Gliadins Polymerized with Cysteine: Effects on the ... - ACS Publications

Biomacromolecules 2004, 5, 1503-1510

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Gliadins Polymerized with Cysteine: Effects on the Physical and Water Barrier Properties of Derived Films Pilar Herna´ ndez-Munõz,*,†,‡ Jose´ M. Lagaro´ n,‡ Amparo Lo´ pez-Rubio,‡ and Rafael Gavara‡ School of Packaging, Michigan State University, East Lansing, Michigan 48824, and Institute of Agrochemistry and Food Technology, C.S.I.C., Apdo. Correos 73, 46100 Burjassot, Valencia, Spain Received January 29, 2004; Revised Manuscript Received April 28, 2004

To study the effects of disulfide bonds on certain functional properties of films made from the wheat gluten proteins gliadin and glutenin, cysteine was used to promote the formation of interchain disulfide bridges between gliadins in 70% ethanolic solution. Disulfide-mediated polymerization of gliadins was confirmed by means of SDS-PAGE analysis. After chemical treatment of gliadins, films were solution cast and the effects of both glycerol (used as a plasticizer) and relative humidity were studied on water vapor permeability, moisture sorption isotherms at 23 °C, and the optical properties of the films. The results were compared with those obtained from analogous films made from untreated glutenin macromolecules. Cysteine-mediated polymerization of gliadins improved the water vapor resistance of films achieving values close to those obtained for glutenin films. Development of intra- and interchain disulfide bonds did not change the moisture sorption capacity of the films but transparency was slightly diminished. Introduction Considerable efforts are currently being made in the development of biomaterials based on natural macromolecules such as polysaccharides and proteins in order to obtain films with characteristics similar to those of synthetic polymers commonly used in food preservation. Films based on proteins have been thoroughly reviewed.1-3 These materials are excellent barriers to oils and oxygen in low and intermediate moisture environments; however, they lack mechanical strength and are poor water barriers, the latter being a consequence of their hydrophilic nature. Wheat gluten has received considerable attention as a promising biobased material for use in food packaging and agricultural applications. Although wheat gluten comprises two kinds of proteins, gliadins and glutenins, differentially extractable in aqueous ethanol,4 studies regarding the functional properties of films made from one or other of proteins are scarce.5,6 It is well-known that cysteine residues play an important role in the formation of protein networks of both gliadin and glutenin films since these residues are involved in disulfide bonds within the same polypeptide chain or between polypeptide chains. However, little attention has been paid to the role of disulfide bonds in the final properties of the films produced. Glutenins contain high molecular weight and low molecular weight subunits interconnected by disulfide bonds and are considered to be among the largest naturally occurring protein macromolecules. Gliadins are comprised of single * To whom correspondence should be addressed. Current address: Institute of Agrochemistry and Food Technology, C. S. I. C., Apdo 73, 46100 Burjassot, Valencia, Spain. Tel: (+34) 963 900 022. Fax: (+34) 963 636 301. E-mail: [email protected]. † Michigan State University. ‡ Institute of Agrochemistry and Food Technology.

polypeptide chains associated by hydrogen bonds and hydrophobic interactions in which, unlike glutenins, disulfide bonds only occur intramolecularly. Gliadins are highly soluble in 70% aqueous ethanol and are usually classified into R-, β-, γ-, and ω-gliadins in decreasing order of electrophoretic mobility under acidic conditions and increasing order of relative molecular mass.7,8 Although cysteine residues are absent in ω-gliadins (so-called sulfur-poor gliadins), they are involved in intramolecular disulfide-bonds in R-, β-, and γ-gliadins.9 According to the Belton “train and loop” model for gluten,10 hydrogen bonding between the repeat regions of the high molecular weight glutenin subunits is responsible for the elasticity of gluten while gliadins are considered to contribute to gluten viscosity. Previous studies carried out in our laboratory showed that films made from glutenin- and gliadin-rich fractions extracted from wheat gluten differ in mechanical and water barrier properties.11 In the present work, the promotion of new intermolecular disulfide bonds occurring in gliadins will be evaluated in terms of the moisture sorption capacity and water barrier properties of derived films. For this purpose gliadins were treated with cysteine which due to its reducing character is expected to cleave intramolecular disulfide bonds promoting their rearrangement via disulfide/sulfydryl interchange reactions thus favoring the formation of new intermolecular bonds between monomeric subunits. Finally, the properties of the resulting films will be compared to those of films made from polymeric glutenins. Materials and Methods Reagents. Crude wheat gluten, glycerol, ethanol, and all of laboratory grade, were supplied by Sigma Co. (St Louis, MO).

