Mechanical, Optical, and Barrier Properties of Soy Protein Film As

Oct 27, 2014 - mechanical properties of film from cuttlefish skin gelatin as compared to the unoxidized extracts. Among the phenolic acids studied, GA...
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Mechanical, Optical, and Barrier Properties of Soy Protein Film As Affected by Phenolic Acid Addition Anchana Insaward, Kiattisak Duangmal, and Thanachan Mahawanich* Department of Food Technology, Faculty of Science, Chulalongkorn University, Phayathai Road, Pathumwan, Bangkok 10330, Thailand ABSTRACT: This study aimed to explore the effect of phenolic acid addition on properties of soy protein film. Ferulic (FE), caffeic (CA), and gallic (GA) acids as well as their oxidized products were used in this study. Phenolic acid addition was found to have a significant effect (p ≤ 0.05) on the mechanical properties of the film. GA-containing films exhibited the highest tensile strength and elongation at break, followed by those with added CA and FE, respectively. Oxidized phenolic acids were shown to produce a film with higher tensile strength and elongation at break than their unoxidized counterparts. Phenolic acid addition also affected film color and transparency. As compared to the control, phenolic-containing film samples demonstrated reduced water vapor permeability and water solubility and increased contact angle, especially at high concentrations of oxidized phenolic acid addition. KEYWORDS: protein film, soy protein isolate, phenolic compounds

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Louis, MO, USA). Tris(hydroxymethyl)methylamine and glycerol were acquired from Fisher Scientific (Leicestershire, UK) and Ajax Finechem (New South Wales, Australia), respectively. Film Preparation. The film-forming solution was prepared by slowly dissolving 5 g of soy protein isolate in constantly stirred 75 mL of 0.05 M Tris-HCl buffer (pH 8.0) containing 55% glycerol by weight of protein. The mixture was then homogenized at 22000 rpm for 2 min. After heating at 70 °C for 30 min, the film-forming solution was cooled to room temperature (27 ± 2 °C). Separately, the phenolic acid (FE, CA, or GA) at 0.5, 1.0, and 1.5% by weight of protein was dissolved in 25 mL of 0.05 M Tris-HCl buffer (pH 8.0). The phenolic solution was then added to the soy protein mixture and homogenized again at 22000 rpm for 2 min. Air bubbles present in the film-forming solution were removed using ultrasonic degassing technique. A 45 mL aliquot of the film-forming solution was cast onto a 150 mm × 150 mm acrylic mold and then dried at room temperature for 24 h. The dried film was then stored at 50% relative humidity for a period of 48 h before undergoing further analyses. For the film-forming solutions with oxidized phenolic acid, the phenolic acid was dissolved in 25 mL of Tris-HCl buffer (pH 8.0). Hydrogen peroxide was then added to obtain a concentration of 100 mg H2O2/100 g solution. The mixture was next kept at room temperature for 30 min to allow oxidation to proceed. The pH of the oxidized phenolic solution was then adjusted to 9.0 to remove residual hydrogen peroxide.10 After this, either oxidized ferulic (OX-FE), oxidized caffeic (OX-CA), or oxidized gallic (OX-GA) was added to the soy protein solution. Except for using oxidized phenolic solution, other steps of film preparation were exactly the same as described earlier for the film samples with unoxidized phenolic acid. Thickness and Mechanical Properties. Film thickness was measured using a digital guage (model 7301, Mitutoyo, Tokyo, Japan). Tensile strength and elongation at break of the film samples were

INTRODUCTION Due to their degradability, protein films have been receiving growing attention in recent years as efficient waste management becomes a worldwide issue. Protein films have an advantage as they are good at resisting permeation of gases, organic volatiles, and oils.1 Meanwhile, the films are inferior in terms of resisting water permeation. Another major drawback of protein films is low mechanical strength, especially when compared to their plastic counterparts. Increasing molecular interaction is one method to improve the mechanical strength of a protein film. Protein cross-linking can be achieved using various crosslinking agents. Aldehydes, such as formaldehyde, glutaraldehyde ,and glyoxal, were reported to be effective protein cross-linkers by reacting with amino and sulfhydryl side chains of polypeptides, resulting in intra- and intermolecular crosslinks.2−5 Despite this, the safety of aldehyde-added film is still questionable.6,7 Many phenolic compounds are found in nature with low toxicity. They can react with amino and sulfhydryl side chains of polypeptides, which can lead to the formation of C−N and C−S bonds.8,9 Phenolic compounds of different states of oxidation may differ in their ability to cross-link protein. The capability of a phenol to be oxidized to a quinone structure was reported as an important factor underscoring its reactivity toward a protein.8 Phenolic acids, as well as their oxidized products, are, therefore, potential protein cross-linkers and, thus, can be used to improve or tailor the properties of a protein film. The objective of this study was to investigate the effect of selected phenolic acids and their oxidation products at different concentrations on properties of soy protein isolate film.

