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Effect of different fractions of zein on the mechanical and phase properties of zein films at nano-scale
Journal of Cereal Science 55 (2012) 174e182
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Effect of different fractions of zein on the mechanical and phase properties of zein films at nano-scale Chithra Panchapakesan a, Nesli Sozer b, Hulya Dogan c, Qingrong Huang a, Jozef L. Kokini b, * a
Rutgers University, Department of Food Science, 65 Dudley Road, New Brunswick, NJ 08901, USA Food Science and Human Nutrition, College of Agricultural, Consumer and Environmental Sciences, University of Illinois at Urbana-Champaign, 1304 West Pennsylvania Ave., Urbana, IL 61801, USA c Kansas State University, 104 Shellenberger Hall, Manhattan, KS 66506, USA b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 November 2010 Received in revised form 21 October 2011 Accepted 6 November 2011
The phase behavior of zein films has been investigated at nano-scale using atomic force microscopy (AFM) and compared to the phase behavior of the bulk using a thermal characterization technique. The local surface properties of the films were evaluated as a function of water activity using AFM. The glass transition temperature (Tg) of zein films decreased with increasing water activity. Adhesion forces measured by the AFM force curves increased with increasing water activity. Topography of zein and zein fractions were evaluated both qualitatively and quantitatively by the use of AFM and dedicated software to calculate the surface roughness. It has been found that processing technologies (solvent casting, drop deposition and spin casting) has influence on the surface structures of films. The films which were formed by the alpha zein rich fraction were found to have highest roughness values. Sectional surface profiles revealed that a-zein films have mean roughness (Ra) of 1.808 nm and root mean square roughness (RMS) of 2.239 nm while b-zein films have mean roughness (Ra) of 1.745 nm and root mean square roughness (RMS) of 3.623 nm. The discussions conducted on the differences/similarities in the observations were based on the hydrophobic/hydrophilic properties and interactions of these zein fractions. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Atomic force microscopy (AFM) Zein Molecular organization Surface topography Phase behavior Self-assembly
1. Introduction Biodegradable films which are produced from renewable sources have been the core of food packaging studies for many years. They are encountered as environmentally friendly since they not only decrease the deposit of solid wastes but also save the fossil fuel sources (Chang et al., 2009). Biopolymer based films can be produced from proteins, polysaccharides, lipids or combinations of these. Various plant and animal origin proteins have been investigated for their film forming properties (do A. Sobral et al., 2005; Chang et al., 2009; Gounga et al., 2007; Krochta, 2002; Shi et al., 2009).
Abbreviations: AFM, atomic force microscopy; DSC, differential scanning calorimeter; Tg, glass transition temperature; Ra, mean roughness; RMS, root mean square roughness; PtBuA, poly-(tert butyl acrylate); mw, molecular weight; EtOH, ethanol; (DGtethanol / water), free energy change; Sethanol, solubilities of the amino acid in ethanol; Swater, solubilities of the amino acid in water; n, the number of amino acid residues; k, spring constant; Fad, adhesive force; d, deflection/ displacement of the cantilever. * Corresponding author. Tel.: þ 1 217 333 0240; fax: þ1 217 333 5816. E-mail address: [email protected] (J.L. Kokini). 0733-5210/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.jcs.2011.11.004
Biopolymer based films or coatings have a wide range of applications as being carriers of antimicrobial agents, ant ioxidants, nutraceuticals and coloring materials (Han and Gennadios, 2005; Lee et al., 2008; Ozdemir and Floros, 2001). Natural biopolymer films have limited applications as compared to synthetic polymers due to their poor mechanical and water vapor barrier properties. In the literature there are many studies on improving the barrier and mechanical properties of biopolymer films either by modification of the films or inclusion of reinforcement structures such as filler materials. Even if most of these methods were successful from the perspective of improvement, there are still many gaps in understanding the structural conformations and most of these films are found to be still not satisfactory and limited for many applications (Rhim, 2007). The molecular basis of film-forming is not completely understood and no attempts have been made to determine the relationship between self-assembly of film network and the structure of the protein film. The functional properties of films such as barrier properties and stability are related to the molecular organization of biopolymers used during film formation. Polymer synthesis and self-assembly are the phenomenons which occur at the nanolevel. Analysis of protein topology at the nano- and macro-level will help to
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understand the functional properties of films. Film structure can be revealed by understanding the specific conformational arrangements by the use of AFM. Parameters which can easily be evaluated from AFM data can help to create surface topography, adhesion force and elasticity diagrams of film samples. Up to now many researchers have used AFM for mechanical characterization of soy protein isolate films, zein films, chitosan/clay nanocomposite films, polylactic acid/cellulose fiber films and gellan films (Casariego et al., 2009; Lai and Padua, 1997; León et al., 2009; Ogale et al., 2008; Sanchez-Garcia et al., 2008). State diagrams have been useful tools in characterization of amorphous and crystal states of food components, in particular proteins and carbohydrates (Cocero and Kokini, 1991; Madeka and Kokini, 1994, 1996). Proteins have been found to exist mostly in the amorphous state or in partially crystalline form. The physical properties of many amorphous materials can be related to their glass transition temperature (Tg). Tg defines a second order phase change temperature, where a solid, glassy material becomes a soft, rubbery material (Cocero and Kokini, 1991; Roos et al., 1996; Sperling, 1992). For amorphous polymers, the glass transition temperature is a critical temperature where the mechanical properties undergo a dramatic change. Several factors such as the presence of plasticizers, molecular weight, crystallinity and ionic binding interactions affect the Tg of polymers. Water is known to depress the glass transition temperature of amorphous proteins (Cocero and Kokini, 1991; Hoseney et al., 1986; Madeka and Kokini, 1994), and partially crystalline polymers such as starch and maltodextrins (Roos, 1992; Zeleznak and Hoseney, 1987). The direct effect of moisture content at a constant temperature leads to increased mobility of chains in the amorphous regions, which decreases the glass transition temperature (Karel, 1985; Levine and Slade, 1992; Roos and Karel, 1991). Predicting the changes in mechanical properties that occur as a result of plasticization with water or of temperature is critical to control the physical properties and the resulting quality and stability of the biofilms. Differential scanning calorimetry (DSC) and rheometry, in particular small amplitude oscillatory measurements, are the most common techniques used to study the glass transition of biopolymers. Phase behavior of polymers can also be studied by measuring the adhesive forces between AFM probe tip and the molecules at the surface. As the cantilever approaches the surface, it experiences an attractive force from the sample. A pull-off force is experienced when the cantilever is pulled back to detach the tip from the surface, which is indicated as negative force in an AFM force curve. It can be simply calculated through the sum of the van der Waals and the capillary forces between the AFM tips and the substrate (Eastman and Zhu, 1996). AFM force curves have been used by many researchers as an aid in investigating the viscoelasticity and surface properties of polymeric materials such as surface heterogeneity (Bliznyuk et al., 2002; Eaton et al., 2000, 2002; Marti et al., 1999; Mizes et al., 1991; Tsui et al., 2000). Tsui et al. (2000) demonstrated that AFM adhesion measurements offer a powerful probe to examine the surface viscoelastic properties of a polymer compared to those of the bulk determined by conventional methods. They studied the mechanical properties of thin films of PtBuA (poly-(tert butyl acrylate)) by increasing the temperature and measuring the adhesive force between the sample and the cantilever. In the vicinity of Tg, the adhesive force measured was found to increase rapidly. They also found equivalence between temperature and probe rate and stated that the adhesion was found to increase with increasing temperature and decreasing probe movement rate. The dynamic behavior and molecular relaxation of the polymer sample was found to be not very different from the bulk properties for the PtBuA samples.
