Characterization, Functional Properties, and Resistant Starch of Freshwater Macrophytes

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.


Introduction
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 [1]. 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 [2]. 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 [3], 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 [6]. Lotus seeds, consumed boiled or processed into powder, are also used in the pharmaceutical industry to treat inflammation, arrhythmia, cancer, and skin diseases [7]. Most of the starch's diverse uses are from cultivated species [8][9][10][11], and research on the wild species is still scarce [12].
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 highamylose starch. RS is poorly digested starches and absorbed in healthy individuals' small intestine due to its complex molecular structure [15]. 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.

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 [16] 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% (w/v) 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).

Polarized Optical Microscopy.
A small amount (0.2 mg) of starch powder was placed on a microscope slide (25:4 mm × 76:2 mm) 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).

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 doublesided adhesive tape and sputtered with a 20-30 nm gold layer using a sputter coater before observation.

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 [17].
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. [15].

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 (To), peak temperature (Tp), conclusion temperature (Tc), and gelatinization enthalpy (ΔH) (J/g dry starch) were determined in triplicate.  [20]. 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 [19] 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 [21]. 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. [22] 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 [23]. The rest of the species were as simple granules. Besides, some starch granules may still attach to other nonstarch components like fiber, lipid, or protein fragments despite undergoing isolating process, as shown in T. angustifolia seed starch (Figure 1(h)). According to Svihus et al. [24], these components, especially lipid and protein associated with granules, may influence digestion by reducing the contact between starch and digestive enzymes, thus slowing the swelling of the granules and gelatinization process. The position of the hilum or Maltese cross was also considered one of the unique identifications as it described the nucleus around which the starch form [25]. All the aquatic macrophytes starch samples showed distinct Maltese cross across the 3 International Journal of Food Science hilum when viewed under a polarized microscope. Roundand oval-shaped granules were with centric hilum, such as in corm of C. esculenta and E. dulcis, and rhizome of T. angustifolia. On the other hand, hilum was observed eccentrically at the small end of the elongated granules of S. sagittifolia corm and N. nucifera rhizome. Large granule like potato starch displayed pronounced striations around the central hilum where it is the botanical point of origin for the granule to grow [19], which can be seen in the rhizome of N. nucifera and the seed of T. bispinosa (circled granules) even though the layer not as distinct as reported in potato.

