Extraction and Characterization of the Functional Properties of Starch from Miso (Mirabilis expansa [Ruíz & Pav.] Standl.): A Non-Conventional Source

Abstract

Mirabilis expansa root (MER) is an Andean source of starch that could be considered a “lost crop” by the scarcity of its cultivation. Consequently, few studies have reported on its functional properties. To address this gap, we herein analyze and characterize the main components of MER and Mirabilis expansa starch (MES), measuring the water-absorption index (WAI), swelling power (SP), and water solubility index (WSI). We characterized the MES morphological and structural properties by using scanning electron microscopy (SEM). We also examined the starch pasting properties using a Rapid Visco Analyzer (RVA) to determine the peak viscosity (PV), final viscosity (FV), pasting temperature (PT), breakdown (BD), and setback (SB). The thermal properties were determined by differential scanning calorimetry (DSC), the crystallinity by X-ray diffraction, and the molecular structure by Fourier transform infrared spectrometry (FTIR). The main components in the MER and MES were carbohydrates and crude fiber. The MES appeared rich in amylopectin. The functional properties, the WAI, SP, and WSI, were dependent on temperature. The MES showed no morphological changes, a moderate gelatinization temperature, and C-type crystallinity. Finally, the FTIR spectrum presented the typical form for an unmodified starch. Based on these results, Mirabilis expansa may be considered a natural, non-conventional source of starch with potential applications in the food, chemical, and pharmaceutical industries.

1. Introduction

“Miso” or “mauka” (Mirabilis expansa [Ruíz & Pav.] Standl.) is an Andean root used exclusively by inhabitants of the Andean region of Ecuador, Peru, and Bolivia since pre-Columbian times as a source of carbohydrates. Nowadays, this species could be considered a “lost crop” because its cultivation has become scarce, as suggested by the paucity of studies found in the literature. It grows at altitudes of 2700 to 3500 m above sea level (m.a.s.l.) and at temperatures ranging from 5 to 25 °C [1,2]. However, today, the only available information about it is conveyed from an ethnobotanical perspective [3,4]. Otherwise, few studies have reported on its main constituents or the functional properties of this root’s starch [1,4,5,6].

Starch is an important commodity because it is used in the chemical, pharmaceutical, and food industries. Today, starches are a natural and renewable source of biopolymers that could replace those derived from the petrochemical industry [7,8]. Currently, the main sources of starch are corn, wheat, and potato [8], but finding new sources of starch could provide the market with additional alternatives for use in the industries noted above. One such alternative is the starch present in Mirabilis expansa roots (MERs), which is a polysaccharide consisting of amylose and amylopectin, the variable quantities of which, in turn, affect the functional properties of Mirabilis expansa starch (MES) and, hence, its applications in the noted industries.

Nevertheless, most of the current information on miso involves its composition. For instance, it has been reported that miso roots contain proteins (4–7%), ash (4–5%), fat (1–2%), and fiber (1–5%), but they mainly contain carbohydrates (33–80%) [5,6,9,10]. While the high carbohydrate content is related to starch in miso roots, only one study has characterized its functional properties [6]. The same data were reported in [5] in 2004. This study reports an amylose content of 21%, which is similar to those in some conventional starch sources, such as sweet potato, cassava, potato, and corn (17–28%) [11]. It further reports the swelling power (1.89), solubility (0.98), and absorption (1.82) indexes evaluated at 30 °C, all properties similar to those of such common sources as potato or sweet potato. This means that miso could be applied in the food, pharmaceutical, or chemical industries. However, these reported data have some limitations. First, the functional properties were evaluated at a constant temperature of 30 °C (Villacres and Espin (1999), as cited in [5,6]). However, these properties need to be tested against variations in temperature since temperature fluctuations affect potential applications [12]. Therefore, it is a regular practice to report these properties within the same temperature range as that reported for sweet potato, cassava, potato, and corn, i.e., from 60 to 90 °C [11]. Second, the data reported for miso starch were performed using the equipment available in 1999, such as optical microscopy and Brabender amylography, as in the 2004 study by the authors of [5]; however, no studies have reported on MES, and no morphological studies have been performed using modern techniques, such as scanning electron microscopy. Furthermore, no studies have reported on properties determined by thermal analysis or Fourier transform spectroscopy. Neither viscosity nor functional properties have been studied as a function of temperature.

