Isolation of Starch from Rice (Oryza Sativa L.) and its Morphological Study using Scanning Electron Microscopy

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1 International Journal of Agriculture and Food Science Technology. ISSN , Volume 4, Number 9 (2013), pp Research India Publications ijafst.htm Isolation of Starch from Rice (Oryza Sativa L.) and its Morphological Study using Scanning Electron Microscopy D. Kodandaram Reddy 1 and M.G. Bhotmange 2 1 Department of Food Technology, University College of Technology, Osmania University, Hyderabad, Andhra Pradesh, INDIA. 2 Department of Food Technology, Laxminarayan Institute of Technology, R.T.M Nagpur University, Nagpur, Maharashtra, INDIA. Abstract Rice (Oryza Sativa L.) is widely consumed and important cereal crop in India. Rice is one of the excellent sources of starch and starch isolated from rice has been used for various food and non food applications. In this study three Indian rice cultivars viz., HMT, Swarna and Basmati were studied and starch was isolated by alkali extraction method and characterized for morphological studies by Scanning Electron Microscope (Jeol JXA-840A, Jeol Ltd, Tokyo, Japan).Starch yield was found to be in the range 81-84% on fresh weight basis. The highest starch yield of 84% was obtained from Basmati and the lowest yield of 81% was obtained by HMT where as that of Swarna variety was 82%.The scanning electron microscopy (SEM) studies showed that rice starch granules were large to medium sizes with crystalline or irregular in shape. The granules separated from all the cultivars showed a wide size distribution (10 m to 150 m).basmati starch granules has a intact structure having crystalline surface with pores on some parts of the granules, while Swarna and HMT starch granules has loose structure with highly rough surface. This irregular rough surface of the granules was due to damage of starch during isolation process. The images were taken at different magnifications (X X 3500) at a high accelerating voltage of 15 kv with working distance (WD) of 14mm to observe the close structure of granules and pores from the surface. Keywords: Rice starch; alkali extraction; morphological properties; Scanning Electron Microscopy

2 860 D. Kodandaram Reddy & M.G. Bhotmange 1. Introduction Cereal grains play an important role in meeting the nutrient needs of the human population. Like any food, they are good to excellent sources of some nutrients and low or void in other nutrients. They are the cheapest source of the food, particularly for the developing countries. They constitute a higher percentage of calories and protein intake of man and when supplemented with legumes forms almost complete food. Wheat, rice corn, barley, sorghum, rye and oat are the principle cereal crops grown all over the world. The quality of cereal products is determined by a variety of characteristics which may be assigned different significance depending on the desired and use or type of product. These cereals have various food and industrial applications. 1.1 Rice (Oryza sativa L.) Rice (Oryza sativa L.) is one of the world s most important cereals for human consumption. In the densely populated countries of Asia especially Bangladesh, China, India, Indonesia, Iran, Japan, Korea, Pakistan and Sri Lanka rice is the most important staple food. India is the world's second largest producer of white rice, accounting for 20% of all world rice production. Rice is India's prominent crop, and is the staple food of the people of the eastern and southern parts of the country. There are around varieties of rice in India of which around 4,000 are cultivated. India has the world's largest harvesting area (43.6 million ha) for rice and second in production (91.7 million tons) next to China. The type of rice grown in different parts of India depends on the weather, soil, structure, characteristics and purposes. Rice in India is grown under diverse conditions. There are various varieties of rice cultivated in India. Some of them are Samba, Masuni, Vijayantha, Pusa JB, Basmati, Deepakratna, Swarna, HMT etc. For the present investigation three different rice cultivars viz. HMT, Swarna and Basmati were used. 1.2 Starch Starch is the major dietary source of carbohydrates and is the most abundant storage polysaccharide in plants. It is present in high amounts in roots, tubers, cereal grains and legumes and also occurs in fruit and vegetable tissues. Starch is a polymer of glucose linked together by α-d-(1-4) and/or α -D-(1-6) glycosidic bonds. The starch granule mass comprises 70% amorphous regions, which consists of amylose and branching points of amylopectin molecules, and 30% crystalline, which is mainly composed of the outer chains of amylopectin Amylose Amylose is the linear portion of the starch, with glucose residues linked by α-d-(1-4) bonds. Depending on the species, amylose constitutes typically 20 to 30% of starch,has a degree of polymerization (DP) of 500 to 6000,hydro dynamic radius 7-22nm and molecular mass ranging from107 to 109 g/mol. The variable number of 1, 6-branching points, as well as amount of glucose monomers, makes it difficult to determine amylose content in different starches. The long chains of amylose can form single or

Isolation of Starch from Rice (Oryza Sativa L.) and its Morphological Study 861 double-helical structures with hydrophobic cavities that can complex with lipids and iodine.

2 Amylopectin Amylopectin is one of the largest molecules in nature, with a degree of polymerization (DP) averaging 2 million and a molecular mass several fold greater than amylose.

