Food Hydrocolloids 23 (2009) Contents lists available at ScienceDirect. Food Hydrocolloids

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1 Food Hydrocolloids 23 (2009) Contents lists available at ScienceDirect Food Hydrocolloids journal homepage: Short Communication Effect of ph on aqueous structure of maize starches analyzed by HPSEC-MALLS-RI system Ju Hun Lee a, Jung-Ah Han b, Seung-Taik Lim a, * a Graduate School of Life Sciences and Biotechnology, Korea University, Seoul , Korea b Division of Human Environmental Sciences, Sangmyung University, Seoul , Korea article info abstract Article history: Received 16 June 2008 Accepted 23 December 2008 Keywords: Starch Maize Structure ph Light scattering Molecular conformation of maize starches (waxy, normal and high amylose) was examined at different ph values (5 10) using an aqueous medium-pressured size exclusion column chromatography (SEC) connected to multi-angle laser light scattering (MALLS) and refractive index (RI) detectors. The starch molecules were partially degraded at ph 5 and 10, resulting in lower values for molar size and radius of gyration than those in neutral condition. The specific volume data revealed that the structural changes under an alkaline condition such as ph 10 was more significant for amylose than that for amylopectin. Logarithmic ratios between molar size and radius of gyration were similar ( ) for the amylopectins in waxy and normal maize starches, but higher ( ) for the amylopectin in high amylose starch, indicating that the amylopectins in waxy or normal maize starches had sphere forms whereas that in high amylose starch had a random coil shape. Ó 2008 Elsevier Ltd. All rights reserved. 1. Introduction Starch molecules, consisting of amylopecin and amylose, can be fractionated using a size exclusion column chromatography (SEC) (Bello-Perez, Roger, Baud, & Colonna, 1998; Jackson, Choto-Owen, Waniska, & Rooney, 1988) and characterized in situ by various detectors. Laser light scattering detectors are often used to characterize various starches (Bello-Perez, Roger, Colonna, & Paredes-Lopez, 1998; Blennow, Bay-Smidt, & Bauer, 2001; Han & Lim, 2004a, b; Millard, Wolf, Dintzis, & Willett, 1999). The radius of gyration (R g ) and molar size (M w ) measured by the detectors, and the specific volume of gyration (SV g ) calculated from R g and M w provide information about the chain conformation of starch dispersed in a solution (You & Lim, 2000). One parameter useful for characterizing the chain conformation is the R g change induced by M w change, obtained from an equation: R g ¼ km v w (Hanselmann, Burchard, Ehrat, & Widmer, 1996). The logarithmic slope between R g and M w reveals three dimensional chain conformation: sphere, random coil, and rod shapes (Wyatt, 1993). Sample preparation and analytical procedure should be carefully controlled to obtain accurate structural data. Instead of pure * Corresponding author. Tel.: þ ; fax: þ address: limst@korea.ac.kr (S.-T. Lim). water, dimethyl sulfoxide (DMSO) and alkaline solutions are frequently used for the complete dissolution of starch (Jackson, Choto-Owen, Waniska, & Rooney, 1988; Han & Lim, 2004a, b; Chamberlain & Rao, 1999). Thermal treatments such as autoclaving, jet-cooking, and microwave heating accompanied with high shear stirring have been suggested to achieve the complete dissolution (Han & Lim, 2004b; You & Lim, 2000; Fishman, Cooke, White, & Damert, 1995). However, some of those physical treatments cause starch degradation (Han & Lim, 2004b; Barth & Carlin, 1984; Hanselman, Ehrat, & Widmer, 1995; Peng & Perlin, 1987). As the hydroxyl groups in the anhydroglucose units (AGU) of starch are exposed to the dissolution solvent, interactions between the hydroxyl groups of AGU and solvent components then affect the structure of the starch chains. When a starch was dissolved in aqueous media, the ph affected the physical properties and chain structure of starch. Chemical degradation by acids (Hoover, 2000) and alkalis (Han & Lim, 2004a) has been already reported. However, the conformational analyses of starch chains in terms of the solution ph were rarely done. In this study, three maize starches different in amylose content (waxy, normal and high amylose starch) were analyzed to characterize the structural relations of starch with the ph of dissolution medium (5 10), by using a size exclusion column chromatography (SEC) connected with multiangle laser light scattering (MALLS) and refractive index (RI) detectors X/$ see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi: /j.foodhyd

