Utilization of Amylose−Lipid Complexes as Molecular Nanocapsules

Biomacromolecules 2005, 6, 121-130

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Utilization of Amylose-Lipid Complexes as Molecular Nanocapsules for Conjugated Linoleic Acid Inbal Lalush, Hagit Bar, Imad Zakaria, Sigal Eichler, and Eyal Shimoni* Department of Biotechnology and Food Engineering, TechnionsIsrael Institute of Technology, Haifa 32000, Israel Received June 17, 2004; Revised Manuscript Received August 18, 2004

Amylose-conjugated linoleic acid (CLA) complexes were produced by water/dimethyl sulfoxide (DMSO) and KOH/HCl complexation methods. The formation of amylose V form was confirmed by X-ray diffraction (XRD), and complexes formed at 30, 60, and 90 °C exhibit melting temperatures exceeding 88 °C. Atomic force microscopy (AFM) images showed distinct difference in complex organization, with complexes formed in water/DMSO showing spherical shape with typical diameter of 150 nm. Complexes formed by KOH/ HCl showed elongated structure with typical width of 43-160 nm. Water/DMSO complexes exhibit superior protection to CLA against oxidation. All complexes showed high retention of CLA in simulated stomach conditions, and the digestion of complexes by amylases results in high hydrolysis and CLA release by pancreatin and R-amylase. Only moderate release was detected following hydrolysis by amyloglucosidase and β-amylase. It is therefore suggested that amylose-CLA complexes can serve as molecular nanocapsules for protection and delivery of CLA. Introduction The interaction between amylose and lipids is often characterized by amylose chains forming semicrystalline V-forms. The V-form is an amylose chain that forms a helix with a large cavity in which various ligands can be situated, and the size of the ligand determines the number of glucosyl residues per turn (6, 7, or 8).1 It was suggested that the crystalline state of amylose-fatty acid complex involves the V-amylose 6-fold single-chain left-handed helix, well-known among starch polymorphs.2 Accordingly, it is often assumed that the fatty acid is a “stem” (planar zigzag) inside the helix, whose inner surface is hydrophobic because of the carbonhydrogen of the 6-fold helix.3 The insoluble amylose complexes exist in two polymorphic forms, types I and II, each being characterized by the temperature at which dissociation occurs: type I polymorphs have lower dissociation temperatures.4,5 It is thought that V-helical complex segments are interrupted by short sections of uncomplexed amylose that permit random orientation of the helical segments in the type I complexes, and folding into parallel and antiparallel arrays in the crystalline type II complexes.6,7 Most of the interest in amylose-lipid complexes focused on their technological importance in starchy food systems, since it modifies the texture and structural stability of starch based-products (e.g., reduction in stickiness, improved freeze-thaw stability, and retardation of retrogradation).5,6,8-10 Other researches studied amylose-lipid complexes in view of their contribution to the bioavailability of starch, in terms of its enzymatic digestion.11-14 It was shown that the V-forms can be produced from mono- and diglycerides6,9,14-16 and * Corresponding author: tel +972-4-8292484; fax +972-4-8293399; e-mail [email protected].

saturated fatty acids,5,8,17-19 as well as unsaturated fatty acids.1,5,8,9,20 These studies showed that the complexes formed have high melting temperatures, that the complexed fatty acid is efficiently protected from oxidation, and that the digestibility of starch is influenced by complex formation, which decreased the digestibility of starch. In the present study, it is hypothesized that amylose-lipid complexes can be used as a delivery system for polyunsaturated fatty acids (PUFA). It is suggested that these complexes will provide protection during processing and storage and will release the PUFA in the intestine following enzymatic hydrolysis of the amylose. The feasibility of this concept is examined by use of conjugated linoleic acid (CLA) as a model. CLA refers to a group of polyunsaturated fatty acids that exist as positional and stereoisomers of conjugated dienoic octadecadienoate (18:2). Numerous physiological properties have been attributed to CLA including action as an antiadipogenic, antidiabetogenic, anticarcinogenic, and antiatherosclerotic agent. In addition, CLA has effects on bone formation and the immune system as well as fatty acid and lipid metabolism and gene expression in numerous tissues.21 The scope of this study was to develop amylose-CLA complexes, with optimal stability to oxidation and thermal treatments, to dissolution in the stomach, and efficient release by mammalian amylases. The expected results, stable encapsulated PUFA, will enable the supplementation of various staple foods with these important bioactive compounds. Materials and Methods Materials. Potato amylose (DP-900, according to manufacturer), CLA (a mixture of cis- and trans-9,11 and -10,12-