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Special Issue: 1st ACS-AGFD and ACS-Thailand Chapter Joint Symposium

MATERIALS AND METHODS

Received: August 21, 2014 Revised: October 27, 2014 Accepted: October 27, 2014

Materials. Soy protein isolate (90% protein, wet basis) was purchased from Mighty International (Bangkok, Thailand). Ferulic (FE), caffeic (CA), and gallic (GA) acids were products of Sigma (St. © XXXX American Chemical Society

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with silicone vacuum grease. An O-ring was used to hold the film in place. The cup was then placed in a chamber containing distilled water and stored at 25 °C. The weight gain of the cup determined at 2 h intervals for a period of 30 h was used to calculate the WVP. Contact Angle. A film sample, with the side in contact with air upon drying placing upward, was mounted onto the measuring plate of an ODG20 contact angle measuring instrument (DataPhysics Instruments, Filderstadt, Germany). A drop (4 μL) of deionized water was placed on the surface of the film sample, and the contact angle between the water droplet and the film surface was then measured. Water Solubility. Pieces of film sample (20 mm × 20 mm) and Whatman no. 4 filter paper were first dried at 70 °C for 24 h. The dried filter paper and film sample were then weighed to obtain an initial dry weight. Water solubility was determined by immersing the film sample in 20 mL of distilled water and stored at room temperature for a period of 24 h. After filtering through the dried filter paper, the filter paper with the remaining film sample was dried at 70 °C for 24 h. After that, it was weighed to obtain a final dry weight. Total soluble matter content was determined according to eq 1:17

determined using an Instron universal material testing machine (model 5565, Instron, Norwood, MA, USA) equipped with a 5 kg load cell. A 30 mm × 100 mm strip of film sample was tested using a pneumatic side-action grip probe (Instron).The film sample was pulled at a constant rate of 5.0 mm/s until failure. Sodium Dodecyl Sulfate−Polyacrylamide Gel Electrophoresis (SDS-PAGE). The protein pattern of the film samples was analyzed using a method described elsewhere 11 with some modifications. To study the protein pattern, film sample (0.3 g) was dissolved in 4 mL of extracting solution (60 mM Tris-HCl (pH 7.5) containing 2% (w/v) SDS). The mixture was stored at room temperature for 12 h and boiled for 3 min. After that, the mixture was centrifuged at 31154g and 4 °C for 15 min, using a Hettich refrigerated microcentrifuge (model 22R, Hettich, Buckinghamshire, UK) equipped with an A1195-A high-speed angle rotor. The supernatant was determined for its protein content using a modified Lowry method.12 The protein solution was then mixed with a sample buffer (0.5 M Tris-HCl (pH 6.8) containing 10% (w/v) SDS, glycerol, β-mercaptoethanol, and 1% (w/v) bromophenol blue) at a ratio of 7:3 (v/v). Sample (20 μg of protein) was loaded onto the polyacrylamide gel made of 4.5% stacking gel and 10% separating gel. The electrophoresis was carried out in a Hoefer electrophoresis unit (model miniVE, Hoefer, Holliston, MA, USA) using a current of 20 mA. After that, the gel was stained with Coomassie blue R-250 in 50% (v/v) ethanol and 10% (v/v) acetic acid and destained with 25% (v/v) ethanol and 10% (v/v) acetic acid. Wide-range molecular weight protein markers (Sigma-Aldrich, Munich, Germany) were used to estimate the molecular weights of the proteins. Available Lysine Content. Available lysine was determined using trinitrobenzenesulfonic acid (TNBS).13 A film sample was solubilized in 20 mM Tris-HCl buffer (pH 7.0) containing 0.6 M KCl, 8 M urea, 2% SDS, and 10 mM ethylenediaminetetraacetic acid (EDTA). The protein content of the supernatant was determined using a modified Lowry method.12 The protein content was then adjusted to 1.0 mg/ mL using the same buffer. An aliquot (1 mL) of the protein solution was added to 1 mL of 4% NaHCO3 (pH 8.5) and 1 mL of 0.1% TNBS solution. The mixture was then incubated at 40 °C in the dark for 2 h. The absorbance was then taken at 415 nm against a blank containing 1 mL of distilled water instead of protein solution. An extinction coefficient of 1.4 × 104 M−1 cm−1 was used to calculate the concentration of available lysine. Sulfhydryl Content. Total sulfhydryl content was determined using 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB).14 Protein solution was prepared according to the method described above for the determination of available lysine. An aliquot (1 mL) of the protein solution was mixed with 4 mL of 0.2 M Tris-HCl buffer (pH 7.0) containing 8 M urea, 2% SDS, and 10 mM EDTA and then with 100 μL of 10 mM DTNB solution. The mixture was then incubated at 40 °C in the dark for 15 min. The absorbance was then taken at 412 nm against a blank containing 1 mL of distilled water instead of protein solution. An extinction coefficient of 13600 M−1 cm−1 was used to calculate the concentration of total sulfhydryl group. Fourier Transform Infrared (FTIR) Spectra. C−N bond formation was monitored using a FTIR spectrometer (model Spectrum One, Perkin-Elmer, Waltham, MA, USA). Transmittance was measured around a wavenumber of 1100 cm−1, corresponding to the C−N stretching region.15 Color and Transparency. The color of the film samples was determined in a CIELAB system using a chromameter (model CR400, Konica Minolta Sensing, Osaka, Japan). The measurement was done under D65 illuminant with a 10° observer. To evaluate transparency, the film sample was cut to the exact dimension of the side of a glass cuvette and then mounted onto the inside of the cuvette. The transparency of the film samples was expressed in terms of percent transmittance measured at 500 nm using a UV−vis spectrophotometer (model V-530, Jasco, Easton, MD, USA). An empty cuvette was used as a blank. Water Vapor Permeability (WVP). WVP was determined using an ASTM standard method.16 A film sample was mounted over a glass permeation cup containing silica gel (0% relative humidity) and sealed

