Abstract: Soy Proteins Isolate (SPI) based films were prepared. The various factors affecting the formation of these films were studied by measuring the physical (thickness and surface density), chemical (moisture content and water solubility), optical (light transmission and color), mechanical (tensile strength and elongation at break) and barrier (water vapor and oxygen permeability) properties and examination of the ultrastructure of the prepared films. The proper pH value for preparing soy protein film with good mechanical and barrier properties was 10. Addition of PEG400 as a plasticizer at 60% of SPI weight gave better film properties comparing with other used plasticizers. Cross-linking of soy protein film by adding formaldehyde or glutaraldehyde at different level into film forming solution improved the tensile and barrier properties of the obtained films. The appropriate amount of formaldehyde, which gave good mechanical and barrier properties, was 0.3 mg/100 mL film forming solution. Combination of SPI with starch caused noticeable improvement in mechanical and barrier properties of plain SPI film and the best results were obtained at 70/30 w/w ratio of SPI/starch. The examination of soy protein-based films by scanning electron microscopy was measured. Moreover, the IR spectra of these films were obtained and the characteristic IR bands for these spectra were assigned.
INTRODUCTION
Today’s packaging continues to play indispensable role in protecting food from external contamination, preserving the food quality and enhancing marketing. Petroleum-based plastic materials represent an integral part of food packaging, however, its non-degradable behavior, migration of various plastic matrix and interaction with food ingredients have environment pollution, toxicological health risk and off-flavor defects, respectively (Tice and Offen, 1993; Tawfik and Huyghebaert, 1998).
Therefore, edible films have given much attention to overcome those problems. Edible films are biodegradable, able to act as moisture, gas, aroma and lipid barriers and protect food product after the primary package is opened (Kim and Ustunal, 2001).
Various natural substances (e.g., polysaccharides, lipids and proteins) have to prepare edible films (Lindstrom et al., 1992; Kristo and Biliaderis, 2006; Soliman et al., 2007).
Protein-based edible films was found to be superior as gas barrier and mechanical properties compared with lipid and polysaccharide films (Ou et al., 2004). Moreover, blending protein with hydrophobic substances can overcome the disadvantage of its high moisture permeability (Lindstrom et al., 1992).
Protein-based films have a three dimensional network structure. Such structure forms through the following three steps; first, rupture of low-energy intermolecular bonds using rupture agents, secondly, rearrangement and orientation of polymer chains (shaping) and finally, formation of a three dimensional network structure by forming new interactions and bonds (Cuq et al., 1998). Generally, two technological processes are used to make protein-based films; a wet process, which based on dispersion or solublization of proteins and a dry process, which based on the thermoplastic properties of proteins under low moisture conditions (Cuq et al., 1998).
The major soy protein fractions are 7S globulin (conglycinin) and 11S globulin (glycinin). The ratio between both fractions varies among soybean cultivars. Both protein fractions have a quaternary (subunit) structure. They differ greatly in their molecular weight, amino acids pattern, surface characteristics and isoelectric points. Therefore, each of the two fractions contributes different functional properties of soy protein for food and industrial applications include solubility, water and fat binding, texterizing capability, emulsification, whippability (foaming) properties, adhesiveness and cohesiveness (Motoki and Seguro, 1994).
Soy proteins can be modified to be usable for specific industrial applications. The modification techniques are designed to improve functional properties of such protein by altering its molecular structure or conformation through physical, chemical, or enzymatic agents at the secondary, tertiary and quaternary levels (Huang and Sun, 2000) in cooperation of starch, sodium alginate, whey protein, gelatin with soy protein (Rhim et al., 1999; Tang et al., 2003; Cao et al., 2006).
To our knowledge there are no studies conducted to monitor the impact of various factor levels on the physical and mechanical properties of soy edible films.
This study was focused on the preparation and characterization of soy protein edible films by manipulating and interacting between different parameters including pH, plasticizers, cross-linking agents and composting with starch. The produced films were evaluated in terms of physical, mechanical, optical, chemical properties, microstructure as well as infrared spectrum.
MATERIALS AND METHODS
Materials
This study used the following materials showed in Table
1 for preparing soy protein based films.
