Use of High-Performance Size-Exclusion Chromatography for Characterization of Amylose Isolated from Diverse Botanical Sources

Abstract

Amyloses were isolated from diverse botanical sources (apple, mango, maize, and potato), and they were studied in their molecular characteristics (amylose content, molar mass, and molecular weight) using high-performance size-exclusion chromatography as a repetitive and faster protocol. The amylose purity ranged between 85.6–92.6 %, in agreement with the λ_max values (601–610 nm), showing that some impurities with molecules of higher molar mass (amylopectin) were present. The standard curve of pullulan showed a high regression coefficient (0.998) inside of the limits of molar mass of amylose. Chromatograms of amylose showed the presence of components of high molar mass with a principal peak that corresponds to amylose. Molar mass of amylose ranged between 1.2 and 8.5 × 10⁵ g/mol with polydispersity values between 1.3–4.1, indicating a narrow range of molar mass distribution of the amyloses analyzed. The high-performance size-exclusion chromatography coupled with a refractive index methodology used in this study may be considered simple and rapid for molecular studies of amylose.

Keywords:

• High-performance size-exclusion chromatography
• Amylose
• Molar mass
• Molecular weight
• Unconventional sources of starch

Introduction

Starch is composed of two macromolecules of different structures: Amylose is the mainly linear component and amylopectin is the branched molecule. Starch owes much of its functionality and physical organization into a granular structure to these molecules.^[1] It is of great importance to understand the molecular and structural characteristics of starch components so as to suggest possible applications of this polymer in diverse systems. The average molecular weights of amylose and amylopectin influences the functional properties of starch, as demonstrated in gels,^[2] extrusion products,^[3] and starch pastes.^[4] It is, therefore, important to have a reproducible technique for measuring this macromolecular feature. Several studies have been carried out on molecular and structural characteristics of amylopectin using high-performance size-exclusion chromatography (HPSEC),^[5–8] HPSEC coupled with multi-angle laser light scattering (MALLS),^[9–15] or field flow fractionation (FFF) coupled with MALLS,^[15,16] due to this molecule being responsible for diverse physicochemical and functional characteristics of starch, such as viscosity, swelling of the granule, thermal characteristics, crystallinity, and starch digestibility. On the other hand, amylose determines some of the main functional properties of starch including gel formation and starch retrogradation.^[17] These properties are strongly dependent on the macromolecular features of amylose. It was reported that a minimal molecular weight of 40,500 g/mol is necessary for amylose gelation.^[18] Diverse techniques have been reported for amylose characterization, some give mean–average values, such as classical light scattering, other include intrinsic viscosity measurement;^[19–21] however, for a polydisperse in a polymer such as amylose, the average value is not enough to fully predict its physicochemical and functional properties and a technique which a macromolecular distribution is more powerful as HPSEC–MALLS.^[22]

A pre-requisite step for determining the molecular weight distribution of amylose and amylopectin and their average molar mass by any of these techniques is the complete dissolution of the starch sample in an appropriate solvent without any degradation of the constituent macromolecules. High temperatures and high pH increase the solubility of starch in aqueous solvents, but may result in molecular size reduction resulting from degradation, depolymerization, or oxidation.^[23] The fulfillment of this requirement is necessary to get information representative of the initial starch sample. A solubilization procedure was tested using microwave heating and a high solubilization percentage was obtained for amylase, but when the amount of amylopectin in the starch increased the solubilization decreased.^[10] This method has advantages for the sample; it can be solubilized just prior to chromatographic analysis, thus minimizing variation in analysis due to starch aggregation.^[11]

Recently, the same procedure was tested in the molecular study of starches isolated from non-conventional source using a MALLS detector.^[24] However, the size-exclusion chromatography (SEC) system using a HPLC coupled a refractive index (RI; HPLC–RI) might be feasible for molecular studies of amylose for polymers with low polydispersity. Solvent such as dimethyl sulfoxide (DMSO) with LiBr has been used for analysis of starch and its components (amylose and amylopectin) using SEC–MALLS equipment.^[23,25,26] The present work proposed a methodology for molecular studies of amylose using a microwave heating for solubilization of the sample and thereafter, analysis in the HPSEC–RI system.

Materials and Methods

Starch Isolation

Immature apple (Malus domestica cultivar Golden Delicious), determined on the basis of the starch index (the skin color and size were the parameters used for cutting of the fruit) were collected at the local farm “La Campana” in Cuauhtémoc, Chihuahua (México). The methodology reported by Bello-Pérez et al.^[27] was used for starch isolation. Green mangos (Mangifera indica L.) variety “Ataulfo” were also purchased in the local market and the starch was isolated with the procedure previously reported by Agustiniano-Osornio et al.^[28] Potato starch was carefully isolated using a modification of the procedure of Kim et al.^[29] and maize starch was obtained with the method suggested by Adkins and Greenwood.^[30] The white-starch sediments were dried in a convection oven (Lab-Line Instruments Inc., Imperial V, Melrose Park, IL) at 40°C for 48 h, carefully ground with a mortar and pestle to pass through a US No. 100 sieve and stored at room temperature (25°C) in a glass container.^[31,32] Amylose, from the starches previously mentioned, was isolated by the thymol and n-butanol complexation method.^[33] Commercial amylose type III from potato (Sigma Chemical Co., St Louis, MO, USA) was used as a control and subsequently was named as commercial.

