the region of higher concentration wherein peaks of starch samples are commonly observed [26]. The CI was obtained as described by Wakelin, Virgin & Crystal (1959) [27], considering the amorphous starches to be the 100% amorphous area, and the alpha quartz as 100% crystalline material [28]. This was done following Eq. (1): %CI ¼ ðΣjIs −Ia j  ΣjIc −Ia jÞ  100

ð1Þ

in which: |Is − Ia| = absolute difference between the sample [Is] and amorphous [Ia] intensities; and |Ic − Ia| = absolute difference between the crystalline (quartz) [Ic] and amorphous [Ia] intensities. In order to treat native potato, banana, corn, and cassava starches to obtain amorphous samples, the method described by Ratnayake & Jackson, 2006 [29] was employed. Six-gram aliquots of each starch source were separately stirred in a boiling bath, containing 100 mL of distilled water, for 30 min. They were then cooled to −80 °C (Coldlab CL58086V) and freeze-dried (Liotop 148 L101; −43 °C) until dry powder was obtained. 2.5. Absorption spectroscopy The characterization of samples by molecular absorption spectroscopy was carried out based on the method proposed by Zhu, Jackson, Wehling & Geera (2008) [9], with some adaptations. Samples containing 200 mg of mass, previously dried at 40 °C for 24 h, were transferred to a 100 mL volumetric flask, followed by an addition of 1 mL of ethanol and 10 mL of NaOH 1 N. After the solution was kept at rest for 1 h, the volumetric flask was completed with distilled water. An aliquot (2 mL) was transferred to a 100 mL volumetric flask, and 50 mL of distilled water was added. This solution was neutralized with 0.1 N HCl using a pH indicator strip. Then, 2 mL of 2% (m/v) iodine/iodide solution was added to the neutral solution, and the volume was completed with distilled water. The final solution was kept at rest for 30 min for color development and subsequently measured on a Shimadzu spectrophotometer UV–Vis Model 1650 PC. This was done in quartz cuvettes, with an optical path of 1 cm and in a wavelength range of 190–1100 nm. 2.6. High performance size exclusion chromatography coupled to differential refractive index (HPSEC-DRI) The method determined by Charles, Chang, Ko, Sriroth & Huang (2005) [30] HPSEC was used for the analysis of amylose and amylopectin samples, with some adaptations. The 75 mg samples were dispersed in 15 mL of DMSO. The suspension was stirred and heated to boiling in a water bath for 1 h, and then stirred for 24 h at about 25 °C. After stirring, the solution was heated again, filtered through a nylon membrane (0.2 μm), and 100 μL was injected in liquid chromatograph.

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The purity of the amylose and amylopectin fractions was monitored using an HPSEC-DRI system, consisting of Perkin Elmer Series 200 with GPC columns Shodex SB-OH 803, 804, 805, 806 connected in series (in this order). The temperature of these columns was maintained at 55 °C. For the analytical run, new ultra-pure filtered water was used as the mobile phase, with a flow rate of 1 mL min−1. Before chromatographic injection of samples obtained by the Montgomery & Senti (1958) [24] separation procedure, the potato amylose, amylopectin, and maize amylopectin (obtained from Sigma-Aldrich) were analyzed. These starch fractions were injected to assign the amylose and amylopectin peaks based on retention time, and integrated to obtain the normalized areas using TotalChrom® software version 6.3. The molecular weights (Mw) of the amylose and amylopectin fractions were estimated by constructing an analytical curve. Ten dextran's standards (American Polymer Standards, with the following molecular weights: 1.02 × 105; 2.07 × 105; 4.31 × 105; 6.55 × 105; 7.59 × 105; 1.36 × 106; 2.02 × 106; 2.80 × 106; 3.4 × 106; 5.9 × 106 (Da)) were prepared in the same way as the starch fractions and injected. The analytical curve was successfully obtained, and the logarithm of the standards' molecular weight and the average retention time of each triplicate (SD b 0.05) were highly correlated by a linear fit (r2 = 0.9904). The deconvolution of the potato, banana, corn, and cassava amylopectin fractions were performed using OriginPro 2018 until the maximum iterations by the software, assuming the Gaussian, Lorentzian, Voigt, and Log-Normal shapes. The resulting peaks were named fractions FI, FII, FIII, and FIV. In order to evaluate the heterogeneity of the distribution of FI-FIV obtained by each deconvolution, the variation coefficient (%CV) was calculated following Eq. (2): %CV ¼ SDabsolute areas  Aabsolute areas  100