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Bliznyuk et al. (2002) measured the surface glass transition temperature of several amorphous polystyrene samples of different molecular weights (mw) ranging between 3900 and 1,340,000 by force-distance measurements at different temperatures using a scanning force microscope. The results related to existing theories on the glass transition temperature of the bulk describing the decrease in Tg at lower mw as a result of increased free volume in the system. The values of the surface glass transition for the samples with mw > 30,000 have been found to be the same as corresponding bulk values. The effect of temperature on surface phase behavior was also observed to be similar to that of the bulk. Pull-off forces measured at temperatures above the Tg of the samples were significantly lower than those measured at temperatures below Tg. Tranchida et al. (2009) worked on the viscoelastic characterization of a model material as well as the glass to rubber transition by the help of an AFM. They found that the network which had higher molecular weight crosslinks had the glass to rubber transition lower than room temperature. Wu et al. (2009) measured modulus of elasticity of polylactic acid coated stents by AFM. The group found the onset glass transition temperature of the polylactic acid to be around 55 C through the elasticity versus temperature curves. Among many protein sources, zein (the major storage protein in corn endosperm) has been actively investigated for its film forming ability and its potential to produce novel polymeric films (Beck et al., 1996; Kim et al., 2004; Lai and Padua, 1997; Weller et al., 1998). Zein is one of the most hydrophobic proteins among many cereal proteins with an average hydrophobicity of 1.365 J/mol. The high proportion of non-polar amino acid residues is responsible for the hydrophobicity of zein. Order of hydrophobicity of zein fractions is g- and d-zein > a-zein > b-zein (Holding and Larkins, 2009). The mechanical and barrier properties of biofilms depend on many factors such as formulation, processing technique as well as the state of film forming polymer at a particular temperature and water activity level. Understanding the phase behavior of biofilms is very important to control their functionality at their end usage. In this study it was aimed to understand the phase/state behavior of zein films both at macro-scales and at nano-level organizations. Films were investigated for their phase behavior as a function of water activity using DSC and the results were compared to the adhesive force measurements obtained from AFM. 2. Materials and methods 2.1. Materials Zein (F4000) was commercially supplied from Freeman Industries, Inc. (Tuckahoe, NY) containing 92.54 4.10% crude protein, 1.03 0.05% ash and 1.18 0.20% crude fat. Dry milled corn grains were used to extract different fractions of zein which are soluble in 90% (v/v) (Fraction 1) and 60% (v/v) (Fraction 2) ethanol (EtOH). 2.2. Methods 2.2.1. Isolation of zein fractions The a- and b-zein fractions were isolated from dry milled corn flour. Zein is a prolamin which has been previously classified to a, b, g fractions based on mass and ethanol solubility (Esen, 1986). Alpha zein has a molecular weight range 20e24 kDa soluble in 40e95% (v/v) EtOH, whereas beta zein and gamma zein have 17e18 kDa/30e80% (v/v) EtOH solubility, 27 kDa/0e80% (v/v) EtOH, respectively. Attention should be given that beta zein is insoluble in 90% (v/v) EtOH and gamma zein is soluble in EtOH only in the
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presence of 2-Mercaptoethanol (Esen, 1986). The protocols used for separation of a- and b- zein fractions have been adapted from Matsushima et al. (1997) and Esen (1986) were summarized in Fig. 1. Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) of fractions of corn protein was carried out in order to estimate the molecular weight compositions of the purified fractions. 2.2.2. Hydrophobicity measurement Hydrophobicity is one of the major factors affecting structure, solubility, fat binding properties, etc., of proteins. One of the most commonly used ways to estimate relative hydrophobicities of amino acids side chains involves experimental determination of free energy changes for dissolution of amino acid chains in water and in ethanol (Damodaran, 1996). The free energy change ðDGtethanol / water Þ of transfer of the amino acid from ethanol to water for amino acid side chains of zein at 25 C was obtained from Damodaran (1996). The free energy for transferring 1 mol of the amino acid from the aqueous solution into the ethanol solution is given by:
DGtethanol/water ¼ RTln
Sethanol Swater
where Sethanol and Swater represent solubilities of the amino acid in ethanol and water, respectively. Amino acid side chains with large positive DGt values are considered to be hydrophobic and they tend to be in the organic phase rather than in the aqueous phase. Amino acid residues with negative DGt values are hydrophilic. The average hydrophobicity of a protein is calculated as n P
DGt ¼
DGt;sidechains n
where n is the number of amino acid residues. Defatting using hexane (1:5, repeated twice)
Solubilization at 60 % ethanol
Centrifugation (9000 rpm, 10 min, 4 0C)
Pellet
Supernatant ( - and - zein)
(discarded) Adjust EtOH concentration to 90%
Centrifugation (9000 rpm, 10 min, 4 0C)
Supernatant
Pellet
( - zein rich fraction)
( - zein rich fraction)
(Fraction 1)
(Fraction 2)
Fig. 1. Schematic diagram for the separation of a- and b- zein fractions.
In order to understand the organization and arrangement of the zein Fraction 1 and Fraction 2 on the surface of the silicon wafer, average hydrophobicity of zein fractions were calculated. 2.2.3. Film formation Zein films were produced by the use of unpurified and purified zein fractions. Three different methods which are solvent casting, drop deposition and spin casting were applied for the production of unpurified zein films whereas the spin casting method was used for production of films from different fractions of zein. Detailed descriptions of the protocols are given below. 2.2.3.1. Film formation methods for unpurified zein and fractions of zein (fraction 1 and fraction 2) films 2.2.3.1.1. Solvent casting. Approximately 16 g of unpurified zein (F4000, Freeman Inc.) was dissolved in 84 ml of 75% (v/v) ethanol by stirring constantly at 75e80 C. The solution was allowed to cool at room temperature and then poured on plastic petri plates to form a thin layer on the surface of the plate. The films were allowed to oven dry at 30 C for 18e24 h to remove excess ethanol, and such a slow drying procedure ensures the formation of even films. Thickness of cast films was approximately 1.4 0.05 mm. Films were stored under ambient dry conditions until further processing. 2.2.3.1.2. Drop-deposition. Initial experiments with AFM topography were conducted with zein films made by drop deposition using the unpurified (F4000, Freeman Inc.) zein fraction. Zein was dissolved in concentrations of 40 mg/ml, 20 mg/ml, 10 mg/ml, 5 mg/ml, 2 mg/ml and 1 mg/ml in 75% (v/v) ethanol. The solution was mixed on a vortex at the highest speed setting (7) for approximately 5 min. The films were formed by dropping the solution onto a silicon wafer which had a hydrophobic coating (manufactured by Veeco Instruments, CA). The sample was then allowed to dry at 30 C to form a thin film layer on the surface of the wafer. 2.2.3.1.3. Spin casting. Zein (F4000) and its subfractions (Fraction 1 and Fraction 2) were spin-cast onto silicon wafers following dissolution in 70% (v/v) ethanol for w2 h with constant stirring. Concentrations of 1 mg/ml (0.1%) were used to ensure both fractions could be completely dissolved. Spin cast conditions were maintained constantly at 3000 rpm for 10 s. Approximately 200 ml of the solution was dispensed onto the wafer and spin-cast to form a thin-layer of zein. The films were then dried for w1 h at 40 C in a conventional oven. The thicknesses of the films were measured by making an artificial scratch on the film surface. Atomic force microscopy images were taken by scanning across the edge of the scratch. The thicknesses of the spin-cast films were found to be approximately 17 nm. 2.2.4. Water activity adjustments of the solvent casted films. Solvent casted films were stored at various water activity levels till they equilibrated. They were evaluated for their physical and mechanical properties by measuring the glass transition temperature and adhesive force. Films were cut into small pieces and stored in saturated desiccators containing different salt solutions (LiCl, K2CO3, NaNO2, KCl, KNO3) which produced nominal water activities of 0.12, 0.43, 0.64, 0.84 and 0.93 respectively, at 25 C (Nyqvist, 1983). The water activity of the samples was measured using the AquaLab water activity meter (Series 3 TE, Decagon Devices, WA). The actual and nominal water activities were found to agree within 15%. 2.2.5. Thermal measurements A Differential Scanning Calorimeter TA 4000 Thermal Analysis System with a DSC 30-S Cell/TC11 TA Processor (Mettler Instrument Inc., Highstown, NJ) was used to evaluate the glass transition
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Fad ¼ k*d where Fad is the adhesive force, k is the cantilever spring constant and d is the deflection/displacement of the cantilever. Samples at high water activity levels were contained in an “atmospheric hood” made of polycarbonate which allowed the sample to be enclosed in a chamber to avoid any moisture loss from the sample. The measurements were completed approximately within 15 min, and the water loss over this period of time was determined to be insignificant.
Zein isolated by ethanol extraction from dry milled corn contains four major fractions which are a-zein (MW ¼ 21e25 kDa), b-zein (MW ¼ 17e18 kDa), g-zein (MW ¼ 27 kDa), d-zein (MW ¼ 9e10 kDa) (Esen, 1986). Fraction 1 was found to be rich in
Aw= 0.28
a Frequency
2.2.7. Adhesive force measurements Adhesive forces of solvent cast, water activity equilibrated zein films were measured as a function of increase in water activity. Force measurements were conducted in the “contact mode” using the force analysis software. Since the adhesion force measured by the AFM force curves depends on the sample topography (Mizes et al., 1991), repeated force measurements were conducted on film surfaces. Equilibrated zein films (Aw ¼ 0.28e0.93) were adhered to the sample holders and around 90e100 force curves were generated on each sample in order to obtain an average adhesive force measurement over the sample surface. In order to provide a good representation of the local surface adhesive force characteristics of the zein film samples, three different film samples at the same water activity level were analyzed and the average of resulting 270e300 adhesive force readings was reported. The same cantilever with spring constant (k) of 0.58 N/m was used in the measurements to minimize the error that may be caused by differences in cantilever tip properties and dimensions. The force between the cantilever and the sample was minimized by maintaining the set point always at “zero”. Adhesive force (Fad) was calculated by the following equation:
3. Results and discussion
60 40 20 0
b Frequency
2.2.6. Atomic force microscopy (AFM) A Multimode Scanning probe Microscope with a NanoScope IIIa SPM Controller (Digital Instruments/Veeco Instruments, CA) was used with a J or E scanner attached (Lateral XeY range: 125 mm 125 mm/10 mm 10 mm; Zerange of 5 mm/2.5 mm respectively for J and E). Silicon nitride cantilevers with a nominal spring constant of either 0.12 N/m or 0.58 N/m were used. The dimensions of the cantilever as specified by the manufacturer were nominal thickness ¼ 0.6 mm, thickness ¼ 0.4e0.7 mm, tip height ¼ 2.5e3.5 mm, maximum tip radius ¼ 60 nm and tip angle (front, side, back) ¼ 35 . Typical scan rates as suggested by the manufacturer were employed and ranged between 0.5 and 1.5 Hz.
