Morphological and Rheological Properties of Starches Separated from Cultivars of Rice (Oryza sativa L.) from North East India

Starch granules isolated from different accessions of rice from North East India showed a typical polygonal morphology with size ranging from 3.4 ± 0.8 μm to 6.4 ± 1.2 μm. The apparent amylose content (AAC) of the granules varied from 1.9% to 28.33%. Our results identify starch from IC583088 as “waxy” and IC-583085, IC-583038, DPRR-168 as “very low” amylo-. The coefficient of resistance to flow (n) for starch pastes from the varieties of rice studied in the present investigation recorded a value of <1.0. The observed “n” value deviates from the Newtonian flow indicating the pseudoplastic nature of starches isolated from these varieties. While starch pastes from the accession IC-583088 recorded the highest ‘‘n’’ value of 0.6, that from the accession IC-545197 showed the lowest value of 0.03. The variations in coefficient of resistance to flow clearly revealed a higher ‘‘n’’ value for starch pastes from the “waxy” and ‘‘low” amylo-cultivars than the “intermediate” or ‘‘high” amylo-cultivars. Our results clearly established characteristic rheological properties for starches from the accessions SKY-AK-1608, IC-583035, YS-RC-219, IC-564939 and IC-332963, which exhibited greater resistance to thinning and shearing than other varieties.


Introduction
Starch, an insoluble polymer that occurs in plant cells in the form of a complex granular mixture of amylose and amylopectin, is one of the major dietary sources of carbohydrates and the most abundant storage polysaccharide in plants.Starch is synthesized in amyloplasts in the form of granules with a partially crystalline texture.De-N.K. Chrungoo, A. G. Devi 2020 pending on the number of starch granules initiated in each amyloplast, they have been classified as simple, compound and semi-compound [1].Compound starch granules are composed of several sub-granules, each of which is synthesized simultaneously within a single amyloplast.Depending upon the species and the tissue, the granules vary in size from 1 to 100 µm and in shape from round to polygonal [2].Starch granules of wheat, barley, rye and triticale have been reported to show bimodal size distribution in the endosperm tissues and have been classified into two types viz., "A" type which is lenticular in shape with diameter ranging between 10 and 20 µm and "B" type which is spherical in shape with a diameter of <10 µm [3]- [7].Starches of rice and oat tend to exist in clusters of individual granules designated as compound granules [8]- [11]."C" type starches are considered to be a mixture of A-and B-type polymorphs.While the "A" and "C" type starches are characteristic of cereals and legumes, respectively, the "B" type structures are characteristics of starches from roots and tuber tissues [12]- [14].
The functional properties of starches from different sources vary considerably and are therefore unique for each type.These differences arise from variations in amylose to amylopectin ratio [15] [16], presence of lipids [17] and in morphology [18].According to Tester and Morrison [19] while starch swelling is a property associated with amylopectin, amylose in starch restricts it [15] [16].Thus, while low amylose flour is good for preparation of oriental noodles as well as for extended shelf life of bread, flour with high amylose content is good for fried foods [20] [21].
As the primary dietary source of carbohydrates for human nutrition, rice plays an important role in meeting energy requirements and nutrient intake [22].Juliano [8] has highlighted the unique characteristics of rice starch and has emphasized that it can be a better substitute for many other different types of starches including that from corn.North East India, being at the confluence of Indo-Malayan and Indo-Chinese biogeographic realms, covering an area of 2.55 × 10 5 km 2 from 21˚57' to 29˚28'N latitude and from 89˚40' to 97˚25'E longitude, represents one of the "biodiversity hotspots" of the world.Even though the region houses a large diversity of both upland and lowland cultivars of rice which show large variations in some of the key quality attributes including kernel length, aroma and cooking quality [23], there is no information on the quality characteristics of starches from cultivars of rice from this region.The paper reports the physico-chemical and rheological properties of starch from cultivars of rice from North East India differing in their amylose-amylopectin ratio.

Plant Material
Grains of different accessions of rice (Oryza sativa L.) from North East India, representing cultivars with varying apparent amylose content (AAC) of starch, were procured from the rice germplasm bank of North Eastern Regional Station of National Bureau of Plant Genetic Resources, India.The collection comprised of 24 upland and 20lowland accessions from the region.The collection was maintained ex situ in the experimental field of the University.Grains of each accession were raised to seedling stage in 6 cm deep plastic trays filled with 50:50 mixture of soil and compost which was irrigated with water to cover the soil surface.The seedlings were subsequently transferred to 2 meter × 2 meter low level beds raised for the purpose in the experimental fields of the University and maintained ex situ under irrigation till maturity at an average day temperature ranging between 19˚C to 22˚C.Transplanting of seedlings was done at a spacing of 20 × 15 cm with the soil submerged in 1 to 2 cm of water up to 10 days after transplanting.The seedlings were dipped in chloropyriphos: water (1:1000) solution before transplanting for preventing insect attack.Fertilizers were applied @ 80:60:40 kg NPK/ha in split dozes with P, K and 50 % of N applied as the basal dose, 25% N at maximum tillering and rest 25% N at panicle initiation stage.Grains were harvested at maturity from plants of each accession maintained in the experimental field.