ð2Þ

in which SD = standard deviation area, and A = average of the absolute area. The peaks assigned with “a” were integrated and included in normalizations without the fittings (well-resolved peaks). The starch separation procedure was also performed without employing 1-butanol as a precipitation agent, in order to obtain pure potato amylose. This fraction was injected and collected based on the retention time of potato amylose (S-A). In order to ensure the efficiency of this procedure, the color change of an aliquot of 2% iodide-iodide solution (as employed in the spectrophotometric method) was monitored, while the mobile phase dropped from the instrument's peek tube. Some injections were performed until about 5 mg of the sample was obtained. The resulting solution (mostly water) was freeze-dried, and the pure potato amylose extracted without 1-butanol was recovered. 2.7. Differential scanning calorimetry (DSC) Experiments were conducted on a differential scanning calorimeter (TA-60, Shimadzu Corp.), previously calibrated, to detect the incremental change in heat capacity Cp associated with glass transition (Tg). Samples (20 mg) were stored in a vial containing 2 μL of distilled water and carefully homogenized. They were kept for 72 h until completely moistened, as suggested by Yu (2001) [31], and as performed in previous studies [21]. The moistened samples (2 mg) were sealed in aluminum pans and heated at 15 °C min−1 from 25 to 270 °C. The first scan was performed to erase the thermal memory of the amylose and amylopectin, obtained in the starch separation procedure. After cooling, a second scan was performed with the same heating rate, from −50–270 °C. All scans were performed at a flow of 50 mL min−1 (N2). To evaluate the melting endotherm of the amylose and amylopectin fractions, the method of Carmona-García, Aguirre-Cruz, Yee-Madeira & Bello-Pérez, 2009 [32] was employed, with an adaptation on the amount of water used. Two milligrams of each sample was placed in aluminum pans, followed by the addition of 10 μL of distilled water.

The DSC pan was sealed and kept at rest for 1 h, to complete the hydration of polysaccharides. The hydrated samples were heated in the calorimeter from 30 to 100 °C at a heating rate of 10 °C min−1. The DSC parameters were as follows: the onset temperature (To); the peak temperature (Tp); the conclusion (Tc); and the melting enthalpy (ΔHgel). These values of the endotherms were obtained by TA-60® software. All scans were performed at a flow of 50 mL min−1 (N2). 2.8. Thermogravimetry (TG) The measurements were obtained using a Perkin Elmer thermogravimetric analyzer, Pyris1 TGA, previously calibrated. Based on the Stawski (2008) [2] method, with some modifications, a platinum crucible was used under the following conditions: sample mass of approximately 5 mg, N2 flow rate of 40 mL min−1, heating rate 15 °C min−1, and temperature range of 25–600 °C. The moisture of the samples was calculated by Pyris1 TGA® software. This was done by measuring the mass lost during the first thermal event of each TG curve (25–200 °C). Measurements related to degradation events as Tonset DTG, T50%, and Tpeak DTG were calculated by the same software. T50% represents the temperature at 50% of mass loss. The slopes were calculated using the Tonset DTG (left limit) to T50% (right limit), both seen on the TG curves. 3. Results and discussion 3.1. Separation efficiency of potato, banana, corn, and cassava starches to obtain amylose and amylopectin fractions of each source The Sigma-Aldrich (S-A) amylose and amylopectin and the fractions obtained by the starch separation procedure (S-P) were submitted to iodine binding and evaluated by absorption spectroscopy at the maximum absorbance values (λmax). This was done to evaluate the efficiency of the starch separation method. The results are presented in Table 1. Unsurprisingly, the absorbance values of potato, banana, corn, and cassava amyloses (between 0.525 and 0.764 (dimensionless unit)) were higher than those of the amylopectin samples obtained from the same starch sources (between 0.068 and 0.168 (dimensionless unit)). This was because only the amylose fraction forms inclusion complexes with iodine in aqueous solutions, changing the color to dark purple. The HPSEC-DRI was used as complementary technique. In Fig. 1(a), (b), (c), and (d), the peaks around 20 and 32 min were attributed to the amylopectin and amylose fractions, respectively. Only one expressive peak can be observed in the chromatograms of amylopectin samples at around 20 min, indicating high amylopectin content with some amylose remaining. However, a small amyloserelated peak was observed, justifying the displacement of lambda max from 570 to 620 nm. The normalized areas of separated amylopectin samples show N90% purity (Table 1). Peaks around 20 min were also noticeable on the chromatograms of amylose samples. Although these peaks were observed in the amylopectin range of the retention time, these likely refer to the intermediate material. This consists of branched molecules with molecular weights lower than that of amylopectin, eluted between the amylose and amylopectin fractions [33]. The purities of potato, banana, maize, and cassava amyloses by normalized areas were higher than 85% (Table 1). Fig. 1(c) shows that maize amylose was less contaminated with branched material compared with those obtained from other sources. This could be justified by the higher crystallinity of 1-butanol complexes formed during selective precipitation in the separation procedure. It has already been established that amylose forming molecular inclusion complexes with 1-butanol makes it insoluble. Consequently, it then precipitates from the aqueous supernatant. A purer fraction of amylose should therefore be obtained from more insoluble amyloses. Starch chain solubility is related to the degree of crystallinity and amylose

