Study on Amylose Iodine Complex from Cassava Starch by Colorimetric Method

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1 Study on Amylose Iodine Complex from Cassava Starch by Colorimetric Method Sirinat Boonpo and Sukjit Kungwankunakorn Department of Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand {sirinat.yoy, Abstract The absorbance pattern of amylose iodine complex from cassava starch sample was examined by scanning of UV visible spectra between nm. The cassava starch sample was analyzed at the different conditions to investigate maximum wavelength and absorbance. To study about the elimination of lipid from cassava starch, the defatting process was operated on 3 types of solvent (methanol, ethanol, and petroleum ether) by stirring for 24 hours with solvent. The result indicated that the maximum wavelength of amylose iodine complex of cassava starch sample depended on the amount of iodine that was used to form amylose iodine complex. Moreover, the amount of iodine has the effect on absorbance of amylose iodine complex. The analysis of amylose iodine complex for defatted starch provided the higher absorbance than that from nondefatted starch. Index Terms amylose, cassava, colorimetric method, starch I. INTRODUCTION Carbohydrate is a major staple food for people around the world. It is one of biopolymer that contains a number of glucose units. The combination of glucose units supply two main structures as amylose and amylopectin [1] and [2]. The ratio of amylose and amylopectin are the significant factor to indicate quality of starch because it controls starch physicochemical properties. Amylose is a linear structure of carbohydrate whereas amylopectin is branched structure. The ratio of amylose and amylopectin in starches is depended on the source [3] and [4]. Cassava (Manihot esculenta Crantzis) is one of the most significant source of carbohydrate. The root of cassava is the important part that contains numerous carbohydrate. It composes of moisture, carbohydrate, fat, fiber, protein and trace amount of vitamins and minerals [5]. For estimation of amylose content in starch sample, the blue amylose iodine complex was applied. This method was known as colorimetric method that has been widely used in many laboratory of agriculture until nowadays. The principle based on the forming of complex between amylose and iodine that stains blue solution [6]-[8]. After that quantitative analytical technics such as potentiometric titration [9] and [10] or ultraviolet-visible spectrophotometry [11]-[13] were operated to determine the blue amylose iodine complex that related to amylose Manuscript received January 19, 2017; revised June 24, content. Hence, the forming process of amylose iodine complex is very importance to investigate. Commonly, starch from the different source not only provide different amount but also a dissimilar property of amylose that is the noteworthy indicator of amylose content determination. Therefore, the suitable amount of iodine should be studied for each type of starch sample to acquire accurate data. However, the forming of amylose iodine complex has a problem from natural interferences which are the components of starch such as long branched amylopectin, protein and lipid. These components interfere the efficiency of amylose determination by colorimetric method. As a result, the improvement of sample preparation process is necessary for increasing the efficience. Lipid is one of natural interference in amylose determination. It binds with amylose structure in nature that compete with iodine to form complex. It should be eliminated from sample before it analyzing by colorimetric method [14]. A. Materials II. MATERIALS AND METHODS All of cassava starch sample in this work was acquired from the markets in Thailand. The sample was dried in oven at 105 C for 24 hours and kept in desiccator. The amylose standard from potato was purchased from Sigma Chemical and was used for the investigation of amylose iodine complex color. The 0.2% of iodine reagent was prepared by mixing 0.2 g of iodine and 2.0 g of potassium iodide. Then the volume of iodine solution was made up to 100 ml with deionized water. B. Starch Defatting For study on the suitable condition to remove lipid from starch sample, 3 types of solvent (methanol, ethanol, and petroleum ether) were applied by solvent extraction. The starch sample was stirred with each type of solvent for 24 hours by magnetic stirrer. After that the mixed solution was filtrated and air dried. The starch sample was heated in oven at 105 C for 24 hours and kept in desiccator. C. Preparation of Amylose Iodine Complex The analysis of blue amylose iodine complex of cassava starch sample was adapted from the method of McCready (1943) [15]. A 100 mg of cassava starch 2017 Journal of Advanced Agricultural Technologies 345 doi: /joaat