% total soluble matter = (initial dry weight − final dry weight)/initial dry weight × 100

(1) Statistical Analysis. Experiments were done in triplicate. A completely randomized design was used for all experiments. Data were analyzed using analysis of variance. Duncan’s new multiple-range test was used to determine the difference among sample means at p = 0.05.

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RESULTS AND DISCUSSION Thickness and Mechanical Properties. All film samples had thicknesses in the range of 0.132−0.134 mm. Film thickness was found to be unaffected (p > 0.05) by type, oxidation, or concentration of the phenolic acids. Therefore, the difference, if any, in the film properties should not be the result of film thickness. Phenolic addition was found to pose a significant effect (p ≤ 0.05) on tensile strength as shown in Figure 1. For the films containing unoxidized phenolic acid

Figure 1. Tensile strength of soy protein films with added unoxidized and oxidized phenolic acids at 0.5, 1.0, and 1.5% by weight of protein.

(FE, CA, and GA), it was found that tensile strength tended to increase with increasing phenolic concentration. In the case of the films with oxidized phenolic acid, all OX-FE and OX-CA samples possessed tensile strengths similar to those samples containing 1.5% unoxidized phenolic acid (p > 0.05). OX-GA films, on the other hand, exhibited increasing tensile strength with increasing phenolic concentration from 0.5 to 1.5%. The sample with 1.5% OX-GA demonstrated the highest tensile strength of 1.47 MPa. All samples possessed higher elongation B

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decrease in intensity of the bands representing lower molecular weight protein, implying extensive cross-linking in the samples. This was in agreement with the reduction in available lysine and sulfhydryl contents (Table 1). Both available lysine and

at break as compared to the control, although only those with OX-GA at 1.0 and 1.5% exhibited higher elongation at break of significant difference (p ≤ 0.05) (Figure 2). The increase in

Table 1. Available Lysine and Sulfhydryl Contents of Soy Protein Films with Added Unoxidized and Oxidized Phenolic Acids at 0.5, 1.0, and 1.5% by Weight of Proteina film sample control FE 0.5% FE 1.0% FE 1.5% CA 0.5% CA 1.0% CA 1.5% GA 0.5% GA 1.0% GA 1.5% OX-FE 0.5% OX-FE 1.0% OX-FE 1.5% OX-CA 0.5% OX-CA 1.0% OX-CA 1.5% OX-GA 0.5% OX-GA 1.0% OX-GA 1.5%

Figure 2. Elongation at break of soy protein films with added unoxidized and oxidized phenolic acids at 0.5, 1.0, and 1.5% by weight of protein.