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Table 1 Efficiency of starch separation procedure by HPSEC-DRI based on normalized areas and the absorbance values obtained from molecular absorption spectroscopy at λmax. Starch

Potato

Banana Maize Cassava

Starch fraction

Normalized areas evaluated by HPSEC-DRI (%)

LogMw (Da)

Absorbance values (dimensionless unit)

λmax (nm)

86.5 65.9 94.8 94.0 95.5 94.7 97.2 91.2 99.9 85.3 97.8

5.88 4.45 8.49 8.11 5.71 8.23 5.40 8.25 8.26 5.96 9.04

0.579 0.525 0.133 0.053 0.658 0.123 0.764 0.168 0.021 0.535 0.068

646 646 620 570 646 620 646 620 570 646 620

Amylose (S-P) Amylose (S-A) Amylopectin (S·P) Amylopectin (S-A) Amylose (S-P) Amylopectin (S-P) Amylose (S-P) Amylopectin (S-P) Amylopectin (S-A) Amylose (S-P) Amylopectin (S-P)

S-A: obtained from Sigma-Aldrich. S-P: obtained by the starch separation procedure.

content [34]. The purities of amylose-1-butanol complexes are discussed in more detail in Sections 3.3 and 3.4.2. The normalized area and absorbance values obtained from the amylose and amylopectin samples were correlated. A high coefficient was found for both fractions, according to the following decreasing order for amylose: corn N banana N potato N cassava (r = 0.9394); and amylopectin: cassava N potato ≥ banana N corn (r = −0.9748). Thus, HPSEC-DRI and spectrophotometric evaluations show that the starch separation procedure provided high purity amylopectin and amylose

fractions of potato, banana, corn, and cassava. However, these fractions were not free of branched material. Both amylose and amylopectin separated from potato starch had higher purity than those obtained from Sigma-Aldrich. In contrast, maize amylopectin separated from corn starch had less purity (91.2%), when compared to that obtained from Sigma-Aldrich (99.0%). All data are presented in Table 1. These differences in sample purities were attributed to the starch separation methods, which were mainly related to bath temperature, ultracentrifugation speed, and the precipitant employed.

Fig. 1. Chromatographic (HPSEC-DRI) profiles of amylose and amylopectin samples.

P.V.F. Lemos et al. / International Journal of Biological Macromolecules 132 (2019) 32–42

3.2. Molecular weight evaluations of amylose and amylopectin fractions The molecular weights (Mw) of the separated fractions and those obtained from Sigma-Aldrich were estimated and expressed as the logarithm of Mw (Table 1). In order to evaluate the molecular weights of the separated amylose and amylopectin fractions, HPSEC-DRI was employed. All comparisons of Mw were related to the majority peak of each sample. The peaks at around 20 min (corresponding to branched material) appeared to be co-eluted (Fig. 1(a), (b), (c) and (d)). Chen & Bergman, 2007 [13] reported problems related to the accuracy of HPSEC-DRI molecular weight determinations when performing column calibration. Despite this, the Mw values of the potato [35,36], banana (no comparable data) corn [35,37], and cassava [35,36] amyloses obtained in the present work were comparable to those obtained in other reports, which employed MALLS or LALLS detectors. The measurements ranged 2.5–9.1 × 105 Da, in the decreasing order of cassava N potato N banana N corn. The potato amylose from Sigma-Aldrich (2.8 × 104 Da) presented with a smaller value than the separated amyloses (Table 1). Even when employing a large extrapolation of the analytical curve (at about 103 Da), the Mw measurements obtained for the potato [38], banana [38,39], corn [38,39], and cassava [38] amylopectin fractions were comparable to those obtained in other reports, which employed the MALLS detectors (Fig. 1; Table 1). The measurements of the present work ranged from 2.0 × 108 –2.0 × 10 9 (Da) and followed the decreasing order of cassava N potato N maize N banana. The potato and maize amylopectin from Sigma-Aldrich (1.3 and 1.8 × 108 Da) had values close to that of the amylopectin fractions separated from the same source.

