In vitro digestibility of edible films from various starch sources

Banana, maize, potato and sagu starches were boiled in the presence or absence of plasticizer (glycerol), producing edible films. In vitro digestibility features, amylose content and amylopectin gel filtration behavior of films and parent starches were evaluated. Available starch contents were lower in glycerol-containing films, due to dilution by the plasticizer. Total resistant starch increased in the maize starch-based film but decreased markedly in those prepared from the other starches. Amylose content of banana starch (40%) was about double those of the other starches. Nonetheless, all starch films exhibited similar retrograded resistant starch content. Although film production led to increased -amylolysis rates, these were further augmented by additional film heating, thereby indicating that film-manufacture did not promote complete starch gelatinization. Gel filtration chromatography suggested amylopectin depolymerization after film-making, which may also increase digestion kinetics. The presence of glycerol in the films slowed down starch digestion, a feature of potential dietetic use.     

Crystallinity and morphology in films of starch, amylose and amylopectin blends

Films of potato starch, amylose, and amylopectin and blends thereof were prepared by solution casting and examined using X-ray diffraction, light microscopy, transmission electron microscopy, and differential scanning calorimetry. Amylose films had a relative crystallinity of about 30% whereas amylopectin films were entirely amorphous. Blending of amylose and amylopectin resulted in films with a considerably higher degree of crystallinity than could be predicted. This is explained by cocrystallization between amylose and amylopectin and possibly by crystallization of amylopectin. The crystallized material gave rise to an endotherm detected with differential scanning calorimetry. The enthalpy and peak temperature of the transition also increased as the water content decreased. When the amylose proportion in the blends was low, separate phases of amylose and amylopectin were observed by light microscopy. At higher amylose proportions, however, the phase separation was apparently prevented by amylose gelation and the formation of a continuous amylose network. The amylose network in the films, observed with transmission electron microscopy, consisted of stiff strands and open pores and became less visible as the amylose proportion decreased. The water content of the films was dependent on the microstructure and the crystallinity.     

Effects of cholesterol on surface activity and surface topography of spread surfactant films

Pulmonary surfactant forms a monolayer of lipids and proteins at the alveolar air/liquid interface. Although cholesterol is a natural component of surfactant, its function in surface dynamics is unclear. To further elucidate the role of cholesterol in surfactant, we used a captive bubble surfactometer (CBS) to measure surface activity of spread films containing dipalmitoylphosphatidylcholine/1-palmitoyl-2-oleoylphosphatidylcholine/1-palmitoyl-2-oleoylphosphatidylglycerol (DPPC/POPC/POPG, 50/30/20 molar percentages), surfactant protein B (SP-B, 0.75 mol %), and/or surfactant protein C (SP-C, 3 mol %) with up to 20 mol % cholesterol. A cholesterol concentration of 10 mol % was optimal for reaching and maintaining low surface tensions in SP-B-containing films but led to an increase in maximum surface tension in films containing SP-C. No effect of cholesterol on surface activity was found in films containing both SP-B and SP-C. Atomic force microscopy (AFM) was used, for the first time, to visualize the effect of cholesterol on topography of SP-B- and/or SP-C-containing films compressed to a surface tension of 22 mN/m. The protrusions found in the presence of cholesterol were homogeneously dispersed over the film, whereas in the absence of cholesterol the protrusions tended to be more clustered into network structures. A more homogeneous dispersion of surfactant lipid components may facilitate lipid insertion into the surfactant monolayer. Our data provide additional evidence that natural surfactant, containing SP-B and SP-C, is superior to surfactants lacking one of the components, and furthermore, this raises the possibility that the cholesterol found in surfactant of warm-blooded mammals does not have a function in surface activity.     