3 Isolation of Starch from Rice (Oryza Sativa L.) and its Morphological Study 861 double-helical structures with hydrophobic cavities that can complex with lipids and iodine. Amylose does not dissolve easily in water and forms rigid gels. Figure 1: Representative structures of Amylose and Amylopectin Amylopectin Amylopectin is one of the largest molecules in nature, with a degree of polymerization (DP) averaging 2 million and a molecular mass several fold greater than amylose. It easily dissolves in hot water and does not form a gel. Starches that contain only amylopectin are termed waxy starches. Most amylopectin molecules have three branch chain fractions that differ in lengths. 2. Materials and Methods 2.1 Rice Grains Three cultivars of Rice (Oryza sativa L.) viz., Basmati, HMT and Swarna were procured from local market of Nagpur city. The rice grains were cleaned, ground in mixture grinder and stored properly at room temperature prior to their use in actual experiment. 2.2 Isolation of Starch (Alkali Extraction Method) The residue obtained after protein extraction was sequentially extracted with 1 liter of each distilled water and 2% NaCl (each for 24 hrs at 4 o C) followed by extraction with 300 ml of 0.1 N NaOH twice (48 hrs at 4 o C). Subsequent to each of above the extraction, the slurry was centrifuged at rpm for 30 min. at 4 o C and supernatant was discarded. The residue from the second NaOH extraction was further extracted with 80 % aqueous ethanol (100 ml) at 80 0 C for 1hr, cool to room temperature and allowed to settle for 4 hr at 4 0 C. The supernatant was discarded, and the residue was dehydrated and powdered.

4 862 D. Kodandaram Reddy & M.G. Bhotmange Flow sheet: gm Rice Flour (Disperse in 1500 ml D.W. Adjust to ph 10 with 1 N NaOH; stand for 1 hr with moderate stirring.) Centrifuge at 5000 rpm for 30 min. Supernatant Residue (Processed for protein extraction) (Extracted with 1 Liter D.W. for 24 hrs at 4 o C) Centrifuge at rpm for 30 min. Residue (Extracted with 1 liter 2 % NaCl, for 24 hrs, at 4 o C) Centrifuge at rpm for 30 min. and Discard Supernatant Residue (Extracted with 300 ml of 0.1 N NaOH twice for 48 hrs at 4 o C) Centrifuge at rpm for 30 min. Discard the supernatant, collect white starch layer Re suspend in 80 % Ethanol (Blended for 1 min using stirrer) Heating on water bath at 80 0 C for 1 hr. Settle for 4 hr at 4 o C and discard supernatant Residue Freeze Dehydrate Starch Powder 2.3 Morphological Studies by Scanning Electron Microscopy (SEM) Scanning electron micrographs were obtained with a scanning electron microscope (Jeol JXA-840A, Jeol Ltd, Tokyo, Japan). Starch samples were applied on an aluminum stub using double sided adhesive tape, and the starch was coated with gold (using auto quick gold coater). An acceleration potential of 15 kv was used during micrography.

Isolation of Starch from Rice (Oryza Sativa L.) and its Morphological Study 863 3. Results and Discussions 3.1 Isolation of Starch The three different rice cultivars viz.

Table 1: % Yield of starch from Rice Flour. Sr. No.

The highest for Basmati (84%) followed by Swarna (82%) and HMT (81%). The starch yield found directly correlated to the dry matter contents of the cereals.

5 Isolation of Starch from Rice (Oryza Sativa L.) and its Morphological Study Results and Discussions 3.1 Isolation of Starch The three different rice cultivars viz., Basmati, HMT and Swarna were processed in the present investigation for isolation of Starch and % yield of obtained starch was compared and shown in Table 1. Table 1: % Yield of starch from Rice Flour. Sr. No. Variety of Rice Yield of Starch (%) 1 Basmati 84 2 HMT 81 3 Swarna 82 Table 1 Indicates the % yield of starch varied from 81% to 84% on fresh weight basis. The highest for Basmati (84%) followed by Swarna (82%) and HMT (81%). The starch yield found directly correlated to the dry matter contents of the cereals Starch % Basmati HMT Swarna 3.2 Microscopic Study of Starch by SEM The morphological properties of starch granules were studied by Scanning Electron Microscope. The scanning images of starch granules of three different cultivars of rice viz., Basmati, HMT and Swarna were shown in Fig. 2, 3and 4 respectively Morphology of rice starch (Basmati)

6 864 D. Kodandaram Reddy & M.G. Bhotmange Figure 2: Morphology of Basmati Rice starch at X100, X500, X1000, X2000, X2500 and X Morphology of rice starch (HMT) Figure 3: Morphology of HMT Rice starch at X100, X500, X1000, X2000, X2500 and X Morphology of Rice starch (Swarna)

Isolation of Starch from Rice (Oryza Sativa L.) and its Morphological Study 865 Figure 4: Morphology of HMT Rice starch at X100, X500, X1000, X2000, X2500 and X3500. 4. Conclusions As the Rice (Oryza Sativa L.

, Basmati, HMT and Swarna are good source of starch for food and other industrial uses.

The results obtained during the course of present investigation are given below: The three different rice cultivars showed significant variation in % yield of starch and other properties.