3.3. Evaluation of amylopectin polymodal distribution The fine structure of amylopectin has already been reported by Hizukuri, 1985 [40] and Hizukuri, 1986 [41]. It has been described as a polymodal distribution of the amylopectin fractions of a variety of starches. Arbitrary boundaries were used for the peaks due to overlapping. A few years later, Ong and colleagues, 1994 [35] improved the HPSEC system previously proposed by Hizukuri, 1986 [41], employing five size exclusion columns and deconvolution using a Gaussian function. These improvements allowed better-resolved chromatograms for evaluating the occurrence of at least five amylopectin chain lengths in debranched starches: A (shortest), B1, B2, B3, and B4 (longest). Several authors [30,33,42,43] demonstrated the amylopectin fine structure by HPSEC techniques. These authors agreed that differences in the fine structure results in the starch granules having different chemical and physical properties, such as paste viscosity and retrogradation properties. In previous studies, pullulanase and isoamylase were employed to debranch native starches. The debranching enzymes specifically hydrolyze α-(1,6)-D-glycosidic inter-chain linkages, but have no action on the major α-(1,4)-D-glycosidic linkages. Due to this hydrolysis, debranched amylopectin HPSEC elutions presented greater amounts of small chains [30,35,40–42]. In the present paper, the amylopectin cluster was fractionated without enzymatic debranching. The repeated aqueous heating (100 °C) and continuous mechanical stirring (200 rpm) in the starch separation procedure were enough for this fractionation. The amylopectin chromatographic profiles (Fig. 1) tended to decrease from high to low molecular weights. This is in contrast to enzymatic debranching, which typically display chromatographic profiles that increase from high to low molecular weights. Interestingly, the same trend was previously reported in GPC analysis of native and debranched maize starches [33].

Some authors have employed Gaussian peak deconvolution, not only in starch structural studies, but also for other polysaccharides. As these compounds are all biological derivatives, this appears to be appropriate [35]. For the first time, the polymodal distribution of amylopectin was investigated by peak deconvolution, employing the Lorentzian, Voigt, and Log-Normal algorithms. This was done in addition to the Gaussian algorithm. Both the Gaussian and Lorentzian algorithms present symmetrical shapes [44,45], and the Voigt algorithm presented a combination of both [46,47]. On the other hand, the Log-Normal algorithm (asymmetric) has been used quite often to represent chromatographic, and sometimes spectroscopic, peaks [44]. Considering the main applications of these algorithms [44] and the most common peak shapes of the chromatographic data [45], these four functions were chosen to investigate the amylopectin polymodal distribution. Fig. 2 represents a typical peak deconvolution and fitting performed on cassava amylopectin. All amylopectin fractions have presented four distributions of different molecular sizes assigned as FI-IV (Table 2), except for banana (only three distributions were detected). The Mw evaluations estimated by the Gaussian, Lorentzian, Voigt, and Log-Normal algorithms did not display expressive differences between them (Table 2). As displayed, all Mw ranged from 107 to 109 Da, which is in agreement with previous literature. On the other hand, some differences were noticed on the polymodal distribution of amylopectin obtained by the algorithms, measured as %CV (Table 2). Considering the Gaussian and Lorentzian functions, potato and cassava amylopectin had the highest molecular sizes (FI) and were the most abundant (Fig. 1 (a, d) - Supplementary Material). This is in contrast to banana and maize, which had higher amounts of FII and FIV (Fig. 1 (b, c) – Supplementary Material), respectively. Accordingly to the Voigt fitting, banana and cassava had higher amounts of FI, which was the most abundant (Fig. 1 (b, d) - Supplementary Material). In contrast, potato (FII) and maize (FIV) had lower amounts present (Fig. 1 (a, c) – Supplementary Material). When considering the Log-Normal algorithm, FII was more abundant in potato and banana (Fig. 1 (a, b) - Supplementary Material), in contrast to corn (FIV) and cassava (FI) (Fig. 1 (c, d) – Supplementary Material). Therefore, it is clear that these four different algorithms influence the proportion of the amylopectin polymodal distribution. As seen in Fig. 1(a–d), the intermediate material eluted in the FIV region was also observed in amylose samples. This occurs because the molecular weight of fraction IV is similar to that of amylose [33]. However, coherent correlations between the FIV fractions and the amylose purities were not detected.

FI

Cassava amylopectin Fit (Voigt)

Intensity (

36

FII FIII

15

17

18

20

FIV

22

23

Time (minutes) Fig. 2. Typical deconvolution and fitting of cassava amylopectin by a Voigt function.

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Table 2 Polymodal distributions of amylopectin (S-P) and molecular weights (Mw) obtained by different deconvolution algorithms. Amylopectin

Algorithm of deconvolution Gaussian

Lorentzian

LogMw (Da)

Potato Banana Maize Cassava

Peak Area

FI

FII

FIII

FIV

9.10 8.32 8.37 9.30

8.66 8.16 8.17 8.73

8.45 7.87 7.98 8.47

7.64a n.d 7.03a 7.84

%CV 62.39 42.41 68.42 144.74

Voigt

LogMw (Da)

Peak Area

FI

FII

FIII

FIV

9.13 8.29 8.39 9.30

8.66 8.13 8.22 8.58

8.46 7.72 7.95 7.86

7.64a n.d 7.03a 6.97

%CV 134.59 26.69 47.03 98.23

Log-Normal

LogMw (Da)

Peak Area

FI

FII

FIII

FIV

8.23 8.23 8.34 9.28

7.99 7.99 8.20 8.63

7.78 7.78 7.84 8.24

7.64a n.d 7.03a 7.84

%CV 59.85 86.77 81.50 158.57

LogMw (Da)

Peak Area

FI

FII

FIII

FIV

9.42 8.23 8.36 9.30

8.86 7.78 8.13 8.64

8.50 7.59 7.75 8.27

7.64a n.d 7.03a 8.02

%CV 66.86 59.86 45.64 134.55

F (I,II,III and IV): Amylopectin fractions I, II, III and IV. a Data obtained without fitting; n.d.: not detected.

