Physical and mechanical properties of pea starch edible films containing beeswax emulsions PRIOR APPLICATION INFORMATION

This application claims the benefit of Canadian Patent Application 2,552,179, filed July 6, 2006.

BACKGROUND OF THE INVENTION

Starch is one of the most abundant, inexpensive and commonly used natural polysaccharides (Guilbert and Gontard 2005; Narayan 1994), and in both its native and modified forms, has been playing important roles in the food industry. Starches and their derivatives have been used to modify physical properties of food products, which include texture, viscosity, gel-formation, adhesion, binding, moisture retention, product homogeneity and film-formation (Thomas and Atwell 1997; Liu 2005). The use of polysaccharides as film and coating materials has grown extensively in recent years because of their edibility, low permeability to oxygen and contribution to quality preservation (Mark and others 1966; Roth and Mehltretter 1970; Lourdin and others 1995; Palviainen and others 2001 ; Forssell and others 2002; Han and Gennadios 2005). Edible films and coatings are generally prepared using edible biopolymers such as polysaccharides, proteins and lipids, and food-grade additives (Gennadios and others 1997; Han and Gennadios 2005). Especially, starch is an attractive raw material for edible packaging because of its renewability, biodegradability and low cost compared with protein (Guilbert 2000; Guilbert and Gontard 2005; Liu 2005).

Starch is normally a mixture of amylose and amylopectin polymers. Most starches, such as those from wheat, corn and potato, contain about 25% amylose and 75% amylopectin (Haase 1993; BeMiller and Whistler 1996). However, some legume starches including those of peas are characterized by a high-amylose content (24-65%, varies with species) (Hoover and Sosulski 1991). High-amylose starch is a very useful film-forming material because it normally improves mechanical strength including tensile strength and gas barrier properties (Wolff and others 1951 ; Lourdin and others 1995; Palviainen and others 2001). This is probably due to the higher degree of crystallinity of amylose-rich region after dehydration (Garcia and others 2000; Liu and Han 2005).

Pea is an important grain legume, as an animal feed and human food, which is cultivated in many regions of the world and ranks fourth in terms of world production of food legumes behind soybean, peanuts and dry beans (FAO 2000). Pea starch is mainly used in industrial applications, but not much in food applications. Therefore, it is economically important to explore possible avenues for improving the functional properties of pea starch for it to be successfully utilized in the food industry.

The efficiency and functional properties of edible film and coating materials are highly dependent on the inherent characteristics of film-forming materials including a variety of polysaccharides and their derivatives (Liu 2005; Lacroix and Le Tien 2005). Films from polysaccharides possess excellent oxygen barrier properties due to their tightly packed and ordered structure through intermolecular hydrogen bonds (Fang and Hanna 2000; Dϊaz-Sobac and others 2001), but their barrier properties against water vapor is poor due to their hydrophilicity (Guilbert 1986; Kester and Fennema 1986; Gennadios and others 1994; Lacroix and Le Tien 2005). Many functions of edible films and coatings are similar to those of synthetic packaging films; however, edible film and coating materials must be chosen according to the specific food application, the types of food products, and the major mechanisms of quality deterioration (Petersen and others 1999; Guilbert 2002; Guilbert and Gontard 2005).

Edible films composed of polysaccharides have suitable mechanical and optical properties, but are highly sensitive to moisture and are poor water vapor barrier materials (Perez-Gago and Krochta 2005). An approach to improving water vapor barrier properties of the films is to produce a composite film by adding hydrophobic components such as lipid and wax materials, which results in bi-layer or emulsion films (Debeaufort and others 1993). Significant variables for modifying moisture resistance of emulsion composite films, which have been studied, were lipid types, location, volume fraction, polymorphic phase, and drying conditions (Krochta 1997; Perez-Gago and Krochta 2005). Emulsion composite films require only a single casting process for film-formation without any further process of lamination, ^" however their physical and mechanical properties are highly dependent on lipid content, lipid particle size and visco-elasticity of the lipid (Debeaufort and others 1993; Perez-Gago and Krochta 2005).

Emulsion of lipids in starch films can modify the surface properties of the films to be more hydrophobic, which can affect the wettability, water resistance, grease resistance, static discharge, surface energy and ink printability.