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octadecadienoic acids. Linoleic acid R-amylase > amyloglucosidase > β-amylase. Complete hydrolysis (100%) was obtained by pancreatin, whereas R-amylase hydrolyzed up to 87% of the complexes. Amyloglucosidase exhibited low rate of hydrolysis (up to

Figure 6. Extent of hydrolysis (%) (A), and CLA release (%) (B) of amylose-CLA complexes created in water/DMSO solution at 90 °C (gray bars), 60 °C (white bars), and 30 °C (black bars). Hydrolysis was performed by pancreatin (pan), amyloglucosidase (gluco), R-amylase (R), and β-amylase (β) at concentrations of 35 units/mL. The control contained no enzyme.

36%), and the β-amylase hardly hydrolyzed the complexes (up to 8.5%), despite extremely high enzyme activity (700 units/mL). For pancreatin, amyloglucosidase, and β-amylase, there was insignificant difference in the hydrolysis between complexes prepared by the two methods being used. The exception was R-amylase, with a higher extent of hydrolysis of complexes created by KOH/HCl solution at 60 °C. To test the release of CLA due to the enzymatic degradation, we first incubated a control experiment including the complexes with no enzyme. Here, only mild release of CLA was observed; for complexes made in water/DMSO solution it ranged from 3% to 6%, and for complexes created by KOH/HCl solution, from 7% to 11%. When incubated in the presence of amylases, among complexes created by KOH/ HCl solution, the maximum release due to amylolytic activity was obtained with pancreatin in complexes produced at 90 °C (p < 0.05). Unlike pancreatin, R-amylase caused higher hydrolysis of complexes produced at 60 and 30 °C. The release of CLA by pancreatin and amyloglucosidase increased with the increase in crystallization temperature (p < 0.05), and the maximal release by both enzymes was obtained in complexes produced at 90 °C. The crystallization temperature had no significant effect on the release of CLA from these complexes when R-amylase was used. The release after the action of all enzymes was similar for complexes made in water/DMSO solution at the three crystallization temperatures. When the activity of different

Amylose-Lipid Complexes as Molecular Nanocapsules

Figure 7. Extent of hydrolysis (%) (A) and CLA release (%) (B) of amylose-CLA complexes created by KOH/HCl solution at 90 °C (gray bars), 60 °C (white bars), and 30 °C (black bars). Abbreviations are as described for Figure 6.

enzymes on complexes created by different complexation methods was compared, a significantly different release was obtained for complexes created by KOH/HCl solution at 90 °C by use of amyloglucosidase. This difference occurred in spite of the fact that no significant difference in hydrolysis was obtained for this enzyme. Furthermore, for complexes creates by the two methods at 60 and 30 °C, no significant differences in the release of CLA were obtained as a result of hydrolysis by amyloglucosidase. It should be noted, however, that regardless of the system tested, there was a correlation between the release and extent of hydrolysis. Discussion Percent Yield and CLA Content of the Complexes. Amylose-CLA complex formation is strongly influenced by the type of lipids involved.5 It is known that amylose can form complexes with free fatty acids (FA). Various studies obtained reasonably efficient complexing with unsaturated free FA and monoglycerides.5,7,9,26-28 However, early reports claim that cis-unsaturated FA complex poorly with amylose, giving low yields and enthalpies of dissociation.9,28-31 This has been attributed to inefficient complexing by such FAs, which is depicted as nonlinear or kinked due to the cis double bond. The results of this study show that amylose can also complex with the unsaturated CLA.