Effect of glycerol on behaviour of amylose and amylopectin films

The effect of water and glycerol on sorption and calorimetric T_gs of amylose and amylopectin films were examined. The mechanical properties of the films were also analysed under varying glycerol content at constant RH and temperature. Based on changes observed in sorption and tensile failure behaviour glycerol was strongly interacted with both starch polymers. Even though water was observed to be more efficient plasticiser than glycerol, glycerol also affected the T_g. But in spite of the observed decrease in T_g under low glycerol contents brittleness of the films increased based on changes in elongation. The increase in brittleness of both polymers was also in agreement with their actual behaviour. At around 20% glycerol great change in the rheological properties occurred. Above 20% glycerol amylose film showed much larger elongation than the low glycerol content films and was still strong but the amylopectin produced a very week and non-flexible film.     

The molecular deposition of transgenically modified starch in the starch granule as imaged by functional microscopy

The molecular deposition of starch extracted from normal plants and transgenically modified potato lines was investigated using a combination of light microscopy, environmental scanning electron microscopy (ESEM) and confocal laser scanning microscopy (CLSM). ESEM permitted the detailed (10 nm) topographical analysis of starch granules in their hydrated state. CLSM could reveal internal molar deposition patterns of starch molecules. This was achieved by equimolar labelling of each starch molecule using the aminofluorophore 8-amino-1,3,6-pyrenetrisulfonic acid (APTS). Starch extracted from tubers with low amylose contents (suppressed granule bound starch synthase, GBSS) showed very little APTS fluorescence and starch granules with low molecular weight amylopectin and/or high amylose contents showed high fluorescence. Growth ring structures were sharper in granules with normal or high amylose contents. High amylose granules showed a relatively even distribution in fluorescence while normal and low amylose granules had an intense fluorescence in the hilum indicating a high concentration of amylose in the centre of the granule. Antisense of the starch phosphorylating enzyme (GWD) resulted in low molecular weight amylopectin and small fissures in the granules. Starch granules with suppressed starch branching enzyme (SBE) had severe cracks and rough surfaces. Relationships between starch molecular structure, nano-scale crystalline arrangements and topographical-morphological features were estimated and discussed.     

Influence of amylose content on starch films and foams

After extraction of smooth pea starch and waxy maize starch from pure amylose and amylopectin fractions, films with various amylose contents were prepared by casting in the presence of water or water with glycerol. For unplasticized films, a continuous increase in tensile strength (40–70 MPa) and elongation (4–6%) was observed as amylose increased from 0 to 100%. Discrepancies with values obtained for native starches with variable amylose content and different botanical origins were attributable to variations in the molecular weights of components. Taking cell wall properties into account, the values obtained in the laboratory were used to improve the relation between the flexural behavior of extruded foams and the model of cellular solids with open cavities.

The properties of plasticized films were not improved by the presence of glycerol and remained constant when amylose content was higher than 40%. Results are interpreted on the basis of topological differences between amylose and amylopectin.     

Internal structure and physicochemical properties of corn starches as revealed by chemical surface gelatinization

The organization of amylose and amylopectin within starch granules is still not well elucidated. This study investigates the radial distribution of amylose and amylopectin in different corn starches varying in amylose content (waxy corn starch (WC), common corn starch (CC), and 50% and 70% amylose corn starches (AMC)). Corn starches were surface gelatinized by 13 M LiCl at room temperature to different extents (approximately 10%, 20%, 30%, and 40%). The gelatinized surface starch and remaining granules were characterized for amylose content, amylopectin chain-length distribution, thermal properties, swelling power (SP), and water solubility index (WSI). Except for the outmost 10% layer, the amylose content in CC increased slightly with increasing surface removal. In contrast, amylose was more concentrated at the periphery than at the core for 50% and 70% AMC. The proportion of amylopectin A chains generally decreased while that of B1 chains generally increased with increasing surface removal for all corn starches. The gelatinization enthalpy usually decreased, except for 70% AMC, whereas the retrogradation enthalpy relatively remained unchanged for CC but increased for WC, 50% and 70% AMC with increasing surface removal. The SP and WSI increased with increasing surface removal for all corn starches, with WC showing a significant increase in SP after the removal of the outmost 10% layer. The results of this study indicated that there were similarities and differences in the distribution of amylose and amylopectin chains along the radial location of corn starch granules with varying amylose contents. More amylose-lipid complex and amylopectin long chains were present at the periphery than at the core for amylose-containing corn starches.     