Methods
Plain Soy Protein Films
Soy Protein Isolate Preparation
Soy Protein Isolate (SPI) was prepared from defatted soy flasks (Table
1) by extracting the protein with an aqueous NaOH solution following
by HCl precipitation according to Wu et al. (1999).
Soy Proteins Isolate Film Preparation
It was prepared according to the method of Gennadios et al.
(1997). The following trails were done to determine;
The Appropriate pH Value of Film-forming Solution
SPI was firstly dissolved in distilled water, then glycerol as a plasticizer
was added at concentration 30% w/w of SPI quantity. The obtained solutions
were homogenized at 3000 rpm for 5 min using an Ultra-Turrax T-25 homogenizer
and filtered through cheesecloth to remove insoluble matters. The pH value
of the prepared homogenized solution was adjusted to 2, 3, 6, 8, 10, or
12 with 1 N NaOH before heating to 80-85°C in 20 min on hot plate
with stirring. After filtration through stainless steel screen (120 mesh)
to remove any small lumps, the solutions were poured onto 20x20 cm2
glass plates resting on leveled granite surface, then left for 20 h at
ambient temperature (22±2°C) for drying.
| Table 1: | Materials used in the study |
The Proper Type and Level of Plasticizers
To overcome the brittleness of soy protein-based films, different
concentrations of each of the following plasticizers were added to the
film forming solution before casting and drying; glycerol at concentration
30 and 40% w/w of SPI quantity, sorbitol at concentration 50 and 60% w/w
of SPI quantity and polyethylene glycol400 (PEG400)
at concentration 50 and 60% w/w of SPI quantity, glycerol/PEG400
(50:50) at concentration 50% w/w of SPI quantity, sorbitol/PEG400
(50:50) at concentration 50% w/w of SPI quantity. Whereas, the pH of film-forming
solutions in this case was adjusted to 10 and the remaining steps were
done by the same methodology mentioned earlier.
Cross-linked Soy Protein Films
Such films prepared with a modification of Park’s (2000) method. Formaldehyde
and glutaraldehyde were used as cross-linking agents. They were added
to the prepared soy protein film-forming solution after adjusting it’s
pH to 10 with 1 N NaOH at the concentrations 0.1-0.5 mg/100 mL of film
forming solutions. The solutions were heated to 85°C in 20 min with
stirring and then subjected to casting and drying.
Soy Protein-Starch Blends
These films were prepared by substituting definite proportions of
soy protein isolate with high amylose corn starch (HAS) and using 50:50
w/w glycerol/polyethylene glycol400 mixture as a plasticizer.
Combination of Soy Protein Isolate (SPI) and High Amylose Starch (HAS)
were undertaken to prepare soy protein-starch blend films using the following
combination ratios; 90/10, 80/20, 70/30, 60/40 and 50/50 w/w SPI/HAS.
After homogenization, the pH of the solutions was adjusted to 10 with
1 N NaOH. The obtained blends were heated and subjected to casting and
drying.
Physical and Mechanical Properties
Film Preparation for Analysis
Before measurements of thickness, surface density, tensile strength
and elongation at break, the prepared films were conditioned for 48 h
in a desicator containing saturated calcium nitrate solution to maintain
the Relative Humidity (RH) at 50±5% and room temperature 20±2°C.
Thickness
Film thickness was measured using Tri-Circle 25 hand-held micrometer
(China).
Surface Density
The weight of 16 specimens (5x5 cm2) of each film were
weight to the nearest 1 mg. Average weight value divided by the area of
the sample (25 cm2) to calculate the surface density (mg cm-2).
Tensile Strength and Elongation
Tensile Strength (TS) and elongation percentage at break (E%) of 100
mm long x25 mm wide film specimens were determined according to the American
Standard Testing Methods (ASTM, 1991) using an Instron Universal Testing
Machine (Instron Engineering C-operation, Canton, MA).
Optical Properties
Light Transmission
It was using a modified standard procedure for British Standards Institution
(BSI, 1968). Samples of films were cut into a rectangle and placed on
the internal side of spectrophotometer cell. The light absorbance values
between 400-800 nm at 10 nm intervals were recorded for each sample using
a UV-Vis Recording Spectrophotometer UV-160A (Shimadzu Scientific Instrument
Corp., Columbia, Md).