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Although, given the chemical structure of CLA, it is difficult to think of a formation of a helical inclusion, it is possible that only part of the aliphatic segment, adjacent to the conjugated bonds of the CLA, is inside the helical amylose and the rest of the CLA molecule is outside the helix. This suggestion is in agreement with the structure of amyloseFA complexes suggested by computer modeling.17 It was shown that the cis-trans part of the FA could be outside the helix. Moreover, complexation with other bulky molecules such as naphthol was demonstrated,6,15 which supports the possibility of inclusion of the CLA inside the helix. In addition, the XRD results also support the formation of an inclusion complex with CLA, especially for complexes created in water/DMSO solution. As for complexes created by KOH/HCl solution, the additional two peaks observed in the XRD results may imply the presence of an additional form of amylose-CLA complexes, as will be discussed later. Another major factor governing the formation of complexes is the solubility/dispersability of the FA in the complexation medium, and this in turn depends on variables such as temperature and FA molecular weight.8,28,32 The complexation in DMSO/water solution occurs in pure DMSO, and the water added is used to precipitate the complexes formed. Since DMSO is an organic solvent, the solubility of the FA is very high; hence the FA molecules are available to create inclusion complexes with amylose, and only a V-type is formed, as seen by the XRD results. For the complexation by KOH/HCl solution, the FA is dissolved in aqueous solution of KOH before mixing with the amylose solution. But, although the alkaline improves the solubility of FA by ionization, the solubility/dispersability of the CLA in water is less than in organic solvent. Therefore, less FA is available to form helical inclusion complexes, as is also supported by the XRD analysis. The weight yield of the complexes obtained in the different conditions ranged from 52% to 60%, with CLA content of 2%-3.5% (Table 1). Previously reported yields of amylose complexes were 60%-83% with FA content of about 4% (complexes created with a mixture of mainly 18:1, 18:2, and 18:3).20 Others obtained complexes with FA content of 4.9%-8.3% depending on the crystallization temperature.5 Apparently, the yield and CLA content obtained for the different complexes were lower than reported for other unsaturated free FAs. This may arise from the structure of CLA. Computer modeling of amylose-FA complex demonstrated that the “all-trans” segment for the FA, which is energetically favored (global minimum), fit into the helix.19 Free rotation about C-C bonds adjacent to CdC bonds allows the unsaturated FA to adopt a quasi-linear conformation around the double bond, which would reduce the steric hindrance for complexation.5 Unlike other unsaturated FA, the conjugated double bonds of CLA are not separated by C-C bonds; hence CLA cannot adopt a fully linear conformation and the molecule is bent by the conjugated CdC bonds. It is therefore possible that, due to the bend of CLA molecules, a greater steric hindrance for complexation is created, thus resulting in lower yield and content compared to other unsaturated FA.