Herein we identify physical and chemical effects of the addition of lipid emulsions into high-amylose pea starch for the purpose of specific modification of the chemical structure-related properties of pea starch films.

SUMMARY OF THE INVENTION According to the invention, there is provided an edible film comprising a high amylose starch and an edible, hydrophobic lipid.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Microscopic image of pea starch films with 0% beeswax (A and B), 20% beeswax (C and D), and 40% beeswax (E and F). A, C and E are with

4x magnification, and B, D and F are with 10x magnification. Scale bars show 100 μm.

Figure 2. Diameter and shape factor distributions of beeswax particles in pea starch films. Figure 3. Tensile properties of pea starch films with various beeswax contents.

Figure 4. Water vapour permeability (g mm rτϊ ² h ^"1 kPa ^"1 ) and oxygen permeability (cc μm m ^"2 d ^"1 kPa ^"1 ) of pea starch films with various beeswax contents. Figure 5. DSC thermograms of pea starch and pea starch films. native pea starch (A), pea starch film without glycerol and beeswax (B), pea starch film with glycerol and without beeswax (C), and pea starch film with glycerol and

40% beeswax (D, E).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Although any methods and materials similar

or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference.

The invention comprises a combination of two materials: (1) starch and (2) lipids. Preferably, the starch contains a certain amount of amylose. Conventional low amylose starches (for example corn starch, wheat starch, tapioca starch, rice starch, potato starch, cassava starch and other starches) make partially soluble or fully soluble films which are not good moisture barriers. High-amylose starches from high amylose rice, high amylose corn or high amylose peas possess stronger barrier properties against moisture and water than conventional starches because the amylose component of starch increases crystallinity of gelatinized starch films. Starch containing more than 25% of amylose (i.e., less than 75% amylopectin) can form quite good moisture barrier films. Increasing the percentage of amylose in starch creates better moisture barrier as well as stronger films with higher tensile strength and elastic modulus. However, starch containing more than 40% of amylose requires high pressure chambers (or containers) in order to gelatinize the high amylose starch. Starch with more than 40% starch has its gelatinization temperature over 100C, and to achieve this temperature for starch dispersion in water a high pressure container is required. Starch with lower than 40% amylose has its gelatinization temperature below 100C and the boiling of starch dispersion will gelatinize the starch. Accordingly, in some embodiments, the starch has an amylose content of at least 20% or at least 25%. In other embodiments, the starch has an amylose content between 20-75%, 25-75%, 20-40% or 25-40%.

The second material of this invention are hydrophobic lipids such as waxes, paraffins or Shellac. Any edible lipids can be used for the hydrophobic materials in starch films; however, in some embodiments of the invention, solid lipids are suggested to be used. At room temperature (around 20 - 25C) or the temperature of end product use (such as the refrigeration temperature), this hydrophobic material should not be liquid oil. Liquid oil are leached out (or sweated out) from the starch film if they are used as hydrophobic lipid materials. The solid lipids can be melted at the temperature used for the gelatinized of the starch solution, and then solidified during cooling and drying following homogenization in the starch solution. Preferably, the solid lipid is selected from the group consisting of

beeswax, carnauba wax, (and other natural waxes), paraffin wax (or other petroleum based edible waxes known in the art), animal fats (i.e., tallow, lard or butter-fat), Shellac and other edible lipids. The addition of hydrophobic materials in hydrophilic polymeric materials is generally attempted to increase the water resistance of the hydrophilic films. When hydrophilic starch films contain hydrophobic solid lipid particles, for example, beeswax or alternatively any suitable wax from various sources, for example, paraffins and Shellac as well as others discussed above, the tensile strength, elongation-at-break, water vapor permeability and oxygen permeability did not change significantly until the beeswax content reaches more 20 - 30% of pea starch. However, above 30% of beeswax content, the tensile strength, elongation-at-break, and water vapor permeability decreased. Elastic modulus has been increased when the film contains beeswax. The oxygen permeability was increased at 40% of beeswax content in the film. DSC results identified the existence of amylose-lipid complex endothermic peak of the starch films containing 40% beeswax. Overall the addition of beeswax in pea starch films alters the mechanical, physical and thermal properties of the starch films when the films contain very high concentration of beeswax. The addition of beeswax under the concentration range of 30% was not effective to increase the water resistance of the hydrophilic starch films. Accordingly, in some embodiments, the invention comprises an edible film comprising: a high amylose starch and an edible, hydrophobic lipid. It is of note that as used herein, the meaning of the term 'edible' will be readily understood by one of skill in the art as referring to lipids and a film comprising said lipids that can be digested. As discussed above, in some embodiments, the starch has an amylose content of at least 20% or at least 25%. In other embodiments, the starch has an amylose content between 20-75%, 25-75%, 20-40% or 25-40%. The high amylose starch is from, that is, may be extracted from, high amylose rice, high amylose corn or peas. Preferably, the hydrophobic lipid is solid at the intended temperature of use for the film. The hydrophobic lipid may be selected from the group consisting of beeswax, carnauba wax, paraffin wax, animal fat and shellac.