2.2.8. Topographical measurements The different fractions of zein (unpurified, Fraction 1 and Fraction 2) were analyzed for topographical and surface features using the contact mode of the AFM. Solvent cast, spin cast and drop-deposited films were evaluated and analyzed for surface characteristics and mechanics of film formation. Scan sizes of images ranged from 1 to 10 mm and several images (w10e15) were generated. The thickness of the spin-cast films was evaluated by scanning an “artificial scratch” created over the surface of the film. The silicon wafers and the sample holders were placed in 95% (v/v) ethanol for 24 h and then cleaned alternately using ethanol, methanol and distilled water several times (5 min each) and dried at 30 C before usage. If the formation of any film on the surface was observed after drying, the cleaning step was repeated. The exact same cantilever was used for the adhesive force measurements (kc ¼ 0.58 N/m). The cleaned silicon wafers were imaged before use, in order to confirm the cleaning process. If images indicated the presence of any organic matter, the cleaning procedure was repeated and re-imaged until the wafer indicated a clean surface.
30
60
90
120 150 Fad (nN)
180
210
180
210
180
210
Aw= 0.55 60 40 20 0
30
60
90
120 150 Fad (nN)
Aw= 0.90
c Frequency
of the solvent cast zein films at various water activity levels. Approximately 20 2 mg of sample was weighed into 40 ml hermetically sealed medium pressure stainless steel crucibles to prevent moisture loss on heating. An empty crucible was used as a reference. Calibration of the instrument was performed using indium as a standard. Before sealing the DSC crucibles they were placed into the respective desiccators for w24 h to compensate for any moisture losses incurred during sample transfer. Samples were scanned between 20 C and 120 C with a heating rate of 5 C/min. Rescans were performed immediately after each scan, in order to erase the thermal history of the samples and to confirm the location of the Tg, based on the reversibility of this second order transition. The glass transition temperature was determined from the DSC rescans, at the midpoint in the shift of the heat flow baseline, which corresponded to the temperature at which one-half of the change in the heat capacity, DCP, occurred. The reported data are the averages of at least two replicate measurements.
177
60 40 20 0
30
60
90
120 150 Fad (nN)
Fig. 2. Adhesive force distribution curves at (a) Aw ¼ 0.28, (b) Aw ¼ 0.55, and (c) Aw ¼ 0.93.
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a-zein having molecular weight ranging between 20 and 24 kDa. The presence of dimers at minor amounts (MWw44e50 kDa) was also observed. The SDS-PAGE confirmed the current literature about zein stating that primary fraction lies between 17 and 18 kDa (a-zein) and some other weaker bands around 25 kDa. The second fraction was found to be rich in b-zein together with minor amounts of both a- (20e24 kDa), g-(27 kDa) and probably also the d-zein fraction. 3.1. Characterization of nano-scale phase behavior of zein films Glass transition temperature is the characteristic transition temperature from the amorphous phase of polymers. Below the Tg, the polymer chain is incapable of diffusing within a random matrix due to intermolecular interactions and thus has a rigid structure whereas above the Tg, the kinetic forces are strong enough to
overcome intermolecular forces and can diffuse, resulting in a rubbery material (Robeson, 2007). Molecular weight, crosslinking within polymers, presence of plasticizers affect the phase behavior of polymers. Therefore the molecular structure at the surface may be different in structure compared to the bulk. In the current study, we investigated the phase behavior of zein films as bulk by measuring their Tg using DSC. Adhesive force measurements using AFM were utilized as an indicator for molecular mobility. The DSC results were correlated with the adhesive force measurements to investigate the surface properties of zein films at the nano-scale. This set of information will enable us to understand the properties of zein films at the nano-scale and relate them to those at macroscopic levels. The effect of water activity on both adhesive force and glass transition temperature of solvent casted films were determined. By the increase of water activity, glass transition temperature dropped
Fig. 3. a) 10 mm scan of 16% w/v cast zein film, data scale ¼ 758.4 nm, top view b) 1 mg/ml (0.1%) drop-deposited film images of unpurified zein 600 nm scan. Data scale ¼ 30 nm top view (left) and 3D view (right) c) 1 mg/ml (0.1%) image of unpurified, spin-cast film at 3000 rpm. Scan size ¼ 2.5 mm, data scale ¼ 10.0 nm top view (left) and 3D view (right).
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Fig. 4. Ra - mean roughness and Rq- RMS roughness values of spin coated unpurified, fraction 1 and fraction 2 zein films.
from 75 C to 25 C whereas adhesive force increased from 67.18 nN to 138.79 nN which is due to the plasticization effect of water. The mechanism of plasticization of water can be explained in terms of a free volume effect (Brandt et al., 1960; Cocero and Kokini, 1991; Slade and Levine, 1991). The free volume is inversely proportional to the number average molecular weight (Sperling, 1992). Therefore, the presence of a low molecular weight substance like water leads to an increased free volume, allowing increased mobility of the polymer backbone chains. An increased mobility is then manifested as a lower glass transition temperature of the polymer/ plasticizer glass (Madeka, 1996). The variation of Tg with the
plasticization effect of water in zein films was compared with the existing literature on Tg of zein powders (Gillgren et al., 2009; Madeka, 1996). Madeka (1996) found that the glass transition temperature for zein powder dropped from 83 C to 21 C as water activity increased from 0.46 to 0.93. Between the zein powder and zein films Tg values shifted with an average deviation of 14.32% which can be attributed to residual amounts of ethanol in the films and different sample preparation methods. The increase in adhesive forces was attributed to the increased mobility in the molecules of the samples analyzed. When the biopolymer film exists in its “glassy state”, there is less mobility in
Fig. 5. Fraction 1 (a) and fraction 2 (b) (0.1% in 70% ethanol) as imaged by the AFM; top view (left), 3D view (right), scan size ¼ 2.5 mm, data scale ¼ 100 nm.
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the chains of the polymer. The molecules are fixed in their positions, and there is less flexibility of the polymer chains. This causes less adhesion to the AFM tip when the tip contacts the surface of the film. Several other studies confirmed that increasing molecular mobility by increasing the temperature results in larger adhesive forces (Marti et al., 1999; Muthuselvi and Dhathathreyan, 2006; Tranchida et al., 2009; Tsui et al., 2000). A range of adhesive forces were measured between zein film surfaces and AFM tip due to the inhomogeneity in sample
topography The range of distributions was presented in the form of adhesive force histograms. Fig. 2 shows the adhesive force distributions measured on zein films equilibrated to different water activity levels. It is evident that measurements at low water activity levels result in low adhesive force readings ranging between 45 and 105 nN (Fig. 2a). Adhesive force measurements conducted at higher water activity levels (Fig. 2c) showed a wider distribution between 65 and 220 nN with an average value around 140 nN. Since the adhesive force is a sum of van der Waals interaction and capillary
Fig. 6. 1 mm scan of 0.1% spin cast film of a) fraction 1 and b) fraction 2 at 50 nm data scale surface plot (top image), top view (left image), section analysis and section analysis results (right image).
