P-STARCH-15 Gelatinization and retrogradation properties of hypochlorite-oxidized cassava starch
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1 P-STARCH-15 Gelatinization and retrogradation properties of hypochlorite-oxidized cassava starch Kunruedee Sangseethong a, Niti Termvejsayanon a and Klanarong Sriroth b,c a Cassava and Starch Technology Research Unit, National Center for Genetic Engineering and Biotechnology, Bangkok, Thailand b Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University, Bangkok, Thailand c Kasetsart Agricultural and Agro-Industrial Product Improvement Institute, Kasetsart University, Bangkok, Thailand Abstract Gelatinization and retrogradation properties of hypochlorite-oxidized cassava starch as influenced by reaction ph under alkaline conditions (ph 9 and 11) were investigated. Generally hypochlorite oxidation introduced carbonyl and carboxyl groups to starch molecules as well as reduced starch viscosity. The formation of carbonyl and carboxyl groups was more favorable under mild alkaline condition (ph 9). Oxidation conducted at ph 11 produced both functional groups at a much slower rate and to a lesser extent. Viscosity of starch paste decreased markedly with the degree of oxidation and was dependent on reaction ph. Thermal behavior studied by differential scanning calorimetry (DSC) demonstrated that oxidation decreased gelatinization temperature and enthalpy of the starch. A decrease in gelatinization temperature was strongly dependent on the carboxyl content. The more the oxidized starch contained carboxyl groups, the lower was the gelatinization temperature. Retrogradation enthalpy of oxidized starch was not much different from that of native starch, suggesting that hypochlorite oxidation did not decrease amylopectin retrogradation significantly. On the other hand, results from the studies of light transmittance of starch paste during refrigerated storage suggested that retrogradation was retarded in oxidized starch. While the light transmittance of native starch drastically decreased during storage, the changes observed in oxidized starches were less pronounced. Oxidized starch produced under mild alkaline condition (ph 9) which had higher carboxyl content exhibited lower extent of retrogradation as revealed by light transmittance studies. Introduction Oxidized starch is widely used in many industries, particularly in applications where film formation and adhesion properties are desired. The major application of oxidized starch is as surface sizing agents and coating binders in paper industry. Oxidized starch is commonly produced by reaction of starch with an oxidizing agent under controlled temperature and ph. Although many oxidizing agents are available, hypochlorite is the most commonly used chemical in the commercial production of oxidized starch. During the oxidation of starch, different reactions occur. These reactions lead to the introduction of carbonyl and carboxyl groups, and to the degradation of starch molecules. The desired properties of oxidized starch are mainly lower viscosity and improved stability of starch paste. Several investigations on oxidation of amylose, amylopectin and native starches from various origins have been reported (Whistler et al., 1956; Whistler and Schweiger, 1957; Forssell, et al., 1995; Kuakpetoon and Wang, 2001; Kuakpetoon and Wang, 2006). It has been claimed that the ph range during oxidation process is a critical factor. At low and intermediate ph, aldehyde and keto groups tend to be formed; while at high ph carboxyl groups are formed. In order to favor the production of carboxyl group which is a key component in stabilizing the 263
2 viscosity of starch dispersion and minimizing retrogradation, hypochlorite oxidation of starch is normally performed under mild to moderate alkalinity (Wuzburg, 1986). Relatively few reports are available in the literature concerning the physicochemical and functional properties of oxidized starch especially those prepared under various levels of reaction alkalinity. The present investigation was undertaken in order to study gelatinization and retrogradation properties and their relations to the chemical nature of oxidized starch modified under different alkalinity levels. Materials and Methods Preparation of oxidized starch Cassava starch slurry containing 40% dry solid was prepared and the ph was adjusted to 9 or 11 with sodium hydroxide