Chemical
Properties. The starch paste behaviour and functionality depend on the composition of amylose and amylopectin and the microcomponent as such affects the final food quality. Table 2 shows that the moisture content of the starch flours from the present study is in the range between 8.91% of T. angustifolia pollen and 17.23% of E. dulcis corm starch. A higher total starch was in corm samples, including E. dulcis (90.87%), C. esculenta (82.35%), and S. sagittifolia (71.71%), whereas the T. angustifolia pollen has a significantly least starch content (7.02%). Based on previous studies, Typha sp. generally had low starch contents ranging between 13.01% and 14.5% compared to other commercial starch such as Zea mays (22.40%) [26,27]. The composition of amylose and amylopectin gives variable effects of starch swelling capacity, water solubility, water-binding capacity, and microscopic characteristics, hence carried different industrial applications [28]. Normal starch generally contains 75% of amylopectin and 25% of amylose, while waxy starch contains <15% amylose and 20-35% amylopectin. Regular starch and high-amylose starches contain >40% amylose [22]. The present study shows that N. nucifera seed had high-amylose content with 71.45%, whereas T. angustifolia pollen and S. sagittifolia corm were categorized as regular starches with an amylose content of 36.47% and 19.80%, respectively. The rest were classified as waxy starch,   [30]. A starch with high-amylose content usually had a small and narrow size range with nonsymmetrically shape granules [31]. Similarly, such characteristics of starch granules of N. nucifera seed and T. angustifolia pollen also possess a higher amylose content. The high amylose of N. nucifera seed starch can be applied as a gelling agent or in the biodegradable plastic film industry. The chemical properties of the less-branched amylose molecule also provide beneficial effects to human health. Meals prepared with high amylose slow the rate of digestion, as it possesses the higher crystallinity which generated a postprandial glucose response in the attributed blood and lessened the fasting concentration of triacylglycerol and cholesterol [32]. Consequently, highamylose N. nucifera seed starch may reduce the blood glucose and insulin level and enhance the body's fat burning.
In contrast, waxy starch with higher amylopectin which possesses branched and long chain length greater than 30 chains yielded higher molecular weight. Thus, this branched chain hindered each particle from reassociating and formed an ordered structure that resulted in weaker hydrogen bonding and the strength of the gel [20]. Amylopectin with the clustered structures reduced retrogradation, gelation, and syneresis (loss of water). However, they are better at building viscosity and the production of more slimy paste. This suggested that waxy starch in this study are suitable in making soft-textured food such as dessert-like products, baby foods, gravies, sauces, and thickening soup due to its excellent water retention properties, porous texture, and stickiness criteria [33]. Generally, foods with higher amylopectin have a higher glycemic index as they are readily digestible and absorbed after consumed. Thus, it is advisable to opt for food lower in amylopectin to prevent the raising of blood sugar, insulin, and cholesterol levels. However, recent studies show that scientists have devised various ways to minimise food digestibility for waxy starches via an enzymatic process, encapsulation, extrusion cooking, recrystallization, and starch chemical modification [33]. Under this new development, the modified waxy starches such as wheat, rice, and maize have great potential as novel functional food ingredients, which also equally applied for waxy aquatic macrophytes starches.