L-cysteine,

10.1021/bm0499381 CCC: $27.50 © 2004 American Chemical Society Published on Web 06/04/2004

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Biomacromolecules, Vol. 5, No. 4, 2004

Film Preparation. 100 g of crude wheat gluten was dispersed in 400 mL of 70% (v/v) ethanol/water, stirred overnight at room temperature, and centrifuged at 10 000×g for 30 min at 23 °C. The resulting supernantant containing the gliadin-rich fraction was collected and used as the gliadin film-forming solution. The precipitate, consisting mostly of glutenins, was resuspended in a solution of 50% (v/v) ethanol/water and 0.05 N acetic acid and the mixture stirred at 40 °C for 1 h. The insoluble portion containing starch and protein aggregates was eliminated by centrifugation at 10 000×g for 10 min at 23 °C. Glutenins were separated from residual gliadins remaining in the supernatant by precipitation with ethanol. Ethanol was added to a final concentration of 70% (v/v) and the mixture was left for 12 h at 2 °C. The precipitated glutenins were obtained by centrifugation at 10 000×g for 30 min and dispersed in 50% (v/v) ethanol/water thus yielding the glutenin film-forming solution. The pH of each fraction was brought to 5 with acetic acid. The initial protein content of the gliadin- and gluteninrich fractions was 34% and 18.5% (g/100 g gluten), respectively, and was later adjusted to 7.5% (w/w) in the film-forming solutions. Protein content was determined using the micro-Kjeldahl method12 after previous evaporation of the solvent. Chemical modification of gliadins was conducted by adding cysteine (CYS) at 2% (g/100 g protein) into the filmforming solution and incubation at 40 °C for 30 min under gentle stirring. Preliminary studies showed that neither the addition of greater amounts of cysteine to gliadins nor incubation times longer than half an hour modified the studied properties of the resulting films. Addition of glycerol (GLY) as plasticizer was required to avoid cracking of the films during handling. Glycerol was added to the film-forming solution in a proportion ranging from 11% to 66% (g/100 g dry protein) and stirred for 20 min. Moisture sorption isotherms were determined for unplasticized films and films containing 33% glycerol. 40 g of the film-forming solution were poured onto a horizontal flat Teflon tray (37 × 24 cm2), and the water and ethanol were allowed to evaporate. Films were dried at 23 ( 2 °C and 50 ( 5% relative humidity for 10 h and peeled off the casting surface. Film thickness was measured using a micrometer (Fisher Scientific, Pittsburgh, PA) with a sensitivity of 2.54 µm. The mean thickness was calculated from measurements taken at five different locations on each film sample. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE). Cysteine-treated and control gliadin-rich material extracted from wheat gluten were analyzed by SDS-PAGE performed in a vertical electrophoresis unit (Hoefer Scientific Instrument, San Francisco, CA). The procedure was that of Laemmli13 with some minor modifications based on the procedure of Ng and Bushuk.14 100 µL of the film-forming solution from control or cysteine-treated gliadins were mixed with 100 µL of SDS-sample buffer (0.063 M Tris-HCl, pH 6.8, 2% w/v SDS, 0.01% (w/v) Pyronin) with or without reduction of disulfide bonds with 5% (v/v) 2-mercaptoethanol (2-ME). Each sample-buffer mixture was allowed to stand at room temperature for 2 h

Herna´ ndez-Munõz et al.

with occasional shaking and centrifuged at 13 800×g for 10 min. 10 µL of the clear top layer of each sample was loaded into each slot in the gel. After electrophoresis, the gel was stained with Coomassie Brilliant Blue G-250. The molecular weights of the standard protein mixture ranged from 36 kDa (glyceraldehyde-3-phosphate dehydrogenase) to 205 kDa (myosin) and were obtained from the Sigma Chemical Co (St. Louis, MO). Water Solubility. Films were dried in a desiccator containing dry calcium sulfate. Preweighed dry film samples of about 500 mg were immersed in beakers containing 50 mL of distilled water at 23 °C for 24 h with periodic gentle manual agitation. Films were removed from the water and placed back in the desiccator until they reached a constant weight which was used as the final dry weight of the film. Water solubility was defined as the percentage of dry film mass that dissolved divided by dry film initial mass. Water solubility test for each type of film was replicated three times. Moisture Sorption Isotherms. Films were cut into small pieces weighing 4-5 g, placed in aluminum dishes, and allowed to reach moisture equilibrium with eight different salt solutions in 10 L sealed containers placed in an environmental chamber conditioned at 23 °C. The relative humidity values in the containers were 11.3 ( 0.3, 22.5 ( 0.3, 32.8 ( 0.2, 43.2 ( 0.4, 52.9 ( 0.2, 66 ( 0.05, 75.3 ( 0.1, and 93.6 ( 0.2% and were obtained by using saturated salt solutions of LiCl‚H2O, KC2H3O2, MgCl2‚6H2O, K2CO3‚ 2H2O, Mg (NO3)2‚6H2O, NaNO2, NaCl, and KNO3, respectively.15 Once constant weight was obtained (after ca. two weeks), the samples were removed and dried in a vacuum oven at 60 °C for 24 h.16 Moisture content was calculated on a dry basis at each relative humidity condition and reported as the average of three replicates taken from different batches of film. Water Vapor Permeability. Water vapor transmission rate [g/(m2s)] through the films was measured using a Permatran W3/30 (Mocon Inc., Minneapolis, MN) at 23 °C and three different relative humidity gradients of 50%, 75%, or 90% to 0% (in dry nitrogen) across the film. Each film sample was mounted between two aluminum masks leaving a circular uncovered film area of 5 cm2 and placed in the permeation cell. At least three specimens of each film type were measured. Permeability values are reported as water vapor permeability coefficient in [(g m)/(m2 s Pa)]. Opacity and Color. Film apparent opacity was evaluated according to the method described by Gontard et al.17 based on a modified standard procedure.18 The absorbance spectra of films were determined by a Perkin-Elmer Lambda 25 UV-vis spectrophotometer (Perkin-Elmer Instruments, Shelton, CT) for the range of 400-800 nm. Apparent opacity was defined as the area under the curve as determined by integration with UV-WIN-Lab software and expressed as the product of the absorbance value and wavelength (AV nm). Samples were measured in triplicate. Films were conditioned at 50% relative humidity for 72 h prior to testing. Film color was determined with a hand-held Minolta Chroma Meter CR300 (Minolta Camera Co., Ltd., Osaka, Japan) set to D65 illuminant/10° observer. Film specimens were placed on the surface of a white standard plate and the