tensile strength and elongation at break upon adding phenolic acids could be due to the formation of cross-linked structure due to interactions between protein and phenolics. Upon oxidation, a phenolic compound is converted to its corresponding quinone, which is an efficient protein crosslinker. The quinone could react with an amino or sulfhydryl side chain of a polypeptide to form a covalent cross-link,8,9,18 resulting in an increase in tensile strength and elongation at break of the protein film. In this study, SDS-PAGE was carried out to monitor crosslinking of soy protein (Figure 3). The control and those samples with 1.5% unoxidized or oxidized phenolic acids were chosen as representatives to investigate the effect of phenolic addition on protein cross-linking. Incorporation of phenolic acids, especially OX-CA and OX-GA, resulted in a noticeable

available lysine (10−6 mol/g protein) 544.76 502.38 453.33 416.19 447.14 428.57 411.43 394.76 372.62 365.00 389.05 361.91 350.95 354.76 343.33 348.10 333.81 331.67 290.48

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

6.06 a 16.84 a 17.51 b 20.20 bcd 13.47 b 14.82 bc 1.35 bcde 29.63 cdef 36.13 fg 9.09 defg 30.98 cdef 11.45 efg 12.12 fg 26.26 fg 35.02 fg 28.28 fg 29.63 gh 5.72 gh 5.39 h

total sulfhydryl (10−6 mol/g protein) 13.38 13.09 12.65 12.21 11.76 10.88 9.56 9.71 7.06 5.88 10.88 10.59 9.26 9.12 6.91 6.91 8.82 5.29 5.29

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.21 1.46 2.08 0.21 0.83 1.25 1.87 1.66 2.91 2.08 2.08 0.42 0.21 1.66 1.87 0.21 2.08 0.83 0.42

a ab abc abcd abcd abcd bcdef bcdef efgh gh abcd abcdef cdef cdef fgh fgh defgh h h

a

Sample means within a column that do not share a common lower case letter differ significantly at p = 0.05.

sulfhydryl contents were found to decrease upon the addition of a phenolic acid, particularly the oxidized ones. GA- and OXGA-containing samples demonstrated significantly lower available lysine and sulfhydryl contents as compared to those samples with FE and CA and their oxidized products (p ≤ 0.05). To confirm the protein cross-linking, FTIR was used to monitor the formation of C−N bonds. In the characteristic C− N stretching region (a wavenumber of around 1100 cm−1), a reduction in transmittance was observed with increasing GA content and also upon the addition of OX-GA (Figure 4). It should be noted that changes in the C−S bond could not be observed using FTIR due to the weak spectrum signal.15 Nondisulfide covalent bonds and disulfide bonds as well as other weak bonds appeared to contribute to the filmstrengthening effect.19 According to an earlier study,18 oxidized phenolic-containing extracts of cinnamon, clove, and star anise were reported to be more efficient in terms of improving mechanical properties of film from cuttlefish skin gelatin as compared to the unoxidized extracts. Among the phenolic acids studied, GA and its oxidative product (OX-GA) resulted in a film with the highest tensile strength and elongation at break (Figures 1 and 2). It was proposed that one factor affecting the capability of a phenolic compound to interact with a protein is the number of hydroxyl groups. A phenolic compound with a greater number of hydroxyl groups tends to bind more strongly to a protein.9 GA with its three hydroxyl groups thus efficiently cross-links the soy protein when compared to CA and FE, which contain two and one hydroxyl group, respectively.

Figure 3. Protein patterns of soy protein films with added unoxidized and oxidized phenolic acids. Lanes: M, protein marker; C, control; film samples with added 1.5% ferulic acid (FE), 1.5% oxidized ferulic acid (OX-FE), 1.5% caffeic acid (CA), 1.5% oxidized caffeic acid (OX-CA), 1.5% gallic acid (GA), and 1.5% oxidized gallic acid (OX-GA). C

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Figure 5. Appearance of soy protein films with added unoxidized and oxidized ferulic, caffeic, and gallic acids at 0.5, 1.0, and 1.5% by weight of protein. Figure 4. FTIR spectra of soy protein control film and those with added 0.5 and 1.5% gallic acid (GA) and 1.5% oxidized gallic acid (OX-GA).