3.4. Characterization of amylose and amylopectin fractions obtained by Montgomery & Senti (1958) starch separation procedure 3.4.1. X-ray diffraction (XRD) The molecular association of amylose with ligands, under suitable conditions, can produce crystalline structures [48]. The guest molecule is thought to occupy the central axis of a helix consisting of 6, 7, or 8 glucosyl residues per turn, with a repeat spacing (pitch) of 0.8 nm [49]. The complexes have been extensively studied by XRD and crystallography, and give rise to the V-pattern of amylose [49]. As previously reported, the V-amylose polymorph presents a relatively hydrophobic helix interior, which plays a major role in stabilizing inclusion complexes with organic molecules [50].

The diffractions (2θ) of peaks in potato amylose (S-P) were 8.0°, 12.0°, and 19.8°. In contrast, amylose (S-A) was amorphous (Fig. 3(a)). An amylose semi-crystalline structure was also found for the banana, maize, and cassava amyloses (S-P). These results suggest that the starch separation procedure yielded amylose inclusion complexes with 1butanol, the precipitant employed. The peaks found were typical of the V-type crystal structure of amylose with small molecular guests [51–53]. The crystalline index of amylose-1-butanol complexes decreased in the following order: maize N banana N potato N cassava (Table 3). Interestingly, the %CI calculated presented were directly proportional and highly correlated to their respective purities by linear fit (r = 0.9893), evaluated using HPSEC (Table 1). These findings are in accordance with the separation procedure, performed by selective precipitation with 1-butanol. As the degree of order of amylose-1-butanol complexes increases, their water solubility decreases. The lower solubility observed in more ordered polymers is related to their conformation and configuration [54]. Caira, 1998 [55] studied the polymorphism of organic compounds, and reported that crystalline compounds are less soluble than amorphous compounds. This can be explained by the tendency of amylose to form crystalline regions, utilizing extensive intra-helix hydrophobic interactions with 1-butanol, in addition to forming intramolecular and intermolecular hydrogen bonds. As the crystalline state has a lower energy than the amorphous one, its dissolution is more difficult [56]. Fig. 3(b) displays the amylopectin fraction from Sigma-Aldrich, obtained by the starch separation procedure. The maize amylopectin presented peaks on 11.2°, 15.1°, 17.0°, 18.0°, and 22.8° (2θ), and had a similar native granule structure to corn starch, as previously studied [21]. This is likely a waxy maize variety. This sample presented the highest %CI (74.8%). On other hand, the potato amylopectin (S-A) and those obtained by starch separation (S-P) lost their native crystalline profiles (Fig. 3(b)) beyond the native granular structures, as observed by SEM techniques. Though they are not to scale (Fig. 3(b)), there are some low intensity peaks observed at around 17.0° (2θ). These were mainly associated with the remainders of their respective amyloses after starch separation, resulting in retrogradation. 3.4.2. Differential scanning calorimetry (DSC) It is well established that the molecular motion of polymeric chains is “frozen” in amorphous polymers at low temperatures, thus rendering Table 3 Crystalline index and peaks of amylose-1-butanol complexes. Amylose-1-butanol

Fig. 3. XRD of (a) amylose and (b) amylopectin fractions.

Potato Banana Maize Cassava

Crystalline index (%)

Peaks (2θ degrees)

17.9 43.5 56.5 11.4

8.0;12.9;19.8 7.5;13.5;19.7 7.4;13.0;19.6 7.5;12.9;19.8

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them in the immobile glassy state. When sufficient heat is applied, molecular motion is initiated, and the polymeric chains have sufficient energy to slide past one another. The polymer therefore becomes rubbery and flexible. These changes refer to glass transition (Tg) [57], which was extensively discussed at the end of last century. This is important for understanding the physical state and physicochemical properties of polymeric materials [58]. All semi-crystalline amylose-1-butanol complexes (S-P) presented with changes in heat capacity associated with the Tg (Fig. 4(a) and Table 4). The %CI and Tg decreased in the following order: maize N banana N potato N cassava. The decreases were directly proportional and high correlated (r = 0.9229). These results were in accordance with other reports [57,59] that evidenced a Tg increase accompanied by an increase in %CI, mainly due to chain end effects, as well as branching point flexibility related to higher conformational entropy [59]. In other words, the more ordered structures presented less chain mobility, and consequently, higher Tg. The potato amylose S-P had a large shift (11.8 °C) when compared to the result obtained from S-A. These discrepancies were attributed to the sample purities, the presence of branched material, and crystallinity. The Tg values of amylose-1-butanol complexes were correlated with LogMw, and an inversely proportional relationship was detected (r = −0.9578). This result is not in consensus with the observed decreases in chain mobility when the molecular weight increases [59,60]. However, this may be justified by the presence of small molecules, such as water and 1-butanol, housed between the polymer chains and thus