solution. The temperature of the slurry was maintained at 30 C, and sodium hypochlorite (3% active chlorine based on starch) was added dropwise over a period of 20 min with stirring. During the addition of the reagent and the course of reaction, the ph of the slurry was maintained with sodium hydroxide and/or hydrochloric acid solution. Then the mixture was stirred under the defined conditions and the sample was then collected at the reaction time of 30, 60, 120 and 300 min. The reaction in the collected samples was terminated by addition of sodium bisulphite and the ph was adjusted to The sample was then filtered and thoroughly washed and oven dried at 50 C. Determination of carbonyl content The carbonyl content was determined as described by Kuakpetoon and Wang (2001). Starch sample (4 g) was slurried in 100 ml of distilled water. The slurry was gelatinized in a boiling water bath for 20 min, cooled to 40 C, adjusted to ph 3.2 with 0.1 M HCl, and 15 ml of hydroxylamine reagent was added. The flask was stoppered and agitated in a water bath at 40 C. After 4 hours, the sample was rapidly titrated to ph 3.2 with 0.1 M HCl. A blank determination with only hydroxylamine reagent was performed in the same manner. The hydroxylamine reagent was prepared by dissolving 25 g hydroxylamine hydrochloride in 100 ml of 0.5 M NaOH. The final volume was then adjusted to 500 ml with distilled water. Determination of carboxyl content The carboxyl content of hypochlorite-modified cassava starch was determined following FAO method (2001). Starch sample (5 g) was stirred in 25 ml of 0.1 M HCl for 30 min. The slurry was then filtered and washed with distilled water until free of chloride ions. The filtered cake was transferred to 300 ml water and the starch slurry was heated in a boiling water bath with continuous stirring until gelatinized and continue stirring at that temperature for another 15 min. The hot sample was titrated with 0.1 M NaOH using phenolphthalein as an indicator. A blank determination was run on the original sample in the same manner but stirred in 25 ml distilled water instead of 0.1 M HCl. Viscosity measurement The measurement of viscosity of oxidized starch was performed on a rotational Physica MCR 300 rheometer (Physica Messtechnik GmbH, Ostfildern, Germany) using a concentric cylinder. Samples for all measurements were prepared by heating 15% starch suspension in a water bath at 95 C for 15 min with continuous stirring. The samples were then immediately transferred to the rheometer. The viscosity of oxidized starch was measured over a shear rate range of
3 500 s -1 at 80 C. The viscosity value at 22 s -1 was used for comparison between different samples. Gelatinization and retrogradation properties by DSC The gelatinization and retrogradation of native and modified starches were measured using a Perkin-Elmer Differential Scanning Calorimeter (DSC7, Norwalk, CT). Starch was weighted into a stainless steel DSC pan and deionized water was added to give 70% moisture content. The pan was sealed, equilibrated at room temperature overnight, and scanned from 0 to 120 C at a rate of 10 C/min. After scanning, the gelatinized sample was stored at 4 C for 7 days, after which the sample was left at room temperature for 1 hour and rescanned under the same condition with the first scanning. An empty pan was used as the reference and the DSC was calibrated with indium. The onset (T o ), peak (T p ) and conclusion (T c ) temperatures and the enthalpy of gelatinization ( H g ) and retrogradation ( H r ) were determined. Light transmittance of starch paste Light transmittance of starch paste was measured as described by Jacobson et al. (1997) with some modification. A 5% (w/w) aqueous suspension of starch in screw-capped test tube was heated in a boiling water bath for 30 min with constant shaking for the first 5 min and occasional shaking afterward. After cooling to room temperature, transmittance was determined at 650 nm against water blank using a SPECTRONIC GENESYS-5 (Milton Roy Company, USA). Also to monitor tendency for retrogradation, samples were stored at 4 C in a refrigerator and transmittance of samples was determined during cold storage for up to 7 days. Results and Discussion The properties of oxidized cassava starches modified under different reaction alkalinity (ph 9 and 11) were compared. The carbonyl and carboxyl contents of oxidized starches are