Therefore, the aim of this study was to characterize the physicochemical, functional, structural, and thermal properties, as well as the technological characteristics, of the starch obtained from miso (Mirabilis expansa [Ruíz & Pav.] Standl.), a potential new source of carbohydrates.

2. Materials and Methods

2.1. Raw Materials and Starch Isolation

Miso roots (MERs) were acquired from local farms located in the community of Cubinche, Pichincha Province, Ecuador (geographic position: 0°1’ north, 78°14’ west, 2800 m.a.s.l.). A sample of plant material was authenticated by an expert from the Misael Acosta Solís Herbarium of Ecuador (Universidad Técnica de Ambato, Ecuador), and a voucher specimen (N°. YFRC23-01) was deposited at the Yurakuna Food Research Centre (Quito, Ecuador). Samples were selected and washed, and those exhibiting any mechanical damage were discarded.

Ripeness was checked with a refractometer (Atago, PAL-BX/RI, Tokyo, Japan) to identify the quantity of dissolved solute, such as carbohydrates, in the roots (AOAC, method 935.25) [13].

Extracting starch from MERs was performed as described in [14]. Using a knife, samples were cut into cubes (around 6 mm), immersed in a 0.075% sodium metabisulfite solution (Na₂S₂O₅) (1:1) for 5 min, and ground in a screw mill (Landers, Corona L14200, Medellín, Colombia). The resulting mass was then filtered through a muslin cloth, adding water to remove the starch. The retentate was ground again with water in a 1:1 ratio, filtered once more, adding water to remove the starch, and mixed with the first filtrate.

The filtrate was left to settle at room temperature (18 °C) for 4 h until a consistent layer of starch was obtained at the bottom. At the end of the first decantation, the supernatant was removed, and water was added to the starch in a 1:2 ratio and left to stand for 4 h. The supernatant was then removed, and water was added to the starch in a 1:1 ratio. The sample was again left to settle for 4 h, and the supernatant was removed. Finally, the extracted starch was placed on filter paper and placed in an oven (Memmert, SF-30PLUS, Büchenbach, Baden-Württemberg, Germany) at 45 ± 2 °C for 48 h, followed by grinding with a centrifugal mill (Vieira, MCS 280, São Paulo, SP, Brazil) and sieving through a 100-mesh sieve to obtain the MES.

2.2. Starch Characterization

2.2.1. Proximate Analysis

The chemical compositions of the MER and MES were determined using Association of Official Analytical Chemists (AOAC) analytical methods [13] for moisture (method 934.01), crude fat (ether extract) (method 920.39), ash content (method 923.03), total fiber (method 962.09), total protein (method 920.103), and total carbohydrates by difference. All assays were performed in triplicate and expressed as a mean value ± standard deviation.

2.2.2. Starch Extraction Yield and Amylose Content

The starch extraction yield was obtained by the gravimetric method since the relationship between the mass of starch obtained and the raw material used on a dry basis (DB) is critical to extraction and processing [15]. The amylose content of the MES was determined by colorimetry [16].

2.2.3. Scanning Electron Microscopy (SEM)

Morphological properties of the MES were visualized by means of SEM (Aspex, PSEM Express, Delmont, PA, USA), and the analysis conditions were as follows: acceleration voltage: 20 kV; filament charge: 59.6%; current output: 89.2 μA; vacuum pressure: 2.6 × 10⁻⁵ Torr [17].

2.2.4. Functional Properties of Starch

• The water-absorption index (WAI), swelling power (SP), and water solubility index (WSI) were determined at temperatures of 60 °C, 70 °C, 80 °C, and 90 °C [18,19]. A mass of starch (Ws) of 1.25 g (DB) was suspended in a volume of 30 mL water, stirred intermittently over a 30 min period, and centrifuged at 3000 rpm for 10 min. The supernatant was poured carefully into a tared evaporating dish and dried to a constant weight (W1). The remaining gel was weighed (Wg). The WAI, SP, and WSI were calculated as follows:

  WAI = Wg/Ws × 100%

  (1)

  WSI = W1/Ws × 100%

  (2)

  SP = Wg/[Ws × (100% − WSI)] (g/g)

  (3)
• The pasting properties of the MES were determined with the Rapid Visco Analyzer (PerkinElmer, Perten-RVA, model super4, Shelton, CT, USA) [18]. A mass of 2 g, 11.8% moisture (DB), was mixed with 25 mL of distilled water in the RVA sample canister. The programmed heating and cooling cycle began with the samples held at 50 °C for 1 min, heated to 95 °C in 7.5 min, held at 95 °C for 5 min, cooled back to 50 °C in 7.5 min, and then held at 50 °C for 2 min. The parameters obtained were as follows: the peak viscosity (PV), final viscosity (FV), moisture (DB), and setback (SB). The viscosities are presented in centipoise (cP).