The hydrophobic lipid may be present at 10-40%, 20-40%, 20-30% or 30-

40% (w/w) of the starch. As will be appreciated by one of skill in the art, this means that for every 10 g of high amylose starch, 1-4 g of hydrophobic lipid may be added, as discussed herein.

In other embodiments, a plasticizer is added. As discussed below, the plasticizer may be glycerol. Accordingly, an effective amount of glycerol may be added as a plasticizer. As discussed below, the glycerol may be added to the starch at a ratio of 2:3 or 40% glycerol: 60% starch.

The addition of more wax may increase the resistance of water-related properties of starch films. However, an extreme amount of wax in starch will reduce the advantageous properties of starch. Starch is acting as the structural polymer of the film matrix. Therefore, a high concentration of wax in starch without damaging the structural properties of the starch polymer matrix is the most desirable product.

Microstructure

All film samples were observed before using a microscope. They were homogeneous and translucent. Figure 1 shows the increase in particle numbers when beeswax content increased (Fig. 1A, C and E). Beeswax particles are distributed in the continuous starch matrix. Particle size and shape will affect the allowable amount of wax in the starch, as well as the physical and mechanical properties of starch films. Smaller particle size is beneficial for the minimization of the interference of wax with physical and mechanical properties of starch film. Also smaller particle size (lower than 1000 nm) will create new application area of this invention in the nanotechnology applications. Specifically, it is noted that the use of reduced size beeswax particles may change properties of the resulting films. For example, smaller sized beeswax particles would decrease permeability of gases and moisture through the film. Furthermore, more smaller sized beeswax particles could be incorporated into the film without significantly changing the film properties. It is of note that the size of the beeswax particles can be controlled or varied by many means known in the art, for example, through the use of a homogenizer or by adding emulsifiers, wherein a faster homogenizer speed or increased emulsifier concentration will produce smaller particles.

Figure 1 B is the picture of very thin part of pea starch film that is not a usual part of the film, therefore, can not be compared with other pictures in Fig. 1. Figure 1 B shows many filament structures which are connecting together. The filament structure of Fig. 1 B is composed mainly of amylose extracted from starch granules during heat gelatinization process. Liu and Han (2005) reported the fibrous structure formation during drying of the amylose solution resulting in dendrite crystals. This fibrous structure was also observed from Fig. 1 B.

Table 1 listed film thickness and particle size. Film thickness was increased after the addition of beeswax compared to 0% wax film; however, from 10% to 40% of beeswax concentration, the film thickness remained the same. With fairly large standard deviation, the average emulsion size did not show any difference at 10% significance interval. However, the coefficient of variation increased from 58.1 %, 59.1 %, 66.8% to 66.9% for 10%, 20%, 30% and 40% beeswax, respectively. Increase in coefficient of variation means more heterogeneity (lower homogeneity) in the film structure. This heterogeneity will increase variability of film characteristics of end products. Low concentration of beeswax could generate more homogeneous distribution (i.e., smaller coefficient of variation) of emulsion particle size. Total 250 - 350 particles were identified from one frame of image. Figure 2A shows the distributions of particle size and indicates that the peak height at 4 - 5 μm is decreasing as the beeswax concentration increased, which supports the increase in the coefficient of variation. From Fig. 2B, the major peak of the emulsion shape factor was shifted from 0.9, for 10% beeswax, to 0.3 - 0.4 for 20%, 30% and 40% beeswax concentration, indicating the spherical shape of beeswax emulsion particles at low concentration of beeswax changed to rod shape of emulsion particles when the beeswax concentration increased. The spherical shape will minimize the effect of the particle on the starch film properties because the sphere takes the smallest volume per weight of wax. However, rod shape particles would be beneficial to the moisture vapor or other hydrophilic barrier properties because they will increase the tortuosity of the hydrophilic migrant through the starch film. Increasing beeswax concentration may raise the shearing effect of the high speed homogenizer to the particles and create more rod shape when the beeswax was hardened during cooling process. To obtain the more homogeneous spheres the termination time of the homogenizer operation and the