of the ability to prevent light transmission, which could be beneficial for products that are light-sensitive. The OX-GA samples, in particular, demonstrated remarkably low percent transmittance. The decrease in film transparency could be a result of the color of polymerized quinone,20 the colored product of phenolic−protein reaction,20 as well as the increase in protein interaction.21−23 Water Vapor Permeability. The effect of phenolic acid addition on the WVP of soy protein film is shown in Table 3. In general, incorporation of unoxidized phenolic acids did not significantly affect the WVP of the film samples (p > 0.05). Contrastingly, those films with oxidized phenolic acids demonstrated a decrease in WVP with increasing phenolic concentration. This could be due to the fact that oxidized phenolic acids effectively establish interactions, such as hydrogen and covalent bonding, with reactive groups of soy protein. The increase in the number of intermolecular bonds resulted in a polymeric network with decreasing free volume. Water vapor could thus permeate through the film matrix at a reduced rate and this, in turn, resulted in a lower WVP of the film.23,24 The decrease in water vapor permeability was also

Color and Transparency. Color parameters and transparency of soy protein isolate films as a result of type, oxidation, and concentration of phenolic acids are shown in Table 2. The control film demonstrated the highest L* (lightness) of 86.78. Upon the addition of a phenolic acid, L* tended to decrease with increasing phenolic acid concentration, and this was pronounced in those samples with added oxidized phenolic acid. The decrease in L* coincided with the increase in a* (redness) and b* (yellowness) as the phenolic-containing samples appeared to be more intense in brown color (Figure 5). Similar changes were also reported for films from fish myofibrillar protein incorporated with caffeic acid, tannic acid, ferulic acid, and catechin.19 It was suggested that the color of the product of phenolic−protein interaction was affected mostly by the type and amount of phenolic compounds.20 The transparency of the film samples was expressed as percent of transmittance (Table 2). It was found that all phenolic-containing soy protein films were improved in terms

Table 2. Color Parameters and Transparency of Soy Protein Films with Added Unoxidized and Oxidized Phenolic Acids at 0.5, 1.0, and 1.5% by Weight of Proteina color parameters film sample control FE 0.5% FE 1.0% FE 1.5% CA 0.5% CA 1.0% CA 1.5% GA 0.5% GA 1.0% GA 1.5% OX-FE 0.5% OX-FE 1.0% OX-FE 1.5% OX-CA 0.5% OX-CA 1.0% OX-CA 1.5% OX-GA 0.5% OX-GA 1.0% OX-GA 1.5% a

L* 86.78 86.34 86.13 85.09 80.67 75.20 72.73 71.24 62.35 54.31 85.50 85.71 82.40 74.97 72.34 70.39 71.67 62.31 53.43

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.37 0.47 0.47 0.58 0.47 0.47 0.58 0.47 0.47 0.47 0.47 0.58 0.58 0.47 0.47 0.47 0.47 0.47 0.47

a* a ab ab b d e f fg h i ab ab c e f e g i i

−1.29 −1.48 −0.93 −0.55 3.60 4.50 6.79 9.11 10.97 16.51 −0.92 −1.03 −0.47 5.69 6.44 7.54 4.38 8.90 15.12

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.07 0.80 0.08 0.03 0.24 0.58 1.90 0.94 0.66 1.30 0.05 0.22 0.39 1.35 0.87 1.73 0.51 0.58 2.09

b* jkl l jkl jk i h f d c a jkl jkl j g f e h d b

16.86 16.93 18.15 21.57 22.60 25.35 27.22 39.01 41.56 43.35 19.85 19.06 24.94 27.59 26.58 32.34 32.63 41.62 44.55

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.76 1.50 1.21 1.34 0.69 0.75 5.38 2.48 1.73 0.49 0.39 2.11 0.88 2.36 2.19 4.62 1.32 0.24 1.50

% transmittance k k jk h h fg e c b a i ij g e ef d d b a

75.41 71.66 71.92 69.76 58.87 55.46 53.23 48.56 33.60 33.52 75.38 72.06 69.64 61.59 51.83 50.29 49.97 34.29 28.73

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.33 1.55 2.67 3.54 1.33 1.38 2.89 5.59 1.63 6.19 1.44 5.60 3.65 3.79 3.44 4.92 3.04 2.33 5.91

a ab bc c d e ef g h h ab bc c d fg fg fg h i

Sample means within a column that do not share a common lower case letter differ significantly at p = 0.05. D