Color
Hunter color values (L, a and b) were determined from CIE units (Francis
and Clydesdale, 1975).
Barrier Properties
Film Preparation for Analysis
Before measurement of water vapor and oxygen permeabilities, the prepared
films were conditioned for 48 h in a desicator containing saturated lithium
chloride solution to maintain the Relative Humidity (RH) at 11±5%
and room temperature 20±2°C.
Water Vapor Permeability
ASTM E-96 method (ASTM, 1990) was used to determine water vapor permeability
(WVP) using cups described by Brandenberg et al. (1993). All WVTR
values were corrected for the air gap between the water surface and film
underside according to McHugh et al. (1993).
Oxygen Permeability
It was determined as described by Davis and Huntington (1977).
Chemical Properties
Moisture Content
It was determined according to ASTM D 644-94 method (ASTM, 1994).
Film Solubility
The method modified by Stuchell and Krochta (1994) was used to estimate
the film solubility.
Microstructure
Scanning Electron Microscope (SEM) type Joel JSM 5300 (Joel Ltd.,
Tokyo, Japan) was used to investigate the microstructure of the prepared
films. Samples of these films were attached to the aluminum stubs with
double sided tape and then coated with 60:40 gold-palladium alloy by a
Joel JFC-1100E sputter coater to a thickness of 100 A°. Samples were
examined using an accelerating voltage of 15 Kv (Sawyer and Grubb, 1987).
Infrared Spectrum
Genesis II Fourier Transform Spectrophotometer (FTIR) (Mattson Instruments,
Madison, WI) equipped with a deuterated triglycine sulfate detector was
used for spectral scanning of bio-based films in 4000-400 cm-1
range at a resolution of 2 cm-1 using 200 scan. The spectrometer
controlled by an IBM-compatible Pentium 200 MHZ PC running under Windows
based Winfirst Software (Microsoft Corporation). Background spectra were
collected every 30 min and each sample spectrum was ratioed against the
most recently collected background spectrum (Jaenfils and Galloy, 1990).
RESULTS AND DISCUSSION
Soy Protein-Based Films
To study the influence of pH, the SPI film-forming solution was adjusted
to different pH values, 2-12 (Table 2). It was found
that no films were produced at 4-5 pH range. This range of pH is near
from the isoelectric point of soy proteins, 4-5 (Wolf, 1977). At IEP,
the soy proteins become insoluble and precipitated. Far from this range
of pH either an acidic or an alkaline side, the SPI dispersed in solution.
At pH 8-10, the protein is fully dissolved forming a viscous solution.
At pH 2-4, the protein aligned into separate longitudinally filamentous
(Rahma, 1997). According to Wolf (1977), a rapid shift in the sedimentation
constant from 2, 7, 11 and 13.8 to mainly 3S as alkalinity of SPI solution
raised from pH 8 to 12. At this range of pH 8-12, the protein dissociated
and unfolded. Also, the sulfhydryl-disulphide interchange reactions are
favored the alkaline conditions. Such change enhances formation of new
S-S bonds between the aligned protein polypeptide chains. Table
2 and Fig. 1 shows the different characteristics
of SPI films prepared at different pH values. Results of Table
2 showed that adjusting the pH of soy protein-film forming solution
to pH 10 produced films with good mechanical and barrier properties. The
prepared SPI films at various pH values had yellow color owing to the
soy flavonoid compounds and the texture of the prepared films at pH less
than 6 and more than 8 was smooth. However, it was slightly rough at pH
6-8. This roughness can be attributed to incomplete solubility of soy
protein as a result to slight breaking in S-S bonds and formation of unfolded
protein chains. Films prepared at pH 8 had the lowest thickness and surface
density, while the highest values of both parameters were observed for
films prepared at pH 12 followed by those formed at pH 10, 6, 3 and 2,
respectively. Furthermore, films prepared at acidic pH values (2 and 3)
had higher moisture content, solubility, water vapor and oxygen permeability,
lower tensile strength and elongation at break comparing to those prepared
at alkaline pH values (Table 2).
Plasticizer Type and Level
Comparing the influence of the used plasticizers indicated that the
best film properties obtained when PEG400 was added at 60%
of SPI weight (Table 3). This addition decreased the
film brittleness without a noticeable effect on its barrier properties.