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force between the tip and the film surface, higher water activity cause the capillary force increase, thus leads the adhesive force increase (Eastman and Zhu, 1996). 3.2. Characterization of film topography The effect of various film formation methods on the surface characteristics was evaluated by AFM. Surface features of commercial zein films were evaluated at concentrations in the range of 1e40 mg/ml in aqueous ethanol solutions. We found that the surface structure of zein films can be altered by the use of different casting techniques (Fig. 3). Solvent casting resulted in films which did not show any characteristic structure except the presence of several pores (Fig. 3a). The diameter of the pores was found to be approximately 2 mm with an average depth of 300 nm by the use of “sectional analysis” function of the AFM software. We observed more well-defined structures for the films which were produced by drop-deposition (Fig. 3b) and spin casting (Fig. 3c) techniques. Fig. 3b shows some of the distinct structures which are below 30 nm height and are not aggregating to form large structures. Spin casting of zein films resulted in more controlled and uniform structures due to high speeds (3000 rpm) performed during film formation. The molecules across the film surface spread more evenly, decreasing the rate of clumping. Mean roughness values were used to quantitatively analyze the structures which occurred on the surface of zein films. It is the arithmetic average of the absolute values of the surface height deviations measured from a mean value on the line. The mean value is usually predetermined by the AFM software. Roughness measurements were conducted over 25 images of each sample. Fig. 4 shows the roughness values for unpurified and two fractions of zein which is an indicator of how well zein particles organize themselves over the silicon wafer. It was found that films formed by the unpurified zein fraction yielded the least roughness whereas alpha zein rich films had the maximum roughness values. The aggregation/agglomeration rate of zein on the surface of films is controlled by the properties and the changes in the zein fraction. Among the four different fractions of zein (a-, b-, g-, d-), b- zein fraction is the most hydrophilic fraction. Fig. 5 qualitatively shows the effect of different fractions of zein on the film surface properties. The alpha zein rich fraction (Fig. 5a) resulted in a zein film which is less smooth than the beta-zein rich fraction (Fig. 5b). However, we would like to highlight that even though the second fraction obtained from purification of zein was rich in the beta-zein fraction it was contaminated with some amounts of a-zein, g-zein and d-zein. Therefore, the film forming properties of this fraction were altered by the presence of fractions mentioned above. In order to understand the organization and arrangement of the zein Fraction 1 and Fraction 2 on the surface of the silicon wafer, average hydrophobicity of zein fractions was calculated as described in Methods. Based on the amino acid composition of zein fractions (Larkins et al., 1993), and free energy of amino acid residues (Damodaran, 1996), the average hydrophobicities of a-, b-, g- and d-zein fractions were calculated to be 4.958, 3.825, 5.329 and 5.316 kJ/mol, respectively. In films formed from spin casting of the a-zein rich fraction, the film formation depends on formation of layers depending on the hydrophobic/hydrophilic indices which results in cylindrical elongated tube like structures. Wang et al. (2004) also found that zein had a greater affinity for hydrophilic than for hydrophobic surfaces. The possibility of forming a smooth film is therefore less likely in this case, and we would expect to see more cylindrical/ovoid projections on the surface of the film, thus contributing to more roughness. The ability of the a-zein to interact with the hydrophobic surface of the silicon wafer is reduced, resulting in more elongated-cylindrical “pin-like” structures being
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formed on the surface of the film (Fig. 6a). The hydrophobicity of the b-zein rich film was increased due to contamination of this fraction by more hydrophobic fractions. When cast on a hydrophobic surface, making use of its high hydrophobicity, it is able to spread itself on the surface of the silicon wafer and form a relatively smooth film (Fig. 6b), because of the formation of hydrophobic and di-sulphide bonds. This process of film formation using hydrophilic and hydrophobic interactions has also been studied and explained by other authors (Kogan et al., 2002). The difference in the hydrophobicity of the two fractions affected their self-assembly behavior on silicon substrate surfaces. Surface features of zein films were evaluated for films resulting from zein solutions at concentrations in the range of 0.1e10 mg/ml. Significant differences were observed in the topography of a- and b-zein films. Sectional surface profiles revealed that a-zein films have mean roughness (Ra) of 1.914 nm and root mean square roughness (RMS) of 2.247 nm while b-zein films have mean roughness (Ra) of 1.745 nm and root mean square roughness (RMS) of 3.623 nm (Fig. 6). It suggests that because of higher hydrophobicity, a-zein can self-assemble to form rougher but more homogeneous films compared to b-zein. 4. Conclusions Experimental findings indicate that the phase behavior of zein, depicted by the change in its glass transition temperature, is clearly affected by the presence of water. The glass transition of zein films decreased with increasing water activities, confirming the plasticizing effects of water. This glass to rubber transition driven by increasing water activity was also observed using the atomic force microscope, in the form of increased adhesive forces observed at higher water activities. Topography of zein films revealed differences based on the method of film preparation and the chemistry of zein fractions. The differences in the hydrophobicity of two zein fractions affected their self-assembly behavior on silicone substrate surfaces. Clear understanding of the self-assembly and molecular organization of zein is important for devising strategies on how to manipulate the self-assembly process to obtain favorable film properties. References Beck, M.I., Tomka, I., Waysek, E., 1996. Physico-chemical characterization of zein as a film coating polymer: a direct comparison with ethyl cellulose. International Journal of Pharmaceutics 141, 137e150. Bliznyuk, V.N., Assender, H.E., Briggs, G.A.D., 2002. Surface glass transition temperature of amorphous polymers. A new insight with SFM. Macromolecules 35, 6613. Brandt, W., Berko, S., Walker, W.W., 1960. Positronium decay in molecular substances. Physical Review 120, 1289e1295. Casariego, A., Souza, B.W.S., Cerqueira, M.A., Teixeira, J.A., Cruz, L., Díaz, R., Vicente, A.A., 2009. Chitosan/clay films’ properties as affected by biopolymer and clay micro/nanoparticles concentrations. Food Hydrocolloids 23, 1895e1902. Chang, P.R., Yang, Y., Huang, J., Xia, W., Feng, L., Wu, J., 2009. Effects of layered silicate structure on the mechanical properties and structures of protein based bionanocomposites. Journal of Applied Polymer Science 113 (2), 1247e1256. Cocero, A.M., Kokini, J.L., 1991. The study of the glass transition of glutenin using small amplitude oscillatory rheological measurement and differential scanning calorimetry. Journal of Rheology 35, 257e270. Damodaran, S., 1996. In: Fenemma, O. (Ed.), Amino Acids, Peptides and Proteins, in Food Chemistry. Marcel Dekker Inc., pp. 321e425. do A. Sobral, P.J., dos Santos, J.S., Garcı’a, F.T., 2005. Effect of protein and plasticizer concentrations in film forming solutions on physical properties of edible films based on muscle proteins of a Thai Tilapia. Journal of Food Engineering 70, 93e100. Eastman, T., Zhu, D.M., 1996. Adhesion forces between surface-modified AFM tips and a mica surface. Langmuir 12 (11), 2859e2862. Eaton, P., Graham, P., Smith, J.R., Smart, J.D., Nevell, T.G., Tsibouklis, J., 2000. Mapping the surface heterogeneity of a polymer blend: an adhesion-forcedistribution study using the atomic force microscope. Langmuir 16, 7887e7890.
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Eaton, P., Smith, J.R., Graham, P., Smart, J.D., Nevell, T.G., Tsibouklis, J., 2002. Adhesion force mapping of polymer surfaces: factors influencing force of adhesion. Langmuir 18, 3387e3389. Esen, A., 1986. Seperation of alcohol soluble proteins (zeins) from maize into three fractions by differential solubility. Plant Physiology 80, 623e627. Gillgren, T., Barker, S.A., Belton, P.S., Georget, D.M.R., Stading, M., 2009. Plasticization of zein: a thermomechanical, FTIR, and dielectric study. Biomacromolecules 10, 1135e1139. Gounga, M.E., Xu, S.Y., Wang, Z., 2007. Whey protein isolate-based edible films as affected by protein concentration, glycerol ratio and pullulan addition in film formation. Journal of Food Engineering 83, 521e530. Han, J.H., Gennadios, A., 2005. Edible films and coatings: a review. In: Han, J.H. (Ed.), Innovations in Food Packaging. Elsevier Academic Press, San Diego, CA, pp. 239e262. Holding, D.R., Larkins, B.A., 2009. Zein storage proteins. In: Kriz, A.L., Larkins, B.A. (Eds.), Molecular Genetic Approaches to Maize Improvement, vol. 63. Springer, Berlin Heidelberg, pp. 269e286. Hoseney, R.C., Zeleznak, K.J., Yost, D.A., 1986. A note on the gelatinization of starch. Starch/Stärke 38, 407e409. Karel, M., 1985. Effects of water activity and water content on mobility of food components, and their effects on phase transitions in food systems. In: Simatos, D., Multon, J.L. (Eds.), Properties of Water in Foods. Martinus Nijhoff, The Netherlands, pp. 153e169. Kim, C.S., Hunter, B.G., Kraft, J., Boston, R.S., Yans, S., Jung, R., Larkins, B.A., 2004. A defective signal peptide in a 19-kD a-zein protein causes the unfolded protein response and an opaque endosperm phenotype in the maize De*-B30 mutant. Plant Physiology 134, 380e387. Kogan, M.J., Dacol, I., Gorostiza, P., Lopez-Iglesias, C., Pons, R., Pons, M., Sanz, F., Giralt, E., 2002. Supramolecular properties of the prolinerich gamma-zein N-terminal domain. Biophysical Journal 83, 1194e1204. Krochta, J.M., 2002. Proteins as raw materials for films and coatings: definitions, current status, and opportunities. In: Gennadios, A. (Ed.), Protein-Based Films and Coatings. CRC Press, Boca Raton, FL, pp. 1e41. Lai, H.M., Padua, G.W., 1997. Properties and microstructure of plasticized zein films. Cereal Chemistry 74 (6), 771e775. Larkins, B.A., Lending, C.R., Wallace, J.C., 1993. Modification of maize-seed-protein quality. American Journal of Clinical Nutrition 58 (2), 264Se269S. Lee, J.W., Son, S.M., Hong, S.I., 2008. Characterization of protein-coated polypropylene films as a novel composite structure for active food packaging application. Journal of Food Engineering 86, 484e493. León, P.G., Chillo, S., Conte, A., Gerschenson, L.N., Del Nobile, M.A., Rojas, A.M., 2009. Rheological characterization of deacylated/acylated gellan films carrying l-(þ)-ascorbic acid. Food Hydrocolloids 23 (7), 1660e1669. Levine, H., Slade, L.,1992. Glass transitions in foods. In: Schwartzberg, H.G., Hartel, R.W. (Eds.), Physical Chemistry of Foods. Marcel Dekker, New York, pp. 83e221. Madeka, H., 1996. Characterizing phase changes in gliadin and zein as a function of moisture and temperature using rheology and DSC. PhD thesis. in Food Science. Rutgers, The State University of New Jersey. Madeka, H., Kokini, J.L., 1994. Changes in rheological properties of gliadin as a function of temperature and moisture: development of a state diagram. Journal of Food Enginering 22, 241e252. Madeka, H., Kokini, J.L., 1996. The effect of glass transition and cross-linking on rheological properties of zein: development of a preliminary state diagram. Cereal Chemistry 73 (4), 433e438. Marti, O., Stifter, T., Waschipky, H., Quintus, M., Hild, S., 1999. Scanning probe microscopy of heterogeneous polymers. Colloids and Surfaces A: Physicochemical and Engineering Aspects 154, 65e73. Matsushima, M., Danno, G., Takezawa, H., Izumi, Y., 1997. Three-dimensional structure of maize a-zein proteins studied by small-angle x-ray scattering. Biochimica et Biophysica Acta 1339, 14e22.