Fig. 4. Glass transition (Tg) temperature evaluations of (a) amyloses and (b) amylopectin fractions obtained by the separation procedure.

moving them away from each other (plastifying effect) [59]. The amount of moisture (%) detected in dry samples (Table 4) reinforces this possibility, as the Tg increased with decreasing moisture. The Tg of the amylopectin fractions (S-P) decreased in the following order: banana N cassava N maize N potato (Fig. 4(b) and Table 4). The Tg of the amylopectin fractions (S-P) was not correlated with crystallinity, as all fractions presented as amorphous. The Tg measurements could also not be correlated with their respective Mw, suggesting that other factors, such as branching points [59] and the polymodal distribution, influenced these results. When heated with high moisture content (N50%), the starch (semi crystalline) polymers tend to absorb water and swell. Water uptake by the amorphous regions destabilizes their crystalline structures. Upon continuously heating all crystallites, which are mostly related to amylopectin structures, these structures tend to melt. This results in high molecular motion between polymeric chains, allowing the separation of the amylose and amylopectin fractions [29,61]. This phenomenon is observed by DSC techniques and measured with endothermic peaks. The absence of the melting endotherm of the potato, banana, maize, and cassava amylose-1-butanol complexes is presented in Fig. 2 – supplementary material and Table 4. These data show that melting of the crystalline portions did not occur in this temperature range (30–100 °C). This was likely due to the strong hydrophobic interactions between the ligand and the helix, thus blocking water inclusion. This is in accordance with similar findings in other reports [62,63]. Le Bail, Rondeau & Buleón, 2005 [64] noticed two endotherms at 66.5 and 80 °C for the potato amylose-1-butanol complex. These were attributed to the different methods used for obtaining inclusion complexes, employed by the aforementioned authors and the Montgomery & Senti (1958) [24] starch separation procedure. More specifically, these were attributed to the temperature conditions. The melting endotherms of the amylopectin fractions (S-P) were also not demonstrated (Fig. 5; Table 4). This was attributed to the successive dispersions of these samples, done in boiling water with continuous agitation during the starch separation procedure. Thus, the crystalline domains, composed by amylopectin crystals, were lost. The loss of the native crystalline profile is supported by both the previously described XRD data (Section 3.4.1) and other reports [29,61]. The maize amylopectin (S-A) presented an endothermic peak associated with melting, while potato amylopectin (S-A) did not. This occurred because potato amylopectin had an amorphous structure. The melting temperature and enthalpy were quite similar to that of corn starch [21,65]. The broadening peak of the melting endotherms (Tc-To) can be considered a measurement that indicates the heterogeneity (size and degrees of perfection) of amylopectin crystallites [66]. Furthermore, the melting endotherm refers to the melting of crystalline regions [21]. Measurements of Tc -T o (the temperatures of conclusion and onset of starch gelatinization endotherms) have already been studied previously, with the same instrumental conditions as the present work. Measurements of 8.3, 9.8, 9.5, and 13.2 °C were obtained for potato, banana, corn, and cassava native starches, respectively [21]. These data were directly proportional and high correlated with the measurements of the amylopectin polymodal distribution, obtained by Gaussian, Voigt, and Log-Normal algorithms. In the present paper, this was described as %CV (Fig. 6). The normalized variation (%CV) represents an absolute value obtained in order to evaluate variations in FI-FIV proportions, obtained by deconvolution. Surprisingly, the Voigt algorithm (r = 0.9992) gave a higher correlation value than the other algorithms. These data show that variations in the amylopectin polymodal distribution, measured by %CV, are directly proportional to heterogeneity of crystallites' sizes and shapes. This was assessed by DSC techniques and measured as Tc-To (°C). Therefore, the more heterogeneous the amylopectin polymodal distribution, the broader the starch gelatinization endotherms tended to be.

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39

Table 4 Moisture, T50%, Tonset DTG, Tpeak DTG, Tg, Tp and ΔHgel of amyloses and amylopectin fractions. Starch

Potato

Banana Maize Cassava

Fraction

Aml–S-A Aml–S-P Aml–S-P (No butanol) Amp–S-A Amp–S-P Aml–S-P Amp –S-P Aml–S-P Amp –S-P Amp–S-A Aml–S-P Amp–S-P

Moisture (%) Dry

Hydr.