Isolation of Starch Granules
Starch granules were isolated from mature grains of rice according to Takaoka et al. [24].The granules were homogenized to fine powder under liquid nitrogen in a chilled pestle and mortar.The powder was subsequently suspended in deionised water and the suspension maintained at 4˚C for 4 hours with occasional stirring.The suspension was filtered through a single layer of muslin and the filtrate centrifuged at 10,000 ×g at 4˚C for 10 min.The pellet was resuspended in appropriate volume of SDS sample buffer containing 60 mM Tris-HCl (pH 6.8), 10% (w/v) sodium dodecylsulfate (SDS), 3% (v/v) β-mercaptoethanol and 10% (v/v) glycerol and then pelleted by centrifugation at 30,000 g at 4˚C for 10 min.The resultant pellet was resuspended again in SDS sample buffer, pelleted by centrifugation at 30,000 ×g at 4˚C for 10 min and finally washed twice with deionised water followed by three washes with chilled acetone.The washed pellet comprising of purified starch granules was dried at room-temperature and stored at −20˚C.

Scanning Electron Microscopy of Isolated Starch Granules
Surface morphology of the starch granules was determined under a scanning electron microscope after spraying a thin layer of purified starch granules on 'brass stubs' and coating with a thin layer of gold vapour (300 Å) as per David et al. [25].The starch granules were subsequently observed in a JOEL-1850 SEM at magnification ranging between 7500× and 10000× operated at 20 kV.

Determination of Apparent Amylose Content
The apparent amylose content of starch was determined by the method described by Juliano [26].A suitable mass of starch was suspended in an appropriate volume of 95% ethanol: 1N NaOH (1:9).The suspension was mixed thoroughly and heated at 100˚C for 10 min.in a dry bath.A suitable aliquot of the suspension was mixed with 1N acetic acid and I 2 -KI solution and allowed to stand for 20 min. in dark at room temperature.Absorbance of the solution was recorded at 620 nm in Lambda650 UV/Vis Spectrophotometer (Perkin Elmer, U.S.A.) against a reagent blank with potato amylose (Sigma) as the reference standard.Starch isolated from grains of var.NDR97, a traditional variety of indigenous "Bororice" which is known to have low (4%) AAC [27], was used as a reference for purposes of comparison.

Determination of Rheological Properties
The rheologicalproperties of starches suspended in distilled water were determined by Rotational rheometer (C-DG26.7/QC,RheolabQC) according to Li et al. [28].An 8% (w/v) suspension of native rice starch was prepared by dispersing a suitable mass of dried starch granules in appropriate volume of MilliQ deionized water with constant stirring.After loading the starch suspension between the cone and plate, it was equilibrated to 50˚C for 1 min at 200 s −1 followed by increase in the temperature to 95˚C at the rate of 12˚C•min.−1 .The sample was held at 95˚C for 2 min.and 30 seconds after which the temperature was decreased to 50˚C at the rate of 12˚C•min.−1 .The sample was finally held at 50˚C for 1 min.The shear rate was maintained at 200 s −1 throughout the experiment.The pasting properties including onset temperature, peak temperature, peak viscosity, trough viscosity, final viscosity, breakdown viscosity and setback viscosity of each sample were inferred from acquired data.

