Several aquatic macrophytes such as Colocasia esculenta, Eleocharis dulcis, Nelumbo nucifera, Sagittaria sagittifolia, Trapa bispinosa, and Typha angustifolia possessed carbohydrate mainly in their storage and reproductive parts. Starch morphology, total starch, and amylose content of these six freshwater plant species were determined. Their functional properties, i.e., starch crystallinity, thermal properties, and rheological behaviour were assessed. Large starch granules were in N. nucifera rhizome (>15 μm), medium-sized was N. nucifera seed (8-18 μm), while the rest of the starches were small starch granules (<8 μm). Shapes of the starch granules varied from oval and irregular with centric hilum to elongated granules with the eccentric hilum. Eleocharis dulcis corm starch had significantly higher total starch content (90.87%), followed by corms of C. esculenta (82.35%) and S. sagittifolia (71.71%). Nelumbo nucifera seed starch had significantly higher amylose content (71.45%), followed by T. angustifolia pollen (36.47%). In comparison, the waxy starch was in N. nucifera rhizome (7.63%), T. bispinosa seed (8.83%), C. esculenta corm (10.61%), and T. angustifolia rhizome (13.51%). Higher resistant starch was observed mostly in rhizomes of N. nucifera (39.34%)>T. angustifolia (37.19%) and corm parts of E. dulcis (37.41%)>S. sagittifolia (35.09%) compared to seed and pollen starches. The XRD profiles of macrophytes starches displayed in all the corms and N. nucifera seed had A-type crystallinity. The T. bispinosa seed had CA-type, whereas the rest of the starches exhibited CB-type crystallinity. Waxy starches of C. esculenta corm had higher relative crystallinity (36.91%) and viscosity (46.2 mPa s) than regular starches. Based on thermal properties, high-amylose of N. nucifera seed and T. angustifolia pollen resulted in higher gelatinization enthalpy (19.93 and 18.66 J g⁻¹, respectively). Starch properties showed equally good potential as commercial starches in starch-based food production based on their starch properties and functionality.
Starch plays a vital role in food and nonfood industries, e.g., pharmaceutical, paper, textiles, biomedical, and polymer, because of its gelling characteristics, thickening, water binder, and food system stabilizing capacities . Research on the structure and physicochemical properties of starch in cultivated plants, Zea mays (maize), Manihot esculenta (cassava), and Solanum tuberosum (potato) resulted in their extensive utilization in food industries. However, other plants besides those mentioned above may possess potential and promising alternative starch sources.
Over the years, aquatic plants’ usage has become increasingly important; for example, rice, Oryza sativa, is a human staple diet . Detailed studies of starch isolated from aquatic macrophytes are increasing and mostly focused on specific plants such as water chestnut, lotus, rice, and taro. Asian countries such as China and Japan had cultivated aquatic macrophytes such as lotus (Nelumbo nucifera), Chinese water chestnut (Eleocharis dulcis), water caltrop (Trapa bispinosa), taro (Colocasia esculenta), and arrowhead (Sagittaria sp.) for starch-based food. The research conducted showed that starches from macrophytes could also be a promising candidate as an energy source in the food-related industry. Water chestnut corm flour as a thickening agent and dusting powder in food preparation , arrowhead corms, and water caltrop fruits are eaten boiled or cooked and can be dried and ground into a powder [4, 5]. Taro tuber possesses low fat, high carbohydrate, and minerals content and suitable as a food ingredient for baby food, chips, and bread . Lotus seeds, consumed boiled or processed into powder, are also used in the pharmaceutical industry to treat inflammation, arrhythmia, cancer, and skin diseases . Most of the starch’s diverse uses are from cultivated species [8–11], and research on the wild species is still scarce .
Research on starch isolated from freshwater macrophytes such as cattails, arrowhead, yellow nutsedge, and duckweed and their physicochemical properties are also available and less prevalent [13, 14]. Although consumed by local communities, their local utilization was seldom reported in the literature. Nowadays, consumers are engaging with resistant starch (RS) to promote health benefits similar to high-amylose starch. RS is poorly digested starches and absorbed in healthy individuals’ small intestine due to its complex molecular structure . They are either entirely or partially fermented as a food source for bacteria, primarily inhabiting the colon. There are limited studies conducted regarding RS in aquatic macrophytes starches.