The variations in the effect of these plasticizers on the SPI film characteristics
may be attributed to the difference in their polarity, molecular size
and weight.
The results are in agreement with those of Park et al. (1994) and McHugh and Krochta (1994). They showed that addition of glycerol as
a plasticizer to whey protein isolate and wheat gluten protein films increased
elongation and reduced tensile strength of the resultant films. According
to Gontard et al. (1993), water vapour permeability of wheat gluten
films increased with increasing the level of plasticizer addition. Labuza
(1984) ranked sorbitol, glycerol and polyethylene glycol400
according to their polarity in the following increasing order, sorbitol,
polyethylene glycol400 and glycerol, respectively. The disruption
of the hydrogen bonds between polypeptides chains increased with addition
of polar plasticizers (Gekko and Timasheff, 1981). Wiggins (1995) stated
that due to low molecules size of glycerol, it is easily inserted into
the protein matrix causing disruption to the hydrogen bonds which bind
the polypeptide chains. In contrast, PEG400 with large molecular
size is less able to penetrate the protein matrix. Therefore, its effect
on disruption of the protein-protein association is weak (Siew et al.,
1999).
| Table 2: | Influences of pH value of film-forming solutions on
properties* of soy protein films |
*Reported values for each property are means of three
replications±standard deviation but for tensile strength and
elongation at break are means of five replications±standard
deviation |
|
| Table 3: | Influences of plasticizer type and level on properties*
of soy protein films |
GLY: Glycerol; S: Sorbitol; PEG400: Polyethylene
glycol400; *: Reported values for each property are means
of three replications±standard deviation but for tensile strength
and elongation at break are means of five replications±standard
deviation |
|
| Fig. 1: | SEM photomicrographs of surfaces of soy protein films
prepared at pH (A) 6, (B) 10, or (C) 12 and (D) microstructure of
the film prepared at pH 10 |
Cross-Linking
To improve the mechanical and barrier properties of SPI-based films,
different concentrations of each of formaldehyde and glutaraldehyde were
added as cross-linking agents into film-forming solutions. Cross linking
of soy protein film with 0.3 mg formaldehyde/100 mL of film forming solution
was suitable for preparing films with good mechanical and barrier properties
(Table 4). The data in Table 4 showed
that; incorporation of cross-linking agents, formaldehyde and glutaraldehyde,
increased the thickness, surface density, tensile strength, elongation
at break, lowered moisture content, solubility, water vapor and oxygen
permeability of the resultant SPI based films. This means that aldehyde
addition increased the association between polypeptide chains of SPI.
According to Wolf (1977), unfolding of soy proteins and cross-linking
of unfolded chains occur at expense of the S-S interchange reactions of
protein polypeptide chains.
The SEM photomicrographs in Fig. 2 and 4 showed the change in the microstructure of the control SPI films and SPI films cross-linked with formaldehyde or glutaraldehyde. The following points could be concluded from the examination of these figures. Native SPI consist of highly folded polypeptides stabilized by the interactions between amino acids residues relatively far apart in the sequence. Such interactions are responsible for the presence of the folded protein polypeptides (tertiary structure) in the film matrix (Fig. 2). Thermal treatment of SPI caused swelling, enlargement, melting and dissociation of folded polypeptides of SPI (Fig. 2). According to Bradley et al. (1975), the changes taking place during thermal treatment of soy proteins solution including dissociation of protein subunits to from soluble polypeptide chains. The dissociation of the protein involved the cleavage of disulphide bonds and formation of new associations that mainly are co-operative combination of hydrophobic and ionic interactions. As shown from the photomicrograph (Fig. 2), SPI films composed of associated random distributed strands (polypeptide chains) giving fiber like structure. In accordance, Rahma (1997) stated that during thermoplastic extrusion, the soy protein bodies re-formed into longitudinally strands forming extruded material with fiber like structure.
Addition of glutaraldehyde into SPI film forming solution caused the
same changes mentioned above with formaldehyde (Fig. 2).
