OPTIMIZATION OF GCWS SAGO STARCH PREPARED BY ALCOHOLIC-ALKALINE TREATMENT
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1 OPTIMIZATION OF GCWS SAGO STARCH PREPARED BY ALCOHOLIC-ALKALINE TREATMENT Jau-Shya Lee 1)*), Kuan Yau Hoong 1) and Aishah Bujang 2) 1) School of Food Science and Nutrition, Universiti Malaysia Sabah Locked Bag No. 2073, Kota Kinabalu, Sabah, Malaysia 2) Faculty of Applied Science, Universiti Teknologi MARA Shah Alam, Selangor, Malaysia *) Corresponding author: Tel: ; Fax: ABSTRACT A two-factor three-level Faced-Centered Cube (FCD) Design consisting of thirteen runs was employed to optimize the preparation of granular cold water-soluble (GCWS) sago starch using alcoholic-alkaline treatment. The variables studied were ethanol concentration (40%, 50% and 60%) and the ratio of sodium hydroxide (1.5 M): starch (1:1, 2:1 and 3:1 at 100g db starch). Response Surface Methodology (RSM) was used to evaluate the effect of process variables and their interactions on the apparent amylose content, cold water solubility and swelling power, apparent viscosity and the granule mean particle size of the GCWS starches produced. In general, sodium hydroxide (NaOH) exhibited more pronounced effect on most of the responses studied; it was found significantly increased the amylase leaching, cold water solubility and swelling power (p<0.05) of the products. On the other hand, ethanol restricted excessive swelling by decreasing the cold water solubility (p<0.05). At high NaOH concentration, the starch granules swelled to rupture. GCWS sago starch pastes also exhibited higher viscosity (p<0.05) than the native counterpart. Cold water solubility and granule mean particle size were selected to optimize the treatment, and the optimum conditions identified consists of % ethanol and g NaOH. To validate, the condition at the middle point of this optimum area was used to produce GCWS sago starch. The experimental values obtained for cold water solubility and mean particle size were 52.2 ± 1.5% and 25.4 ± 1.2 µm respectively, agreed well with the predicted values. This indicates suitability of the model employed and the success of RSM in optimizing the GCWS sago starch preparation using alcoholic-alkaline treatment. Keywords: GCWS sago starch, alcoholic-alkaline, optimization INTRODUCTION Granular cold-water-soluble (GCWS) starch is a type of pre-gelatinized starch where it could form dispersions or pastes or gels with cold water. The advantage of this starch is it simplifies and shortens the food preparation process and has been widely used in our daily instant convenience food products, such as instant pudding, dessert, gravies, sauces and so on. Generally there are two major types of pre-gelatinized starch distinguished by the granule integrity after processing (Peterson, 1975). The GCWS starch with intact granules exhibits quality comparable to traditional cook-up starches than pre-gelatinized starch which contains fully rupture granules. Problems encountered in fully rupture pre-gelatinized starch such as graininess, less sheen, reduced consistency paste, reduced strength gel and less flexibility to processing conditions (Rajagopalan & Seib, 1992; Chen & Jane, 1994) could be minimized using GCWS starch. GCWS starch exhibits greater cold water viscosity, smoother texture and higher processing tolerance (Light, 1990).