Mizes, H.A., Loh, K.-G., Miller, R.J.D., Ahuja, S.K., Grabowski, E.F., 1991. Submicron probe of polymer adhesion with atomic force microscopy: dependence on topography and material inhomogeneities. Applied Physics Letters 59, 2901e2909. Muthuselvi, L., Dhathathreyan, A., 2006. Contact angle hysteresis of liquid drops as means to meausure adhesive energy of zein on solid substances. Pramana, Journal of Physics 66 (3), 563e574. Nyqvist, H., 1983. Saturated salt solutions for maintaining specified relative humidities. International Journal of Pharmaceutical Technology and Product Manufacturer 2, 42e48. Ogale, A.A., Cunningham, P., Dawson, P.L., Acton, J.C., 2008. Viscoelastic, thermal, and Microstructural characterization of soy protein isolate films. Journal of Food Science 65 (4), 672e679. Ozdemir, M., Floros, J.D., 2001. Analysis and modeling of potassium sorbate diffusion through edible whey protein films. Journal of Food Engineering 47, 149e155. Rhim, J.W., 2007. Potential use of biopolymer-based nanocomposite films in food packaging applications. Food Science and Biotechnology 16, 691e709. Robeson, L.M., 2007. Polymer Blends: A Comprehensive Review. Hanser Gardner Publications, Inc., Ohio, USA, p. 253. Roos, Y.H., 1992. Phase 530 transitions and transformations in food systems. In: Heldman, R.D., Lund, D.B. (Eds.), Handbook of Food Engineering. Marcel Dekker, Inc., New York, pp. 145e197. Roos, Y., Karel, M., 1991. Phase transitions of mixtures of amorphous polysaccharides and sugars. Biotechnology Progress 7, 49e53. Roos, Y.H., Karel, M., Kokini, J.L., 1996. Glass transitions in low moisture and frozen foods: effects on shelf life and quality. Food Technology 50 (11), 95e108. Sanchez-Garcia, M.D., Gimenez, E., Lagaron, J.M., 2008. Morphology and barrier properties of solvent cast composites of thermoplastic biopolymers and purified cellulose fibers. Carbohydrate Polymers 71 (2), 235e244. Shi, K., Kokini, J.L., Huang, Q., 2009. Engineering zein films with controlled surface Morphology and Hydrophilicity. Journal of Agriculture and Food Chemistry 57 (6), 2186e2192. Slade, L., Levine, H., 1991. A food polymer science approach to structure-property relationships in aqueous food systems: non-equilibrium behavior of carbohydrate-water systems. In: Levine, H., Slade, L (Eds.), Water Relationships in Foods. Plenum Press, New York, pp. 29e102. Sperling, L.H., 1992. Introduction to Physical Polymer Science. Wiley-Interscience Inc., New York. Tranchida, D., Kiflie, Z., Acierno, S., Piccarolo, S., 2009. Nanoscale mechanical characterization of polymers by atomic force microscopy (AFM) nanoindentations: viscoelastic characterization of a model material. Measurement Science and Technology 20, 1e9. Tsui, O.K.C., Wang, X.P., Ho, J.Y.L., Ng, T.K., Xiao, X., 2000. Studying surface glass-torubber transition using atomic force Microscopic adhesion measurements. Macromolecules 33, 4198e4204. Wang, Q., Geil, P., Padua, G., 2004. Role of hydrophilic and hydrophobic interactions in structure development of zein films. Journal of Polymers and the Environment 12 (3), 197e202. Weller, C.L., Gennadios, A., Saravia, R.A., 1998. Edible bilayer films zein and grain sorghum wax or carnaubu wax. Lebensmittel-Wissenschaft und-Technologie 31, 279e285. Wu, M., Kleiner, L., Tang, F.W., Hossainy, S., Davies, M.C., Roberts, C.J., 2009. Nanoscale mechanical measurement determination of the glass transition temperature of poly (lactic acid)/everolimus coated stents in air and dissolution media. European Journal of Pharmaceutical Science 36, 493e501. Zeleznak, K.J., Hoseney, R.C., 1987. The glass transition in starch. Cereal Chemistry 64, 121e124.
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Journal of Cereal Science journal homepage: www.elsevier.com/locate/jcs
Effect of different fractions of zein on the mechanical and phase properties of zein films at nano-scale Chithra Panchapakesan a, Nesli Sozer b, Hulya Dogan c, Qingrong Huang a, Jozef L. Kokini b, * a
Rutgers University, Department of Food Science, 65 Dudley Road, New Brunswick, NJ 08901, USA Food Science and Human Nutrition, College of Agricultural, Consumer and Environmental Sciences, University of Illinois at Urbana-Champaign, 1304 West Pennsylvania Ave., Urbana, IL 61801, USA c Kansas State University, 104 Shellenberger Hall, Manhattan, KS 66506, USA b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 24 November 2010 Received in revised form 21 October 2011 Accepted 6 November 2011
The phase behavior of zein films has been investigated at nano-scale using atomic force microscopy (AFM) and compared to the phase behavior of the bulk using a thermal characterization technique. The local surface properties of the films were evaluated as a function of water activity using AFM. The glass transition temperature (Tg) of zein films decreased with increasing water activity. Adhesion forces measured by the AFM force curves increased with increasing water activity. Topography of zein and zein fractions were evaluated both qualitatively and quantitatively by the use of AFM and dedicated software to calculate the surface roughness. It has been found that processing technologies (solvent casting, drop deposition and spin casting) has influence on the surface structures of films. The films which were formed by the alpha zein rich fraction were found to have highest roughness values. Sectional surface profiles revealed that a-zein films have mean roughness (Ra) of 1.808 nm and root mean square roughness (RMS) of 2.239 nm while b-zein films have mean roughness (Ra) of 1.745 nm and root mean square roughness (RMS) of 3.623 nm. The discussions conducted on the differences/similarities in the observations were based on the hydrophobic/hydrophilic properties and interactions of these zein fractions. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Atomic force microscopy (AFM) Zein Molecular organization Surface topography Phase behavior Self-assembly
1. Introduction Biodegradable films which are produced from renewable sources have been the core of food packaging studies for many years. They are encountered as environmentally friendly since they not only decrease the deposit of solid wastes but also save the fossil fuel sources (Chang et al., 2009). Biopolymer based films can be produced from proteins, polysaccharides, lipids or combinations of these. Various plant and animal origin proteins have been investigated for their film forming properties (do A. Sobral et al., 2005; Chang et al., 2009; Gounga et al., 2007; Krochta, 2002; Shi et al., 2009).