Size distributions of amylose and amylopectin solubilized from corn starch granules

Size distributions of extracts derived from starch were investigated to aid in elucidating structure-function relationships of these polymers in water. Starch granules derived from waxy maize and amylomaize VII were dissolved in water by microwave heating in a high pressure vessel. Transmission electron microscopy of starch deposited from dilute solution and rotary shadowed with platinum, revealed that amylopectin imaged from waxy maize could be broadly classified as about 28% circular space filling patches containing branched clusters and 72% asymmetric linear containing branched clusters. Lengths of asymmetric linear amylopectin components ranged from about 37 to 980 nm whereas the diameter of circular amylopectin components ranged from about 44 to 200 nm. Although the starch in amylomaize VII is about 70% amylose, its narrow asymmetric structure when visualized by microscopy enabled us to image amylose even though amylopectin was present. Lengths of components ranged from about 46 to 254 nm. After smoothing and curve fitting, we found that all size distributions investigated could be treated as if they were multimodal in nature. The most abundant amylose component had a linear density of 8.2 × 10³ molar mass units/ nm. This value could be explained if amylose had an aggregation number of about 5.9.     

A scanning force- and fluorescence light microscopy study of the structure and function of a model pulmonary surfactant

The structure of an artificial pulmonary surfactant was studied by scanning force- and fluorescence light microscopy (SFM, and FLM, respectively). The surfactant – a mixture of dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG) and recombinant surfactant-associated protein C (SP-C) – was prepared at the air-water interface of a Langmuir film balance and imaged by FLM under various states of compression. In order to visualize their topography by SFM, the films were transferred onto a solid mica support by the Langmuir-Blodgett (LB) technique. We found that a region of high film compressibility of the spread monolayer close to its equilibrium surface pressure (π=50 mN/m) was due to the exclusion of layered protrusions with each layer 5.5 to 6.5 nm thick. They remained associated with the monolayer and readily reinserted upon expansion of the film. Comparison with the FLM showed that the protrusions contained the protein in high concentration. The more the film was compressed, the larger was the number of layers on top of each other. The protrusions arose from regions of the monolayer with a distinct microstructure that may have been responsible for their formation. The molecular architecture of the microstructure remains to be elucidated, although some of it can be inferred from spectroscopic data in combination with the SFM topographical images. We illustrate our current understanding of the film structure with a molecular model. Received: 20 September 1996 / Accepted: 22 May 1997     

Mechanical,barrier and morphological properties of pea starch and peanut protein isolate blend films

Mechanical, barrier and morphological properties of edible films based on blends of Pea starch (PS) and Peanut protein isolate (PPI) plasticized with glycerol (30%, w/w) were investigated. As PPI ratio in PS/PPI blends increased, the thickness of films decreased, the opacity slightly elevated and color intensified. The addition of PPI to the PS film significantly reduced tensile strength from 5.44 MPa to 3.06 MPa, but increased elongation from 28.56% to 98.12% with the incorporation of PPI into PS at 50% level. Film solubility value fell from 22.31% to 9.78% upon the incorporation of PPI ranged from 0 to 50% level. When PPI was added into PS film at 40% level, the WVP and WVTR of the films markedly dropped from 11.18% to 4.19% and 6.16 to 1.95%, respectively. Scanning electron microscopy (SEM) of the surface of films showed that many swollen starch granules were presented in the 100% PS film, while 100% PPI film was observed to have rougher surfaces with presence of pores or cavities. The PS/PPI blend films upon the incorporation of PPI at 20% and 50% level were not homogeneous. However, the smoother film surface was observed in PS/PPI blend films with the addition of PPI at 40% level. SEM image of the cross-sections of the films revealed that the 100% PS film showed a uniform and compact matrix without disruption, and pore formation and 100% PPI film displayed a smooth structure. Rougher and flexible network was shown in blend film with the addition of PPI reaching 40% level.     

