Extraction, characterization, physicochemical and rheological properties of two different varieties of chickpea starch

The variations in physiochemical, rheological, morphological, and thermal properties of extracted starches of two chickpea varieties (PDG‐5 and BG‐1076) were evaluated. Both the varieties resulted in starch yields 28.2–31.2 g/100 g. Amylose content was 30.2–31.2%. Morphological study by scanning electron microscopy of starches of both the varieties of chickpea revealed the presence of oval, spherical shaped granules with smooth surfaced, with mean granular length of 2 to 30 μm and width of 3 to 10 μm. Rheological properties of extracted starches at selected concentration (1–5%) were evaluated in flow viscometry and dynamic oscillatory mode to assess flow properties in terms of shear stress, shear rate, apparent viscosity, elastic, and viscous modulus. Fourier transform infrared spectroscopy showed the presence of polysaccharides bands. X‐ray diffraction patterns showed that extracted starches are mixture of both A‐ and B‐type crystal. Thermal and pasting properties of chickpea starches were determined using differential scanning calorimeter (DSC) and rapid visco analyzer, respectively. Onset (T0), peak (Tp), end (Tc) temperature, enthalpy of gelatinization (ΔH), peak height index, and gelatinization temperature range were measured by DSC. DSC thermograms revealed that T0, Tp, and Tc values of chickpeas were ranged from 65.6°C to 66.5°C, 69.6°C to 71.1°C, and 75.6°C to 76.2°C, respectively, whereas the corresponding enthalpy values varied between 14.8 and 15.6 J/g. The viscosity values at the peak, hold, final, breakdown, and setback viscosity for PDG‐5 variety were 2,562, 2,200, 3,646, 852, and 1,456 cP, respectively, and the corresponding values for BG 1076 were 4,848, 3,416, 6,384, 1,452, and 2,978 cP. It was observed that starches from BG‐1076 variety with low swelling power had higher solubility and pasting temperature.

good physical and sensory properties, as well as an enhanced nutritional composition.
After cellulose, starch is the second most plentiful organic compound in the earth. Starch is also the most essential polysaccharide of human diet. Starches from different sources vary its composition in qualitative and quantitative makeup as well as in some of their physicochemical properties. Apart from its dietetic uses in the food industry, starches also have significant nonfood industrial applications (Ashogbon, 2018).
Utilization of starch in food matrices are chiefly controlled by its ease of aqueous solubilization, swelling, gel formation, gelation temperature, flow behavior, viscosity, pasting properties, digestibility, and thermal property. Whereas starch is the most essential constituent of chickpea, comparatively very few methodical studies have been reported. Therefore, our specific objectives were to isolate the starch from chickpea varieties PDG-5 and BG-1076 and its characterization for its appropriate food and nonfood applications. Physiochemical, rheological, X-ray diffraction, Fourier transformation infrared (molecular properties), scanning electron microscopy (morphological properties), and differential scanning calorimeter (DSC; thermal properties) were determined.
The entire reagent used was of analytical grade and obtained from Loba Chemie laboratory reagents and fine chemicals, Mumbai, India.

| Methods
Both varieties of chickpea seeds were dried in a tray dryer at 60 C for 16-17 hr to reduce moisture content. Dried sample was then grinded into fine powder for further processing. Moisture, ash, protein (N × 6.25), fat, and crude fiber were estimated using Association of Official Analytical Chemists method, and carbohydrate was determined by difference. Experiment was done in triplicates.

| Extraction of starch from chickpea seeds
Two different cultivars of chickpea seeds (BG-1076 and PDG-5) were purchased from the Panjab Agriculture University, Ludhiana, Panjab, India. Starch was extracted from the chickpea seeds using minor modification of alkaline soaking method (Gunaratne et al., 2018). A total of 100-g chickpea seeds were soaked in a 500 ml of 0.05% sodium hydroxide solution at 4 C for 15 hr in a refrigerator to break down the starch-protein matrix. Soaked seeds were hulled and blended with sodium hydroxide solution for 4 min using the laboratory blender. The resulting mixture was screened through 100 mesh sieve or nylon cloth (100 mesh), and supernatant was discarded after 16 hr of precipitation at 4 C. Neutralization of starch precipitates was done with 0.01% Hydro Chloric Acid (HCL) followed by centrifugation at 3,000 g for 30 min at room temperature. The supernatant was removed and further purified with repeated suspension in distilled water and centrifugation for 3-4 times. The extracted starch was washed 3-4 times with distilled water and purified starch was dried at 40 C for 12 hr and stored at cool and dark place for further use. Extraction was done in triplicates.