13.81 7.58 9.70 9.97 7.45 8.63 8.35 8.88 7.51 7.66 8.56 8.51

22.6 16.5 – 18.8 16.4 17.5 17.3 16.3 16.3 16.5 16.0 17.3

T50% (°C)

Tonset DTG (°C)

Tpeak DTG (°C)

Slope (% °C−1)

Tg (°C)

Tp (°C)

ΔHgel (J/g)

313.2 355.8 358.9 353.6 353.3 359.4 362.7 353.4 362.5 364.9 362.2 360.2

224.6 311.8 288.6 296.7 313.1 316.1 309.5 313.6 311.6 310.6 306.0 311.0

302.8 351.2 363.2 351.2 345.5 349.3 346.8 356.1 368.6 364.2 349.0 348.9

−0.5 −1.1 −0.4 −1.0 −1.2 −1.2 −1.1 −1.3 −1.4 −1.3 −1.2 −1.1

36.4 24.6 – 31.0 17.3 26.2 35.2 32.1 31.7 21.5 19.3 34.5

n.d n.d n.d n.d n.d n.d n.d n.d n.d 70.1 n.d n.d

n.d n.d n.d n.d n.d n.d n.d n.d n.d 9.7 n.d n.d

Hydr.: Samples hydrated as described in method session. Aml-S-A: Amylose obtained from Sigma-Aldrich. Aml-S·P: Amylose obtained by the starch separation procedure. Amp-S·P: Amylopectin obtained by the starch separation procedure. n.d: not detected.

3.4.3. Thermogravimetry (TGA) In order to observe the thermal differences between the separated starch fractions and verify the feasibility of the thermogravimetric method proposed by Stawski (2008) [2], the potato, banana, corn, and cassava amyloses (precipitated with 1-butanol) and their respective amylopectin fractions were scanned by TGA. The profiles are presented in Fig. 7, and the measurements for Tonset DTG, T50%, Tpeak DTG, and the slopes of the TGA curves are presented in Table 4. For the construction of the analytical curve proposed by Stawski (2008) [2], amylose and amylopectin were used for the solid binary mixtures. However, particle size standardization was necessary in order to avoid this parameter influencing TG curves [67,68]. The first thermal event (30–200 °C, Fig. 7) of all the samples corresponds largely to the evaporation of water molecules and some remaining slightly bonded 1-butanol. This is due to 1-butanol volatilization, which occurs around 100 °C [69]. This thermal event ranged from 7.45 to 9.70%, considering the separated samples after freeze-drying. The second thermal event can be attributed to the thermal decomposition of the studied samples, which started above 200 °C. Considering the separated amyloses, this event has been associated with dissociation of the amylose chains at high temperatures, thereby leading to breakage of the structure and the quick volatilization of 1butanol [70]. The decomposition mechanism of all of the studied

samples involved the elimination of polyhydroxyl groups, chain depolymerization, and decomposition, yielding carbonyl compounds (likely aldehydes), CO, CO2, CH4, C2H4, and C2H2O [71–73]. All amylose fractions obtained by starch separation had similar profiles and temperatures, related to the thermal decomposition of their respective amylopectin. In the potato starch fractions, it was found that the amylose S-A had a difference in T50% at about 42 °C when compared to amylose S-P (Fig. 7(a), Table 4). For amylopectin, this difference was only 1 °C (Fig. 7(a), Table 4). The behavior was similar to maize amylopectin, presenting no expressive difference in T50%. The amylose and amylopectin fractions derived from banana and cassava starches had no differences in T50% (Table 4). The maize amylose and amylopectin S-P showed a difference of 9.1 °C (Table 4). The data presented in Table 4 show that the fractions obtained by starch separation had very close values at T50%, making the construction of the analytical curve proposed by Stawski (2008) unfeasible [2]. In order to elucidate the similarities between amylose and amylopectin thermal decomposition temperatures, obtained by the method of Montgomery & Senti (1958) [24], starch separation was performed to separate potato starch without using 1-butanol as the precipitation agent. As seen in Fig. 7(b), the resulting amylose obtained in the absence of 1-butanol presented a different TG profile and different measurements when compared to the potato amylose-1-butanol complex. This

Fig. 5. Melting endotherm evaluations of separated amylopectin fractions (S-P) and from Sigma-Aldrich (S-A).

Fig. 6. Correlation between the broadening peak of the native starches gelatinization endotherm and %CV obtained by deconvolution of amylopectin (S-P) molecular weights.