presented in Figure 1 and 2. Oxidized starches modified under both conditions contained higher carboxyl content than carbonyl content which is in agreement with the previous reports that starch oxidized under alkaline conditions tended to produce higher amount of carboxyl than carbonyl groups. (Wuzburg, 1986). The formation of both functional groups was more favorable under milder alkaline condition (ph 9). Oxidation conducted at ph 11 produced both functional groups at a much slower rate and to a much lesser extent Carbonyl content (%) Native ph 9 ph Reaction time (min) Figure 1 Carbonyl content of native and oxidized cassava starches prepared under different reaction ph and times. 265
4 1 0.9 Carboxyl content (%) Native ph 9 ph Reaction time (min) Figure 2 Carboxyl content of native and oxidized cassava starches prepared under different reaction ph and times. Apparent viscosity of oxidized starch modified under different ph is shown in Figure 3. Data for native cassava starch are not presented since the paste of native starch was too viscous for viscosity determination at this high solid content (15% w/w). In general, viscosity of oxidized starch decreased with increasing reaction time. The decrease in starch viscosity was caused by oxidative cleavage of glycosidic linkages, resulting in a reduction of molecular size of starch molecules. The rate and extent of viscosity reduction was much greater when oxidation was conducted at ph 11. Beside oxidative cleavage, alkaline degradation might take part in the viscosity reduction of oxidized starch modified under more vigorous alkalinity Viscosity (mpa.s) ph 9 ph Reaction time (min) Figure 3 Viscosity of oxidized cassava starches prepared under different reaction ph and times. Gelatinization properties of native and oxidized cassava starches measured by DSC are summarized in Table 1. In general, oxidized starch had lower onset gelatinization temperature (T o ) and gelatinization enthalpy ( H g ) than native starch. The decreased T o in oxidized starch 266
5 could be attributed to the presence of carboxyl group whose negative charge could readily absorb water and facilitate hydration and swelling of starch granules, thus; gelatinization occurred at a lower temperature. The observed change in T o of oxidized starches seemed to be in accordance with the amount of carboxyl group in the starch samples. Oxidized starch produced under milder alkaline condition (ph9) which contained higher amount of carboxyl group exhibited greater reduction in T o. The decrease in H g was possibly from degradation of crystalline lamellae which seemed to be increased with degree of oxidation under both reaction conditions. DSC data of retrograded starches stored at 4 C for 7 days are shown in Table 2. During refrigeration storage of starch paste, recrystallization of amylopectin occurred and reheating of a stored sample in a DSC produced an endothermic transition and the enthalpy observed during this transition could be used as a measure of retrogradation extent of amylopectin in starch samples. In this study, we observed that retrogradation enthalpies ( H r ) of oxidized starches were not significantly different from that of native starch and in the case of starches modified at ph 11 the enthalpies were even slightly higher (Table 2). It seemed that the introduction of carboxyl groups into starch molecules by hypochlorite oxidation could not prevent amylopectin retrogradation. Possibly, the site of carboxyl group formation might be close to the branching point as suggested by Kuakpetoon and Wang (2006); in that position, they were not very effective in preventing the recrystallization of the amylopectin chains. Table 1 Gelatinization properties of native and oxidized cassava starches by DSC Reaction ph Reaction time (min) Transition temperature ( C) Gelatinization enthalpy (J/g) T o T p T c Native