2.2.5. Thermal Properties by Differential Scanning Calorimetry (DSC)

The thermal properties of the MES were determined by DSC, as described in [18]. A mass of MES was mixed with distilled water (1:3 w:w) and left in a hermetically sealed crucible for at least 1 h. The heating profile ranged from 20 °C to 100 °C at a rate of 5 °C/min in an inert atmosphere (20 mL/min of N₂), and a sealed empty pan was used as a reference. The data obtained were as follows: the gelatinization enthalpy (ΔH), initial or onset temperature (To), peak temperature of gelatinization (Tp), and conclusion temperature (Tc).

2.2.6. Fourier Transform Infrared Spectroscopy (FTIR)

FTIR spectra were obtained using an FTIR spectrometer (Perkin Elmer, L1600312 Spectrum Two, Waltham, MA, USA) equipped with an attenuated total reflectance (ATR) accessory. Sixty-four scans were recorded within the spectral range of 400 to 4000 cm⁻¹ and were presented in absorbance (%) against wavelength. The resolution was set at 4 cm⁻¹ [20]. Perkin Elmer Spectrum Version 10.5.2 software was used for peak positioning.

2.2.7. X-Ray Diffraction (XRD)

This analysis was carried out as described in [21] using a diffractometer (MalvernPanalytical, Empyrean, Malvern, Worcestershire, UK) equipped with a linear X’Celerator Scientific detector and configured in Bragg–Brentano geometry using CuKα radiation of 1.540598 Å with typical operating conditions of 45 kV and 40 mA. The diffraction patterns were recorded between 5° and 80° using a scan rate of 0.02°/s. The data were obtained using Data Collector 5.1 software.

2.2.8. Microbiological Profile

Microbiological analyses were carried out according to AOAC methods [13] for total mesophilic aerobic bacteria (method 990.12), yeast and molds (method 997.02), and total and fecal coliforms (method R.I. 110402). Values were expressed as CFU/g (colony-forming units per gram).

2.3. Statistical Analysis

Results were expressed as a mean value ± standard deviation (DB) of three repetitions. The statistical analysis was performed using STATISTICA version 10 (Statsoft, Tulsa, OK, USA) applying analysis of variance (one-way ANOVA), and a Tukey test was used to determine significant differences between means (α = 0.05).

3. Results and Discussion

3.1. Raw Material and Starch Extraction

The miso roots (Figure 1a) presented a very irregular shape with a yellowish color, as reported in [1,6]. The Brix index was 13.6 °Brix, as an indicator of ripeness. The starch obtained (MES) presented a white color (Figure 1b). Compounds responsible for the yellowish color were removed by the starch extraction technique upon discarding the supernatant, and the MES was subjected to several washing processes.

3.2. Starch Characterization

3.2.1. Proximate Analysis

The proximate analysis of the MER and MES is presented in

Table 1. Physicochemical parameters of Mirabilis expansa roots (MERs) and starch (MES).

Parameter (%)	MER 1	MES 1
Moisture (WB)	71.90 ± 0.02 a	11.80 ± 0.05 b
Ash	7.97 ± 0.10 a	0.23 ± 0.03 b
Fat	0.28 ± 0.02 a	0.30 ± 0.01 a
Protein	5.20 ± 0.23 a	0.75 ± 0.01 b
Crude fiber	17.40 a ± 0.55 a	0.25 ± 0.01 b
Total carbohydrates	69.0 ± 0.23 a	86.67 ± 0.14 b
Amylose	-	26.44 ± 0.14
Amylopectin	-	73.56 ± 0.57
Starch extraction yield (%)	-	8.56 ± 0.28

¹ The values expressed are the mean ± standard deviation in DB except for moisture. Same superscript letters in the same row indicate no significant difference when comparing different starches at p < 0.05.

. Information regarding this species is scarce; however, the parameters of the moisture, protein, and total carbohydrates are similar to those reported for miso by the Instituto de Investigaciones Agropecuarias of Ecuador (INIAP) [5,6], but our values for ash and crude fiber are higher compared with those from the INIAP.