2 Alcoholic-alkaline treatment is one of various processes/treatments reported to produce GCWS starch. It is concerned with a process wherein granules of starch are treated with alcohol and strong base to cause swelling of the granules and conversion to a form having increased cold water solubility (Jane & Seib, 1991). To date, Malaysia is the world largest producer of sago starch. It has been chosen as the subject of study in this project due to its abundance availability. More importantly, this is one of the efforts to diversify the downstream processing of sago starch into a value-added product, and also an attempt to widen sago starch application and increase its market value. The objective of this study is employing Response Surface Methodology (RSM) to optimize the amount of sodium hydroxide (NaOH) and ethanol required to produce GCWS sago starch with highest possible cold water swelling power and solubility. The effect of NaOH and ethanol on the apparent amylose content, cold water swelling and solubility, apparent viscosity as well as granule particle size of sago starch also quantified. MATERIALS AND METHODS Materials Native sago starch was manufactured by Nee Seng Ngeng & Sons Sago Industries Sdn. Bhd., Sibu, Sarawak, Malaysia. Amylose from potato (Type III, A0512) and amylopectin from potato starch (A8515) were obtained from Sigma Chemical Company. Other chemicals were all reagent grades and were used without further treatment. Experimental Design Response Surface Methodology using two-factor and three-level Faced-Centered Cube (FCD) Design was employed to produce GCWS sago starch using alcoholic-alkaline treatment. This design produced thirteen experimental runs included four replicates at the centre point. The generalized second-order polynomial model used in the response surface analysis was as follow: η = β k k k 1 k βixi + βiixi + βijxi i= 1 i= 1 i= 1 j = i+ 1 where η is the dependent variable (response variable) to be modelled, x i and x j are the independent variables (factors), β 0, β i, β ii, β ij are the regression coefficients for intercept, linear, quadratic, and interaction terms. The design variables were the ethanol concentration (x i, %, w/w) and ratio of NaOH : starch (x j ); while response variables were apparent amylose content, cold water solubility, cold water swelling power and mean granule particle size. The effects of unexplained variability in the observed response due to extraneous factors were minimized by randomizing the order of experiments. The lower, middle, and upper design points for the factors in coded and actual values are shown in Table 1. Table 1: The treatment combinations for preparation of granular cold water-soluble sago starch generated by Two-Factor, Three-Level Face-Centered Cube Design (FCD). Randomized Ethanol Concentration (%) NaOH : starch ratio Run Order Coded value Actual value Coded value Actual value : : : : : : :1 x j
3 : : : : : :1 Preparation of GCWS Sago Starches The method of Chen & Jane (1994) was lightly modified to prepare GCWS sago starches. Exactly 100 g (dsb) of starches were suspended in 700 g of aqueous ethanol solution (40%, 50% and 60% w/w), followed by transferred to a preheated water bath at 40 C. Aqueous NaOH solution 1.5 M (100 g, 200 g and 300 g) was added at about 4 g/min using a burette. The mixtures were then allowed to stand for 15 min with gentle stirring. Additional aqueous ethanol solution was added slowly to the starch suspension. After 10 min of stirring, the slurry was then left at room temperature until the starch granules had settled at the bottom. Fresh ethanol solution was used to wash the starch. After being washed, the starch was re-suspended in aqueous ethanol solution and neutralized with HCl (3 M in ethanol). The neutralized starch was washed twice with ethanol solution and then subjected to oven drying at 80 C for 3 hr. The dried starch was sieved with a sieve (150 μm) and stored in a screw-capped bottle at room temperature for further analysis. Moisture Content and Apparent Amylose Content Moisture content was analyzed by using HG 53 Moisture Analyzer (Mettler Toledo, Greifensee, Switzerland), whilst apparent amylose content was determined using method reported by Hoover & Ratnayake (2001). Cold Water Solubility and Swelling Power The procedure described by Schoch (1964) and Jane & Seib (1991) was slightly modified to determine the cold water solubility and cold water swelling power of GCWS sago starches. Exactly 1 g (dsb) of pregelatinized sago starch was dispersed in 50 ml of distilled water for 30 min in the centrifuge tube with continuous stirring. After 30 min, distilled water was topped up to 100 ml. The slurry was centrifuged for 15 min at 3100 rpm. A 25 ml aliquot of the supernatant was then transferred to a weighing dish and then dried at 100 C for 4 hr and weighted. The cold water solubility was calculated using the following formula: (Weight of solids in 25 ml) 4 Solubility (%) = 100 Weight of sample (dsb) To obtain the cold water swelling power, the supernatant was discarded and calculation was done using the following formula: Sediment (g) Swelling Power = Weight of sample (dsb) Apparent Viscosity Apparent viscosity of GCWS sago starched and native sago starch were determined using a Brookfield Viscometer, Model HA DVE 230 (Stoughton, Massachusetts). Starch slurry (3% dsb) was prepared in 200 ml of distilled water. The sample was cooked in a boiling water bath for 30 min and then cooled to 30 C. Cold paste viscosity was determined using spindle no. 3 at 30 C at 2, 4, 10 and 20 rpm. Laser Scattering Particle Size Distribution The particle size distribution of the GCWS sago starches and native sago starches were measured using LA-300 Laser Scattering Particle Size Distribution Analyzer (Horiba Instruments,