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Complexes created in water/DMSO solution exhibited an increase in the yield as the crystallization temperature decreased. This is in agreement with effective complexation of unsaturated monoglycerides and FAs in ambient temperatures.5,9 In contrast, complexes created by KOH/HCl solution did not show any significant difference between the crystallization temperatures. Characterization of the Complexes. Amylose-FA complexes exist in two polymorphic forms, namely, types I and II. The type I forms are considered to be amorphous while the semicrystalline type II forms exhibit X-ray diffraction patterns characteristic of the 6-fold single helices in crystallites.1,6 At low temperatures (Tc), formation of complex I is the favored process, whereas form II is favored at high Tc.6,18 In this study all complexes created by the two complexation methods, at the three crystallization temperatures, exhibited a characteristic diffraction patterns of the V-complex, meaning that at all crystallization temperatures (90, 60, and 30 °C) form II complexes were produced, which is in contradiction with other observations.5,6 Complexes created by KOH/HCl solution exhibited narrower peaks than those obtained from complexes created in water/DMSO solution, suggesting rather larger crystallites, but according to the AFM characterization, the morphology of the complexes created by the two methods was different (rods vs globular); hence no comparison of crystallite sizes can be done from the AFM images. It should be noted that as the crystallization temperature increased, complexes created by KOH/HCl solution exhibit narrower peaks, suggesting the formation of larger crystallites, as seems to be supported by the dimensions of the complexes as analyzed by cross-section analysis of the AFM software (results not presented). For complexes created in water/DMSO solution, the crystallization temperature seemed to have no influence on the peak width, which is in contrast to the AFM results (decrease in height of complexes as the crystallization temperature decreased; results not presented). This may be explained if the crystallite size increase occurs along the macromolecular chain axis, which may not be visible on the width of major diffraction lines observed.33 Two additional diffraction peaks are seen in samples prepared by KOH/HCl complexation. Other studies that observed additional peaks to the peaks characteristic to the V-form17,32 suggested that these peaks reflect the presence of pure crystalline FA, which can also be demonstrated by appearance of another endotherm in the DSC spectrum that corresponds to the melting of the FA itself. These studies used FA (C12-C18) with melting temperatures higher than 44.2 °C.8 These FAs are solid at room temperature, whereas in the present research we used CLA, which has a very low melting temperature and thus is liquid at room temperature. Therefore, the additional peaks seen for complexes created by KOH/HCl complexation are unlikely to stem from pure crystalline FA. An alternative possible explanation could be the presence of another type of amylose-FA complexes, as seen by Fourier transform infrared (FTIR) analysis of starchhydrocarbon complexes.34 The FTIR analysis revealed that complexing hydrocarbons expelled part of the amorphous content of starch to form internal empty domains. In such

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domains, hydrocarbons were held with involvement of local van der Waals and dispersion forces of D-glucose units rather than by formation of helical complexes with amylose. Form II is believed to have a lamellarlike organization of amylose complexes; that is, the polysaccharide chains are so folded as to have their chain axes perpendicular to the surface of the lamella.6,15,35 This molecular organization seems to be similar to the lamellar stacks shown by amylose-palmitic acid complexes in TEM; their thickness was 4.6 nm, which corresponds to the total length of two palmitic acid molecules.10 The AFM images of the current study cannot support the lamellarlike organization, mainly since much higher magnification is needed. Their observation, however, can be supported by the AFM image of amylose-polyether inclusion complexes at a magnification of 100 nm × 100 nm, revealing structures separated by distances of 3-3.5 nm.36 The structures (both rods and globular) presented in the AFM images (Figures 2 and 3) exhibit a diameter of about 100 nm, which is supported by AFM images (scan 3 µm × 3 µm) of amylose complexes created with carbon nanotubes,37 that exhibit a width of about 75 nm. The melting temperature (Tm) of all the complexes was higher than 88 °C (Table 2). It is known that complexes of form II give high melting temperatures and enthalpies. Linoleic acid (LA)-amylose complexes, type II, have Tm of about 100 °C.5 Since the position of the double bonds of the unsaturated FA influences the melting point of the complexes, the lower Tm of complexes created with CLA can be attributed to the conjugated double bonds of CLA. No significant influence of the crystallization temperature on the Tm was observed, which is in contrast to the increase of Tm with the increase of crystallization temperature.6 The melting enthalpies of the complexes ranged from 7 to 17.4 J/g (Table 2), which is in contrast to the higher enthalpies reported for type II complexes but yet in agreement with the influence of the structure of CLA compared to LA (maximum enthalpy of 24 J/g at all crystallization temperatures).5 An interesting outcome of the measured Tm is that amylose-CLA complexes may be used to protect CLA during thermal processing such as pasteurization. Stability of the Complexed CLA against Oxidation. The protection against oxidation afforded to CLA by its inclusion in an amylose complex (Figure 3) demonstrates the potential of the complexes, especially those created in water/DMSO solution, to efficiently protect CLA from oxidation. These results are in agreement with other studies.20,38 Interestingly, the extent of CLA oxidation correlates well with their enthalpy of melting (Figure 8). As the enthalpy increased, the extent of oxidation decreased. Since the enthalpy reflects the complexes’ degree of crystallinity, it may be suggested that the protection against CLA oxidation depends on the degree of crystallinity. It seems that complexes created in water/DMSO solution exhibited higher crystallinity than complexes created by KOH/HCl solution. Hence, it is in agreement with the fact that complexes created in water/DMSO solution exhibited better protective ability from oxidation. Also, it is possible that the differences between the morphology of the com-