7 Isolation of Starch from Rice (Oryza Sativa L.) and its Morphological Study 865 Figure 4: Morphology of HMT Rice starch at X100, X500, X1000, X2000, X2500 and X Conclusions As the Rice (Oryza Sativa L.) is rich in starch, in the present investigation efforts have been made to explore the possibility of isolation of starch from three different rice cultivars viz., Basmati, HMT and Swarna are good source of starch for food and other industrial uses. Attempts have also been made to study the different Morphological a properties and to compare the yield and characteristics among the three varieties. The results obtained during the course of present investigation are given below: The three different rice cultivars showed significant variation in % yield of starch and other properties. The percent yield of starch ranging from 81 to 84%. The scanning images of starch granules of three different cultivars of rice viz., Basmati, HMT and Swarna were shown in Fig. 2, 3 and 4 respectively. The surfaces of the granules from the various starches were observed to be crystalline when view under SEM at different magnification range (X100 to X3500). The native starches of different cultivars consisted of mixed population of large, medium and small granules with the diameter range of 10 m m. The small granules were spherical or ellipsoidal while the medium and large granules were ellipsoidal to irregular or cubical in shape. Basmati starch granules has a intact structure having crystalline surface with pores on some parts of the granules, while Swarna and HMT starch granules has loose structure with highly rough surface. This irregular rough surface of the granules was due to damage of starch during isolation process. The images were taken at different magnification to observe the close structure of granules and pores from the surface. The structure and morphology of starch granule affect the swelling, solubility and water absorption capacity of the flour, as these affect the final application of the starch in food products. Scanning Electron Micrographs of starch granules of three different varieties showed variations in their size. The starch granules of all of the three varieties were irregularly shaped with crystalline granules being predominant. The morphological properties showed uneven distribution of granules sizes in the starches of different rice cultivars is due to the hereditary characteristics and is mainly responsible for the variation in their properties. Granule fractions with desired properties are suitable for distinct industrial applications.

8 866 D. Kodandaram Reddy & M.G. Bhotmange References [1] AOAC (1996) AOAC, Official methods of analysis, Association of Official Analytical Chemists, Washington, DC (1996). [2] Asaoka et al., 1985 M. Asaoka, K. Okuno, Y. Sugimoto, M. Yano, T. Omura and H. Fuwa, Structure and properties of endosperm starch and water-soluble polysaccharides from sugary mutant of rice (Oryza sativa L), Starch 37 (1985), pp [3] Ashogbon, A.O. and Akintayo, E.T, Morphological, functional and pasting properties of starches separated from rice cultivars grown in Nigeria,International Food Research Journal 19(2): (2012) [4] Bao et al., 2004 J. Bao, H. Corke and M. Sun, Genetic diversity in the physicochemical properties of waxy rice (Oryza sativa L) starch, Journal of The Science of Food and Agriculture 84 (2004), pp [5] Basak S., Tyagi R. S., Srivastava K. N., 2002, Biochemical characterization of aromatic and Non-aromatic rice cultivars. Journal of Food Sci. Technol. Vol. 39 No. 1, pp [6] Han and Hamaker, 2002 X.-Z. Han and B.R. Hamaker, Location of starch granule-associated proteins revealed by confocal laser scanning microscopy, J. Cereal Science 35 (2002), pp [7] Jiang Chen, Junjie Zhang, Yinghong Liu and Yubi Huang, Morphologies and microstructures of maize starch granules differed in hardness during development of kernels, Journal of Food, Agriculture & Environment Vol.11 (3& 4 ) : [8] King J. M., An H. J., 2007, Thermal characteristics of ohmically heated rice starches and rice flour. Journal of Food Sci. Vol. 72, No. 1 pp. C84-C87. [9] Leach and Schoch, 1961 H.W. Leach and T.J. Schoch, Structure of the starch granule, Cereal Chemistry 38 (1961), pp [10] Navdeep Singh Sodhi, Narpinder Singh, Morphological, thermal and rheological properties of starches separated from rice cultivars grown in India, Food Chemistry 80 (2003) [11] Patindol and Wang, 2002 J. Patindol and Y.-J. Wang, Fine structures of starches from long-grain rice cultivars with different functionality, Cereal Chemistry 79 (2002), pp [12] Sodhi and Singh, 2003 N.S. Sodhi and N. Singh, Morphological, thermal and rheological properties of starches separated from rice cultivars grown in India, Food Chemistry 80 (2003), pp [13] Wu & Seib (1990) Y. Wu and P.A. Seib, Acetylated and hydroxy-propylated di-starch phosphates from waxy barley: Paste properties and freeze-thaw stability, Cereal Chemistry 67 (1990), pp [14] Yamamoto et al., 1973 K. Yamamoto, S. Sawada and T. Onogaki, Properties of rice starch prepared by alkali method with various condition, Journal of the Japanese Society of Starch Science 20 (1973), pp [15] Yang et al., 1984 C. Yang, H. Lai and C.-Y. Lii, The modified alkaline steeping method for the isolation of rice starch, Food Science 11 (1984), pp

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