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Table 3. Water Vapor Permeability, Contact Angle, and Water Solubility of Soy Protein Films with Added Unoxidized and Oxidized Phenolic Acids at 0.5, 1.0, and 1.5% by Weight of Proteina film sample control FE 0.5% FE 1.0% FE 1.5% CA 0.5% CA 1.0% CA 1.5% GA 0.5% GA 1.0% GA 1.5% OX-FE 0.5% OX-FE 1.0% OX-FE 1.5% OX-CA 0.5% OX-CA 1.0% OX-CA 1.5% OX-GA 0.5% OX-GA 1.0% OX-GA 1.5% a

water vapor permeability (g m/m2 h Pa) 0.76 0.69 0.72 0.75 0.71 0.60 0.63 0.65 0.67 0.73 0.61 0.62 0.77 0.76 0.74 0.62 0.71 0.66 0.61

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.06 0.06 0.04 0.07 0.06 0.10 0.07 0.10 0.11 0.07 0.10 0.07 0.04 0.05 0.08 0.07 0.09 0.12 0.13

contact angle (°)

a abcde abc ab abcd e de cde bcde abc e de a a abc de abcd cde e

37.20 44.01 52.88 52.54 25.56 33.35 37.67 46.66 42.68 55.28 44.36 47.52 48.66 35.20 47.95 47.63 48.81 55.36 65.28

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.80 4.32 3.50 3.77 0.50 1.60 3.66 4.76 1.66 2.55 8.65 5.76 7.82 4.69 3.11 1.87 5.61 5.85 3.52

efg bcdefg bcdefg bc h gh defg bcde cdefg ab bcdef bcde bcd fgh bcde bcde bcd ab a

total soluble matter (%) 69.46 50.41 45.60 40.85 51.73 50.61 54.17 47.93 47.06 42.93 45.88 44.64 44.56 40.69 37.63 39.12 40.30 36.95 33.38

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.75 4.19 2.81 5.43 4.68 5.45 7.96 2.66 6.30 5.36 5.29 5.24 7.11 7.09 3.74 2.71 3.92 2.03 4.55

a bcd bcdefg defgh bc bc b bcde bcdef cdefg bcdefg bcdefg bcdefg efgh fgh efgh efgh gh h

Sample means within a column that do not share a common lower case letter differ significantly at p = 0.05.

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noted in chitosan film fortified with phenolic-containing green tea extract.25 Contact Angle. The contact angle of a water droplet reflects the hydrophobicity of the film surface. Low levels of unoxidized phenolic acid addition (0.5 and 1.0%) seemed to pose no effect on contact angle of the soy protein film (p > 0.05) (Table 3). However, an increase in contact angle was observed at a high level of unoxidized phenolic acid addition (1.5%). For the oxidized phenolic-containing samples, the increase in contact angle was evident, especially in OX-GA films. This increase in surface hydrophobicity may be due to the interaction of phenolic acids with amino and sulfhydryl groups, thus limiting the availability of these hydrophilic groups to form a hydrogen bond with water, subsequently leading to a decrease in the affinity of soy protein film toward water.10 The increase in surface hydrophobicity was reported earlier for sunflower protein isolate film with added tannin26 and gelatin−pectin coacervates with added phenolic-containing grape juice and coffee extract.9 Water Solubility. Water solubility, expressed as percent total soluble matter, of the soy protein films is shown in Table 3. The control possessed water solubility of 69.46%. The solubility decreased significantly upon addition of a phenolic acid (p ≤ 0.05). Oxidized phenolic acids were shown to produce a film with lower solubility as compared to their unoxidized counterparts. Cross-linking of protein due to the action of phenolic acids could cause an effect on the amount of polar groups.27 This, in turn, may render the protein film less soluble. In conclusion, phenolic acids, with their protein cross-linking ability, could be used to improve/tailor soy protein film properties. With the difference in the number of hydroxyl groups, oxidation, and concentration, different phenolic acids may differ in their potential to cross-link and alter the properties of soy protein film.

AUTHOR INFORMATION

Corresponding Author

*(T.M.) Phone: +66 2 218 5247. Fax: +66 2 254 4314. E-mail: [email protected] Funding

This work was supported by Chulalongkorn University Special Task Force for Activating Research (STAR), Project GSTAR 56-006-23-003. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED FE, ferulic acid; CA, caffeic acid; GA, gallic acid; OX-FE, oxidized ferulic acid; OX-CA, oxidized caffeic acid; OX-GA, oxidized gallic acid; SDS-PAGE, sodium dodecyl sulfate− polyacrylamide gel electrophoresis; FTIR, Fourier transform infrared spectroscopy; WVP, water vapor permeability

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REFERENCES

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dx.doi.org/10.1021/jf504016m | J. Agric. Food Chem. XXXX, XXX, XXX−XXX