| Amylose content
Amylose content of extracted starch was determined using the method described by . The absorbance was read at 620 nm using ultraviolet-visible spectrophotometer (UVPC 2410, Simudzu, Japan) and calculated using Equation (1). Experiment was done in triplicates.

| Swelling power and solubility
Swelling power and solubility of starch was measured according to  using 1% aqueous solution of starch. Experiment was done in triplicates.

| Water binding capacity
Water binding capacity (WBC) of the starches from both chickpea varieties was determined in triplicate using the method described by  with minor modification. A suspension of 4 g of starch (dry weight) in 60-ml distilled water was agitated for 1 hr and centrifuged (3200 x g) for 10 min. The free water was removed from wet starch, drained for 15 min, and wet starch was weighed.
Experiment was done in triplicates.

| Turbidity
Turbidity of starches from both the chickpea varieties was measured in triplicate, as described by Singh and Kaur (2017) by measuring absorbance at 640 nm against water in a spectrophotometer (UVPC 2410, Simudzu, Japan).

| Thermal properties
Gelatinization parameters of starch were measured using DSC equipped with thermal analysis data software (Fox Pro Netzsch, Germany) and was analyzed according to Ghoshal and Mehta (2019) with minor modification. A total of 10 mg of sample was taken in the aluminum pan, and 20-μl distilled water was added, sealed hermetically. The scanning temperature range was 10-90 C, and heating rate was 2 C/min. An empty pan was used as reference, and gelatinization, onset temperature, peak temperature, end temperature, enthalpy, and specific heat were determined from the thermal properties results.
Experiment was done in triplicates.

| Pasting properties
Pasting properties of starch was measured using a rapid visco analyzer (RVA, Pertain Instrument, Australia) according to the method of Gunaratne et al. (2018) with minor modifications. A total of 3 g starch (10% moisture basis) was mixed with distilled water (25 g) in the RVA canister to obtain a total constant sample weight of 28 g (10.7% starch concentration). The slurry was then manually homogenized using the plastic paddle to avoid lump formation before the RVA run.
A programmed heating and cooling cycle was set for 24 min, where it was first held at 50 C for 1 min, heated to 95 C in 9 min, hold at 95 C for 2 min, cooled to 50 C within 9 min, and held at 50 C for 1 min. Heating and cooling rate was 5 C/min. All measurements were done in triplicate. Pasting parameters such as pasting temperature, peak viscosity, hot paste viscosity, and cold paste viscosity were directly obtained from the instrumental software. The breakdown viscosity and setback viscosity were calculated by the difference of pasting temperature, peak viscosity, hot paste viscosity, and cold paste viscosity. Experiment was done in triplicates. was also done to determine the gelatinization temperature and to check the thermal stability of the sample. The temperature was enhanced from 10 to 90 C at a heating rate of 2 C/min, constant frequency of 1 Hz at 0.1% strain, and G 0 and G 00 were determined. The rheological measurements were conducted in triplicate on different days and rheological parameters elastic modulus G 0 and viscous modulus G 00 , complex viscosity ɳ* were obtained directly from the software attached with the rheometer (MCR 102, Anton paar, Austria) and were analyzed by rheoplus software (Ghoshal, Shivhare, & Banerjee, 2017). Experiment was done in triplicates.

| Power law modeling
Polymeric solution and molten polymers are characterized by the complex viscoelastic properties. This phenomenon is determined by power law equation.
where γ is shear rate, τ is shear stress, K is consistency index, and n is power law exponent (flow behavior index). If n < 1.0 corresponds to shear thinning behavior or pseudoplastic behavior, n > 1.0 corresponds to shear thickening (Ghoshal & Mehta, 2019).

| Fourier transform infrared spectroscopy
The infrared spectra were recorded at room temperature (28 ± 2 C) using a Fourier transform infrared spectroscopy (FTIR) spectrophotometer (Tensor-27 model, Bruker Germany) in the range of 400-4,000 cm −1 by accumulating 16 scans of 4 cm −1 resolution.
About a pinch of powder chickpea starch was placed on ATR for measurement. After that, peak intensity of each samples were measured, and these entire spectra acquisition procedure took 1 min per sample (Ghoshal et al., 2017;Ghoshal & Mehta, 2019).

| X-ray diffraction
X-ray diffraction (XRD) of chickpea starch were performed using (X'pert PANalytical) equipped with Cu Kα1 (λ = 1.5406 Å). The instrument was equipped with graphite monochromator and operated at 40 kV and 30 mA.
The X-rays get scattered from a crystalline solid interfere constructively and produce a diffracted beam of light. 2θ is the angle of scanning for a sample, and its range is different for different samples, and for chickpea starch sample, it was scanned from 10 to 80 (2θ).