6 Table 2 Retrogradation properties of native and oxidized cassava starches by DSC Reaction ph Reaction time Transition temperature ( C) Retrogradation (min) T o T p T c enthalpy (J/g) Native The tendency for retrogradation of native and oxidized starches was also determined by following the changes in light transmittance of starch paste during storage at 4 C for 7 days, and the results are shown in Figure 4. Native and different oxidized starches showed variation in light transmittance of fresh pastes (0 day of storage). This could be due to the difference in their molecular sizes as well as the amount, type and the position of functional groups present in each starch sample. Unlike results from DSC, light transmittance study demonstrated that oxidized starches showed the decreased tendency for retrogradation. While the light transmittance of native starch drastically decreased during cold storage, the changes observed in oxidized starches were less pronounced. Oxidized starches produced under milder alkaline condition (ph 9) which had higher carboxyl contents exhibited constant light transmittance throughout the storage period, which indicated that retrogradation was minimized in this group of samples. The discrepancy of results from DSC and light transmittance is possibly due to the different natures of these two analytical techniques. To analyze retrogradation by DSC, high starch concentration is required (30% in this case). The gelatinization of starch in a calorimeter pan produces swollen but nondisrupted granules. The pan is then subjected to retrogradation conditions, and the contents are analyzed by DSC (Jacobson and BeMiller, 1998). In such conditions, the localized carboxyl groups formed in starch molecules might not be very effective in preventing the recrystallization of amylopectin molecules. In contrast, to analyze retrogradation tendency by light transmittance, low concentration of starch slurry was heated in a boiling water bath. This condition facilitates granule swelling and promotes starch molecular dispersion into solution. The starch molecules carrying carboxyl groups are mingled with molecules that do not contain functional groups. In this way, the carboxyl groups could sterically hinder the aggregation of starch molecules, resulting in lower retrogradation. Conclusion Hypochlorite oxidation under alkaline conditions tended to favor the formation of carboxyl than carbonyl groups. Oxidation conducted under mild alkaline condition (ph 9) produced carboxyl group at a much faster rate and to a greater extent. However, the rate and extent of viscosity reduction was much greater when oxidation was conducted under condition with higher alkalinity (ph 11). The gelatinization temperatures and the enthalpies of gelatinization of oxidized starches were lower than those of the native starch. The presence of carboxyl group appeared to be responsible for the decrease in the gelatinization temperature of oxidized 268
7 starches. DSC study revealed that hypochlorite oxidation could not prevent amylopectin retrogradation. On the other hand, results from light transmittance of starch paste during cold storage suggested that retrogradation in oxidized starches could be retarded. 120 Light transmittance (%) Native ph 9-30min ph 9-60min ph 9-120min ph 9-300min ph 11-30min ph 11-60min ph min ph min Storage time (days) Figure 4 Light transmittance of native and oxidized cassava starches (prepared under different reaction ph and times) during storage at 4 C Acknowledgement This work was supported by National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Ministry of Science and Technology, Thailand. References Food and Agriculture Organization (FAO) FAO Food and Nutrition Paper 52 Addendum 9. Food and Agriculture Organization of the United Nations. Forssell, P., Hamunen, A., Autio, K., Suortti, T. and Poutanen, K Hypochlorite oxidation of barley and potato starch. Starch/Starke 47: Kuakpetoon, D. and Wang, Y.J Characterization of different starches oxidized by hypochlorite. Starch/Starke. 53: Kuakpetoon, D. and Wang, Y.J Structural characteristics and physicochemical properties of oxidized corn starches varying in amylose content. Carbohydr. Res. 341: Jacobson, M.R. and BeMiller, J.N Method for determining the rate and extent of accelerated starch retrogradation. Cereal Chem. 75: Jacobson, M.R., Obanni, M., and BeMiller, J.N Retrogradation of starches from different botanical sources. Cereal Chem. 74: Whistler, R.L., Linke, E.G. and Kazeniac, S Action of alkaline hypochlorite on corn starch amylose and methyl 4-O-methyl-D-glucopyranosides. J. Am. Chem. Soc. 78: Whistler, R.L. and Schweiger, R Oxidation of amylopectin with hypochlorite at different hydrogen ion concentrations. J. Am. Chem. Soc. 79: Wurzburg, O.B Converted starches. In Modified Starches : Properties and Uses. Ed. O.B. Wurzburg. CRC Press, Inc., Boca Raton, Florida. pp
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