2 Materials and methods 2.1. Materials J.H. Lee et al. / Food Hydrocolloids 23 (2009) Waxy and normal maize starches were provided by Samyang Genex Company (Seoul, Korea), and high amylose maize starch (70% amylose) was provided by Cerestar USA, Inc. (Hammond, IN). Starch (1 g, dry solids) was dissolved in an aqueous DMSO solution (90%, 100 ml) by boiling for 1 h, and then mildly magnetic-stirred at room temperature for 24 h. The starch in the solution was precipitated and washed by absolute ethanol (300 ml, three times), and acetone (300 ml, once) (Klucinec, & Thopmson, 1998). The purified starch obtained was dried in a convection oven at 30 C, overnight Starch sample solutions The purified starch (10 mg, dry solids) was wetted with ethanol (50 ml), and dissolved in 1 M NaOH solution (1 ml) by vigorous vortex. The starch solution was neutralized by adding 1 M HCl (1 ml) (You, Fiedorowicz, & Lim, 1999), and diluted with SEC eluent (100 mm NaCl 18 ml). The solution was autoclaved (121 C) for 20 min, and then the ph of the solution was adjusted to 5 10 by adding 1 M NaOH or 1 M HCl SEC-MALLS-RI system The autoclaved sample was filtered through a membrane filter (5 mm pore size, Advantec Inc, Japan) and injected (1 ml) into the SEC-MALLS-RI system. The eluent (100 mm NaCl) that was adjusted to ph 5 10 with 1 M NaOH or 1 M HCl was filtered through a mixed cellulose ester membrane (0.1 mm pore size, Advantec Inc, Japan), and degassed prior to use. The SEC column (2.6 cm diameter, 70 cm length) was filled with Toyopearl HW-65F resins (TOSOH Corporation, Japan, analytical M w range from 10 4 to 10 6 g/mol based on dextran). A pump (P2000, Spectra System, San Hose, CA), an injector with a 1 ml loop, the SEC column, a MALLS (632.8 nm, DAWN DSP, Wyatt Technology, Santa Barbara, CA) and an RI detector (Optilab DSP, Wyatt Technology, Santa Barbara, CA) were connected in series. The flow rate of the eluent was 1.5 ml/min (You & Lim, 2000) Data analysis As the amylopectin fraction was prior to separate through the SEC, amylopectin and amylose could be distinguished. Molecular information of separated starches and the whole fractions (total) through the SEC were calculated and analyzed by the ASTRA program (Wyatt Technology, Co, Santa Barbara, CA). All measurements were repeated three times for each separate dissolved sample, and the average values and standard deviations were calculated. 3. Results and discussion Acids tend to hydrolyze the glycosidic linkages, producing dextrins of reduced size (Hoover, 2000), and alkalis may also degrade the starch chains through more complicated structural rearrangement (Han & Lim, 2004a; Nagamine and Komae, 1996). Mild alkalinity, however, is commonly used for dissolution because it readily disrupts the hydrogen bonds between starch chains. Amylopectin and amylose were fractionated through the SEC and molecular characteristics of the starch molecules could be examined (Fig. 1). As shown in Fig. 2A, Mw of the maize starches was changed by the solution ph. Although there were some differences among the starches tested, the M w became smaller RI output (volts) Volume (ml) Fig. 1. RI chromatograms of waxy ( C ), normal ( : ) and high amylose ( - ) maize starches at ph 5. when the solution ph was either 5 or 10. It indicates that the starch was degraded even in mild acidic or alkaline conditions. During the chromatographic separation, starch chains were subjected to shearing with pressure. It was reported that physical treatments applied for starch dissolution such as heating and high shearing might cause chain degradation (Han & Lim, 2004b). The degradation of starch in this experiment might be facilitated by the shearing and pressure of the analytical system. The M w of the amylopectin in waxy and normal maize starches was similar, ranging approximately from