Investigating the aquatic macrophytes starches, among others, is to create awareness of their various uses and economic values. For those involved in aquatic macrophytes, it can be part of their added income generation. This study also investigated the potential use of aquatic macrophytes starches in other applications in a starch-based industry. Thus, the present study was to systematically evaluate the starch structure, composition, functional properties, and also their resistant starch (RS) content isolated from selected commonly consumed aquatic macrophytes such as taro, lotus, and water chestnut and rarely consumed, e.g., arrowhead, water caltrop, and cattail in Malaysia.
2. Materials and Methods
2.1. Plant Materials
Two kilograms (2) kg of edible storage organ from five macrophytes species; corms of E. dulcis, S. sagittifolia, and C. esculenta; rhizomes of N. nucifera and T. angustifolia; seeds of N. nucifera and T. bispinosa; and pollen of T. angustifolia were peeled, washed, and isolated for starches.
2.2. Isolation of Native Starch
Native starch was isolated following a method described by Vasanthan  with a slight modification. The plant materials were added to water in a ratio of 1 : 10. The mixture was then blended for 5-10 minutes until a smooth slurry is formed. Approximately 0.01% () sodium metabisulfite was added into the slurry and left for 30 minutes before filtering using 100 μm nylon mesh cloth. The filtrated starch was centrifuged at 8000 rpm at 20°C for 20 minutes. The supernatant was discarded, and the pellet was oven-dried at 40°C for 24 hours. The dried starch was ground using mortar and pestle, sieved (250 μm), labelled, stored in a tightly closed container, and kept dry in a desiccator (10% relative humidity).
2.3. Polarized Optical Microscopy
A small amount (0.2 mg) of starch powder was placed on a microscope slide () by using a spatula. The starch was stained with 0.25% Lugol’s solution. The slide was then covered with a coverslip and observed under a compound light microscope (DM 750, Leica Microsystem, Wetzlar, Germany) equipped with a camera set (ICC50 W, Leica Microsystem, Wetzlar, Germany), polarized filter and analyzer. Images of starch granule and hilum were observed and captured. The granule sizes were measured using the ImageJ software (NIH, US).
2.4. Scanning Electron Microscopy (SEM)
Structural characteristics of the starch granules were examined with scanning electron microscope Jeol JSM-6400 (Jeol Ltd., Tokyo, Japan) and analyzed with an energy dispersive X-ray analyzer (EDS) PGT Spirit at an acceleration of 20 keV. Samples of starch were mounted on aluminium specimen stubs with double-sided adhesive tape and sputtered with a 20-30 nm gold layer using a sputter coater before observation.
2.5. Chemical Properties and Resistant Starch
Macrocomponents (total starch and amylose) and resistant starch were determined using the Megazyme assay kit with given procedures (Megazyme International Ireland Ltd., Bray, Ireland). Microcomponents, i.e., protein, lipid, and phosphorus, were determined the content by following the Official Method of AOAC International .
2.6. X-Ray Diffraction
Starch powders were scanned through the 2θ of 5°-45° using X-ray diffractograms (Xpert Pro MPD, Philips, Netherlands). Traces were obtained using a Cu-Kα radiation detector with a nickel filter and scintillation counter operating under the following conditions: 40 kV, 30 mA, scattering slit 25 nm, K-Alpha1 wavelength 1.78901 Å, K-Alpha1 wavelength 1.7929 Å, Ratio K-Alpha2/K-Alpha1 0.5, and scanning rate of 0.02°/min. The degree of crystallinity of samples was estimated and analyzed following the method of Zhang et al. .