Surface morphology and chemistry of<Emphasis Type="Italic"> Prunus laurocerasus</Emphasis> L. leaves: a study using X-ray photoelectron spectroscopy,time-of-flight secondary-ion mass spectrometry,atomic-force microscopy and scanning-electron microscopy

The surface properties of the plant cuticle play a crucial role in plant–pathogen interactions and the retention and penetration of agriculturally important chemicals. This paper describes the use of X-ray photoelectron spectroscopy (XPS), time-of-flight secondary-ion mass spectrometry (ToF-SIMS), tapping-mode atomic force microscopy (TM-AFM) and scanning electron microscopy (SEM) to determine surface-specific chemical and material properties of the adaxial surface of Prunus laurocerasus L. leaves. XPS data, derived from the uppermost few nanometres (<10 nm) of the leaf surface, were consistent with the wax components and functionality known to be present within the waxes. ToF-SIMS provided molecular speciation from the outermost monolayer of the leaf surface, indicating the importance of a family of acetates with chain lengths ranging from C₂₀ to C₃₄. The presence of alkanes with C₂₉ and C₃₁ chain lengths was also confirmed. SEM and TM-AFM topography images revealed a textured granular surface, while simultaneously recorded AFM phase images revealed heterogeneous material properties at the nanoscale. The relevance of these data to plant cuticle development, allelochemistry and agrochemical delivery is discussed.     

Model films from native cellulose nanofibrils. Preparation, swelling, and surface interactions

Native cellulose model films containing both amorphous and crystalline cellulose I regions were prepared by spin-coating aqueous cellulose nanofibril dispersions onto silica substrates. Nanofibrils from wood pulp with low and high charge density were used to prepare the model films. Because the low charged nanofibrils did not fully cover the silica substrates, an anchoring substance was selected to improve the coverage. The model surfaces were characterized using atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS). The effect of nanofibril charge density, electrolyte concentration, and pH on swelling and surface interactions of the model film was studied by quartz crystal microbalance with dissipation (QCM-D) and AFM force measurements. The results showed that the best coverage for the low charged fibrils was achieved by using 3-aminopropyltrimethoxysilane (APTS) as an anchoring substance and hence it was chosen as the anchor. The AFM and XPS measurements showed that the fibrils are covering the substrates. Charge density of the fibrils affected the morphology of the model surfaces. The low charged fibrils formed a network structure while the highly charged fibrils formed denser film structure. The average thickness of the films corresponded to a monolayer of fibrils, and the average rms roughness of the films was 4 and 2 nm for the low and high charged nanofibril films, respectively. The model surfaces were stable in QCM-D swelling experiments, and the behavior of the nanofibril surfaces at different electrolyte concentrations and pHs correlated with other studies and the theories of Donnan. The AFM force measurements with the model surfaces showed well reproducible results, and the swelling results correlated with the swelling observed by QCM-D. Both steric and electrostatic forces were observed and the influence of steric forces increased as the films were swelling due to changes in pH and electrolyte concentration. These films differ from previous model cellulose films due to their chemical composition (crystalline cellulose I and amorphous regions) and fibrillar structure and hence serve as excellent models for the pulp fiber surface.     

The effects of amylose content on the molecular size of amylose, and on the distribution of amylopectin chain length in maize starches

The amylose to amylopectin ratios in six maize starch samples of differing amylose contents were measured by enzymatic debranching, followed by high performance size exclusion chromatography (HPSEC). The molecular size of amyloses, estimated by -log K_wav, shows progressive decrease with the increase in amylose content in maize starches. The gel permeation chromatographs of the corresponding amylopectins, debranched with isoamylase, showed bimodal distributions containing long and short chains. The average chain length of amylopectin has a correlation with amylose content. The correlation coefficients between amylose content and average chain length, long chain length, weight ratio and the mole ratio of long and short chain length, were 0.97, 0.92, 0.96, 0.94 respectively. The maize starch with the highest amylose content has the lowest amylose molecular size and the longest chains, with a high ratio of long to short chains in its amylopectin fraction. Comparing the values of amylose content determined by HPSEC of starch or debranched starch with those of the iodinecomplex method, we conclude that long chains of amylopectin in high amylose starches contribute significantly to apparent amylose content.     