Abbreviations: AFM, atomic force microscopy; DSC, differential scanning calorimeter; Tg, glass transition temperature; Ra, mean roughness; RMS, root mean square roughness; PtBuA, poly-(tert butyl acrylate); mw, molecular weight; EtOH, ethanol; (DGtethanol / water), free energy change; Sethanol, solubilities of the amino acid in ethanol; Swater, solubilities of the amino acid in water; n, the number of amino acid residues; k, spring constant; Fad, adhesive force; d, deflection/ displacement of the cantilever. * Corresponding author. Tel.: þ 1 217 333 0240; fax: þ1 217 333 5816. E-mail address: [email protected] (J.L. Kokini). 0733-5210/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.jcs.2011.11.004
Biopolymer based films or coatings have a wide range of applications as being carriers of antimicrobial agents, ant ioxidants, nutraceuticals and coloring materials (Han and Gennadios, 2005; Lee et al., 2008; Ozdemir and Floros, 2001). Natural biopolymer films have limited applications as compared to synthetic polymers due to their poor mechanical and water vapor barrier properties. In the literature there are many studies on improving the barrier and mechanical properties of biopolymer films either by modification of the films or inclusion of reinforcement structures such as filler materials. Even if most of these methods were successful from the perspective of improvement, there are still many gaps in understanding the structural conformations and most of these films are found to be still not satisfactory and limited for many applications (Rhim, 2007). The molecular basis of film-forming is not completely understood and no attempts have been made to determine the relationship between self-assembly of film network and the structure of the protein film. The functional properties of films such as barrier properties and stability are related to the molecular organization of biopolymers used during film formation. Polymer synthesis and self-assembly are the phenomenons which occur at the nanolevel. Analysis of protein topology at the nano- and macro-level will help to
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understand the functional properties of films. Film structure can be revealed by understanding the specific conformational arrangements by the use of AFM. Parameters which can easily be evaluated from AFM data can help to create surface topography, adhesion force and elasticity diagrams of film samples. Up to now many researchers have used AFM for mechanical characterization of soy protein isolate films, zein films, chitosan/clay nanocomposite films, polylactic acid/cellulose fiber films and gellan films (Casariego et al., 2009; Lai and Padua, 1997; León et al., 2009; Ogale et al., 2008; Sanchez-Garcia et al., 2008). State diagrams have been useful tools in characterization of amorphous and crystal states of food components, in particular proteins and carbohydrates (Cocero and Kokini, 1991; Madeka and Kokini, 1994, 1996). Proteins have been found to exist mostly in the amorphous state or in partially crystalline form. The physical properties of many amorphous materials can be related to their glass transition temperature (Tg). Tg defines a second order phase change temperature, where a solid, glassy material becomes a soft, rubbery material (Cocero and Kokini, 1991; Roos et al., 1996; Sperling, 1992). For amorphous polymers, the glass transition temperature is a critical temperature where the mechanical properties undergo a dramatic change. Several factors such as the presence of plasticizers, molecular weight, crystallinity and ionic binding interactions affect the Tg of polymers. Water is known to depress the glass transition temperature of amorphous proteins (Cocero and Kokini, 1991; Hoseney et al., 1986; Madeka and Kokini, 1994), and partially crystalline polymers such as starch and maltodextrins (Roos, 1992; Zeleznak and Hoseney, 1987). The direct effect of moisture content at a constant temperature leads to increased mobility of chains in the amorphous regions, which decreases the glass transition temperature (Karel, 1985; Levine and Slade, 1992; Roos and Karel, 1991). Predicting the changes in mechanical properties that occur as a result of plasticization with water or of temperature is critical to control the physical properties and the resulting quality and stability of the biofilms. Differential scanning calorimetry (DSC) and rheometry, in particular small amplitude oscillatory measurements, are the most common techniques used to study the glass transition of biopolymers. Phase behavior of polymers can also be studied by measuring the adhesive forces between AFM probe tip and the molecules at the surface. As the cantilever approaches the surface, it experiences an attractive force from the sample. A pull-off force is experienced when the cantilever is pulled back to detach the tip from the surface, which is indicated as negative force in an AFM force curve. It can be simply calculated through the sum of the van der Waals and the capillary forces between the AFM tips and the substrate (Eastman and Zhu, 1996). AFM force curves have been used by many researchers as an aid in investigating the viscoelasticity and surface properties of polymeric materials such as surface heterogeneity (Bliznyuk et al., 2002; Eaton et al., 2000, 2002; Marti et al., 1999; Mizes et al., 1991; Tsui et al., 2000). Tsui et al. (2000) demonstrated that AFM adhesion measurements offer a powerful probe to examine the surface viscoelastic properties of a polymer compared to those of the bulk determined by conventional methods. They studied the mechanical properties of thin films of PtBuA (poly-(tert butyl acrylate)) by increasing the temperature and measuring the adhesive force between the sample and the cantilever. In the vicinity of Tg, the adhesive force measured was found to increase rapidly. They also found equivalence between temperature and probe rate and stated that the adhesion was found to increase with increasing temperature and decreasing probe movement rate. The dynamic behavior and molecular relaxation of the polymer sample was found to be not very different from the bulk properties for the PtBuA samples.
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Bliznyuk et al. (2002) measured the surface glass transition temperature of several amorphous polystyrene samples of different molecular weights (mw) ranging between 3900 and 1,340,000 by force-distance measurements at different temperatures using a scanning force microscope. The results related to existing theories on the glass transition temperature of the bulk describing the decrease in Tg at lower mw as a result of increased free volume in the system. The values of the surface glass transition for the samples with mw > 30,000 have been found to be the same as corresponding bulk values. The effect of temperature on surface phase behavior was also observed to be similar to that of the bulk. Pull-off forces measured at temperatures above the Tg of the samples were significantly lower than those measured at temperatures below Tg. Tranchida et al. (2009) worked on the viscoelastic characterization of a model material as well as the glass to rubber transition by the help of an AFM. They found that the network which had higher molecular weight crosslinks had the glass to rubber transition lower than room temperature. Wu et al. (2009) measured modulus of elasticity of polylactic acid coated stents by AFM. The group found the onset glass transition temperature of the polylactic acid to be around 55 C through the elasticity versus temperature curves. Among many protein sources, zein (the major storage protein in corn endosperm) has been actively investigated for its film forming ability and its potential to produce novel polymeric films (Beck et al., 1996; Kim et al., 2004; Lai and Padua, 1997; Weller et al., 1998). Zein is one of the most hydrophobic proteins among many cereal proteins with an average hydrophobicity of 1.365 J/mol. The high proportion of non-polar amino acid residues is responsible for the hydrophobicity of zein. Order of hydrophobicity of zein fractions is g- and d-zein > a-zein > b-zein (Holding and Larkins, 2009). The mechanical and barrier properties of biofilms depend on many factors such as formulation, processing technique as well as the state of film forming polymer at a particular temperature and water activity level. Understanding the phase behavior of biofilms is very important to control their functionality at their end usage. In this study it was aimed to understand the phase/state behavior of zein films both at macro-scales and at nano-level organizations. Films were investigated for their phase behavior as a function of water activity using DSC and the results were compared to the adhesive force measurements obtained from AFM. 2. Materials and methods 2.1. Materials Zein (F4000) was commercially supplied from Freeman Industries, Inc. (Tuckahoe, NY) containing 92.54 4.10% crude protein, 1.03 0.05% ash and 1.18 0.20% crude fat. Dry milled corn grains were used to extract different fractions of zein which are soluble in 90% (v/v) (Fraction 1) and 60% (v/v) (Fraction 2) ethanol (EtOH). 2.2. Methods 2.2.1. Isolation of zein fractions The a- and b-zein fractions were isolated from dry milled corn flour. Zein is a prolamin which has been previously classified to a, b, g fractions based on mass and ethanol solubility (Esen, 1986). Alpha zein has a molecular weight range 20e24 kDa soluble in 40e95% (v/v) EtOH, whereas beta zein and gamma zein have 17e18 kDa/30e80% (v/v) EtOH solubility, 27 kDa/0e80% (v/v) EtOH, respectively. Attention should be given that beta zein is insoluble in 90% (v/v) EtOH and gamma zein is soluble in EtOH only in the
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presence of 2-Mercaptoethanol (Esen, 1986). The protocols used for separation of a- and b- zein fractions have been adapted from Matsushima et al. (1997) and Esen (1986) were summarized in Fig. 1. Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) of fractions of corn protein was carried out in order to estimate the molecular weight compositions of the purified fractions. 2.2.2. Hydrophobicity measurement Hydrophobicity is one of the major factors affecting structure, solubility, fat binding properties, etc., of proteins. One of the most commonly used ways to estimate relative hydrophobicities of amino acids side chains involves experimental determination of free energy changes for dissolution of amino acid chains in water and in ethanol (Damodaran, 1996). The free energy change ðDGtethanol / water Þ of transfer of the amino acid from ethanol to water for amino acid side chains of zein at 25 C was obtained from Damodaran (1996). The free energy for transferring 1 mol of the amino acid from the aqueous solution into the ethanol solution is given by:
DGtethanol/water ¼ RTln
Sethanol Swater
where Sethanol and Swater represent solubilities of the amino acid in ethanol and water, respectively. Amino acid side chains with large positive DGt values are considered to be hydrophobic and they tend to be in the organic phase rather than in the aqueous phase. Amino acid residues with negative DGt values are hydrophilic. The average hydrophobicity of a protein is calculated as n P
DGt ¼
DGt;sidechains n
where n is the number of amino acid residues. Defatting using hexane (1:5, repeated twice)
Solubilization at 60 % ethanol
Centrifugation (9000 rpm, 10 min, 4 0C)
Pellet
Supernatant ( - and - zein)
(discarded) Adjust EtOH concentration to 90%
Centrifugation (9000 rpm, 10 min, 4 0C)
Supernatant
Pellet
( - zein rich fraction)
( - zein rich fraction)
(Fraction 1)
(Fraction 2)
Fig. 1. Schematic diagram for the separation of a- and b- zein fractions.
