(500g) Molecular Organization, Topography and Phase Properties of Zein Films

Biodegradable films help reduce the use of synthetic polymer films which take decades if not centuries to completely degrade. Recent research efforts to develop biopolymeric alternatives to polymeric packaging materials have focused on utilization of proteins. The molecular basis of film-forming is not completely understood. No attempts have been made to determine the relationship between the extent of formation of film network and the structure of protein film.

Among many protein sources, zein (the major storage protein in corn endosperm) has been actively investigated for its film forming properties and potential to produce novel hydrophobic films with good water vapor and oxygen barrier properties (Lai and Padua, 1997). The mechanical and barrier properties of thin biofilms are a strong function of their molecular organization. The relationship between the functionality of the zein film and its molecular organization requires consideration of molecular organization during film formation. Atomic force microscopy is a relatively new and useful method for characterization of self-assembled zein structures which strongly influence the functionality of zein films (Panchapakesan, 2005).

Phase behavior of biopolymers is very important in determining the physicochemical properties of several food components, primarily proteins and carbohydrates (Cocero and Kokini, 1991; Madeka and Kokini, 1994, 1996). 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 to 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.

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 and molecular organization of biofilms is very important to control their functionality at their end usage. In this study, we focused on the aspects of the macromolecular organization and film making properties of zein. We utilized Atomic Force microscopy to study phase transitions at nano-level and showed that mobility at the nano-scale is well correlated with mobility at the macro-scale. We studied the self-assembly of zein to understand the molecular organization at the nano-level in order to devise strategies on how to manipulate the self-assembly process to obtain favorable film properties.

Atomic Force Microscopy (AFM) was used for adhesive force measurements and film topography. A multimode Scanning Probe Microscope with a NanoScope IIIa SPM Controller (Digital Instruments/ Veeco Instruments, CA) was used with a J/E scanner attached. Silicon nitride cantilevers with a nominal spring constant of 0.58 N/m were used. The dimensions of the cantilever as specified by the manufacturer were nominal thickness= 0.6 mm, thickness= 0.4-0.7 mm, tip height= 2.5-3.5 mm, maximum tip radius= 60 nm and tip angle= 35°. Typical scan rates ranged between 0.5-1.5 Hz.

Commercial food grade zein (Freeman Industries, NY) were used to form zein films with three different methods: Solvent cast films were prepared by dissolving zein at 16% concentration in 75% ethanol and heating to 75-80°C. Solution was cooled, cast onto petri plates and dried at 30°C. Films by drop deposition were prepared by dissolving 0.1, 1, 2 and 4% zein in 75% ethanol and heating to 75-80°C. A drop of solution was placed on silicone wafers and allowed to dry. For spin-casting, zein was dissolved in 75% ethanol and spin-cast on silicon wafers at 3000 rpm for 10 seconds.

Fractions of commercial zein were extracted from commercial zein based on differential solubility of zein in 90% ethanol and 60% ethanol. Topographical measurements were done on film surfaces using AFM to study the macromolecular organization of zein films. Surface features of both purified and unpurified fractions were evaluated at concentrations in the range of 1-40 mg/ml in aqueous ethanol solutions. Zein films were investigated for their phase behavior as a function of water activity using differential scanning calorimetry (DSC) and the results were compared to the adhesive force measurements obtained from atomic force microscopy (AFM). Force measurements were made on 16% w/v solvent cast films to study the effect of plasticizer content (Aw: 0.12-0.97) on the molecular mobility of the film. Adhesive force of the films was used as a yardstick for molecular mobility. The properties of zein films at the nanoscale were correlated to those at macroscopic levels as measured using traditional methods such as DSC.

The adhesive forces measured over the surfaces of the zein films indicated a glass to rubber transition with the increase in the water activity. The average adhesive force increased from 67.2 nN at Aw= 0.28 to 138.8 nN at Aw= 0.93. The most significant increase in Fad was observed between Aw= 0.82 to 0.93. This sudden or large increase in adhesive force was attributed to the zein changing from a glass to a rubber at this particular water activity. DSC results showed that zein films exhibit a Tg around room temperature (~24°C) when their Aw= 0.93. There is a good agreement between the properties that were observed on zein film surfaces at the nano-scale levels and the properties exhibited by the zein films as bulk, when measured by traditional rheological measurements.

Significant differences were observed in the topography of commercial zein films prepared by different techniques. Solvent cast films were featureless, except the presence of several pores on the surface of the film. The zein film formed by spin casting was observed to form a more uniform distribution across the surface of the film. Film forming properties of the 90% ethanol and 60% ethanol soluble zein fractions were compared. Slight differences in surface features of these films were attributed to the hydrophobic/hydrophilic properties two zein fractions.

REFERENCES

Cocero, A.M., and Kokini, J.L., 1991. The study of the glass transition of glutenin using small amplitude oscillatory rheological measurement and differential scanning calorimetry. J. Rheology, 35: 257-270.

Lai, H.M., Padua, G.W., 1997, Properties and microstructure of plasticized zein films. Cereal Chemistry, 74(6): 771-775.

Madeka, H., and Kokini, J.L., 1994. Changes in rheological properties of gliadin as a function of temperature and moisture: Development of a state diagram. J. Food Eng., 22: 241-252. 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): 433-438.

Panchapakesan, C.P., 2005. Analysis of the topography, molecular organization and phase properties of films formed from zein and its fractions, MS Thesis, Rutgers University, NJ.