2 sample was dissolved with 1 ml of 95% ethanol and 10 ml of 1 M sodium hydroxide. After that the sample was refrigerated at 4 C for 24 hours then volume of sample was adjusted to 100 ml with deionized water at the same temperature. The prepared solution was allowed to stand for hours at 4 C. The 5 ml of the prepared solution was used to develop the blue amylose iodine complex. The ph of the prepared solution was adjusted by 0.05 M of hydrochloric acid. After that 0.2% iodine reagent was added into the solution and the volume was made up to 100 ml with deionized water. D. Characterization of Defatted Starch The UV visible spectrometer (Perkin Elmer, model Lambda 25) was operated to study the spectra of amylose iodine complex from cassava starch sample. To investigate the effect of iodine amount, a 0.2% iodine was varied from 1 to 4 ml to form amylose iodine complex. Then the complexed solutions were analyzed by UV visible spectrometer in scan mode. The solution of amylose iodine complex from nondefatted and defatted cassava starches using 2 ml of 0.2% iodine were analyzed in fixed mode for study about absorbance. The surface morphology of the nondefatted and defatted cassava starch samples were investigated by Scanning Electron Microscope (SEM; JOEL, model JSM-5910). The sample was dried in oven at 105 for 24 hours and kept in desiccator before it was examined by SEM. Figure 1. The spectra of amylose iodine complex of cassava starch sample 1, ((a), (b), (c), (d) are 1, 2, 3 and 4 ml of 0.2% iodine volume to form the blue complex, respectively). III. RESULTS AND DISCUSSION A. The Effect of Iodine Volume for the Characteristic of Amylose Iodine Complex Eferences The amylose iodine complex using different amount of iodine reagent was investigated by the adapted method of McCready using 0.2% iodine as a reagent. The 3 types of cassava starch from the markets were used as studied samples. A 5 ml of the solution including 100 mg of cassava starch sample in total volume of 100 ml, was used for developing the blue complex. After that the prepared solution was analyzed by UV visible spectrometer. The spectra of amylose iodine complex of cassava starch using 1, 2, 3 and 4 ml of 0.2% iodine volume were investigated by UV visible spectrometer in scan mode between nm. The spectra are shown in Fig. 1-Fig. 3. The spectra show that the maximum wavelengths of amylose iodine complex shift to shorter wavelength when increase amount of 0.2% iodine reagent. The range of maximum wavelength and absorbance at the maximum wavelength of amylose iodine complex using different 0.2% iodine reagent volume from 3 types of cassava starch samples are shown in Table I. Figure 2. The spectra of amylose iodine complex of cassava starch sample 2, ((e), (f), (g), (h) are 1, 2, 3 and 4 ml of 0.2% iodine volume to form the blue complex, respectively. Figure 3. The spectra of amylose iodine complex of cassava starch sample 3, ((i),(j), (k), (l) are 1, 2, 3 and 4 ml of 0.2% iodine volume to form the blue complex, respectively. 1 Sample TABLE I. X SD THE MAXIMUM WAVELENGTH AND ABSORBANCE AT MAXIMUM WAVELENGTH OF AMYLOSE IODINE COMPLEX I ml* I ml* I ml* I ml* Abs at λ max λ max (nm) Abs at λ max λ max (nm) Abs at λ max λ max (nm) Abs at λ max λ max (nm) Journal of Advanced Agricultural Technologies 346

3 2 3 *N=7 % RSD X SD % RSD X SD % RSD The 1 ml of iodine reagent provided the maximum wavelength in the range of nm, nm and nm for sample 1, 2 and 3, respectively. The 2 ml of iodine reagent provided the maximum wavelength in the range of nm, nm and nm for sample 1, 2 and 3, respectively. The 3 ml of iodine reagent provided the maximum wavelength in the range of nm, nm and nm for sample 1, 2 and 3, respectively and 4 ml of iodine reagent provided the maximum wavelength in the range of nm, nm and nm for sample 1, 2 and 3, respectively. The absorbance of the blue complex from 3 types of cassava samples with 1, 2, 3 and 4 ml of 0.2% iodine were specific detected at 610 nm and the average (X ) of the absorbance at 610 nm was compared by statistics method (Mann-Whitney). The result in Table II indicated that the adding of different 0.2% iodine volume from 1-4 ml in the sample that contain the same amount of amylose, provided statistically significant difference in absorbance. TABLE II. THE ABSORBANCE OF AMYLOSE IODINE COMPLEX AT 610 NM USING DIFFERENT VOLUME OF 