| Scanning electron microscope
Scanning electron microscope (SEM) analysis of starch was performed using scanning electron microscope (S-3400N, Hitachi, Japan). Each sample was coated with gold in a sputter coater before scanning. Gold

| Statistical analysis
The data reported in the table were the average of triplicate observation and were analyzed by one-way analysis of variance using Microsoft Excel. Statistical significance was determined taking 95% confidence level and p < .05.

| RESULTS AND DISCUSSION
The   (2019) studied the effect of particle size of quinoa starch on proximate analysis and they found that starch, protein, fat, dietary fiber, and moisture loss increased with decreasing particle size.
Amylose content for PDG-5 and BG-1076 are 30.2% and 31.3%, respectively. BG-1076 had higher amylose content than that of PDG-5. Earlier report exhibits the range of chickpea starches was in between 20.7 to 35.5%. Swelling power and solubility of extracted starches are 11.92%, 13.47% and 12.11%, 13.53% for PDG-5 and BG-1076 varieties, similar as reported by . Swelling power of starches was found to be lower might be due to the presence of large number of crystallites formed in association with amylopectin chains also increase stability of starch granules (Tester & Morrison, 1990). BG-1076 has slightly higher swelling power and solubility than PDG-5, and it is directly proportional to amylose content.
According to Schoch and Maywald (1968), chickpea has limited swelling power, solubilization, and stability against mechanical shearing due to the presence of large number of long chain amylopectin crystals.
Turbidity value was determined at 640 nm in ultraviolet-vis-spectrophotometer, and the absorbance values were 2.367 and 2.432 for PDG-5 and BG-1076, respectively. Perera and Hoover (1999) observed increased turbidity during storage of chickpea starch.  .

| Pasting property
The pasting properties of both PDG-5 and BG-1076 starch cultivars have been determined by using rapid visco analyzer. Pasting temperature of PDG-5 and BG-1076 were found at 81 C and 76.5 C, respectively. Srivastava, Harse, Gharia, and Mudia (1970)

| Thermal analysis of chickpea starch
Thermal properties of chickpea starch were measured using DSC (Fox Pro Netzsch, Germany) equipped with a Proteus software for data analysis. The scanning temperature range was 10-90 C, and

| Rheological property of chickpea starch
The flow behavior of extracted starch PDG-5 at different concentrations (1%, 2%, 3%, 4%, and 5% w/v) was shown in Figure 2, it showed pseudoplastic behavior of extracted starch PDG-5, when shear rate was increased, the viscosity of extracted starch show a significant decrease for all the concentration. This study shows that pseudoplastic behavior has been observed for BG-1076 extracted starch at (1%, 2%, 3%, 4%, and 5% w/v) concentration at room temperature. Figure 2a,

| Power law modeling
The graphs have been plotted between log γ versus log τ, it is linear and it exhibited the applicability of power law equation and also well- F I G U R E 2 Comparisons of flow curve of 1%, 2%, 3%, 4%, and 5% of (a) PDG-5 and (b) BG 1076 the varieties. Table 3 represents that the value of n and R 2 are less than 1, showed non-Newtonian pseudoplastic behavior at all concentration. Using the coefficient K and n, the shear stress values were recalculated, and it was found that the values are almost similar and described the adequate fitting of power law model in flow behavior of extracted chickpea starches.