The MER and MES presented statistical differences (p < 0.05) for all parameters since starch is separated from non-starch components in the extraction process.

3.2.2. Starch Extraction Yield and Amylose Content

The extraction yield of miso (8.56%) was lower than those reported by [5,6], at 12.23% and 10.4%, respectively. These differences could be attributed to the grinding process in the disk mill that was used, or losses during the decantation process because aqueous extraction has a lower starch yield [16]. The starch yield was also lower when compared with those of tubers, such as potatoes, cassava, and taro (Colocasia esculenta), ranging from 10 to 20%, and those of cereals, including wheat at 38% and corn at 79% [8,22]. The starch yield varies depending on its source and the extraction technique used. To improve the starch yield, a pretreatment could be employed, such as enzymes and fermentation, as well as mechanical, heat, or ultrasound treatment [8,22].

The amylose content showed slightly higher values than those previously reported [5,6]. This component is important because higher amylose or amylopectin contents affect the thermal, rheological, and functional properties, specifically viscosity, relevant for the application of starches [18]. The amylose content was also higher than the values reported for rice (23.8%), but lower compared to those reported for tapioca, wheat, normal maize, potato, and lentil (29.1, 30.7, 32.0, 34.0, and 37.0%, respectively) [20].

3.2.3. Scanning Electron Microscopy (SEM)

The morphology of MES granules (Figure 2) displays characteristics of an unmodified starch with a plain and rounded, or spherical, structure. The spherical structure of MES has been reported [5,6], albeit by compound microscope with magnifications of 4×, 10×, and 20×. By using SEM, the spherical morphology can be clearly observed.

3.2.4. Functional Properties of Starch

The functional properties, the WAI, SP, and WSI, are presented in Figure 3. These properties increased as a function of temperature.

At 60 °C, the WAI, SP, and WSI appeared as stable properties because the structure of starch granules was preserved. However, above this temperature, significant differences could be seen relative to the changes that MES undergoes, such as gelatinization, starch granule breakdown, and the liberation of amylose and amylopectin under the combined effect of temperature and heat. Accordingly, the functional properties, such as the WAI, are altered. The WAI measures the volume occupied by the granule or starch after swelling in excess water [23], and both amylose and amylopectin interact with water molecules primarily through the creation of hydrogen bonds by their hydroxyl (-OH) groups. Amylopectin is water-insoluble and contributes to viscosity. MES presents high contents of amylopectin (Table 1) and a relatively low gelatinization temperature of 62 °C [19,20,24]. Consequently, the WAI, SP, and WSI may all be related to different contents of amylose and amylopectin.

The WSI presented minimal increase with temperature, and no statistical difference was observed between temperatures of 60 and 70 °C and 80 and 90 °C. This result could be explained by the interaction between amylose/amylopectin and water molecules such that a higher percentage of amylopectin correlates with higher interaction. The branched structure of amylopectin limits, or hinders, interaction between water molecules and amylose; also, water molecules act as a plasticizer that affects the mobility, or diffusion, of the components in the matrix. The WAI and WSI are important because they can be used to characterize extruded products [23].

According to Figure 4, viscosity, which changes by a combination of time, temperature, and mechanical shear, is presented as a curve characteristic of unmodified starch. The MES presented a PT of 69.30 °C, higher than the gelatinization temperature of 61.8 °C, probably because of the leaching out of small molecules of amylose and amylopectin (Figure 5). The values of the SB (583 cP), Trough (825 cP), BD (151 cP), and FV (1408 cP) were lower when compared to those of wheat, normal maize, potato, and lentil but similar when compared to those of tapioca (70.3 °C), lentil (71.2 °C), and pea (70.3 °C) [20]. Differences may be related to the source of starch, the amylose content, and the possible formation of amylose–lipid complexes (ALCs) [20].

3.2.5. Thermal Properties of MES by DSC

The thermal properties of the MES (Figure 5) show a gelatinization temperature of 61.83 °C, lower than the values reported for rice, tapioca, wheat, normal maize, potato, and lentil sources [20].