Statistical Analysis
All the observations were recorded with 3 technical and 3 biological replicates.Statistical analysis of the data was carried out with SPSS package to obtain the values for standard deviation from the mean as well as least significant difference between accessions for grain size, AAC, and pasting properties of starch at 5% probability level.
5.5 mm were classified as short type.Further, Cruz and Khush [29] classified grains with L/W of >3.0, 2.1 to 3.0 and <2.0 as slender, medium and bold respectively.In conformity with this classification, rice grains from accessions studied in the present investigation can be classified into seven categories viz., long-slender (LS), longmedium (LM), medium-slender (MS), medium-medium (MM), medium-bold (MB), short-medium (SM) and short-bold (SB).The accessions also showed marked variations in the colour of pericarp (Table 1 and Table 2).
Starch granules isolated from different accessions, representing both upland and lowland cultivars of rice from North East India, showed a typical polygonal morphology (Figure 1 and Figure 2) with a mono-modal distribution and size ranging from a minimum of 3.4 ± 0.8 μm in IC-564948 to a maximum of 6.7 ± 1.2 μm in IC-540270 (Table 3).A distinct feature of starch granules isolated from accessions YS-RC-105, IC-321201 and IC-332963 was the presence of pores on the surface of the granules.No such pores were visible on the surface of   starch granules from other accessions.On the basis of their size, Lindeboom et al. [7] have classified starch granules as large (>25.0µm), medium (10.0 to 25.0 µm), small (5.0 to 10.0 µm) and very small (<5.0 µm).In conformity with this classification, starch granules isolated the accessions of rice investigated in the present study fall under the "small" to "very small" size category.Juliano [8], Yeh and Li [30] and Li and Yeh [31] have also reported the presence of small starch granules in rice with size ranging between 2.0 µm and 10.0 µm.Starch granule size has been described as an important parameter which has strong correlation with its pasting behaviour [32]- [34].
Marked differences were observed in the rheological properties of starches isolated from different accessions of rice studied in the present investigation.While the values for shear stress recorded a steady increase with increase in shear rate, the magnitude of increase varied from accession to accession.Thus, starch from the accession IC-583088 showed the lowest of 3.6 fold increase in shear stress and that from VP-44 showed the highest of 119 fold increase in shear stress with increase in shear rate from 0.054 to 100 s −1 (Table 4 and Table 5).The   --------------------------------------------- coefficient of resistance to flow (n) for the starch pastes recorded a value of <1.0, with starch paste from IC-583088 showing the highest value of 0.6 and that from IC-545197 showing the lowest value of 0.03.The observed "n" value deviates from the Newtonian flow, for which n = 1, thereby indicating the pseudoplastic nature of the starches isolated from these varieties.The non-Newtonian behaviour of starch pastes has also been reported by Rao et al. [43] and Nurul et al. [44].Our results on the variations in coefficient of resistance to flow (n) of starch pastes from different accessions of rice clearly revealed a higher "n" value for starch pastes from the "waxy" and "low amylo"-cultivars than the "intermediate" or ''high" amylo-cultivars, thereby indicating a positive relationship between amylose/amylopectin ratio of starch and its flow behaviour.Starch pastes from all the accessions of rice studied in the present investigation showed the shear-thinning phenomenon.This is evident from a decrease in apparent viscosity of the starch pastes with increase in shear rate, thereby increasing its fluidity.Our results on the variations in shear stresses clearly revealed that starch pastes from the "waxy" and ''low" amylo-cultivars exhibited higher shear stress than the "intermediate" or ''high" amylo-cultivars at the same shear rate.This indicates a clear relationship between the amylose/amylopectin ratio of starch grains and their rheological properties.Since higher values for shear stress indicate higher structural stability of the starch granules [45], it can be assumed that "waxy" and "low" amylo-starches have a more stable structure than those with intermediate or high AAC.Marked variations were observed in the pasting properties of the starches from accessions of rice studied in the present investigation.While the pasting onset temperatures of the "waxy" and "very low" amylo-cultivars were within the range of 61.6˚C -62.9˚C, those for the "low" and "intermediate" amylo-cultivars were in the range of 63.47˚C -66.36˚C and 67.26˚C -73.52˚C, respectively (Table 6 and Table 7).Our results on the past- 91.5 ± 0.4(l) 92.6 ± 0.0(1m) 92.5 ± 0.0(m) 92.12 ± 0.0(lm) 92.78 ± 0.0(m) 0.87 ± 0.0(j) 0.83 ± 0.0(ij) 0.82 ± 0.0(i) 0.81 0.65 ± 0.0(h) 0.66 ± 0.0(h) 0.62 ± 0.0(g) 0.61 ± 0.0(g) 0.61 ± 0.0(g) 0.60 ± 0.0(g) 0.58 ± 0.0(g) 0.48 ± 0.0(f) 0.43 ± 0.0(e) .042± 0.0(e) 0.39 ± 0.0(cd) 0.38 ± 0.0© 0.36 ± 0.0(bc) 0.37 ± 0.0(bc) 0.37 ± 0.0(bc) 0.36 ± 0.0(c) 0.   ing peak temperatures of starches from different cultivars of rice are in conformity with the temperature and viscosity pasting data reported for rice starches by Park et al. [15] and Li et al. [28].The highest peak viscosity and lowest setback viscosity shown by starch pastes from the accession IC-583088, which is a waxy cultivar, are consistent with the fact that waxy starch comprises mainly of amylopectin which allows the granules to swell easily.Similar observations have been made by Tester and Morrison [19] and Morrison [46] for other waxy starches.Since setback viscosity reflects gel network formation after starch pasting [28], the near absence of amylose in "waxy" starch will result in fewer interactions among close-packed granules, resulting in low gel network formation.Thus, because of their greater resistance to thinning and shearing, starches from SKY-AK-1608, IC-583035, YS-RC-219, IC-564939 and IC-332963 can be of importance to industry for various applications.

Table 1 .
Morphological features of grains of different upland accessions of rice from Northeast region of India.

Table 2 .
Morphological features of grains of different lowland accessions of rice from Northeast region of India.

Table 3 .
Size and apparent amylose content of starch granules isolated from endosperm tissues of grains of upland and lowland accessions of rice from North East India.† Same letters in parenthesis against data for each parameter indicates statistically insignificant difference (p < 0.05).

Table 4 .
Relationship between shear stress and shear rates of 4% (w/v) aqueous pastes of starches from upland varieties of rice from Northeast India.

Table 6 .
Pasting properties of starch isolated from upland varieties of rice from Northeast India.± represents standard deviation, † Same letters in parenthesis against data for each parameter indicates statistically insignificant difference (p < 0.05).

Table 7 .
Pasting properties of starch isolated from lowland varieties of rice from Northeast India.± represents standard deviation, † Same letters in parenthesis against data for each parameter indicates statistically insignificant difference (p < 0.05).