Biomacromolecules 2003, 4, 166-172
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Surface Composition and Morphology of Starch, Amylose, and Amylopectin Films A° sa Rindlav-Westling and Paul Gatenholm* Biopolymer Technology, Department of Material and Surface Chemistry, Chalmers University of Technology, SE-412 96 Go¨teborg, Sweden Received September 18, 2002
The surfaces of solution-cast films of starch, amylose, and amylopectin were examined with scanning electron microscopy (SEM), atomic force microscopy (AFM), electron spectroscopy for chemical analysis (ESCA), and time-of-flight secondary ion mass spectrometry (ToF-SIMS). The surface topography visualized by SEM showed that amylopectin films were very smooth whereas amylose and starch films were rougher. It appears that crystallinity or phase separation in the bulk of the film affects the surface topography. AFM showed that the outmost surfaces of all films were covered with small protrusions, 15-35 nm wide and 1-4 nm high. Studies with ESCA revealed the presence of 3-8% nitrogen on the surfaces. ToF-SIMS indicated that the nitrogen originates from protein because ionic fragments from amino acids and the peptide backbone were found. Extracts from the top surface layer of the starch film showed protein bands in gel electrophoresis (SDS-PAGE) around 60 kDa, which is in the same molecular weight range as the biosynthesizing enzyme GBSS I present in starch granules. The proteins apparently phase separated during film formation and migrated to the surface, resulting in an extensive enrichment of proteins in the film surface, where about 8% of the protein is present in the top 0.01% of the film. We believe that the protrusions observed with AFM could be one or a few proteins aggregated side by side. Introduction There is growing interest in the use of starch in nonfood applications, such as in the production of a variety of plastic materials. Starch is a (1f4)-linked polysaccharide composed of the essentially linear amylose and the extensively (1f6)branched amylopectin and occurs naturally in plants in the form of granules. Films of starch have been made by casting from a water solution of, for instance, potato starch1 and amylose1-3 or by extrusion processing4-6 with or without the addition of plasticizers. The film formation process is important for the film structure and crystallinity. Our previous studies have shown that a slow process, in which solutioncast films are dried in high air humidity, promotes crystallization of the starch polymers2,7 and affects phase separation.1 Possible applications for starch films are as films in packaging, which can exploit the excellent oxygen barrier of starch.2 In such applications, the surface properties of starch-based materials are of crucial importance. Determinations of surface composition and morphology are commonly made by several techniques, such as electron spectroscopy for chemical analysis (ESCA or XPS), secondary ion mass spectrometry (SIMS), scanning electron microscopy (SEM), and atomic force microscopy (AFM). The surface properties of starch granules have been investigated in several studies. An early ESCA study on starch granules by Varma8 found, besides the starch polymers, unspecified hydrocarbon-like impurities on the granule surface. Russell and co-workers9 studied the granules more extensively and * To whom correspondence should be addressed. E-mail: pg@ pol.chalmers.se.
found that proteins and (phospho)lipids were present on the surface. The amount of protein and its content of specific amino acids have also been studied in seed meals by ESCA.10 SIMS analysis on starch granules was performed by Baldwin and co-workers11 and showed carbohydrates, lipids, and contamination on the surface of the granules. SEM is used to visualize the surface topography. Because the analysis is made in high vacuum, the material is dehydrated. AFM measurements, on the other hand, take place in an ambient atmosphere. An image of the surface topography can be obtained, as can information about variations in surface properties, such as stiffness. The surfaces of potato and wheat starch granules were examined in an AFM study by Baldwin and co-workers.12 These surfaces showed many protrusions on the order of 10-300 nm, which were believed to be carbohydrate in nature. The protrusions were interpreted as “blocklet” structures, comprised of amylopectin side-chain clusters. Thermoplastic starch materials have also been examined with AFM. Aging of glycerol-plasticized starch showed variations in surface roughness and friction.6 In a study by Gauldie et al., broken surfaces of a corn starchzein protein thermoplastic polymer showed that they were composed of amorphous and laminated crystalline domains.13 Surface analysis yields valuable information about the surface-specific properties, which are of great importance in the development of new materials and applications. We have previously studied crystallinity, morphology, and thermal properties, as well as the mechanical and barrier properties, of starch, amylose, and amylopectin films.1,2,7,14 The surface of such films has not yet been thoroughly studied, however. The aim of the present study was to
10.1021/bm0256810 CCC: $25.00 © 2003 American Chemical Society Published on Web 11/12/2002
Surface Composition and Morphology
Biomacromolecules, Vol. 4, No. 1, 2003 167
examine the surfaces of starch, amylose, and amylopectin films and study the influence of bulk morphologies on the surface. Materials and Methods Materials. Native granular potato starch was supplied by Lyckeby Sta¨rkelsen, Sweden. The potato amylose (104561) was purchased from Merck, Germany. The amylopectin was in the form of granular amylopectin potato starch and was supplied by Lyckeby Sta¨rkelsen, Sweden. This potato was developed by Lyckeby Sta¨rkelsen and Svalo¨f Weibull by genetic engineering to suppress amylose synthesis.15 Film Formation. The amylose was dissolved in ultrafiltered deionized water (3% w/w) by heating in a pressuretight autoclave in an oven. The autoclave interior temperature was kept above 135 °C for 2 h and eventually reached a maximum of 145 °C. A clear, viscous solution was obtained. The granular amylopectin starch and native potato starch were gelatinized during stirring and heating from 30 to 75 °C (1 min at 75 °C) prior to dissolution in an autoclave in the same way as amylose. In one case, starch was mixed with amylopectin in a weight proportion of starch/amylopectin of 40/60. The granular starch and amylopectin were mixed, gelatinized, and dissolved in an autoclave in the same way as described above. The total polysaccharide concentration in solution was always 3% w/w. The solutions obtained after autoclaving were poured onto polystyrene Petri dishes and left to dry at 23 °C and 50% relative humidity (RH) in a climate room. The drying time, in which an equilibrium water content of the films was reached, was 2 days. The film formation of starch films in 50% RH was varied by drying some films under convection in a hood and some under cover, which resulted in drying times of