2.7. Starch Gelatinization
Thermal properties of starches were studied using differential scanning calorimeter, DSC (Model-823e, Mettler-Toledo, Switzerland). Starch (~10 mg, dry weight) was placed into a 40 μL capacity aluminium pan with the addition of 70% distilled water to achieve starch-water suspension. The DSC analyzer’s calibration was conducted using indium, and an empty aluminium pan was used as a reference. Sample pans were heated from 25 to 120°C at the rate of 10°C/min. Onset temperature (), peak temperature (), conclusion temperature (), and gelatinization enthalpy () (J/g dry starch) were determined in triplicate.
2.8. Rheological Behaviour
Rheological properties of starches suspended in distilled water were determined by rotational rheometer (C-DG26.7/QC, RheolabQC, Anton Par Ltd, Germany). 6% () suspension of native starches were prepared by dispersing a suitable mass of dried starch granules in distilled water by a ratio of 1 : 17 with constant stirring. The viscosity (mPa s) and shear stress (Pa) were determined following the method by Chrungoo and Devi .
2.9. Statistical Analysis
The data recorded in all the tables were mean values and standard error. Analysis of variance (1-way ANOVA) was performed for the data and, if significant, followed by a post hoc Duncan’s multiple range test (DMRT) () using the SPSS 16.0 Statistical Software Program, IBM, Chicago, IL.
3. Results and Discussion
3.1. Starch Granule Morphology
Starch granules of plant species varied in size from 1 mm up to 100 mm, for taxonomic discrimination to be possible. Nelumbo nucifera rhizome had significantly larger starch granules with 20.96 μm. In contrast, the smaller granules were in C. esculenta corm and T. angustifolia pollen with 2.95 and 2.09 μm, respectively (Table 1). Pomeranz  categorized the starch granule size based on commercial starch into three groups; large, 15-100 μm (potato starch), medium-sized, 10-25 μm (maize or corn starch), and small, 3-8 μm (rice starch). From the starch classification, N. nucifera rhizome possesses large starch granules while N. nucifera seed has medium-sized starch granules that ranged 8.11-17.78 μm. The corms of C. esculenta, E. dulcis and S. sagittifolia, T. bispinosa seed, T. angustifolia rhizome, and pollen have small starch granules ranging 0.4-13.44 μm. The size of starch granules affects starch granules gels and paste performance as the larger the granule, the faster it swells, due to less molecular bonding than smaller granule . For example, potato starch possessed a large granule (15-100 μm), which resulted in faster gelatinization range (56-69°C). In contrast, the smaller granules of regular corn (5-25 μm) resulted in a slightly slower gelatinization range with 62-80°C (Pomeranz, 2019). In this present study, a large starch granule of N. nucifera rhizome gelatinizes faster than others. Besides, Pomeranz  also reported that small starch granules are relatively rare, which are suitable in dusting starches used in candy dusting, cosmetics, filling agent for the biodegradable polyethylene film, and tyre molding release agents. Also, in taro, its small starch has been proven to be easily digested, hence a potential commercial value in baby foods and patients with gastrointestinal problems for ease of bioassimilation . The granular structure and shape were also varied, as shown in Figure 1. Small granules of C. esculenta corm and T. angustifolia had predominantly polygonal and irregular shapes with few oval shapes. In contrast, the larger granules normally were observed with predominantly longitudinal and rod-shaped such as in N. nucifera rhizome. Tester et al.  reported that starch granules were as simple or compound. Some plant contains compound granules (C. esculenta corm and N. nucifera rhizome) due to the fusing of different granules developing simultaneously within a single amyloplast during biosynthesis . The rest of the species were as simple granules.
Granules size (μm)
The shape of starch granule
Small starch granule (3-8 μm)
Polygonal, irregular, and oval
Big elongated granule, small oval granule, and spherical with smooth surfaces
Round and oval with smooth surfaces
Centric and eccentric
Elongated with smooth surfaces
Round and oval with smooth surfaces
Polygonal, irregular, and oval
Medium-sized starch granule (10-25 μm)
Oval and ellipsoidal with a smooth surface
Large starch granule (15-100 μm)
Small oval granules, large longitudinal, rod-shaped granules
Centric and eccentric
Data are mean values of , and different superscripts (a>b>c>d>e) are significantly different (DMRT, ).