Amylose-Lipid Complexes as Molecular Nanocapsules

Figure 8. Correlation between the maximal extent of oxidation (moles of O2/moles of CLA), obtained after 70 h at 37 °C, of different complexes and their enthalpies of melting.

plexes (rods vs globular) affect the extent of oxidation due to the surface exposed to oxidation, which is greater for rods (complexes created by KOH/HCl solution). Another possible explanation could stem from better molecular arrangement, as well as more compact structure of the complexes created in water/DMSO solution. Stability of Amylose-CLA Complexes in Simulated Stomach Conditions. The complexation method appears to have a significant effect on the protection afforded by complexation to CLA from release in stomach conditions. Complexes created in water/DMSO solution exhibited higher stability to stomach conditions than complexes created by KOH/HCl solution (Figure 4). One explanation could be the presence of another type of amylose-CLA complex for complexes created by KOH/HCl solution (suggested by the XRD), which is probably less stable to pH changes. Another explanation could be related to possible conformational differences between complexes created in the different methods; complexation in DMSO depends on solvation, meaning that the complex formation is driven mainly by the dissolution of amylose and CLA in the organic solvent, whereas complexation by KOH solution is restricted by steric hindrance due to charged FA, and hydrostatic forces created by the water molecules (hydration), which may result in fewer interactions between amylose and CLA and hence less stable CLA inside the helix. Therefore, the conformation of these complexes can be affected more easily by pH changes than complexes created in DMSO. In general, the hypothesis that the stability of the complexes is affected by their physical properties was approved. Thus, smart control on CLA release can be enabled, and the CLA release probably will not occur in stomach conditions. Enzymatic Digestion and Release of CLA. Complex formation is thought to decrease the digestibility of starch and modulate the glycemic response to ingested carbohydrates.13 The in vitro digestibility of amylose complexes depends on enzyme concentration, incubation time, nature of lipid, and conformational hindrance to enzymatic attack. Most studies investigated the digestibility of amylose-lipid complexes by amylolytic enzymes at concentrations much lower than in physiological conditions, where the amylolytic activity values typical to the intestine are 35 units/mL (minimal) and 120 units/mL (average).23 These studies showed that complexes containing cis-, mono-, or diunsat-

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urated monoglycerides were significantly less stable toward enzymatic breakdown than complexes containing saturated monoglyceride9 and that amylose-oleic acid complexes were hydrolyzed to a greater extent than amylose-palmitic acid complexes.12 The results of the present study show that amylose-CLA complexes can be fully digested in physiological conditions, because of the nature of CLA. This can also be supported by the in vivo study, which showed practically complete absorption within 120 min of amyloselipid complexes that had reached the small intestine,12 and also by the fully degraded complexes under prolonged digestion time and high enzyme levels.14 In this study enzyme hydrolysis of the complexes was performed by the exoamylases glucoamylase and β-amylase (not found in mammals) and by the endoamylases R-amylase and pancreatin (amylolytic activity related to R-amylase). The hydrolysis of complexes from both complexation methods increased in the following order: pancreatin > R-amylase > amyloglucosidase > β-amylase. For R-amylases, no significant differences were obtained in the hydrolysis of complexes created by the two methods (p < 0.05), despite differences observed by the AFM and X-ray diffraction. This is in agreement with similar degradation kinetics despite the apparent differences in thermal stability and supermolecular organization.6 Significantly low hydrolysis was obtained by the exoamylases glucoamylase and β-amylase, indicating better digestibility by endoamylases. While hydrolysis by endoamylases occurs randomly along the amylose chain, exoamylases begin their action from the nonreducing terminus. It is possible that a steric hindrance, caused by the structure of the complexes, hampers the action of exoamylases, resulting in low hydrolysis. Interestingly, the rate and extent of hydrolysis of the complexes are inversely related to the degree of organization: complexes with greater crystallinity were more resistant to enzymatic degradation.14 The results of the present study found no significant differences (p < 0.05) in the crystallinity of the different complexes (DSC) and hence no differences in hydrolysis of complexes created by the two methods. It should be noted that some spontaneous CLA release from the complexes was detected. This release is not likely to occur from uncomplexed CLA, since the complexes were washed twice by a water/ethanol mixture to remove access of CLA. The location of FA molecules within the crystal lattice between the amylose helices is not possible, since the small cavities present between helices cannot accommodate a FA molecule.18 The phosphate buffer has an effect of stabilization on the complexes;39 hence it is also not likely that the spontaneous release is due to the buffer. A possible explanation for the spontaneous release could be the stabilization of the CLA inside the helix. The bend in the CLA molecule created by the conjugated double bonds might prevent the formation of some of the interactions between the amylose and the CLA, resulting in a less stabilized CLA inside the helix. The results of the present work support the hypothesis that hydrolytic activity will release the CLA from the complexes. It is evident that the hydrolysis and the release of CLA are a function of the specific digesting enzyme. Despite the fact