to g/ mol as the ph changed from 5 to 10, whereas the amylopectin in high amylose maize starch showed much smaller M w (approximately g/mol). These data were similar to those reported by Han and Lim (2004a). It was found that the amylopectin in normal maize starch was more susceptible to the acidic and alkaline conditions than the waxy maize amylopectin. Assuming that the chain structures of the amylopectins in both starches were not much different, the presence of amylose in normal starch might affect the susceptibility of amylopectin. We assume that associations between amylose and amylopectin which might exist even in the diluted sample solutions mutually affected the stability of the starch chains. The amylopectin in high amylose maize starch displayed a linear increase of M w as the ph increased from 7 to 9 ( g/mol), whereas normal and waxy amylopectins showed similar values at ph 7 and 9. The average chain length of the amylopectin in A-type starches (normal and waxy starches in this case) is usually smaller than that in B-type starches such as high amylose starch (Hizukuri, 1985). The proportion of short chains (DP < 12) in A-type starches is about twice as much as that in B-type starches, but the proportion of long chains (DP > 25) in A-type starches is about half (Srichuwong & Jane, 2007). Therefore, compared to that in A-type starches, the amylopectin in B-type starches has relatively smaller molar size but consists of longer chains. It was expected that the long side chains of the amylopectin in high amylose starch provided strong associations between the chains, and thus the alkalinity (ph 9) was required to dissolve the amylopectin chains in high amylose starch. Therefore, the amylopectin chains that had not been dissolved in neutral solution could be dissolved at ph 9, and analyzed by the system, resulting in the higher average M w value than that at ph 7. The M w of amylose in normal maize starch ranged from to g/mol, whereas that in high amylose maize starch did from to g/mol. Similar results have been already reported (Han & Lim, 2004a; Takeda, Shitaozono, &

3 J.H. Lee et al. / Food Hydrocolloids 23 (2009) Fig. 2. Changes in molar size (A) and radius of gyration (B) for waxy, normal and high amylose maize starches at different ph; amylopectin ( - ), amylose (, ) and total ( C ). Hizukuri, 1988). In Fig. 2A, the M w of amylose appeared not much changed by the acidic and alkaline ph values than that of amylopectin. Normal maize especially displayed substantial changes in M w : g/mol at ph 5, g/mol at ph 7, g/ mol at ph 9, and g/mol at ph 10, indicating that amylose was also degraded by the acidic ph. However, the amylose in high amylose starch was relatively stable against the ph change (5 10) without a significant change in M w ( g/mol). It was reported that the amylose in high amylose maize starch was smaller and less branched than that in normal maize starch (Takeda, Shitaozono, & Hizukuri, 1988; Takeda, Takeda, & Hizukuri, 1989). The amylose chains smaller sized might have greater mobility and the less branched structure might provide more chance for the chains to associate, which resulted in higher stability to the ph changes. The R g representing the theoretical gyrating volume of chains in solution was also affected by the solution ph (Fig. 2B). The R g of waxy maize amylopectin gradually increased ( nm) as the solution ph increased from 5 to 9, but slightly decreased at ph 10 (245 nm), due to the alkaline degradation. The amylopectin chains in both normal and high amylose starches showed similar trends. The overall changes in R g by the ph changes were similar to those observed in M w, indicating that the gyration volume of amylopectin was dependent on the M w. Likewise, the R g of amylose varied in similar ways to the M w. For normal maize starch, as the ph increased from 5 to 9, the M w increased from to g/mol, and the R g also increased from 85 to 156 nm. In the same theory, as the M w of the amylose in high amylose starch was not much changed, the R g values were also not much changed (65 92 nm). The specific volume for gyration (SV g ), calculated by assuming that the gyration occurs in a sphere pattern, provides the gyration