temperature of film-forming solution should be controlled. The homogenization process should be complete before the beeswax emulsions start to be solidified.

Tensile properties Figure 3 shows that the addition of beeswax emulsions in starch films decreased tensile strength and elongation, and increased elastic modulus of the starch films. This implies that the addition of beeswax reduces the ductility of starch films. If the final product requires more flexibility and elongation of starch film with high level of wax content, more plasticizer may be required. But up to 40% of beeswax content the starch film still possesses very applicable mechanical properties. At 30% and 40% of beeswax, the starch films possessed statistically significant difference in tensile strength, elongation and elastic modulus as compared to those of 0%, 10% and 20% of beeswax contents. However, the magnitude of the difference was less than 3 times. Compared to other tensile properties, tensile strength was reduced least significantly by the addition of beeswax. This implies that the main material to maintain the strength is the starch matrix and beeswax emulsion particles were dispersed in the starch matrix without interfering with the tensile stress distribution in the film structure at the concentration below 20%. Elongation-at-break did not show any difference at the beeswax level ranging from 0% to 20% and started to decrease at 30% of beeswax. The maximum content of beeswax emulsions which did not create any reduction in tensile strength or elongation was 20%. However, the elastic modulus of the starch films responded differently. Elastic modulus increased linearly as the beeswax content increased from 0% to 30% which is the concentration range showing the insignificance of tensile strength and elongation from 0% to 20%. Maximum stress and elongation-at-break were not changed when the beeswax content increased from 0% to 20%. However, the elastic modulus which has been obtained from the initial slope of the stress-strain curve, increased. Figure 3 did not show any data of yield stress of starch film. However, when starch film contained beeswax, the stress-strain profile showed unambiguous yield stress and after-yield stress deformation, which is the characteristics of heterogeneous elastic materials. The addition of beeswax up to 20 - 30% made the starch film little more stiff and glassy, but did not affect the maximum stress and strain at break (i.e., breaking

force). The starch film becomes a little more stiff but the breaking strength is the same as for starch-only film.

Gas barrier properties Figure 4 shows the changes in permeabilities of water vapor and oxygen through beeswax emulsion films. When beeswax concentration increased, the water vapor permeability (WVP) decreased slightly but significantly at high concentration of beeswax. The films contained 30 and 40% of beeswax have lower WVP than those of 0, 10 and 20%. Beeswax particles are absolutely water resistant compared to starch matrix. Therefore, this slight difference indicates that the film still has many spaces and channels enough to allow the penetration of water vapor through the starch matrix. Due to the large portion of starch matrix in the film compared to the portion of beeswax particles, the WVP changed very little after the incorporation of beeswax. More addition of beeswax may decrease WVP much more significantly. However, the extra amount of beeswax could decrease tensile strength and elongation of starch films, and would create inferior mechanical properties of the film.

Oxygen permeability (OP) did not change until the starch films contained 30% of beeswax. However 40% of beeswax addition in the film increased OP significantly. Since the permeation mechanism of gas consists of diffusion and absorption, this result may be explained by two theoretical hypotheses of the mechanisms. Hypothesis 1 (Diffusion): Oxygen molecules diffuse through hydrophobic beeswax channels. Oxygen molecules are water soluble as well as oil soluble. The starch film did not contain free water that could be utilized as an oxygen diffusion passage. Furthermore, the starch polymer contains many hydroxyl groups which can interact with diffusing oxygen molecules. Oxygen may penetrate through beeswax/starch interfaces which are connected together between beeswax particles providing oxygen penetration channels when the film contains high wax content. High concentration of beeswax in starch films increases the possibility of the contact of beeswax particles and oxygen penetration passages, which resulted in larger value of diffusivity. Hypothesis 2 (Absorption): Increased amount of beeswax in the starch films exposes more beeswax on the surface of starch film and consequently decreases the surface energy (i.e.,