0.2% IODINE Sample Absorbance at 610 nm I ml* 2.00 ml* 3.00 ml* 4.00 ml* X a b c d 1 SD %RSD X d e f g 2 SD %RSD X h i j k 3 SD %RSD *N=7 An alphabet is the different data of the average absorbance that were analyzed by Mann-Whitney method: a difference of alphabet mean that average absorbance is difference. However, the observation of amylose iodine complex solution color showed a green tone when used 0.2% iodine more than 2 ml. In some cases, the solution changed to green color by adding only 2 ml of 0.2% iodine. The scanning of UV visible spectra of amylose iodine complex, as shown in Fig. 1-Fig. 3, illustrated the increasing of disturbance in ultraviolet region ( nm) when increase 0.2% iodine reagent volume. The using of iodine reagent more than 2 ml provided spectra that interfered the characteristic peak of amylose iodine complex in the ultraviolet region. For study about the effect of amylose iodine solution color, the amylose standard from Sigma Chemical was used as representative amylose substrate. The stock standard amylose was prepared by McCready method using the solution of a 100 mg of amylose standard in total volume of 100 ml. The amount of stock standard amylose was varied from 1 ml to 6 ml by using 1 or 2 ml of 0.2% iodine as showed in Table III. By adding 1 ml of 0.2% iodine, the amylose complex solutions showed blue color. A light blue tone presented with the adding of 1-2 ml amylose standard as well as a dark blue tone presented with the adding of 3-6 ml. By adding 2 ml of 0.2% iodine, the amylose complex solutions showed blue and green color. A green tone presented with the adding of 1-3 ml amylose standard and a dark blue presented with the adding of 4-6 ml. The result indicated that using small amount of amylose standard and large amount of iodine reagent provided a green tone solution. The green tone solution may occur by the excess volume of iodine and it also effected spectra in ultraviolet region. From the statistics (Mann-Whitney) result of the amylose iodine complex absorbance at 610 nm (Table II), it showed significant difference absorbance of the blue and green tone solution. Therefore, the adding of the same 0.2% iodine volume for every sample types, might cause the error for amylose analysis. B. The Defatting Process of Cassava Starch Sample To study the elimination of lipid from cassava starch sample, the 3 types of solvent (methanol, ethanol, and petroleum ether) were applied by solvent extraction method for 24 hours. The defatted and nondefatted starches from sample 1 were used to develop the amylose iodine complex using 2 ml of 0.2% iodine reagent. Then the amylose iodine complex solution was analyzed by UV visible spectrometer in fixed mode at 610 nm, as showed in Table IV. Table IV shows the slight increase of amylose iodine complex absorbance when the defatting process was applied with methanol, ethanol, and petroleum ether as solvent. The statistics method (CRD) was operated to compare the average absorbance between the blue complex solutions from nondefatted and the defatted starches. It presents that the nondefatted and the defatted starch by 3 types of solvent provided nonsignificant difference of absorbance. The surface morphology of the nondefatted and defatted cassava starch were analyzed by scanning electron microscope as shown in Fig. 4. The result showed the dissimilar texture of the nondefatted and defatted starch. The surface of nondefatted starch sample showed rather smooth area. The surface of the defatted starch by the 3 type of solvents showed the bumpy area that due to the disappearance of lipid Journal of Advanced Agricultural Technologies 347

4 TABLE III. THE SOLUTION COLOR OF AMYLOSE IODINE COMPLEX FROM AMYLOSE SUBSTRATE USING DIFFERENT VOLUME OF 0.2% IODINE 0.2% Iodine reagent Volume of amylose standard solution 1.00 ml 2.00 ml 3.00 ml 4.00 ml 5.00 ml 6.00 ml 1.00 ml 2.00 ml TABLE IV. THE ABSORBANCE OF AMYLOSE IODINE COMPLEX AT 610 NM Starch sample Average absorbance at SD* % RSD* 610 nm* nondefatted defatted by methanol defatted by ethanol defatted by petroleum ether *N=3 The result of defatting indicates that the defatted cassava starch samples by solvent extraction using 3 types of solvent for 24 hours provided higher absorbance of amylose iodine complex. However, these absorbances are not significant different when test by CRD method. ACKNOWLEDGMENT The author would like to acknowledge Science Achievement Scholarship of Thailand, SAST and Department of Chemistry, Faculty of Science, Chiang Mai University for financial support and facilities. REFERENCES Figure 4. The SEM image of surface morphology