volume on a basis of unit mass (You & Lim, 2000). Amylopectin exists in more compact shape than amylose, having smaller specific gyration volume (Fig. 3A). For the maize starches tested, the SV g values for amylopectin were not much different, ranging from 0.11 to 0.34, without showing the ph effect. The result suggests that the chain conformation of amylopectin remained unchanged in the aqueous solutions of different ph values (5 10), even though some chain degradation occurred at acidic or alkaline ph values. It also revealed that the chain degradation at ph 5 and 10 occurred in random fashions. For the amylose in normal maize starch, however, the specific volume continually increased as the solution ph increased from 5 to 10 (Fig. 3A). As shown in Fig. 2A, there was acidic degradation at ph 5 for the amylose in normal maize starch: g/mol at ph 5 vs g/mol at ph 7. The SV g,

4 1938 J.H. Lee et al. / Food Hydrocolloids 23 (2009) Fig. 3. Changes in SV g (A) and slope of log M w vs. log R g (B) for waxy, normal and high amylose maize starches at different ph; amylopectin ( - ) and amylose (, ). however, remained unchanged (0.91) as the ph changes from 5 to 7, indicating that the acidic hydrolysis for the amylose also occurred in a random fashion. The SV g continually increased as the ph increased from 7 to 10, although the M w change was not significant at the ph above 7. It clearly revealed that the SV g of the amylose was not dependent to the M w. Alkali may deprotonize starch chains which may reduce the level of chain association. The amylose in the high amylose maize starch, however, did not show this trend, showing a slightly higher specific volume at neutral ph than that at ph 9 or 10. As previously discussed with M w data, amylose might not be fully dissolved at ph 7, the SV g value measured at ph 7 was not accurate. However, more research should be followed to clarify this. Three dimensional conformation of starch chains in an aqueous medium may also be estimated with the slope in logarithmic plot between R g and M w (Bello-Perez, Roger, Colonna, & Paredes-Lopez, 1998), based on an equation: R g ¼ km w v. It provides conformational information because the R g is proportional to the geometrical size for linear molecules. The estimation is particularly simple when a polymeric sample has sufficiently broad range of molar mass which may be feasible by a chromatographic system (Wyatt, 1993). For a spherical conformation, the relationship between average M w and R g yields a line with a slope of about In the case of rod shaped molecules, while for random coil in a good solvent, the slope should be between 0.5 and 0.6. As shown in Fig. 3B, the amylopectin in high amylose maize starch exhibited higher slope values ( ) than those in waxy and normal maize amylopectin ( ). Although amylopectin chains in the three starches tested had similar SV g values regardless of the solution ph, the amylopectin in waxy and normal maize starches existed in sphere shape in the solution, whereas that in high amylose maize starch was more likely in random coil shape. This result was from the greater chain length of the amylopectin in high amylose maize starch than those in normal and waxy maize starches. Amylose chains had much lower values of the slope than amylopectin showing even some negative values (Fig. 3B). The negative slopes indicate that the R g actually varied in opposite directions to the M w change. The linear chains, even in a dilute solution, may not exist solely independent, but associate each other. As the solution ph increased, however, the slope for the amylose in normal maize starch was gradually increased. It may suggest that with the alkalinity facilitates the dissociation between amylose chains. 4. Conclusions The structure of starch chains in an aqueous medium was affected by the ph of the medium. Even at the acidity or alkalinity like ph 5 or 10, starch chains may be degraded. The amylose was