In order to understand the organization and arrangement of the zein Fraction 1 and Fraction 2 on the surface of the silicon wafer, average hydrophobicity of zein fractions were calculated. 2.2.3. Film formation Zein films were produced by the use of unpurified and purified zein fractions. Three different methods which are solvent casting, drop deposition and spin casting were applied for the production of unpurified zein films whereas the spin casting method was used for production of films from different fractions of zein. Detailed descriptions of the protocols are given below. 2.2.3.1. Film formation methods for unpurified zein and fractions of zein (fraction 1 and fraction 2) films 2.2.3.1.1. Solvent casting. Approximately 16 g of unpurified zein (F4000, Freeman Inc.) was dissolved in 84 ml of 75% (v/v) ethanol by stirring constantly at 75e80 C. The solution was allowed to cool at room temperature and then poured on plastic petri plates to form a thin layer on the surface of the plate. The films were allowed to oven dry at 30 C for 18e24 h to remove excess ethanol, and such a slow drying procedure ensures the formation of even films. Thickness of cast films was approximately 1.4 0.05 mm. Films were stored under ambient dry conditions until further processing. 2.2.3.1.2. Drop-deposition. Initial experiments with AFM topography were conducted with zein films made by drop deposition using the unpurified (F4000, Freeman Inc.) zein fraction. Zein was dissolved in concentrations of 40 mg/ml, 20 mg/ml, 10 mg/ml, 5 mg/ml, 2 mg/ml and 1 mg/ml in 75% (v/v) ethanol. The solution was mixed on a vortex at the highest speed setting (7) for approximately 5 min. The films were formed by dropping the solution onto a silicon wafer which had a hydrophobic coating (manufactured by Veeco Instruments, CA). The sample was then allowed to dry at 30 C to form a thin film layer on the surface of the wafer. 2.2.3.1.3. Spin casting. Zein (F4000) and its subfractions (Fraction 1 and Fraction 2) were spin-cast onto silicon wafers following dissolution in 70% (v/v) ethanol for w2 h with constant stirring. Concentrations of 1 mg/ml (0.1%) were used to ensure both fractions could be completely dissolved. Spin cast conditions were maintained constantly at 3000 rpm for 10 s. Approximately 200 ml of the solution was dispensed onto the wafer and spin-cast to form a thin-layer of zein. The films were then dried for w1 h at 40 C in a conventional oven. The thicknesses of the films were measured by making an artificial scratch on the film surface. Atomic force microscopy images were taken by scanning across the edge of the scratch. The thicknesses of the spin-cast films were found to be approximately 17 nm. 2.2.4. Water activity adjustments of the solvent casted films. Solvent casted films were stored at various water activity levels till they equilibrated. They were evaluated for their physical and mechanical properties by measuring the glass transition temperature and adhesive force. Films were cut into small pieces and stored in saturated desiccators containing different salt solutions (LiCl, K2CO3, NaNO2, KCl, KNO3) which produced nominal water activities of 0.12, 0.43, 0.64, 0.84 and 0.93 respectively, at 25 C (Nyqvist, 1983). The water activity of the samples was measured using the AquaLab water activity meter (Series 3 TE, Decagon Devices, WA). The actual and nominal water activities were found to agree within 15%. 2.2.5. Thermal measurements A Differential Scanning Calorimeter TA 4000 Thermal Analysis System with a DSC 30-S Cell/TC11 TA Processor (Mettler Instrument Inc., Highstown, NJ) was used to evaluate the glass transition
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Fad ¼ k*d where Fad is the adhesive force, k is the cantilever spring constant and d is the deflection/displacement of the cantilever. Samples at high water activity levels were contained in an “atmospheric hood” made of polycarbonate which allowed the sample to be enclosed in a chamber to avoid any moisture loss from the sample. The measurements were completed approximately within 15 min, and the water loss over this period of time was determined to be insignificant.
Zein isolated by ethanol extraction from dry milled corn contains four major fractions which are a-zein (MW ¼ 21e25 kDa), b-zein (MW ¼ 17e18 kDa), g-zein (MW ¼ 27 kDa), d-zein (MW ¼ 9e10 kDa) (Esen, 1986). Fraction 1 was found to be rich in
Aw= 0.28
a Frequency
2.2.7. Adhesive force measurements Adhesive forces of solvent cast, water activity equilibrated zein films were measured as a function of increase in water activity. Force measurements were conducted in the “contact mode” using the force analysis software. Since the adhesion force measured by the AFM force curves depends on the sample topography (Mizes et al., 1991), repeated force measurements were conducted on film surfaces. Equilibrated zein films (Aw ¼ 0.28e0.93) were adhered to the sample holders and around 90e100 force curves were generated on each sample in order to obtain an average adhesive force measurement over the sample surface. In order to provide a good representation of the local surface adhesive force characteristics of the zein film samples, three different film samples at the same water activity level were analyzed and the average of resulting 270e300 adhesive force readings was reported. The same cantilever with spring constant (k) of 0.58 N/m was used in the measurements to minimize the error that may be caused by differences in cantilever tip properties and dimensions. The force between the cantilever and the sample was minimized by maintaining the set point always at “zero”. Adhesive force (Fad) was calculated by the following equation:
3. Results and discussion
60 40 20 0
b Frequency
2.2.6. Atomic force microscopy (AFM) A Multimode Scanning probe Microscope with a NanoScope IIIa SPM Controller (Digital Instruments/Veeco Instruments, CA) was used with a J or E scanner attached (Lateral XeY range: 125 mm 125 mm/10 mm 10 mm; Zerange of 5 mm/2.5 mm respectively for J and E). Silicon nitride cantilevers with a nominal spring constant of either 0.12 N/m or 0.58 N/m were used. The dimensions of the cantilever as specified by the manufacturer were nominal thickness ¼ 0.6 mm, thickness ¼ 0.4e0.7 mm, tip height ¼ 2.5e3.5 mm, maximum tip radius ¼ 60 nm and tip angle (front, side, back) ¼ 35 . Typical scan rates as suggested by the manufacturer were employed and ranged between 0.5 and 1.5 Hz.
2.2.8. Topographical measurements The different fractions of zein (unpurified, Fraction 1 and Fraction 2) were analyzed for topographical and surface features using the contact mode of the AFM. Solvent cast, spin cast and drop-deposited films were evaluated and analyzed for surface characteristics and mechanics of film formation. Scan sizes of images ranged from 1 to 10 mm and several images (w10e15) were generated. The thickness of the spin-cast films was evaluated by scanning an “artificial scratch” created over the surface of the film. The silicon wafers and the sample holders were placed in 95% (v/v) ethanol for 24 h and then cleaned alternately using ethanol, methanol and distilled water several times (5 min each) and dried at 30 C before usage. If the formation of any film on the surface was observed after drying, the cleaning step was repeated. The exact same cantilever was used for the adhesive force measurements (kc ¼ 0.58 N/m). The cleaned silicon wafers were imaged before use, in order to confirm the cleaning process. If images indicated the presence of any organic matter, the cleaning procedure was repeated and re-imaged until the wafer indicated a clean surface.
30
60
90
120 150 Fad (nN)
180
210
180
210
180
210
Aw= 0.55 60 40 20 0
30
60
90
120 150 Fad (nN)
Aw= 0.90
c Frequency
of the solvent cast zein films at various water activity levels. Approximately 20 2 mg of sample was weighed into 40 ml hermetically sealed medium pressure stainless steel crucibles to prevent moisture loss on heating. An empty crucible was used as a reference. Calibration of the instrument was performed using indium as a standard. Before sealing the DSC crucibles they were placed into the respective desiccators for w24 h to compensate for any moisture losses incurred during sample transfer. Samples were scanned between 20 C and 120 C with a heating rate of 5 C/min. Rescans were performed immediately after each scan, in order to erase the thermal history of the samples and to confirm the location of the Tg, based on the reversibility of this second order transition. The glass transition temperature was determined from the DSC rescans, at the midpoint in the shift of the heat flow baseline, which corresponded to the temperature at which one-half of the change in the heat capacity, DCP, occurred. The reported data are the averages of at least two replicate measurements.
177
60 40 20 0
30
60
90
120 150 Fad (nN)
Fig. 2. Adhesive force distribution curves at (a) Aw ¼ 0.28, (b) Aw ¼ 0.55, and (c) Aw ¼ 0.93.
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a-zein having molecular weight ranging between 20 and 24 kDa. The presence of dimers at minor amounts (MWw44e50 kDa) was also observed. The SDS-PAGE confirmed the current literature about zein stating that primary fraction lies between 17 and 18 kDa (a-zein) and some other weaker bands around 25 kDa. The second fraction was found to be rich in b-zein together with minor amounts of both a- (20e24 kDa), g-(27 kDa) and probably also the d-zein fraction. 3.1. Characterization of nano-scale phase behavior of zein films Glass transition temperature is the characteristic transition temperature from the amorphous phase of polymers. Below the Tg, the polymer chain is incapable of diffusing within a random matrix due to intermolecular interactions and thus has a rigid structure whereas above the Tg, the kinetic forces are strong enough to
overcome intermolecular forces and can diffuse, resulting in a rubbery material (Robeson, 2007). Molecular weight, crosslinking within polymers, presence of plasticizers affect the phase behavior of polymers. Therefore the molecular structure at the surface may be different in structure compared to the bulk. In the current study, we investigated the phase behavior of zein films as bulk by measuring their Tg using DSC. Adhesive force measurements using AFM were utilized as an indicator for molecular mobility. The DSC results were correlated with the adhesive force measurements to investigate the surface properties of zein films at the nano-scale. This set of information will enable us to understand the properties of zein films at the nano-scale and relate them to those at macroscopic levels. The effect of water activity on both adhesive force and glass transition temperature of solvent casted films were determined. By the increase of water activity, glass transition temperature dropped
Fig. 3. a) 10 mm scan of 16% w/v cast zein film, data scale ¼ 758.4 nm, top view b) 1 mg/ml (0.1%) drop-deposited film images of unpurified zein 600 nm scan. Data scale ¼ 30 nm top view (left) and 3D view (right) c) 1 mg/ml (0.1%) image of unpurified, spin-cast film at 3000 rpm. Scan size ¼ 2.5 mm, data scale ¼ 10.0 nm top view (left) and 3D view (right).