The minor components analysis showed higher protein accumulated in T. bispinosa seed (26.47%) followed by T. angustifolia pollen (15.70%) starches, whereas the least was from S. sagittifolia corm with 3.06%. Higher lipid contents were in T. angustifolia rhizome and pollen (3.87% and 3.23%, respectively) and N. nucifera seed (2.96%). According to Tester et al. [22], proteins and lipids can limit the starch functionality as they can associate with starch granule on the surface and internal components. Protein contents also influenced the pasting activity as higher protein will build up the interaction's binding forces, thus reducing the pasting temperature [34]. The presence of flexible protein molecules may reduce the water's surface tension and increase the foaming tendency, which is important in maintaining the structure and texture of food products such as baked goods and ice cream [34]. However, higher protein may also have undesired side effects. It can produce browning and off-flavor in foods due to its positive reaction with major food components such as reducing sugars, polyphenols, fats, and other oxidation products [25]. Svihus et al. [24] reported that smaller granules usually have higher lipid contents, which are also observed in small granules of T. angustifolia pollen and rhizome. Starchlipid compositions used in several applications, such as stabilizers and thickener agents, reduced starch stickiness, dough conditioners, enhanced freeze-thaw stability, detain bread, and biscuit staling and bread crumb softener [35]. Table 3 represents resistant (RS) and nonresistant starch (non-RS) content in the freshwater macrophytes starch samples in comparison with previous studies. Higher RS accumulated in the rhizome part of N. nucifera and T. angustifolia (39.34% and 37.19%, respectively). These values are comparable with C. esculenta (35.19%) and Trapa sp. (36.80%) starches from previous studies [35,36]. Lower RS was observed in T. bispinosa seed with 6.92%, whereas S. sagittifolia corm starch had a comparable value of RS (35.39%) and non-RS (37.37%). Besides, C. esculenta starch had significantly higher non-RS compared to 5 International Journal of Food Science others. Generally, high-amylose starch possessed high RS; however, in the present study, the N. nucifera seed had higher digestible starch (44.36%) compared to its RS (27.94%). The present study showed that RS was higher mostly in the rhizome part compared to others due to their B-type crystallinity. Densely packed B-type and CB-type crystallinity patterns are poorly susceptible to hydrolysis containing higher RS compared to A-type [37]. They further reported that high-amylose starch tends to possess higher RS as studied in cereal starch due to low digestibility. This factor can benefit the consumer interested in a lowcarbohydrate dieting regime as slow digestion of RS (digestion occurs after 5-7 hours) can lower insulin and glycemic response and accelerate the satiation level [38]. The variations in resistant starch content between starches of the same species with previous studies (Table 3) were due not only to the chemical parameters (e.g., amylose and phosphorus contents) but also to the physical and structural characteristics such as shape and of granule, molecular interaction and crystallinity pattern of each plant [39]. Also, RS brings benefits in the functional properties of starch in terms of granules swelling, viscosity paste, the formation of gel, and waterbinding capacity [37]. The fine, white, and tasteless characteristic of RS particles and its crispness properties make it easy to process and improve the texture, appearance, and mouthfeel in the final product. Silvi et al. [40] reported that RS is an excellent dietary supplement because it can increase good prebiotic microorganisms and reduce the population of harmless Enterobacteria in the colon. However, excessive consumption of RS, mainly the raw starch, will have adverse  [45] Data are mean values of n = 3 determination ± standard error, and different superscripts (a>b>c>d>e>f) are significantly different (DMRT, p < 0:05).