increase the hydrophobicity) of starch films. This lower surface energy with higher hydrophobicity of the film surface may accelerate the absorption of oxygen from atmosphere and increase the solubility of oxygen. Since permeability consists of diffusivity and solubility, the significantly increased solubility could increase the permeability of oxygen. Both theories explain the significant increase of OP at 40% of beeswax concentration and no change in OP at the low concentration ranging from 0 to 30%.

Differential scanning calorimetry Native pea starch showed traditional gelation endothermic peak (Fig. 5A).

Pea starch film made of 3% suspension after gelatinization and dehydration showed two peaks (Fig. 5B). The range of temperature of the first peak corresponds with the gelation endothermic peak of crystalline melting of starch molecule. The second peak is the range of 143 to 155 ⁰ C, which corresponds with endothermic peak of enzyme-resistant starch (Gruchala and Pomeranz 1993; Sievert and Wursch 1993). A previous study suggested that the enzyme-resistant starch could be formed by the retrogradation of starch, especially high amylose content starch, which resulted from water involved food processing such as cooking, baking and autoclaving (Gidley and others 1995; Gruchala and Pomeranz 1993). After starch gelatinization, α-glucan chains reform double helices and eventually realign the crystalline structure which is called retrogradation (Miles and others 1985). Pea starch film without glycerol should be formed through enzyme- resistant starch formation. In detail, starch molecule involved in recrystallization or retrogradation, and then enzyme-resistant starch formed by retrograded starch during the drying process of the film formation.

Pea starch films treated with glycerol showed a similar endothermic peak in the range of gelation temperature. However, the δH increased from 4.8 to 8.2 (J/g) as compared to the pea starch film without the treatment of glycerol (Fig. 5C). Additionally, there is no second peak after the gelation peak. It should be suggested that glycerol, as an ingredient of film, affects the crystalline structure of film, prevents the formation of enzyme-resistant starch, and increases δH. Glycerol may position between the hydroxyl groups of starch, interfere with the hydrogen

bonds between the hydroxyl groups, and inhibit the formation of retrogradation of starch.

Pea starch film treated with glycerol and 40 % beeswax showed the first gelation endothermic peak and showed the second peak in the range from 120 to 130 ⁰ C (Fig. 5D and E) or 95 - 140 ⁰ C (Table 2), which corresponds with amylose- lipid complexes (Biliaderis and others 1985; Ward and others 1994). This suggests that beeswax plays a role in the formation of amylose-lipid complexes. Since added lipids can form complexes with amylose, less amount of amylose can be used to form enzyme-resistant double helices (Eerlingen and others 1994). The endotherm of amylose-lipid complex with 40% beeswax showed different onset, peak, completion temperature, and δH in each DSC running. This could be the result of the amylose-lipid complex in film forming a heterogeneous structure due to physical reorganization of beeswax in matrices of starch-beeswax during the storage time. The physical reorganization of polysaccharide matrices specially involved in amylose-lipid complexes was also observed in previous study (Tester and Debon 2000).

The 10, 20, and 30% of beeswax do not show amylose-lipid complexes, which indicates a certain amount of beeswax is required to start formation of amylose-lipid complexes. The melting endotherm of beeswax (65°C) was overlapped with the starch crystalline melting area; it results in higher enthalpy change at 50 - 80 ⁰ C as the amount of beeswax is increased. In some embodiments, especially with fine spherical shape and size of beeswax, the starch film may contain more than 60% of beeswax in the starch polymer matrix. In this case starch polymer will works as a glue (adhesive) material between beeswax particles and make them in one piece. This will have a structure similar to that of MDF board containing adhesive, wood chips and sawdust.

The observed values such as thermal properties, oxygen permeability, tensile properties and others were significantly changed in pea starch film with 40% beeswax in both studies of DSC and mechanical properties. DSC study clearly demonstrates the change of thermal properties of pea starch films following the addition of glycerol and beeswax.