from the nondefatted and defatted cassava starch sample, (a) is the image from nondefatted starch and (b), (c), (d) are the image from the defatted starch using methanol, ethanol, and petroleum ether as solvent, respectively. IV. CONCLUSION In this study, the result of UV visible spectra of amylose iodine complex indicated that the adding of different iodine amount showed the maximum wavelength at different wavelength. And the effect of iodine amount also related to the absorbance of amylose iodine complex that absorbance increase when increasing of iodine volume. However, the adding of excess iodine amount presented the green tone solution that provided the significant different absorbance of amylose iodine complex by comparing with the blue tone solution using the statistics method (Mann-Whitney). Hence, the adding of excess iodine volume may cause the error of amylose iodine complex analysis when the wavelength was fixed at 610 nm. Thus, the suitable amount of iodine should be considered for analysis amylose in each type of sample. [1] K. Wuttisela, S. Shobsngob, W. Triampo, D. Triampo, and J. Chil, Amylose/Amylopectin simple determination in acid hydrolyzed tapioca starch, Journal of the Chilean Chemical Society, vol. 53, pp , [2] K. Denyer,P. Johnson, S. Zeeman, and A. M. Smith, The control of amylose synthesis, Journal of Plant Physicology, vol. 158, pp , [3] A. Buleon P. Colonna, V. Planchot, and S. Ball, Starch granules: Structure and b iosynthesis, International Journal of Biological Macromolecules, vol. 23, pp , [4] F. Vilaplana, J. Hasjim, and G. Gilbert, Amylose content in starches: Toward optimal definition and validating experimental methods, Carbohydrate Polymers, vol. 88, pp , [5] T. Mahmod, M. A. Turner, and F. L. Stoddard, Comparison of methods for colorimetric amylose determination in cereal grains, Starch/Stark, vol. 59, pp , [6] V. R. Williams, W. Wu, H. Y. Tsai, and H. G. Bates, Varietal differences in amylose content of rice starch, Journal of Agricultural and Food Chemistry, vol. 6, pp , [7] X. Yu, C. Houtman, and R. H. Atalla, The complex of amylose and iodine, Carbohydrate Research, vol. 292, pp , [8] R. C. Teitelbaum, S. L. Ruby, and T. J. Marks, A resonance Raman/Iodine mossbauer investigation of the starch-iodine structure: Aqueous solution and iodine vapor preparations, Journal of the American Chemical Society, vol. 102, pp , [9] W. Banks, C. T. Greenwood, and D. D. Muir, The characterization of starch and its components. Part 6. A critical comparison of the estimation of amylose-content by colorimetric determination and potentiometric titration of the iodine-complex, Starch/Stark, vol. 26, pp , [10] S. R. Delwiche, M. M. Bean, R. E. Miller, B. D. Webb, and P. C. Williams, Apparent amylose content of milled rice by nearinfrared reflectance spectrophotometry, Cereal Chemistry, vol. 72, pp , [11] C. A. Knutson and M. J. Grove, Rapid method for estimation of amylose in maize starches, Cereal Chemistry, vol. 71, pp , [12] C. A. Knutson, Evaluation of variations in amylose-iodine absorbance spectra, Carbohydrate Polymers, vol. 42, pp , Journal of Advanced Agricultural Technologies 348

5 [13] C. A. Knutson, A simplified colorimetric procedure for determination of amylose in maize starches, Cereal Chemistry, vol. 63, pp , [14] C. A. Lopez, A. H. D. Vries, and S. J. Marrink, Amylose folding under the influence of lipids, Carbohydrate Research, vol. 364, pp. 1-7, [15] R. M. McCready and W. Z. Hassid, The separation and quantitative estimation of amylose and amylopectin in potato starch, Journal of the American Chemical Society, vol. 65, pp , Sirinat Boonpo was born in Mahasarakham, Thailand on March 20, She received her B.Sc degree in the field of chemistry from Mahasarakham University, Mahasarakham, Thailand in 2008 and M.Sc. degree in the field of chemistry from Chiang Mai University, Chiang Mai, Thailand in She is now studying for her Ph.D. degree at Chiang Mai University, Chiang Mai, Thailand. Her research interests focus on the determination of amylose content in starch sample. Sukjit Kungwankunakorn is a lecturer in the Department of Chemistry at Chiang Mai University, and has served as the Head of Forensic Science Program in Graduate School, Chiang Mai University since Her researches have focused on method developments for the analysis of heavy metals using spectrometric methods, the analysis of food and environmental samples and the analysis of forensic samples with advanced analytical techniques. She earned her PhD in Chemistry from The University of Manchester, Manchester Journal of Advanced Agricultural Technologies 349