5 J.H. Lee et al. / Food Hydrocolloids 23 (2009) more susceptible to the solution ph than in the chain degradation than amylopectin. The alkalinity like ph 9 assists chain dissociations in solution, especially for amylose. Amylopectins in waxy and normal maize starches existed in sphere conformation, whereas that in high amylose maize with longer chains had random coil shape in aqueous solutions. Acknowledgement This study was financially supported by a project (No F00068) from the Korea Research Foundation. References Barth, H. G., & Carlin, F. J. (1984). A review of polymer shear degradation in sizeexclusion chromatography. Journal of Liquid Chromatography, 7(9), Bello-Perez, L. A., Roger, P., Baud, B., & Colonna, P. (1998). Macromolecular features of starches determined by aqueous high-performance size exclusion chromatography. Journal of Cereal Science, 27, Bello-Perez, L. A., Roger, P., Colonna, P., & Paredes-Lopez, O. (1998). Laser light scattering of high amylose and amylopectin materials, stability in water after microwave dispersion. Carbohydrate Polymers, 37, Blennow, A., Bay-Smidt, A. M., & Bauer, R. (2001). Amylopectin aggregation as a function of starch phosphate content studied by size exclusion chromatography and on-line refractive index and light scattering. International Journal of Biological Macromolecules, 28, Chamberlain, E. K., & Rao, M. A. (1999). Rheological properties of acid converted waxy maize starches in water and 90% DMSO/10% water. Carbohydrate Polymers, 40, Fishman, M. L., Cooke, P., White, B., & Damert, W. (1995). Size distributions of amylose and amylopectin solubilized from corn starch granules. Carbohydrate Polymers, 26, Han, J. A., & Lim, S. T. (2004a). Structural changes in corn starches during alkaline dissolution by vortexing. Carbohydrate Polymers, 55, Han, J. A., & Lim, S. T. (2004b). Structural changes of corn starches by heating and stirring in DMSO measured by SEC-MALLS-RI system. Carbohydrate Polymers, 55, Hanselmann, R., Burchard, W., Ehrat, M., & Widmer, H. M. (1996). Structural properties of fractionated starch polymers and their dependence on the dissolution process. Macromolecules, 29, Hanselman, R., Ehrat, M., & Widmer, H. M. (1995). Sedimentation field flow fractionation combined with multi angle laser light scattering applied for characterization of starch polymers. Starch/Starke, 46, Hizukuri, S. (1985). Relationship between the distribution of the chain length of amylopectin and the crystalline structure of starch granules. Carbohydrate Research, 141, Hoover, R. (2000). Acid-treated starched. Food Review International, 16(3), Jackson, D. S., Choto-Owen, C., Waniska, R. D., & Rooney, L. W. (1988). Characterization of starch cooked in alkali by aqueous high-performance size-exclusion chromatography. Cereal Chemistry, 65(6), Klucinec, J. D., & Thompson, D. B. (1998). Fractionation of high amylose maize starches by differential alcohol precipitation and chromatography of the fraction. Cereal Chemistry, 75, Millard, M. M., Wolf, W. J., Dintzis, F. R., & Willett, J. L. (1999). The hydrodynamic characterization of waxy maize amylopectin in 90% dimethyl sulfoxide-water by analytical ultracentrifugation, dynamic, and static light scattering. Carbohydrate Polymers, 39, Nagamine, T., & Komae, K. (1996). Improvement of a method for chain-length distribution analysis of wheat amylopectin. Journal of Chromatography A, 732, Peng, O.-J., & Perlin, A. S. (1987). Observations on N.M.R. spectra of starches in dimethyl sulfoxide, iodine-complexing, and salvation in water-di-methyl sulfoxide. Carbohydrate Research, 160, Srichuwong, S., & Jane, J. (2007). Rhysicochemical properties of starch affected by molecular composition structures: a review. Food Science and Biotechnology, 16, Takeda, Y., Shitaozono, T., & Hizukuri, S. (1988). Molecular structure of corn starch. Starch/Starke, 40(2), Takeda, C., Takeda, Y., & Hizukuri, S. (1989). Structure of amylomaize amylose. Cereal Chemistry, 66(1), Wyatt, P. J. (1993). Light scattering and the absolute characterization of macromolecules. Analytica Chimica Acta, 272, You, S. G., Fiedorowicz, M., & Lim, S. T. (1999). Molecular characterization of wheat amylopectins by multiangle laser light scattering analysis. Cereal Chemistry, 76(1), You, S. G., & Lim, S. T. (2000). Molecular characterization of corn starch using an aqueous HPSEC-MALLS-RI system under various dissolution and analytical conditions. Cereal Chemistry, 77(3),