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Fig. 4. Ra - mean roughness and Rq- RMS roughness values of spin coated unpurified, fraction 1 and fraction 2 zein films.
from 75 C to 25 C whereas adhesive force increased from 67.18 nN to 138.79 nN which is due to the plasticization effect of water. The mechanism of plasticization of water can be explained in terms of a free volume effect (Brandt et al., 1960; Cocero and Kokini, 1991; Slade and Levine, 1991). The free volume is inversely proportional to the number average molecular weight (Sperling, 1992). Therefore, the presence of a low molecular weight substance like water leads to an increased free volume, allowing increased mobility of the polymer backbone chains. An increased mobility is then manifested as a lower glass transition temperature of the polymer/ plasticizer glass (Madeka, 1996). The variation of Tg with the
plasticization effect of water in zein films was compared with the existing literature on Tg of zein powders (Gillgren et al., 2009; Madeka, 1996). Madeka (1996) found that the glass transition temperature for zein powder dropped from 83 C to 21 C as water activity increased from 0.46 to 0.93. Between the zein powder and zein films Tg values shifted with an average deviation of 14.32% which can be attributed to residual amounts of ethanol in the films and different sample preparation methods. The increase in adhesive forces was attributed to the increased mobility in the molecules of the samples analyzed. When the biopolymer film exists in its “glassy state”, there is less mobility in
Fig. 5. Fraction 1 (a) and fraction 2 (b) (0.1% in 70% ethanol) as imaged by the AFM; top view (left), 3D view (right), scan size ¼ 2.5 mm, data scale ¼ 100 nm.
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the chains of the polymer. The molecules are fixed in their positions, and there is less flexibility of the polymer chains. This causes less adhesion to the AFM tip when the tip contacts the surface of the film. Several other studies confirmed that increasing molecular mobility by increasing the temperature results in larger adhesive forces (Marti et al., 1999; Muthuselvi and Dhathathreyan, 2006; Tranchida et al., 2009; Tsui et al., 2000). A range of adhesive forces were measured between zein film surfaces and AFM tip due to the inhomogeneity in sample
topography The range of distributions was presented in the form of adhesive force histograms. Fig. 2 shows the adhesive force distributions measured on zein films equilibrated to different water activity levels. It is evident that measurements at low water activity levels result in low adhesive force readings ranging between 45 and 105 nN (Fig. 2a). Adhesive force measurements conducted at higher water activity levels (Fig. 2c) showed a wider distribution between 65 and 220 nN with an average value around 140 nN. Since the adhesive force is a sum of van der Waals interaction and capillary
Fig. 6. 1 mm scan of 0.1% spin cast film of a) fraction 1 and b) fraction 2 at 50 nm data scale surface plot (top image), top view (left image), section analysis and section analysis results (right image).
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force between the tip and the film surface, higher water activity cause the capillary force increase, thus leads the adhesive force increase (Eastman and Zhu, 1996). 3.2. Characterization of film topography The effect of various film formation methods on the surface characteristics was evaluated by AFM. Surface features of commercial zein films were evaluated at concentrations in the range of 1e40 mg/ml in aqueous ethanol solutions. We found that the surface structure of zein films can be altered by the use of different casting techniques (Fig. 3). Solvent casting resulted in films which did not show any characteristic structure except the presence of several pores (Fig. 3a). The diameter of the pores was found to be approximately 2 mm with an average depth of 300 nm by the use of “sectional analysis” function of the AFM software. We observed more well-defined structures for the films which were produced by drop-deposition (Fig. 3b) and spin casting (Fig. 3c) techniques. Fig. 3b shows some of the distinct structures which are below 30 nm height and are not aggregating to form large structures. Spin casting of zein films resulted in more controlled and uniform structures due to high speeds (3000 rpm) performed during film formation. The molecules across the film surface spread more evenly, decreasing the rate of clumping. Mean roughness values were used to quantitatively analyze the structures which occurred on the surface of zein films. It is the arithmetic average of the absolute values of the surface height deviations measured from a mean value on the line. The mean value is usually predetermined by the AFM software. Roughness measurements were conducted over 25 images of each sample. Fig. 4 shows the roughness values for unpurified and two fractions of zein which is an indicator of how well zein particles organize themselves over the silicon wafer. It was found that films formed by the unpurified zein fraction yielded the least roughness whereas alpha zein rich films had the maximum roughness values. The aggregation/agglomeration rate of zein on the surface of films is controlled by the properties and the changes in the zein fraction. Among the four different fractions of zein (a-, b-, g-, d-), b- zein fraction is the most hydrophilic fraction. Fig. 5 qualitatively shows the effect of different fractions of zein on the film surface properties. The alpha zein rich fraction (Fig. 5a) resulted in a zein film which is less smooth than the beta-zein rich fraction (Fig. 5b). However, we would like to highlight that even though the second fraction obtained from purification of zein was rich in the beta-zein fraction it was contaminated with some amounts of a-zein, g-zein and d-zein. Therefore, the film forming properties of this fraction were altered by the presence of fractions mentioned above. In order to understand the organization and arrangement of the zein Fraction 1 and Fraction 2 on the surface of the silicon wafer, average hydrophobicity of zein fractions was calculated as described in Methods. Based on the amino acid composition of zein fractions (Larkins et al., 1993), and free energy of amino acid residues (Damodaran, 1996), the average hydrophobicities of a-, b-, g- and d-zein fractions were calculated to be 4.958, 3.825, 5.329 and 5.316 kJ/mol, respectively. In films formed from spin casting of the a-zein rich fraction, the film formation depends on formation of layers depending on the hydrophobic/hydrophilic indices which results in cylindrical elongated tube like structures. Wang et al. (2004) also found that zein had a greater affinity for hydrophilic than for hydrophobic surfaces. The possibility of forming a smooth film is therefore less likely in this case, and we would expect to see more cylindrical/ovoid projections on the surface of the film, thus contributing to more roughness. The ability of the a-zein to interact with the hydrophobic surface of the silicon wafer is reduced, resulting in more elongated-cylindrical “pin-like” structures being
181
formed on the surface of the film (Fig. 6a). The hydrophobicity of the b-zein rich film was increased due to contamination of this fraction by more hydrophobic fractions. When cast on a hydrophobic surface, making use of its high hydrophobicity, it is able to spread itself on the surface of the silicon wafer and form a relatively smooth film (Fig. 6b), because of the formation of hydrophobic and di-sulphide bonds. This process of film formation using hydrophilic and hydrophobic interactions has also been studied and explained by other authors (Kogan et al., 2002). The difference in the hydrophobicity of the two fractions affected their self-assembly behavior on silicon substrate surfaces. Surface features of zein films were evaluated for films resulting from zein solutions at concentrations in the range of 0.1e10 mg/ml. Significant differences were observed in the topography of a- and b-zein films. Sectional surface profiles revealed that a-zein films have mean roughness (Ra) of 1.914 nm and root mean square roughness (RMS) of 2.247 nm while b-zein films have mean roughness (Ra) of 1.745 nm and root mean square roughness (RMS) of 3.623 nm (Fig. 6). It suggests that because of higher hydrophobicity, a-zein can self-assemble to form rougher but more homogeneous films compared to b-zein. 4. Conclusions Experimental findings indicate that the phase behavior of zein, depicted by the change in its glass transition temperature, is clearly affected by the presence of water. The glass transition of zein films decreased with increasing water activities, confirming the plasticizing effects of water. This glass to rubber transition driven by increasing water activity was also observed using the atomic force microscope, in the form of increased adhesive forces observed at higher water activities. Topography of zein films revealed differences based on the method of film preparation and the chemistry of zein fractions. The differences in the hydrophobicity of two zein fractions affected their self-assembly behavior on silicone substrate surfaces. Clear understanding of the self-assembly and molecular organization of zein is important for devising strategies on how to manipulate the self-assembly process to obtain favorable film properties. References Beck, M.I., Tomka, I., Waysek, E., 1996. Physico-chemical characterization of zein as a film coating polymer: a direct comparison with ethyl cellulose. International Journal of Pharmaceutics 141, 137e150. Bliznyuk, V.N., Assender, H.E., Briggs, G.A.D., 2002. Surface glass transition temperature of amorphous polymers. A new insight with SFM. Macromolecules 35, 6613. Brandt, W., Berko, S., Walker, W.W., 1960. Positronium decay in molecular substances. Physical Review 120, 1289e1295. Casariego, A., Souza, B.W.S., Cerqueira, M.A., Teixeira, J.A., Cruz, L., Díaz, R., Vicente, A.A., 2009. Chitosan/clay films’ properties as affected by biopolymer and clay micro/nanoparticles concentrations. Food Hydrocolloids 23, 1895e1902. Chang, P.R., Yang, Y., Huang, J., Xia, W., Feng, L., Wu, J., 2009. Effects of layered silicate structure on the mechanical properties and structures of protein based bionanocomposites. Journal of Applied Polymer Science 113 (2), 1247e1256. Cocero, A.M., Kokini, J.L., 1991. The study of the glass transition of glutenin using small amplitude oscillatory rheological measurement and differential scanning calorimetry. Journal of Rheology 35, 257e270. Damodaran, S., 1996. In: Fenemma, O. (Ed.), Amino Acids, Peptides and Proteins, in Food Chemistry. Marcel Dekker Inc., pp. 321e425. do A. Sobral, P.J., dos Santos, J.S., Garcı’a, F.T., 2005. Effect of protein and plasticizer concentrations in film forming solutions on physical properties of edible films based on muscle proteins of a Thai Tilapia. Journal of Food Engineering 70, 93e100. Eastman, T., Zhu, D.M., 1996. Adhesion forces between surface-modified AFM tips and a mica surface. Langmuir 12 (11), 2859e2862. Eaton, P., Graham, P., Smith, J.R., Smart, J.D., Nevell, T.G., Tsibouklis, J., 2000. Mapping the surface heterogeneity of a polymer blend: an adhesion-forcedistribution study using the atomic force microscope. Langmuir 16, 7887e7890.
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