Methodology and instrumentation with references
1. Morphological parameters
Grain length and breadth were measured in mm of ten random grains of each variety using digital vernier calliper and then average was calculated[1].
2. Proximate analysis
a. Crude lipid content: Approximately 250 mL of clean boiling flasks are dried and cooled for about 30 minutes in an oven at 105–11 ° C. Around 2.0 g of the sample was correctly weighed into marked thimbles. The dried boiling flasks were adequately weighed and filled with approximately 300 mL of petroleum ether (40-60 ° C boiling point). The thimbles for extraction are tightly stuffed with cotton wool. The Soxhlet unit was installed after that and allowed to reflux for 6 hours. Carefully the thimble was removed, and petroleum ether was gathered from the top and emptied for re-use into another bowl. After that, the flask was dried at 105–110 ° C for 1
hour. It was cooled in a desiccator after drying and measured[2]. The equation below was then used to measure the percentage of fat in the rice sample:
% fat = (Weight of fat / Weight of sample) x 100
b. Crude nitrogen content: Approximately 2g of sample was weighed into digestion flask. To this about 2g of K2SO4 and 90mg of HgO, was added. The sample was digested over digestion rack for 40 min at 37°C. Following this only a limited quantity of water was added. A 100 mL conical flask containing 5mL boric acid solution with mixed indicator was taken. 10 mL of NaOH- sodium thiosulphate solution to the digest in the apparatus through the funnel and rinsed water with water. The boric acid collects the ammonia until there is a colour change from 15- 20 min indicating that ammonia is absorbed. The tip of the condenser is rinsed with water and titrate the distilled sample against HCl or H2SO4 (0.02N) until the appearance of violet colour. To obtain the crude protein content in rice multiplication by conversion factor -5.95[3].
3. Starch extraction:
Starch was extracted from the varieties of - collected rice by the alkaline-steeping method. Rice grains were ground to flour using the mixer grinder, which works at 750 Watts. 10g of Rice flour was soaked in 0.1% NaOH solution and incubated at 4 ºC for 18 hours, followed by homogenization at high speed for 5 mins. The homogenized product was subjected to centrifugation (Eppendorf –centrifuge 5414R, India) at 1500 rpm for 10 mins. The supernatant was discarded, along with the light-yellow layer deposited on the top. The bottom pellet was collected in a separate beaker, and the pH was adjusted to 6.5 using 0.2M HCl. Then it was washed with 30mL distilled water. The residue was centrifuged by maintaining conditions as mentioned earlier. Once the pH was adjusted, the centrifugation was repeated 4-5 times until a clear supernatant was obtained. Finally, the pellet was recovered and subjected to lyophilization using lyophilizer (Power dry LL1500, ThermoScientific USA)[4].
4. Apparent amylose content estimation :
The amylose content in the native starch was estimated using the iodine binding method. 100mg of starch were weighed and mixed with 0.36 of NaOH in mL of distilled water and 1mL of absolute ethanol and the flasks were heated in a boiling water bath for 10 min followed by cooling. Later 1mL acetic acid and 2 mL iodine were added. The volume was made upto 100 mL and left at room temperature for 1 Hr. The 200 μL of the samples were loaded in 96 well plates and the optical density were recorded at 620 nm in in triplicates[4].
5. Morphological characterization:
The aqueous suspension of starch granules was prepared on the clean glass slide. Their micro-morphological structures were visualized using an optical microscope (Olympus BX51, Japan) with a 60X objective lens. The structural differences among the starch granules of different rice varieties were observed before and after enzymatic hydrolysis. The higher resolution images were captured using the scanning electron microscope (JEOL model JSM-6380LA system, Germany). The lyophilized starch samples were attached to the carbon tape on an aluminum stub. It is then silver coated and subjected to a sputtering process before capturing the images. Images of starch were captured at 5000X and 10000X magnifications for high-resolution structural analysis.
Molecular structure and functional group analysis[4].
6. Structural characterization:
Fourier transform Infrared (FTIR) spectroscopy study was employed to monitor the changes in the chemical compositions of starch from different varieties of rice. The lyophilized starch samples were mixed with the KBr to make a pallet and mounted onto the KBr pellet holder. The
spectrum of mounted starch samples was recorded using Bruker alpha FTIR spectrometer (Germany), and the samples were scanned through 4000 to 600 cm -1 [4].
7. Thermal characterization:
Gelatinization properties
The gelatinization parameters of the different starch samples were determined using a differential scanning calorimeter (Shimadzu’s DSC60). The sample preparation for the thermal analysis was done by coating approximately3-4mg of starch samples in an aluminium pan. The pan was manually crimped using a sample-encapsulating press (Shimadzu’s DSC60) and heated at specific temperatures between 30 and 110 ºC with the interval of 10 ºC and monitored for the DSC curves. All samples were maintained at approximately 10% moisture content. The onset, endset, peak temperatures, and transition enthalpy (ΔH) were recorded, and the curves were analysed using the software TA-60WS[4].
FTIR reference table
|
Signal frequency (cm-1 ) |
Band assignment |
References |
|
3550–3200b,m |
O–H stretching of glucopyranose rings in starch |
[5] |
|
3000–2840w |
Asymmetric stretching vibration of C–CH2–C present in glucose |
[6] |
|
1650–1580w |
O–H deformation of an inbound water molecule in starch |
[7] |
|
1600 to 1200w |
C–H bending in –CH2 group and also bending of –OH groups associated with alcohol functional groups |
[8] |
|
1300–1000w |
C–OH stretching and bending vibrations, skeletal vibrations of C–O–C present in glycosidic linkages, C––O and C–C stretching |
[9] |
|
1158w |
C–O stretching of C–OH bonds of starch |
[8] |
|
1080w |
C–C stretching vibration of C–O–C |
[9] |
|
1000m,s |
Bands attributed to amylose and amylopectin of starch |
[10] |
|
926w |
Intramolecular bonding of –OH group at C-6 position in native starch, indicating hydrophilicity of starch |
[11] |
b-broad, m-medium, s-strong, w-weak.
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