4 Inc., Kyoto, Japan). The starch was suspended in distilled water and placed into the dispersion bath unit. The concentration was then adjusted to a specific transmittance range before measurement. Statistical Analysis, Optimization and Verification Design-Expert (Version ) software (Stat-Ease Inc., Minneapolis) was used to analyze the experimental data. Experimental data were fitted to a second-order polynomial model and regression coefficients obtained. The model was simplified by dropping terms which were not statistically significant (p>0.05) by analysis of variance (ANOVA). The Design-Expert software was used to generate response surface and contour plots while holding a variable constant in the second-order polynomial model. Optimization of independent variables for preparation of GCWS sago starch was determined by superimposing the plots for cold water solubility and particle size distribution. The optimum conditions and the predicted values of the response variables were obtained using the graphical method of the software. Verification of the optimum conditions was performed by comparing the selected responses of starch produced to the predicted values using t-test, where terms that statistically insignificant (p>0.05) were accepted. Other data were analysed by either t-test or ANOVA (p<0.05) and the means were separated by Tukey s test, for which Statistical Package for the Social Sciences (SPSS) for Windows Version 12.0 was used. All analyses were performed at least in triplicate and the experimental results were expressed as mean ± standard deviation. RESULTS AND DISCUSSIONS Preliminary tests were performed to determine the suitable levels of NaOH and ethanol used in the design of experiment. Results found that when less than 40% of ethanol and greater than 3:1 (NaOH : starch ratio) were used, the starch slurry formed hard paste and could hardly be stirred. Similarly when 2M of NaOH was used, cold gelatinization of starch also took place before the desired amount of NaOH could be added to the starch. Apparent Amylose Content Native sago starch contained 27.8 ± 0.3% of amylose, while treated starch were generally found to have lower amylose content (20.8 ± ± 1.5%). Alcoholic-alkaline treatment used in preparation of GCWS starch had been reported to cause leaching of amylose (Jane et al., 1986; Chen & Jane, 1994; Singh & Singh, 2003). Results of response surface analysis indicates that twofactor interactions model was found to best fit the data (Table 2) where both ethanol and NaOH exert significant effect (p<0.001) on the amylose content of the treated starch, in which NaOH appeared to have greater effect than ethanol. Meanwhile, the interaction effect between the factors was also found significant (p<0.001). As depicted in Figure 1, the increase of NaOH induced leaching of amylose by swelling the starch granule to ease the release of amylose molecules. This in turn reduced the apparent amylose content detected in the treated samples. On the other hand, addition of ethanol restricted the swelling of starch hence constrained the leaching of amylose. This explains the less pronounce reduction trend in amylose content at higher level of ethanol. Table 2: Estimated regression coefficients and adjusted R 2 of predictive models for few response variables under studied. Factor Apparent Amylose Content Cold Water Solubility Cold Water Swelling Power Apparent Viscosity Granule Particle Size
5 β *** 14.38*** 4.38** * (Intercept) β *** 5.04* (Ethanol concentration) β *** 22.68*** 12.81*** * 3.69** (NaOH : starch ratio) β * 2.42 β *** 12.60** β ** 7.88** Adjusted R * p<0.05; ** p<0.01; *** p< Figure 1: Response surface plot for the effects of NaOH and ethanol (at 40 C) on apparent amylose content of GCWS sago starch. Cold Water Solubility and Swelling Power The cold water solubility and cold water swelling power of GCWS sago starch ranged between 6.7% 66.9% and respectively. Quadratic polynomial model was found best to explain the data collected for cold water solubility and swelling power (Table 2). NaOH was evident to affect both cold water solubility and swelling power in linear and quadratic manner (p<0.01), but ethanol did not affect the cold water swelling power of the samples. It can be observed from the three-dimensional response surface plots (Figure 2 & Figure 3) that addition of NaOH drastically increased the cold water solubility and swelling power of GCWS sago starch. Treatments with higher NaOH content increased the extent of granule swelling by rupturing the hydrogen bonds (Lai et al., 2004) and thus accelerated the cold water solubility. NaOH increases the starch granules affinity to water, induces swelling and results in significant increment in granule size (Lee et al., 2004). According to Chen & Jane (1994), the presence of ethanol in the reaction mixture restricted granule swelling and retarded dissociation of the native, double-helical structure and thus decreases the cold water solubility.
6 When starch molecules were subjected to the alcoholic-alkaline treatment, the crystallite structure was disrupted and water molecules became linked by hydrogen bonding to the exposed hydroxyl groups of amylose and amylopectin, which increased in granule swelling and solubility (Singh et al., 2003). The increase of granule swelling in the presence of alkali might be because of the anions (OH ) that associate at the specific site in the starch and create a large hydration sphere (Yamamoto et al., 2006). Figure 2: Response surface plot for the effects of NaOH and ethanol (at 40 C) on cold water solubility of GCWS sago starch.