6
International Journal of Food Science effects on gastrointestinal performance such as bloating, flatulence, and borborygmi (stomach rumble due to fluid and gas movement in the intestines) and diarrhea. According to Food and Agriculture Organization (FAO) and World Health Organization (WHO), adequate intakes for total fiber are differed based on gender, with 38 g for men, while 25 g for women [41]. Baghurst et al. [42] reported total intakes of approximately 30-40 g per day of RS consumed in developing countries. A 20 g of RS per day consumption is the recommendation by Australia's Commonwealth Scientific and Industrial Research Organization (CSIRO). This amount is much higher than those in Europe of 3-6 g/day due to accessibility in bread and cereals [42] 3.4. Starch Crystallinity. The XRD profiles of aquatic macrophytes are presented in Figure 2, which exhibited wellresolved and intense peaks based on their parts. There are three types of XRD patterns categorized based on their absence of a peak at a certain different angle. The A-type X-ray pattern is characterized with a strong double peak at 2θ ≈ 17°and 18°, and strong diffraction peak at 2θ ≈ 15°and 23°, whereas the B-type pattern showed pronounced peak diffraction peak at 2θ ≈ 15°and 17°, as well broad diffraction peak at 2θ ≈ 23°, with smalls peak around 15°, 20°, 22°, and 24°2θ [22,46]. The C-type is a mixture of A-type and Btype which can be further categorized to C A -type if closer to A-type and C B -type if closer to B-type, or prominent Ctype if the proportion of A-and B-type allomorphs are equal [46]. According to Kim et al. [47], the X-ray diffraction pattern indicates the parallel stranded double helices where Atype structure is more compact than the looser crystallinity of B-type with a more open structure. Furthermore, seed starches generally possessed A-type pattern, whereas tuber and high-amylose cereal starches mostly showed B-type patterns [48].
Based on the present study, corm samples and seed of N. nucifera displayed A-type with the presence of peaks intensity at 2θ ≈ 15°and 23°and strong doublet around 17°and 18°2θ  (Table 4), consistent with the previous studies of arrowhead [49], Chinese water chestnut [50], taro [51], and lotus seed [48]. Trapa bispinosa exhibits A-type characteristic with small peak intensity at 2θ ≈ 20°(24.73%) which results in C A -type similar to the water caltrop species (T. bispinosa, T. quadrispinosa, T. pseudoinisa, and T. taiwanensis) of previous studies [12,[52][53][54]. Furthermore, all the rhizome parts and T. angustifolia pollen starch exhibited C B -type crystallinity with peaks at 5°, 20°, and 24°2θ (5.96). Both B- [55,56] (additional) and Ctype [48,57] XRD patterns in lotus rhizome suggest that temperature and other factors influence the crystalline order of lotus rhizome [56]. The XRD pattern of cattail rhizome is contrasting with previous literature in which there was the presence of A-and B-type XRD; however, higher B-type allomorphs were reported compared to A-type [14]. The XRD pattern of rhizome and pollen of cattails in the present study is poor based on their peak intensity values, caused by other impurities in starch granules.
A relatively higher crystallinity was in C. esculenta corm (36.91%), followed by T. bispinosa seed (35.59%). There was a lower relative crystallinity in T. angustifolia pollen (15.27%) and rhizome (15.56%). The value of relative crystallinity is directly proportional to the amylose content. It expends higher relative crystallinity in the waxy starch of C. esculenta corm and T. bispinosa. The lower amylose content reduced the disruption of crystalline nature and the amorphous region within the starch granules [57].
3.5. Thermal Properties. Table 5 shows a summary of the thermal properties results for the starch samples. The starches gave well-defined single endotherms, where the gelatinization onset (To), peak (Tp), and conclusion (Tc) temperatures of the     [50]. In this present study, a higher To and Tp were in T. angustifolia rhizome, 58.73°C and 96.02°C, respectively. The formation of amylose and lipid complex in starch is insoluble in water. A higher temperature is required to dissociate the formation and increase the individual granules' swelling to diffuse out the amylose from swollen granules [58]. On the other hand, the Tc was observed higher in most of the smaller granules of E. dulcis corm (119.58°C), T. angustifolia rhizome (119.56°C), and C. esculenta corm (119.54°C). The small-sized granules contain a more significant number of granules per unit weight, which needed higher temperatures to disintegrate the starch particles into paste [9]. Tester and Morrison [59] explained that gelatinization enthalpy (ΔH) reflects the loss of molecular order within the granules. The high ΔH lowers the starch crystallinity degree (quality and quantity of crystallites  (Table 6). Both possess a waxy structure characterized by amylopectin, which yielded larger molecules than amylose [20]. On the other hand, lower viscosity was observed in the T. angustifolia pollen (3.2, 3.0, and 2.9 mPa s) due to low-amylose content, as it inhibits the swelling of granules, thus lowering the viscosity [67]. Also, T. bispinosa seed possesses the lowest viscosity and shear stress (8.8 mPa s/13.72 Pa) despite having high amylopectin content. According to Singh et al. [58], higher phosphate monoesters in water caltrop may associate with amylopectin hence increase the starch viscosity while phospholipids caused opaque and decrease the viscosity pastes. The author also mentioned that starch's protein content is contradictory with peak viscosity and correlated with pasting temperature. Thus, the high phosphorus and protein content of T. bispinosa seed starch contribute to its low viscosity.

Conclusions
Starches from T. angustifolia (cattails) and S. sagittifolia (arrowheads) have comparable potentials to commercial aquatic macrophyte starches from N. nucifera (lotus), E. dulcis (Chinese water chestnut), T. bispinosa (water caltrop), and C. esculenta (taro) in food formulation. Although the least starch was isolated from T. angustifolia pollen, its higher amylose content (36.47%) provides excellent human International Journal of Food Science digestive tract benefits. Higher RS content was in tuber parts, i.e., rhizome and corm, than in the seed and pollen of freshwater macrophytes. Lower RS was found in N. nucifera seed starch (27.94%) compared to others despite having higher amylose. Starch isolation procedure of T. angustifolia needs to be improvised, especially on the starch washing to prevent the impurities affecting the functional properties. Simultaneously, the procedure also must be cost-effective and efficient to be commercialized in the large-scale industry. This functionality of the native starches can later be modified and improved physically, chemically, and enzymatically which enhanced its properties and to produce novel molecules and hence can be applied in a broad range of food and nonfood applications. The starch properties in aquatic macrophytes studies showed equally good potential as the commercial starches in starch-based products.

Data Availability
The data used to support the findings of this study are included in the article.

Conflicts of Interest
The authors declare no conflicts of interest.