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that full hydrolysis was not necessarily followed by a complete CLA release from the complexes, the release of CLA was proportional to the hydrolysis. Overall, the CLA release due to amylolytic activity of pancreatin indicates that the CLA-amylose complex system can serve as a vehicle for delivery of CLA to the intestine. Conclusions The results of the presented research show that amylose can complex with CLA. The complexes formed provide stability to oxidation and thermal treatments, such as pasteurization. Control of CLA release is enabled, and the CLA release does not occur in simulated stomach conditions; rather, it is driven by amylolytic activity of pancreatin, which indicates that the location of release in the digestive tract will probably be in the intestine. Complexes created in water/ DMSO solution at 90 or 30 °C provided the maximal stability to oxidation and thermal treatments, dissolution in simulated stomach conditions, and efficient release by mammalian amylases. Overall, the results indicate that the amyloselipid complex system could serve as a vehicle for delivery of polyunsaturated fatty acids (PUFA) to the intestine. Hence, potential use of amylose-lipid complexes can be supplementation of various staple foods with PUFA. Acknowledgment. The research was supported in part by the Israel Science Foundation. References and Notes (1) Snape, C. E.; Morrison, W. R.; Maroto-Valer, M. M.; Karkalas, J.; Pethrick, R. A. Carbohydr. Polym. 1998, 36, 225-237. (2) Buleon, A.; Colonna, P.; Planchot, V.; Ball, S. Int. J. Biol. Macromol. 1990, 23, 85-112. (3) Godet, M. C.; Tran, V.; Delage, M. M.; Buleon, A. Int. J. Biol. Macromol. 1993, 15, 11-16. (4) Horii, F.; Yamamoto, H.; Hirai, A.; Kitamaru, R. Carbohydr. Res. 1987, 160, 29-40. (5) Karkalas, J.; Ma, S.; Morrison, W.; Pethrick, R. A. Carbohydr. Res. 1995, 268, 233-247. (6) Biliaderis, C. G.; Galloway, G. Carbohydr. Res. 1989, 189, 31-48. (7) Karkalas, J.; Raphaelides, S. Carbohydr. Res. 1986, 157, 215-234. (8) Tufvesson, F.; Wahlgren, M.; Eliasson, A. C. Starch 2003, 55, 138149. (9) Eliasson, A. C.; Krog, N. J. Cereal Sci. 1985, 3, 239-248. (10) Godet, M. C.; Bouchet, B.; Colonna, P.; Gallant, D. J.; Buleon, A. J. Food Sci. 1996, 61 (6), 1196-1201.

