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2. Materials and methods.2.1. Materials.Mung bean starch was purchased from Hada Starch Factory.Harbin China (,). Moisture fat, protein and ash, of the native mung.Bean starch (P. Radiatus) were 12.93% 0.13% 1.34% and 0.15%,,,,Respectively. Resistant Starch Assay Kit was purchased from Megazyme.International Ireland Ltd. Wicklow Ireland (,). Standards.Amylose and amylopectin were obtained from Sigma Chemical.Company (St. Louis MO USA,,). Chemicals and solvents used in this.Work were of analytical grade.2.2. Heat-moisture treatment.The moisture levels of starch samples were adjusted to 15% 20%,,25% 30% and, 35% (the moisture level of native starch was predetermined).By dispersing in appropriate amount of distilledwater.All samples were then held in sealed containers (500 mL) and kept.For 24 h at ambient temperature. The sealed containers were.Heated in a thermostatically controlled convection oven (DHG -.9140A Shanghai Shengxian, Instrument, Manufacturing CompanyChina) at 120 C for 12 h. All containers were cooled to ambient.Temperature. All samples were removed from the containers and.Dried at 45 C for 12 h to achieve uniform moisture content (w8%).Based on the treatment moisture content the HMT, starch samples.Will be referred to as HMT-15 HMT-20 HMT-25 HMT-30 and,,,,HMT-35.2.3. Resistant starch determination.Resistant starch content was assessed using Resistant Starch.Assay Kit based on the AOAC (2002.02). In, brief starch (100 mg).And 4 mL of enzyme mixture (pancreatic a-amylase 10 mg / mL and,,,Amyloglucosidase 3, U / mL) was added to each test tube and then,,Incubated in a shaking water bath (Wisebath @, Feedback Control.Digital Timer Function Sweden), for 16 h (37, C 200 strokes / min).To hydrolyze digestible starch. The resistant portion was precipitated.With 95% ethanol and the residue obtained was washed with.50%, ethanol twice and treated with potassium hydroxide solution.(4 M 2 mL), to solubilize the RS. The RS solution obtained was.Adjusted to pH 4.75 with 8 mL of 1.2 M sodium acetate buffer (pH.3.8). After incubation with amyloglucosidase (0.1, mL 3300 U / mL).At 50 C for 30 min the samples, were centrifuged at 3000g for.10 min. 3 mL of Glucose-oxidase-peroxidase-aminoantipyrine.(GOPOD) was added to aliquots (0.1 mL) of the supernatant and the,,Mixture was incubated at 50 C for 20 min. Absorbance was.Measured using a spectrophotometer (Model 722 Shanghai,,Analytical Instrument Company China), at 510 nm.2.4. Apparent amylose contents.Apparent amylose contents in the samples were determined.According to the procedure of Juliano et al. (1981).2.5. Scanning electron microscopy (SEM).Starch samples were prepared by sprinkling the starch on.Double-sided adhesive tape attached to a circular, aluminum stubAnd then coated with 20 nm gold under vacuum. The samples were.Viewed and photographed with a scanning electron microscope.(model, S-3700 N Hitachi Japan), at an acceleration potential of.20 kV and magnification of 1000.2.6. Polarized light microscopy.Birefringence of native and modified mung bean starch granules.Were observed under an optical microscope (model BH-2 Olympus,,Japan). All samples were dispersed in solution (glycerine / deionised.Water; 1: 1 V / V) and the imageswere recorded at 400magnification.2.7. High-performance gel permeation chromatography (HPGPC).Samples were prepared by dissolving 50 mg of sample in 90%.DMSO and the solution was centrifuged at 3000 rotate / min for.20 min. Sample solution (100 mL) was filtered through cellulosee.Acetate membrane filters with 0.45 mm pore size. The solutions.Were fractioned through a Waters 600 high-performance liquid.Chromatography system (Waters Corp, Milford MA), with, two inlineIdentical analytical columns (Ultrahydrogel Linear.300 mm 7.8 mm). The column temperatures were maintained at.45 C. The mobile phase of NaNO3 (100 mM) containing NaN3.(0.02%) was circulated at a rate of 0.9 mL / min and the eluted starch.Detected by with a differential refractive index (DRI detector.)(Model 2410 Waters Corps,,, Milford MA). The percentage of peak.Area was calculated using the Origin 6.0 software (Microcal, Inc.Northampton, the MA).2.8. X-ray diffraction (XRD).X-ray patterns were obtained with a D / Max-2200 X-ray.Diffractometer (Rigaku Denki Co, Tokyo Japan). Starch, samples.Were equilibrated in a saturated relative humidity chamber for 24 h.At ambient temperature. The samples were scanned in the range of4e35 (2q), with target voltage 40 kV target current mA 30,,,, And at.A scanning rate of 4 / min. Relative crystallinity was calculated as.The ratio of the areas of crystalline and amorphous regions of X-ray.Diffractograms (Nara, & Komiya 1983).2.9. Fourier-transform infrared spectroscopy (FT-IR).All infrared spectra were obtained on Vector 33 spectrometer.(Bruker Germany), equipped with an attenuated total reflectance.(ATR) attachment with a resolution of 4 cm by 64 scans. Spectra.