Genotype-specific spatial distribution of starch molecules in the starch granule: a combined CLSM and SEM approach

Starch granule types from a variety of botanical sources were selected to represent differences in crystalline polymorph, amylose and phosphate content, and amylopectin chain length distribution. Equimolar labeling of starch molecules with the fluorophore 8-amino-1,3,6-pyrenetrisulfonic acid (APTS) was used to construct a detailed map of the distribution of amylose and amylopectin within the granule by confocal laser scanning microscopy (CLSM) analysis. Medium- and high-resolution scanning electron microscopy (SEM) were used to provide detailed images of granule surface structures. By using a combined surface and internal imaging approach, interpretations of a number of previous structural observations is presented. In particular, internal images of high amylose maize and potato suggest that multiple initiations of new granules are responsible for the compound or elongated structures observed in these starches. CLSM optical sections of rice granules revealed an apparent altered distribution of amylose in relation to the proposed growth ring structure, hinting at a novel mechanism of starch molecule deposition. Well-described granule features, such as equatorial grooves, channels, cracks, and growth rings were documented and related to both the internal and external observations. A new method for probing the phosphate distribution in native granules was developed using a phosphate-binding fluorescent dye and CLSM.     

Regulation of Starch and Protein Synthesis in Wheat Grains by Feeding Sucrose and Glutamine to Detached Ears Cultured in vitro

Three experiments of in vitro ear culture were conducted to evaluate how the substrates of C (carbon) and N (nitrogen) supply in liquid medium regulate the grain growth and synthesis of protein and starch in two winter wheat cultivars. Increasing glutamine supply with constant sucrose concentration increased the contents of total protein and protein components of albumin and globulin in grain, and the activity of glutamate pyruvate transaminase (GPT) across most treatments, while markedly reducing the contents of total starch and components of amylose and amylopectin as well as the activities of soluble starch synthase (SSS) and granule bounded starch synthase (GBSS). The opposite patterns were observed in the experiment of increasing sucrose supply at constant glutamine concentration. When simultaneously increasing sucrose and glutamine supply at constant ratio, the contents of total protein, albumin and globulin in grain were slowly enhanced, whereas the contents of total starch, amylose and amylopectin and the activities of SSS, GBSS and GPT increased only to a certain extent and then decreased. Negative correlations were found between the contents of protein or protein components in grains and the relative ratio of sucrose to glutamine concentrations in the culture medium, while positive correlations were seen between the contents of total starch or starch components and the ratio of sucrose to glutamine. These results implied that the composition of protein and starch in wheat grain could be readily manipulated by varying the concentrations of sucrose and glutamine and their ratio in the culture medium.     

Osteoblast adhesion on poly(L-lactic acid)/polystyrene demixed thin film blends: effect of nanotopography, surface chemistry, and wettability

Biomaterial surface characteristics are critical cues that regulate cell function. We produced a novel series of poly(l-lactic acid) (PLLA) and polystyrene demixed nanotopographic films to provide nonbiological cell-stimulating cues. The increase in PLLA weight fraction (phi) in blend solutions resulted in topography changes in spin-cast films from pit-dominant to island-dominant morphologies having nanoscale depth or height (3-29 nm). Lower molecular weight PLLA segregated to the top surface of demixed films, as observed by X-ray photoelectron spectroscopy and secondary ion mass spectroscopy (SIMS). For phi > or = 0.5, the topmost film layer was predominantly filled with PLLA (>96% by SIMS at 20-A depth). Nanotextured substrata stimulated osteoblastic cell adhesion to a greater degree than did flat PLLA (phi = 1), and this effect was more pronounced for nanoisland (phi = 0.7 and 0.9) relative to nanopit topographies (phi = 0.5). Demixed films having relatively lower water contact angles generally enhanced cell adhesion and spreading. Our results reveal that cell adhesion is affected by surface chemistry, topography, and wettability simultaneously and that nanotextured surfaces may be utilized in regulating cell adhesion.     