However, addition of glutaraldehyde increased the spaces between the fibers
of SPI formed films as shown from Fig. 3.
| Table 4: | Influences of adding formaldehyde or glutaraldehyde
into film forming solution on properties* of soy protein
films |
| *Reported values for each property are means of three replications±standard deviation but for tensile strength and elongation at break are means of five replications±standard deviation | |
| Fig. 2: | SEM photomicrographs of cross-linked soy protein films
using (a) formaldehyde or (b) glutaraldehyde |
| Fig. 3: | SEM photomicrographs of partly denaturated polypeptides
(subunits) of soy protein shows its denaturation in (A) the presence
of cross-linking agents or (B) its absence |
Generally, the formation of cross-links between soy protein polypeptide
chains is responsible for re-arrangement of protein polypeptide chains
from random distributed to highly ordered structure and subsequently,
increasing the tensile and barrier properties of the film matrix.
| Table 5: | Influences of combination of soy protein with starch
on properties* of soy protein films |
*Reported values for each property are means of three
replications±standard deviation but for tensile strength and
elongation at break are means of five replications±standard
deviation |
|
| Fig. 4: | SEM photomicrographs of (A) plain, (B) formaldehyde
cross-linked and (C) glutaraldehyde cross-linked soy protein films |
| Fig. 5: | SEM photomicrographs of soy protein-starch blend films
using 50/50 w/w SPI/starch (A) surface morphology (B) microstructure |
| Fig. 6: | IR spectra profiles of soy protein-based films |
The changes increased with increasing aldehyde level to 0.4 mg/100 mL of film forming solution. Higher addition reduced the tensile strength of the resulted films. This reduction can be due to an increase in the brittleness of such films resulting from the high association degree between protein polypeptide chains (Table 3). Marquie et al. (1995 and 1997) and Park et al. (2000) stated that both mono-and di-functional aldehydes promote inter-and intra-molecular cross-linking of protein.
Combination with Starch
The data in Table 5 revealed that combination of
starch with soy protein isolate caused a noticeable increase in thickness,
surface density, tensile strength, elongation at break and reduction in
moisture content, solubility, water vapor and oxygen permeability of SPI
films. On the other hand, increasing the level of starch than 43% of soy
protein weight caused a decrease in tensile strength and elongation at
break. This may be due to the reduction in the proportion of soy protein
which is mainly responsible for forming the film matrix.
Figure 5 represents the SEM photomicrographs of soy protein isolate-starch blend films. Examination of these images showed that incorporation of starch in SPI film led to increase the homogeneity of the film matrix (Fig. 5A) and increase the distance between soy protein layers as a result to exist amylose chains which uniformly distributed in the film matrix (Fig. 5B).
Spectra of Soy Protein-based Films
Figure 6 and Table 6 showed the
characteristic IR bands of the different prepared soy protein-based films.
The data presented in both figure and table indicated that the characteristic
IR bands owing to functional groups of soy protein included N-H st., C-H
st., C-O st., C-C st., C-N st., N-H d., C-O-C st. and C-H d. out of plane.
Cross-linking of soy protein with formaldehyde caused slight shift in
the aforementioned IR bands. On the other hand, cross-linking of soy protein
with glutaraldehyde led to increase absorption values of these characteristic
IR bands, appear new peaks owing to C = O st. which due to Fermi resonance
and aldehyde at 2666, 1742 and 1702 cm-1 and slight shift in
characteristic IR bands of the soy protein-based film. These results were
attributed to the variation in the reactions occurring between soy protein
and both formaldehyde or glutaraldehyde.
| Table 6: | Characteristic IR bands of soy protein-based films |
| St. = Stretching, d. = Deformation | |
Combination of starch with soy protein to prepare soy protein-starch blend films caused a noticeable increase in absorption values for all characteristic IR bands of soy protein film due to similarity of the characteristic IR bands of starch with those of soy protein (Fig. 6 and Table 6).
Concerning packaging applications, these bioplastic materials are still limited to short-term use, such as packaging materials (loosefill films), food service items (cutlery, plates, cups), trash bags and other specially items, as none of them has obtained FDA approval for use in long-term food packaging. Few biodegradable films allow long-term packaging of low-moisture foods such as cellophane films coated with nitrocellulose-wax (Krochta and DeMulder-Johnston, 1997).
Accordingly, a more research is still needed to develop economically viable biodegradable packaging films with a relatively long lifetime, especially in moist environments or with moist foods.