7 Figure 3: Response surface plot for the effects of NaOH and ethanol (at 40 C ) on cold water swelling power of GCWS sago starch. Apparent Viscosity Similar to swelling power and solubility, higher quantity of NaOH during preparation raised the apparent viscosity of GCWS sago starch (p<0.05). It is notable that ethanol exerted marked effect and altered the apparent viscosity in quadratic manner (Figure 4). The apparent viscosity of starch increased from 40% ethanol to attain a maximum value and dropped thereafter. Respond surface shows that apparent viscosity peaked at highest NaOH concentration (300g). Figure 4: Response surface plot for the effects of NaOH and ethanol (at 40 C) on apparent viscosity of GCWS sago starch. Results of apparent viscosity obtained are in line with the observation of cold water swelling power. Viscosity of starch dispersion is strongly influenced by swelling of starch granules and the granules integrity as suggested by the results obtained. The viscosity increase at high NaOH concentration can be ascribed to the larger volume fraction of swollen starch in the starch dispersion where friction occurred when these swollen granules squeezed past each other (Singh et al., 2005). At low volume fraction (very low NaOH ratio or very high ethanol concentration), little interaction occurred between the relatively apart granules (Lee et al., 2004), which was then recorded as less viscous dispersion. Starch granules treated with substantially high amount of NaOH and low amount of ethanol experienced granule ruptures (evident by microscopic observation) and partial hydrolysis, imparted as low viscosity dispersion. Broadly speaking, GCWS sago starches were found more viscous (p<0.05) than the native counterpart (data not shown) as reported by other researchers (Bello-Pérez et al., 2000). The development of viscosity is presumably to be the interplay results of swelling power, solubility and granule integrity.
8 Granule Particle Size In order to estimate the granularity of GCWS sago starch, the particle size determination was carried out. The particle size determination only served as an estimation method to investigate the disruption of the sago starch during the alcoholic-alkaline treatment. It is postulated that starch with higher quantity of intact swollen granules will display higher mean of particle size distribution. Only NaOH was found significantly (p<0.01) affecting the mean particle size of starch granules (Table 2) where the particle size reduced with increasing amount of NaOH (Figure 5). The mean particle size for native sago starch was 29.3 µm; closed to the value reported by Ahmad et al. (1999). Alcoholic-alkaline treatment altered the mean granule size to be µm. Granular size bigger than native starch was attributed to the result of swelling, likewise smaller granular size was the consequence of granule rupture. These postulations were further proven by the observation under light microscope (data not shown). Figure 5: Response surface plot for the effects of NaOH and ethanol (at 40 C) on mean particle size of GCWS sago starch. Optimization and Verification The condition for preparation of GCWS sago starch was optimized based on two responses; cold water solubility and particle size distribution. The limit of cold water solubility was fixed based on the fact that GCWS starch must at least has 50% of cold water solubility (Eastman & Moore, 1984). Meanwhile, the limit of granule particle size was decided as the possible highest extent of granularity, taking into consideration the limit of cold water solubility. Contour plots of these two responses were generated and superimposed to obtain an area of optimum treatment condition in accordance to the limits of the responses established earlier. The optimum condition consisted % ethanol and g NaOH. To verify, the middle point (45% ethanol and 285 g of NaOH) under the optimum area was identified and used to prepare the GCWS sago starch. The experimental values of each of the response were compared with the values predicted by the equations of the model (Table 3). The experimental values fell within the predicted range and found to be insignificantly different (p>0.05) with the predicted values. The morphology of GCWS sago starch prepared under