| Frequency sweep
Frequency sweep test was done to calculate the gel structure at linear viscoelatic region. As polysaccharides are viscoelastic material, they exhibit solid and liquid characteristics simultaneously, and moduli G 0 Gel strength increases with increase in starch concentration; at higher concentration, more difference between G´and G´´was observed.
The frequency sweep of PDG-5 ( Figure 3a) starch indicates an increase in the value of both G 0 and G 00 with increasing frequency from 0.1 to 10 Hz. In all the graphs, the value of G 0 is greater than G 00 .
This indicates that storage modulus is prevailing over loss modulus.
Both G 0 and G 00 increased with increase in frequency . The difference between G 0 and G 00 indicates the gel structure. The gel strength increases with increase in sample concentration; the similar results were obtained by Singthong, Cui, Ningsanond, and Goff (2004). Singh and Kaur (2017) reported earlier similar result that G 0 and G 00 are frequency dependent. Figure 3b represents the frequency sweep curve for 1%, 2%, 3%, 4%, and 5% w/v of BG-1076 chickpea starch. At all concentrations (1%, 2%, 3%, 4%, and 5% w/v) the value of G 0 and G 00 increased with increase in frequency as well as G 0 and G 00 increase with concentration. At all the concentration the value of G´is higher than G´´. Both G´and G´´increased slowly and steadily from 0.1 to 10 Hz except 1% concentration after 10 Hz frequency; the G´and G´´both increased sharply. This is very prominent in PDG-5 variety. Ahmed et al. (2008) explained that abrupt increase of G´might be due to the formation of 3D network structure developed by leached out amylose and reinforced by strong interaction among the swollen starch particles.
Until 10 Hz, no crossover was observed. Earlier study on different oat cultivars showed that the value of G 0 and G 00 increased with increase in frequency and the values of G 0 were much greater than G 00 at all frequency values showing the strong elastic behavior of starch sample (Singh & Kaur, 2017). Liu et al. (2018) reported for wheat dough; they explained that the value of G 0 and G 00 increased with increase in frequency. Whereas complex viscosity, ɳ* values gradually decreased with increasing frequency values from 0.1 to 10 Hz (not shown) indicating frequency dependency of starches. Ahmed et al. (2019) reported with increasing frequency, ɳ* value decreases, but during heating after gelatinization, ɳ* values increased with increasing frequency that further decreases with frequency increase due to breakdown of starch structure.

| Temperature sweep
Temperature sweep was done to determine gelatinization and to check the thermal stability of both the varieties of starch. For temperature sweep, test temperature was raised from 10 C to 90 C using heating process at different concentration 1%, 2%, 3%, 4%, and 5% w/v with heating rate of 2 C/min. Significant increase in G 0 might be a formation of 3D network of the swollen granules .  and then formation of 3D network structure. The result shows that if temperature increased further then G 0 and G 00 started decreasing.
Similar result reported by Gupta, Bawa, and Semwal (2009). In PDG-5 variety, peak temperature is little higher than BG-1076 variety, and it is matching with DSC and pasting results. In both the varieties at lower concentration of starch, the peak value was little lower than at higher concentration, and peak area was also higher at higher concentration than at lower concentration. In both the varieties of chickpea starch, amylose content is higher than other cereal flour, and therefore, hard gel formation occurs and credited to higher modulus value G´and G´´. Heating beyond peak temperature cause decrease of Gá nd G´´value that might be due to destruction of gel structure and eventually melting of crystalline region remaining in the swollen particles that soften the particles. According to Ahmed et al. (2019), the 3D network disintegrates due to the failure of interaction between particles and network.

| X-rays diffraction study
X-rays diffraction provides detailed information of the crystal struc-

| Morphological study (SEM)
The Variation of size and shape is more prominent in BG 1076, whereas PDG-5 has less variation in size and shape. The result of SEM revealed that the PDG-5 and BG-1076 starch had the almost same smooth surface; no cracks were observed. Hoover and Ratnayake (2002) Kim et al., 2007).

| FTIR study
FTIR spectrum of chickpea PDG-5 starch (Figure 5c,A) Figure 5c,A,B, F I G U R E 3 Frequency sweep curve of 1%, 2%, 3%, 4%, and 5% of (a)PDG-5 and (b) BG-1076 chickpea starch at 10 Hz numerous small peaks are present that indicates presence of various polysaccharides. The band ranged between 3,435 to 3,294.9 cm −1 has been attributed to stretching of surface hydroxyl (O─H) group and hydrogen bonding of O─H stretching vibration and a moist material characteristics, chemisorbed water and indicates the presence of alcohol and phenols (Akhtar et al., 2018). We can correlate with shear thinning behavior and pseudoplastic behavior of chickpea starch solution. Also in variety BG1076, peak height is smaller than PDG-5 in Figure 5c, and it has low swelling power but higher solubility and pasting temperature eventually higher viscosity than PDG-5 variety. The range 2,932.7-2,935.1 cm −1 has been attributed to ─CH 2 stretching the peak observed at 1,640.8 to 1,645.2 cm −1 is due to bending of water molecules (H─O─H) and at 1,417.2-1,418.7 cm −1 is due to ─CH 2 bending and C─O─O stretching of carboxyl group (Shabaani et al., 2018;Dankar, Haddarah, Omar, Pujolà, & Sepulcre, 2018). The peak range between 1,357.6 and 1,361.7 cm −1 is observed due to C─H (asymmetric) bending of CH 3 . Peak at 1,244 cm −1 is indicating CH 2 OH (side chain)