Genetic control of amylose content in wheat endosperm starch and differential effects of three Wx genes

The endosperm starch of the wheat grain is composed of amylose and amylopectin. Genetic manipulation of the ratio of amylose to amylopectin or the amylose content could bring about improved texture and quality of wheat flour. The chromosomal locations of genes affecting amylose content were investigated using a monosomic series of Chinese Spring (CS) and a set of Cheyenne (CNN) chromosome substitution lines in the CS genetic background. Trials over three seasons revealed that a decrease in amylose content occurred in monosomic 4A and an increase in monosomic 7B. Allelic variation between CS and CNN was suggested for the genes on chromosomes 4A and 7B. To examine the effects of three Waxy (Wx) genes which encode a granule-bound starch synthase (Wx protein), the Wx proteins from CS monosomics of interest were analyzed using SDS-PAGE. The amount of the Wx protein coded by the Wx-B1 gene on chromosome arm 4AL was reduced in monosomic 4A, and thus accounted for its decreased amylose content. The amounts of two other Wx proteins coded by the Wx-A1 and Wx-D1 genes on chromosome arms 7AS and 7DS, respectively, showed low levels of protein in the monosomics but no effect on amylose content. The effect of chromosome 7B on the level of amylose suggested the presence of a regulator gene which suppresses the activities of the Wx genes.     

Rapid High Throughput Amylose Determination in Freeze Dried Potato Tuber Samples

This protocol describes a high through put colorimetric method that relies on the formation of a complex between iodine and chains of glucose molecules in starch. Iodine forms complexes with both amylose and long chains within amylopectin. After the addition of iodine to a starch sample, the maximum absorption of amylose and amylopectin occurs at 620 and 550 nm, respectively. The amylose/amylopectin ratio can be estimated from the ratio of the 620 and 550 nm absorbance values and comparing them to a standard curve in which specific known concentrations are plotted against absorption values. This high throughput, inexpensive method is reliable and reproducible, allowing the evaluation of large populations of potato clones.      

Waxy Chlamydomonas reinhardtii: monocellular algal mutants defective in amylose biosynthesis and granule-bound starch synthase activity accumulate a structurally modified amylopectin.

Amylose-defective mutants were selected after UV mutagenesis of Chlamydomonas reinhardtii cells. Two recessive nuclear alleles of the ST-2 gene led to the disappearance not only of amylose but also of a fraction of the amylopectin. Granule-bound starch synthase activities were markedly reduced in strains carrying either st-2-1 or st-2-2, as is the case for amylose-deficient (waxy) endosperm mutants of higher plants. The main 76-kDa protein associated with the starch granule was either missing or greatly diminished in both mutants, while st-2-1-carrying strains displayed a novel 56-kDa major protein. Methylation and nuclear magnetic resonance analysis of wild-type algal storage polysaccharide revealed a structure identical to that of higher-plant starch, while amylose-defective mutants retained a modified amylopectin fraction. We thus propose that the waxy gene product conditions not only the synthesis of amylose from endosperm storage tissue in higher-plant amyloplasts but also that of amylose and a fraction of amylopectin in all starch-accumulating plastids. The nature of the ST-2 (waxy) gene product with respect to the granule-bound starch synthase activities is discussed.     

Annealing of normal and mutant wheat starches. LM, SEM, DSC, and SAXS studies

Structure and thermodynamic properties of native and annealed wheat starches with different amylose content (from 1.5% to 39.5%) have been studied by high-sensitivity differential scanning microcalorimetry (HSDSC), small-angle X-ray diffraction (SAXS), light (LM), and scanning electron microscopy (SEM). Starch morphology, the values of the melting cooperative unit, the thickness of crystalline lamellae and the size of amylopectin clusters as well as thermodynamic parameters characterizing surface of the face side in starch crystals were determined. Some suppositions based on different physical approaches are used for a discussion of the results concerning structural reorganization of starches on different levels of macromolecular organization.