9 optimum condition was viewed under binocular light microscope; the granules were found to swell to great extent and only slight disruption could be observed (figure not shown). It is thus affirmed that RSM had been successfully used in determining the optimum condition for preparation of GCWS sago starch. Table 3: Predicted and experimental values of the response variables produced at middle point under optimum condition obtained by Response Surface Methodology (RSM). Response Variables Values Predicted Predicted Experimental Range Cold Water Solubility (%) a ± 1.45 a Granule Particle Size (µm) b ± 1.24 b Means with identical alphabet in the same row denotes insignificant difference (p>0.05). CONCLUSIONS Alcoholic-alkaline treatment brought about substantial modification in the chemical and physiochemical properties of sago starch. In general, this treatment reduced the apparent amylose content whilst improved the cold water solubility and swelling power of sago starch. The degree of changes is however highly dependent on the amount of NaOH and ethanol used during the treatment. Results obtained suggest that NaOH exerted greater influence than ethanol on the responses studied. NaOH induced granule swelling, eased the amylose leaching and thus increased the cold water solubility of starch. Optimum amount of NaOH and ethanol used in preparation of GCWS sago starch had been successfully identified and verified in this study. Nevertheless, detailed investigation aiming at improving the cold water swelling of sago starch (yet retain the starch granule integrity) and solubility should be continued as the degree of cold water solubility achieved in this study is deemed less satisfactory. Future work should be focused on attempt to prevent the granule rupture in order to enhance the overall swelling and solubility capacity. ACKNOWLEDGEMENT Financial support from the Ministry of Science, Technology and Innovation, Malaysia (Project code: SF0074) is gratefully acknowledged. REFERENCES Ahmad, F.B., Williams, P.A., Doublier, J.L., Durand, S. & Buleon, A Physico-chemical Characterisation of Sago Starch. Carbohydrate Polymers, 38(4): Bello-Pérez, L.A., Acapulco, R.R.M. & Irapuato, O.P Preparation and properties of physically modified banana starch prepared by alcoholic-alkaline treatment. Starch/Stärke, 52(5), Chen, J. & Jane, J Preparation of granular cold water-soluble starches by alcoholic-alkaline treatment. Cereal Chemistry, 71(6), Eastman, J.E. & Moore, C.O Cold-Water-Soluble Gra nular Starch for Gelled Food Composition. U. S. Patent 4,465,702.
10 Hoover, R. & Ratnayake, W. S Determ ination of total amylose content of starch. In W rolstad, R. E., Acree, T. E., An, H., Decker, E. A., Penner, M. A., Reid, D. S., Schwartz, S. J., Shoemaker, C. F. & Sporns, P. (eds.) Current Pr otocols in Food Analytical Chem istry. New York: John Wiley & Sons Inc, J. S. Lee, Kumar, R.N., Rozman, H.D. & Azemi, B.M.N Flow behaviour of sago starch-g- distilled water and NaOH effect of photografting. Carbohydrate Polymers, poly(acrilyc acid) in 56: Jane, J. L. & Seib, P.A Preparation of granular cold water swelling/soluble starches by alcoholic-alkali treatments. U. S. Patent 5,057,157. Jane, J., Craig, S.A.A., Seib, P. A. & Hoseney, R.C Characterization of Granular Cold Water-Soluble Starch. Starch/Stärke, 38: Lai, L.N., Karim, A.A., Norziah, M.H. & Seow, C.C Effects of Na2CO3 and NaOH on Pasting Properties of Selected Native Cereal Starches. Journal of Food Science, 69(4): Light, J.M Modified food starches: Why, where, and how. Cereal Foods World, 35, Peterson, N.B Edible starches and starch-derived syrups. Food Technology Review NO.24. New Jersey: Noyes Data Corporation. Rajagopalan, S., & Seib, P.A Properties of granular cold-water soluble starches prepared at atmospheric pressure. Journal of Cereal Science, 16: Schoch, T.J Swelling power and solubility of granular starches. In: Whistler, R. L., Smith, R. J., Wolfrem, J. N. M. L. (eds.) Methods in Carbohydrate Chemistry. Orlando: Academic Press, Singh, J. & Singh, N Studies on the Morphological and Rheological Properties of Granular Cold Water Soluble Corn and Potato Starches. Food Hydrocolloids, 17: Singh, N., Sandhu, K.S. & Kaur, M Physicochemical Properties Including Granular Morphology, Amylose Content, Swelling, and Solubility, Thermal and Pasting Properties of Starches from Normal, Waxy, High Amylose and Sugary Corn. Progress in Food Biopolymer Research. 1: Singh, N., Singh, J., Kaur, L., Sodhi, N. S. & Gill, B. S Morphological, Thermal and Rheological Properties of Starches from Different Botanical Sources. Food Chemistry, 81: Yamamoto, H., Makita, E., Oki, Y. & Otani, M Flow Characteristics and Gelatinization Kinetics of Rice Starch under Strong Alkali Conditions. Food Hydrocolloids, 20: 9-20.
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