Materials

Commercial starch of Canadian yellow field peas (Pisum sativum L. Miranda) produced by wet milling process, which contains 35 - 40% amylose was supplied by Nutri-Pea Ltd. (Portage-La-Prairie, Manitoba, Canada).. Starch consists of amylose and amylopectin. Common starch contains low amylose (i.e., high amylopectin). Low amylose starch produces a less water resistant film, while high amylose starch produces a highly water resistant starch. If amylose is higher than 40%, the gelatinization temperature is generally over 100 ⁰ C. Therefore, the gelatinization process requires high pressure chamber to increase the boiling point of water over 100 ⁰ C. Pea starch has a gelatinization temperature bellow 90 ⁰ C. So simple boiling of pea starch dispersion can gelatinize the starch. Increasing the amylose content produces a more resistant starch structure after gelatinization and retrogradation. The resistant starch is not water soluble, and possesses high water resistance. Glycerol (Sigma Chemical Co. Ltd., St. Louis, MO) was used as a plasticizer. Beeswax (refined, melting temperature = 65 ⁰ C) was also purchased from Sigma Chemical Co. Glycerol and beeswax are the most common plasticizer and lipid components, respectively, for the edible emulsion composite film studies, therefore, the results of experiments could easily be compared with those of other researchers.

Film preparation

Aqueous dispersion of 3% (w/w) pea starch (PS) was prepared with de- ionized water. Glycerol (GIy) was added in the starch dispersion at the mass ratio of 60/40 of PS/Gly. Beeswax was added in the starch dispersion at the 10, 20, 30 and 40% (w/w of PS). The PS dispersion with beeswax was heated and held at boiling temperature for 15 min with continuous stirring for complete gelatinization of starch and melt of beeswax. As will be apparent to one of skill in the art, instead of boiling with water, other heat process with water can gelatinize starch. Such processes may include extrusion, microwave heating and steaming cook. After boiling, the gelatinized starch dispersion with molten beeswax was blended by a high-speed homogenizer (Fischer Scientific Ltd., Nepean, ON, Canada) at 20,000 rpm for 15 min. The homogenization process was conducted under vacuum to

avoid coagulation of beeswax and eliminate air bubbles in the final films. The vacuum helps eliminate air bubbles in the beeswax-starch dispersion. If there is no serious effect of air bubbles on the end product properties, vacuum is not essential. Temperature of the gelatinized starch solution (i.e., film-forming solution) after homogenization was 75 - 80 ⁰ C. In general, the homogenization temperature should be above the melting temperature of lipid (beeswax).

About 15 g of film-forming solution was cast onto a polystyrene petri dish

(10 cm in diameter) which was placed on a levelled flat surface. After the solution was allowed to be dried at room temperature for at least 48 h, the films were peeled off from the petri dishes and their thicknesses were measured using a digital micrometer at 5 different random positions of the films.

Image analysis of beeswax particles

Test films were placed onto microscope slides and observed using an inverted phase contrast microscope (Nikon Diaphot TMD, Kanagawa, Japan) equipped with a TV camera (Panasonic WV-1550, Matsushita Electric Industrial Co., Ltd., Osaka, Japan). The transmitted light source in the microscope was a tungsten halogen bulb (12V/50W). An NCB10 (blue) color-correction filter (Nikon) was used in order to reduce the spectral bandwidth of the source to the range of 430 - 530 nm. The DL (dark low) series of objectives (4x, 10x or 100x magnification) were used to produce positive contrast in specimens having a significant difference in refractive index from the surrounding medium. A 2.5x relay lens was placed in the tube adapter to connect the TV camera to the microscope. The image of a test film was recorded by a personal computer equipped with a video capture card. The image resolution was set at 640x480 pixels. The images were recorded to 8-bit greyscale images in bitmap PCX format. An image analysis program (SigmaScan Pro 5.0, Statistical Solutions, Saugus, MA) was used to measure the dimensions of beeswax particles in the images, which were scaled using a stage objective micrometer (1/100 mm per unit scale). From the particle dimension analysis, the diameter and shape factor (SF) of particles were determined. Particle shape factor is a dimensionless constant defined as:

p

where, A and p are area and perimeter of the particle, respectively. A perfect circle, therefore, has a shape factor of 1 , and a rod shape has near zero.