The MES also presented a low gelatinization enthalpy of 3.511 J/g. This value indicates less stability because it requires less energy for gelatinization. The low values of the ΔH are similar to those reported for potato (4.6 J/g); however, they are lower than those reported for cereals, such as rice and corn (9.5 J/g and 10.3 J/g, respectively), lower than those for tubers, such as cassava and sweet potato (10.0 J/g/ and 9.2 J/g, respectively) [11,25], and lower still than the same values reported for rice, tapioca, wheat, normal maize, potato, and lentil sources [20]. Such low values may be related to the amylose content (26.4%), but other factors may be in play, like the starch grain size and the type of crystallinity [26].

3.2.6. FTIR Spectra of MES

Figure 6 shows the FTIR spectrum of the MES in the mid-infrared region from 4000 to 400 cm⁻¹. While no previous comparative data are available, this result shows the characteristic spectra of native, or unmodified, starch, which is a mix of crystalline and amorphous phases. As shown in Figure 6a, the range of 3300 to 500 cm⁻¹ presents widely distributed peaks indicating elemental vibrations associated with the rotational–vibrational structure. At 3304 cm⁻¹, it presents a peak related to hydroxyl groups owing to glucose (-OH). A small peak appears at 2929 cm⁻¹, which is related to the C-H bonds in glucose, CH₂ and CH₃ groups. A small peak appears at approximately 1641 cm⁻¹ indicating bound water within the starch, which is common in native starches. At 993 cm⁻¹, a sharp peak appears to be related to C–O stretching, often associated with the amorphous character of the starch, given its semi-crystalline nature. At approximately 927 cm⁻¹, another small peak is related to the deformation of C–H in the anomeric region of carbohydrates [27].

Figure 6b shows a less smooth low-wavenumber region (400 to 500 cm⁻¹) where the spectra appear in a lattice configuration with the presence of many peaks, possibly related to starch-specific polymorphisms.

3.2.7. X-Ray Diffraction (XRD) Pattern of MES

As seen in Figure 7, the MES exhibited a C-type XRD pattern with diffraction peaks at 5°, 15°, 17°, 18°, 23°, and 24.5° 2θ. The C-type presents a pattern that combines the A-type and B-type, where peaks at 5° and 23° are characteristic peaks of B- and A-type crystallinities associated with the presence of amylose and amylopectin [21,28]. This pattern has not only been reported for starch from roots [12] and tubers, such as sweet potato [28], but also from peach palm fruits [25].

3.2.8. Microbiological Profile

The MES presented total mesophilic aerobic bacteria at 550 CFU/g, yeast at 550 CFU/g, molds < 10 CFU/g, and the absence of coliforms. U.S. Pharmacopeia (USP) standards establish baseline values for microbiological parameters for corn, potato, tapioca, wheat, and rice starches. Total mesophilic aerobic bacteria should not exceed 1000 CFU/g, while total molds and yeasts should not exceed 100 CFU/g and absence of E. coli [29]. MES satisfies the criteria regarding total mesophilic aerobic bacteria and absence of E. coli, but it presents a high count for yeast, likely related to the inappropriate manipulation or poor disinfection of raw materials or equipment or contamination during starch extraction processes from tubers, as previously reported in [30].

MES obtained using the aqueous method presented properties of native, or unmodified, starch with moderate content of amylose (26.44%), type-C crystallinity, round-shaped starch grains, a moderate gelatinization temperature (61.83 °C), and a low gelatinization enthalpy (3.511 J/g). Both the WAI and SP were temperature-dependent. Based on these results, MES could be used in food, as texturizer or gelling, in the chemical industry for producing films or hydrogels, and in the pharmaceutical industry as starch-based hydrogels for drug delivery [11,19,21,26,31,32].

4. Conclusions

Mirabilis expansa [Ruíz & Pav.] Standl. could be characterized as a renewable, non-conventional source of starch, showing a chemical profile and properties quite similar to those of starch from common sources, such as corn and cassava. The starch from Mirabilis expansa [Ruíz & Pav.] Standl. (MES) presented moderate amylose content (26.44%) and relatively high amylopectin content (73.56%). The morphology and vibrational spectra were typical of an unmodified starch with spherical granules and C-type crystallinity, as demonstrated by SEM and FTIR analyses. The MES also presented a low gelatinization temperature (65 °C) and a low enthalpy of gelatinization (ΔH) (3.511 J/g). Functional properties of the MES, such as the WAI, SP, and WSI, were temperature-dependent and increased with temperature. All these results gave evidence of the potential application of this starch in the food, chemical, or pharmaceutical industries and could lead to the preservation of native species.