Amylose Content

The purity of amylose was determined by the total amylose content test of Hoover and Ratnayake^[34] and the wavelength at the maximum absorption (λmax) of the blue iodine–amylose complex.^[35] The total amylose contents of starch samples were determined by above procedure, but with prior deffating with hot n-propanol-water (3:1 v/v) for at least 7 h. In order to correct for over estimation of apparent and total amylose content (due to complex formation between I₂ and the outer branches of amylopectin), amylose content was calculated from a standard curve prepared using mixtures of pure potato amylose and amylopectin (over the range 0–100% amylose).

SEC-RI Study

Pullulan standards (Fluka Chemie GmbH, Steinhein, Switzerland) of diverse molar mass (1.6 × 10⁶, 3.8 × 10⁵, 1.8 × 10⁵, 1.0 × 10⁵, 4.8 × 10⁴, and 1.2 × 10⁴ g/mol) were used to obtain a calibration line. Pullulans were dissolved in HPLC-grade water at 25 ºC overnight, filtered using 0.2 µm nylon syringe filter (Daigger & Company Inc., Vernon Hills, IL) and injected into the HPSEC–RI system. The solubilization procedure of amyloses was carried out as reported by Millan-Testa et al.^[24] using 50 s of microwave heating. The supernatant solution was filtered through a 5 µm nylon syringe filter (Daigger & Company Inc., Vernon Hills, IL). The solution was injected (50 μL) onto the HPLC AT 1100 equipment (Angilent Technology, Deutchland GmbH Waldbronn, Germany) with the gel permeation chromatography-size exclusion chromatography (GPC-SEC) PL aquagel-OH mixed, 8 µm column (7.5 mm ID × 300 mm; Agilent Technologies Deutchland GmbH Waldbronn, Germany). The column and the detector were maintained at 30ºC. The eluant was HPLC-grade water, carefully degassed and filtered before use through Durapore GV (0.2 µm) membranes. The flow rate was 1.0 mL/min. The data analysis was realized using the GPC software of Agilent (Agilent Technologies Deutchland GmbH Waldbronn, Germany). The carbohydrate concentration of the supernatant solution after filtration was measured by the sulphuric acid-phenol colorimetric method.^[10] The procedure was carried at least five times for each sample.

Statistical Analysis

Researchers used a completely random design. An analysis of variance (ANOVA) was used at a 5% significance level (α = 0.05). Results were obtained and normality test was verified using a Sigma-Plot statistical program, version 11.0.^[36] When significant differences were found, researchers applied the Tukey range test for the comparison of means.^[37]

Results and Discussion

Amylose Characteristics

The purity values of the amylose samples evaluated in this work ranged from 85.6 to 92.6% (Table 1), which agrees with their λ_max values (between 601 and 610 nm). Previous works reported λ_max values of pure amylose between 650 and 660 nm.^[22,38,39] However, some contrasting data have been reported. Radosta et al.^[38] determined in one sample of synthetic amylose an amylose content of 99.0% and λ_max of 660 nm, while in a second one they reported 96.3% of purity and λ_max of 667 nm. In the same sense, λ_max values between 652 and 662 nm were reported for amylose isolated from diverse botanical sources.^[22] On further studies,^[40] the investigators previously mentioned determined that these samples had a high molar mass population that contaminated amylose solutions, which were removed by ultracentrifugation. Amylose molar mass values after ultracentrifugation were lower than its original value in the amylose solution.

TABLE 1 Amylose content and λ_max of amyloses from diverse botanical sources

Sample	*Amylose (%)	λmax (nm)
Commercial	85.6 ± 0.019^a	610
Potato	92.6 ± 0.003^d	601
Mango	88.5 ± 0.003^b	605
Maize	90.7 ± 0.008^c	610
Apple	86.0 ± 0.007^a	607

*Values are mean of triplicate determinations ± standard error; Different lowercase letters in the same column show significant difference (p < 0.05).

Standard Curve of Pullulan

A semi-logarithmic plot of molar mass versus elution volume for pullulan standards is shown in Fig. 1. The regression coefficient of the ratio between molar mass and elution volume was 0.998, showing that the SEC system used for elution of pullulan standards is reproducible because in five standard curves was obtained a standard error of 0.003929 with a p < 0.05. The use of the SEC in studies of molecular structure of polymers with medium molar mass is feasible, because HPSEC alone allows the determination of amylose or amylopectin molar mass if pullulan or dextran standards are available.^[22] For polymers with high molar mass (such as amylopectin), HPSEC coupled with MALLS is recommended.^[11,23] However, molar mass determination for amylose was realized avoiding the use of a calibration plot.^[22,40]

FIGURE 1 Semi-logarithmic plot of molar mass versus elution volume for pullulan standards.