Lalush et al. (11) Harmeet, S. G.; Ranjit, S. K.; Elaine, T. C. Nutrition 1997, 74 (5), 561-565. (12) Holm, J.; Bjorck, I.; Ostrowska, S.; Eliasson, A. C.; Asp, N. G.; Larsson, K.; Lundguist, I. Fats (Lipids) Baking Extrusion; Contribution to the LIPIDFORUM Symposium 1984 (meeting date 1983), pp 52-9. (13) Timothy C. C.; Sophie, A. S.; Les, C. J. Nutr. 2000, 130, 20062008. (14) Seneviratne, H. D.; Biliaderis, C. G. J. Cereal Sci. 1991, 13 (2), 129-143. (15) Tufvesson, F.; Eliasson, A. C. Carbohydr. Polym. 2000, 43 (4), 359365. (16) Tufvesson, F.; Skrabanja, V.; Bjorck, I.; Elmstahl, H. L.; Eliasson, A. C. Lebensmittel-Wissenschaft und -Technologie 2001, 34 (3), 131139. (17) Lebail, P.; Buleon, A.; Shiftan, D.; Marchessault, R. H. Carbohydr. Polym. 2000, 43 (4), 317-326. (18) Godet, M. C.; Tran, V.; Colonna, P.; Buleon, A. Int. J. Biol. Macromol. 1995, 17 (6), 405-408. (19) Godet, M. C.; Buleon, A.; Tran, V.; Colonna, P. Carbohydr. Polym. 1993, 21, 91-95. (20) Szejtli, J.; Banky-Elod, E. Starch 1975, 27, 368-376. (21) Belury, M. A. Annu. ReV. Nutr. 2002, 22, 505-531. (22) Dimantov, A.; Kesselman, E.; Shimoni, E. Food Hydrocolloids 2003, 18 (1), 29-37. (23) Dahlqvist. Scan. J. Clin. Lab. InV. 1962, 14, 145-151. (24) Rick, W.; Stegbauer, H. P. In Methods of Enzymatic Analysis, 2nd ed.; Bergmeyer, H. U., Ed.; Verlagchemie: Weinheim, Germany, 1974; Vol. 2, pp 885-890. (25) Kim, S. J.; Park, G. B.; Kang, C. B.; Park, S. D.; Jung, M. Y.; Kim, J. O.; Ha, Y. L. J. Agric. Food Chem 2000, 48, 3922-3929. (26) Morrison, W. R. In New Approaches to Research on Cereal Carbohydrates; Progress in Biotechnology; Hill, R. D., Munck, L., Eds.; Elsevier: Amsterdam, 1985; Vol. 1, pp 61-70. (27) Raphaelides, S.; Karkalas, J. Carbohydr. Res. 1988, 172, 65-82. (28) Riisom, T.; Krog, N.; Eriksen, J. J. Cereal Sci. 1984, 2, 105-118. (29) Krog, N. Starch 1971, 23, 206-210. (30) Lagendijk, J.; Pennings, H. J. Cereal Sci. Today 1970, 10, 354356, 365. (31) Lehmann, G.; Gottschlich, H. Fette, Seifen, Anstrichm 1983, 85, 439443. (32) Fanta, G. F.; Shogren, R. L.; Salch, J. H. Carbohydr. Polym. 1999, 38, 1-6. (33) Rappenecker, G.; Zugenmaier, P. Carbohydr. Res. 1981, 89, 1119. (34) Polaczek, E.; Starzyk, F.; Malenki, K.; Tomasik, P. Carbohydr. Polym. 2000, 43, 291-297. (35) Biliaderis, C. G. Food Technol. 1992, 98-109. (36) Jun-ichi, K.; Kaneko, Y.; Nagase, S. I.; Takahashi, T.; Tagaya, H. Chem. Eur. J. 2002, 8 (15), 3321-3326. (37) Star, A.; Steuerman, D. W.; Heath, J. R.; Stoddart, J. F. Chem. Int. Ed. 2002, 41 (14), 2508-2512. (38) Morrison, W. R. J. Sci. Food Agric. 1978, 29, 365-371. (39) Jovanovich, G.; Anon, M. C. Biopolymers 1999, 49, 81-89.

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