Water vapor permeability (WVP) and oxygen permeability (OP) WVP was determined by the procedure of Choi and Han (2001) which had been modified from McHugh and others (1993). Briefly, 10 mL distilled water was taken into a flat-bottom acrylic cup with a wide rim. The cup was covered with a test film, which was then sealed with a seal ring and silicon sealant (High Vacuum Grease, Dow Corning, Midland, Ml). The whole assembly was then kept inside a closed chamber with a fan, a digital RH-meter and anhydrous calcium sulfate (W.A. Hammond Drierite Co., Xenia, OH) at 25 ⁰ C temperature. The weight changes and RH inside the chamber were monitored after every hour. Once the steady state of weight loss was achieved, the water vapor transmission rate (WVTR in g m ^"2 h ^"1 ) was calculated. The WVP was determined from multiplying the WVTR with the thickness of the film and divided by the RH difference between inside the acrylic cup and the chamber. The RH inside the cup was calculated by the procedure of McHugh and others (1993).

Oxygen permeability (OP) of the films was determined using an oxygen transmission rate test machine (OxT ran 2/20, Mocon, Minneapolis, MN). After a film was placed in a cell and then flushed with nitrogen at 23°C and 50% relative humidity for 1 h, oxygen flow was introduced on one side of the films and the oxygen transmission rate (OTR) was measured. Oxygen permeability (OP) in [cc μm m ^"2 d ^"1 kPa ^"1 ] was calculated from the mean OTR multiplied by the film thickness (μm) and divided by the oxygen gradient in the cell of the testing machine (1 atm).

Tensile test

Film specimens (1 cm wide and 8 cm long) were made from the films. They were conditioned in a controlled relative humidity chamber for 48 h at 50% RH. Tensile strength (TS) was determined from a stress-strain curve using a texture analyzing instrument (Texture Analyser, TA-XT2, Texture Technologies, Corp.,

Scarsdale, NY) based on the procedure outlined in ASTM method D882-91

(ASTM, 1991). The initial grip distance and crosshead speed were 5 cm and 100 mm/min, respectively. TS was calculated by dividing the peak load by the cross sectional area of film (thickness of film x 1 cm) of the initial film. Elongation (E) was calculated by the percentile of the change in the length of specimen to the original distance between the grips (5 cm). Elastic modulus (EM) was calculated from the initial slope of the stress-strain curve. An average of five or six replicates was obtained with a standard deviation.

Differential scanning calorimetry (DSC) Thermal properties of native pea starch and pea starch films were analyzed with a Perkin-Elmer DSC-7 (Norwalk, CT) equipped with an intracooler and Thermal Analysis Controller TAC 7/DX (Perkin-Elmer). Samples (approx. 20 mg each, db) were weighed into stainless steel pans (Perkin-Elmer) designed to withstand high pressures and suppress the volatilization of solvent. The distilled water was added double the weight of sample into the pan using a microsyringe. The stainless steel pan was sealed with an O-ring, and allowed to reach equilibrium of moisture for overnight. The DSC instrument was calibrated with an indium, and an empty DSC pan was used for a reference pan. The heating rate was programmed by holding at -20 ⁰ C for 1 min, followed by ramping the temperature range of -20 ⁰ C to 180 ⁰ C at a rate of 20 °C/min, and holding at 180 ⁰ C for 1 min. Measurements were made at least in duplicate for one treatment.

Statistical analysis

Mean values of each treatment were compared using a least significant difference (LSD) test. The LSD value was calculated by following equation, where Se is pooled standard deviation of all treatment. The mean difference values bigger than LSD value were identified manually as significant differences. The confidence level of t value was 95% and total number of samples (n) was 7 - 9 for each treatment.

While the preferred embodiments of the invention have been described above, it will be recognized and understood that various modifications may be made therein, and the appended claims are intended to cover all such modifications which may fall within the spirit and scope of the invention.

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Table 1. Physical properties of pea starch films containing beeswax emulsions

Values in parentheses are standard deviations. Different superscript letters indicate significant differences of means after the least significant difference test at 10%.

with DSC ^* .

K*

* Mean of duplicates ¹ 1ndividual DSC running