Molecular Characteristics

Complete solubilization of starch components without degradation is important in molecular studies. The solubility of amylose samples ranged from 85.0 to 99.7% (Table 2) and depends on the botanical source as it was in the following descending order commercial > apple > potato > mango > maize. Bello-Pérez et al.^[10] reported an amylose solubility value of 100% using microwave heating, similar values were reported when amylose solutions were ultracentrifuged.^[40] Chromatograms of amyloses (Fig. 2) showed that potato had two small peaks at 4.8 and 6.0 mL as well as commercial sample, whereas apple amylose had only one peak at 6.2 mL. These components of high molar mass contribute to a higher amylose molar mass, because during amylose isolation an amount of amylopectin was not separated. The samples studied presented a main peak at an elution volume between 7.2 and 7.6 mL due to the amylose component. The pattern observed in the chromatograms samples showed an influence in the global molar mass by the chain length distribution of amylose. Components of higher molar mass were found in some amyloses studies, which were separated after ultracentrifugation, contributing to higher molar mass values.^[40] However, SEC–MALLS–RI study of some amyloses did not show the presence of a component of higher molar mass, but appreciable differences in the molar masses were observed.^[38]

TABLE 2 Number-average (Mn) and weight-average (Mw) molar masses of starches amyloses*

Sample	Solubility (%)	Mn (10^–5 g/mol)	Mw (10^–5 g/mol)	Mw/Mn
Commercial	99.73 ± 0.15^b	1.50 ± 0.10^a	2.00 ± 0.10^b	2.70 ± 0.01^c
Potato	98.96 ± 1.70^b	2.20 ± 0.20^b	3.00 ± 0.20^c	4.10 ± 0.05^e
Mango	87.50 ± 2.80^a	1.40 ± 0.10^a	1.90 ± 0.05^b	1.30 ± 0.04^a
Maize	85.00 ± 1.41^a	8.80 ± 0.20^d	1.20 ± 0.01^a	1.40 ± 0.02^b
Apple	99.70 ± 0.42^b	5.70 ± 0.40^c	8.50 ± 0.40^d	2.90 ± 0.09^d

*Values are mean of triplicate determination ± standard error; Different lowercase letters in the same column show significant difference (p < 0.05).

FIGURE 2 Chromatograms of amyloses from diverse botanical source.

Molar masses of amyloses were between 1.2 and 8.5 × 10⁵ g/mol. In general, the values of this study were lower than those obtained for diverse amyloses (between 4.0 × 10⁵ and 1.2 × 10⁶ g/mol) reported by Roger and Colonna,^[22] since potato amyloses of this study presented lower molar mass values (2.0 and 3.0 × 10⁵ g/mol) than that reported for amylose isolated from potato (9.8 × 10⁵ g/mol).^[22] Amylose maize presented a similar behavior; the molar mass obtained in this work (1.2 × 10⁵ g/mol) was smaller than the value (8.7 × 10⁵ g/mol) reported by the same authors.^[22] Nevertheless, these authors determined molar mass values for the same amylose (between 3.4 × 10⁵ and 1.0 × 10⁶ g/mol), using a modified procedure.^[40] Other authors reported molar mass values of potato amylase higher than those obtained here in the same botanical source using HPSEC system (2.1–4.4 × 10⁷ g/mol).^[38] The polydispersity assessed by the ratio Mw/Mn is a distribution measurement of the size of the polymers present in the sample. The Mw/Mn values (Table 2) were in a range from 1.3 to 4.1, showing that the methodology used for determining amylose molar mass can be suggested for the study of its structural characteristics. Similar polydispersity values (1.7–4.4) were reported for amylose of diverse sources.^[22] Roger and Colonna^[40] reported that values did not decrease when ultracentrifugated and a narrower range value of polydispersity was obtained (2.54–3.81).

The solubilization percentage in the samples analyzed was high, indicating that the determination of molecular characteristic of amylose using HPSEC–RI gives information about the totality of the polymer. Some amyloses presented few amounts of polymers of higher molar mass from amylopectin as it was demonstrated by the amylose content, λ_max and the chromatograms. The molar masses of amyloses determined by HPSEC–RI are in the range of values reported for amyloses with low polydispersity, showing a narrow range of molar mass distribution of amylose chains. The use of HPSEC–RI methodology can be feasible for molecular studies of amylose where expensive detectors (such as MALLS, Viscosity, etc.) are not required.

Conclusion

The solubilization percentage in the samples analyzed was high, indicating that the determination of molecular characteristic of amylose using HPSEC–RI gives information about of the totality of the polymer. Some amyloses presented few amounts of polymers of higher molar mass from amylopectin as it was evidenced from the results of amylose content, λ_max and the chromatograms. The molar masses of amyloses determined by HPSEC–RI are in the range of those reported for amyloses with low values of polydispersity, showing a narrow range of molar mass distribution of amylose chains. The HPSEC–RI methodology might be feasible for molecular studies of amylose where expensive detectors (such as MALLS, viscosity) are not required.

Acknowledgments

The authors would like to thank Arturo Ramos Martínez and Emilio Ochoa Reyes for their technical assistance.