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DTG and slope (5.7 °C; 0.2% °C−1), respectively. The amorphous maize amylopectin (S-P), had differences of LogMw (0.01 Da), Tpeak DTG and slope (4.4 °C; 0.1% °C−1), respectively, when compared to semicrystalline S-A. As the potato amylopectin had higher differences in Mw and both were amorphous, these results suggest that Mw may determine the decomposition kinetics of amylopectin when compared to crystallinity (Table 1 and Table 4). Considering both amylose-1-butanol complexes and amylopectin fractions, all findings related to the influence of different molecular sizes and crystallinity on thermal behavior were in agreement with the results obtained in previous studies [21]. 4. Conclusions

Fig. 7. TG profile of amyloses and amylopectin fractions.

molecular inclusion complex increased the thermal stability of amylose. This occurred due to 1-butanol inducing stronger molecular interactions. Therefore, higher energy is required to break up the molecular linkage. These findings provided evidence that the starch separation procedure proposed by Montgomery & Senti (1958) [24] results in amyloses that have no use in thermal analytical studies, nor to the TG method, for determining the amylose ratio proposed by Stawski (2008) [2]. Hu and colleagues (2013) [74] also found similar displacements associated with the presence of 1-butanol in acid hydrolysis of starch. For the first time, these displacements were well characterized by thermogravimetry. The amylose-1-butanol inclusion complex presented not only a displacement to temperatures closer to amylopectin via TG curves, but also promoted some modifications to decomposition kinetics. In other words, the thermal decomposition rate was increased. The decreasing order of Tpeak DTG of amyloses was as follows: maize N potato N banana ≥ cassava. Although the potato amylose S-A had a large difference in Tpeak DTG when compared to the value obtained by starch separation without 1-butanol, the slope of both curves were close to each other. These findings indicate that the amylose-1butanol inclusion complex becomes the amylose crystalline structure. This more organized structure presents higher thermal stability and higher decomposition rates. The inclusion complex had a slope difference of 0.6% °C−1 when compared to amorphous amylose of the same botanical source obtained from Sigma-Aldrich (Fig. 7, Table 4). The Tpeak DTG of amylopectin (S·P) decreased in the following order: maize N cassava N banana ≥ potato. The potato amylopectin S-P and S-A (both amorphous) had differences of LogMw (0.38 Da), Tpeak

The starch separation procedure was successfully performed, and high purity amylopectin and amylose-1-butanol were obtained from potato, corn, banana, and cassava starches. The selective precipitation yielded Vamylose-1-butanol inclusion complexes when using all amyloses. Maize and banana amyloses-1butanol complexes presented more ordered structures than potato and cassava fractions, demonstrated by XRD data. The Tg of amylose-1-butanol complexes were highly correlated with their respective crystalline indexes. The melting of crystalline arrays (Tc-To) measured on native starches in previous works (mainly corresponding to amylopectin) were highly correlated with the heterogeneities of their respective polymodal distribution (%CV), measured in the present work. All amyloses separated from the starches had the same TG profile and very similar measurements (Tonset, T50%, Tpeak DTG) compared to the amylopectin fractions, caused by the yielding of the amylose-1butanol inclusion complex. The crystalline array of Vamylose-1-butanol showed an increase in the thermal stability of this polysaccharide when compared to the amorphous (non-complexed) structure. Research efforts must be concentrated on new starch separation procedures, ideally able to separate high purity amylose and amylopectin fractions in the absence of precipitation agents, such as 1-butanol. Due to the formation of the amylose-1-butanol complex, thermal studies or construction of analytical curves to determine the amylose ratio in starch samples are unfeasible. Acknowledgements The authors are grateful to FAPESB for the scholarship (8892/2015) granted to Paulo Vitor França Lemos, to Dr. Heloysa Martins Carvalho Andrade and MSc. Maurício Brandão (CIEnAm – UFBa) for the XRD analysis, and to Carina Soares do Nascimento (LCM - IfBa) for the SEM and DSC analysis. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.ijbiomac.2019.03.086. References [1] M.C. Garcia, C.M.L. Franco, M.S.S. Júnior, M. Caliari, Structural characteristics and gelatinization properties of sour cassava starch, J. Therm. Anal. Calorim. 123 (2016) 919–926. [2] D. Stawski, New determination method of amylose content in potato starch, Food Chem. 110 (2008) 777–781. [3] J. Zhu, S. Zhang, B. Zhang, D. Qiao, H. Pu, S. Liu, L. Li, Structural features and thermal property of propionylated starches with different amylose/amylopectin ratio, Int. J. Biol. Macromol. 97 (2017) 123–130. [4] A.-M. Hermansson, K. Svegmark, Developments in the understanding of starch functionality, Trends Food Sci. Technol. 7 (1996) 345–352. [5] K.S. Sandhu, N. Singh, N.S. Malhi, Physicochemical and thermal properties of starches separated from corn produced from crosses of two germ pools, Food Chem. 89 (2005) 541–548.

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