Biomacromolecules, Vol. 5, No. 4, 2004 1505

Gliadins Polymerized with Cysteine

CIELAB color space was used to determine the parameters: L* (0 black to 100 white), a* (- greenness to + redness; b* (- blueness to + yellowness). Alternatively, color can also be expressed using polar coordinates L*C*H* where L* is the same as previously, C* is chroma or saturation index and H* is hue. Simple transforms are used to convert L*a*b* coordinates to L*C*H* coordinates C* ) (a*2 + b*2)1/2

(1)

H* ) arctn (b*/a*)

(2)

Five measurements were taken of each sample and three samples of each film were measured. Films were conditioned at 50% relative humidity for 72 h prior to testing. Results and Discussion General Film Properties. The protein contents of unplasticized films made from gliadins and glutenins or gliadins treated with cysteine were greater than 91% on a dry basis, and films had an average thickness of 52 ( 7 µm. All of the films were homogeneous without visual phase separation and had smooth surfaces. Water vapor permeability, color, and opacity of films were evaluated at glycerol concentrations between 11% and 66%. Unplasticized films were too brittle to be tested. Gliadin films could not be made at glycerol concentrations higher than 33% (g/100 g dry protein) since the resulting films were very sticky and did not maintain their integrity during handling. After treatment of gliadins with cysteine, the resulting films could be formed at glycerol concentrations ranging from 11% to 66%. Similar results were also found for glutenin films which indicated that a more robust structure was attained capable of retaining greater quantities of low molecular weight molecules such as glycerol. Water vapor permeability of gliadin films could not be evaluated at glycerol concentrations higher than 22% when the relative humidity gradient was 90%, since the films lost their integrity due to the additional strong plasticizing effect of water. SDS-PAGE Analysis. To verify the ability of cysteine to polymerize gliadins, SDS-PAGE analysis was conducted on untreated (lanes 1 and 3) and cysteine-treated gliadins (lanes 2 and 4) from the film-forming solution (see Figure 1). Bands corresponding to the mobility of gliadins can be observed in lanes 1-4; the formation of protein aggregates that could not enter the gel can be seen at the top of lane 2 and correspond to gliadins treated with cysteine, compared to line 1 associated to untreated gliadins, which evidences the effectiveness of cysteine in promoting gliadin polymerization. Addition of 2-ME to the buffer system resulted in the cleavage of the disulfide bonds between gliadins and hence no differences were apparent between lanes 3 and 4. This observation also indicates that cysteine did not promote the formation of intermolecular covalent bonds other than disulfide bonds. Water Solubility. It is well-known that gluten proteins are poorly soluble in water. Factors that contribute to this behavior are a low content of amino acids with ionizable side chains and a high content of glutamine and nonpolar

Figure 1. SDS-PAGE patterns of gliadins extracted from commercial wheat gluten treated with cysteine in the absence (lanes 1 and 2) or presence (lanes 3 and 4) of 2-mercoaptoethanol. St: molecular standard; lanes 1 and 3: control gliadins; lanes 2 and 4: gliadins treated with cysteine.

Figure 2. Cleavage of intramolecular disulfide bonds and disulfidesulfydryl interchange reactions between gliadins. Cy-SH represents cysteine and R-SS-R the intramolecular disulfide bonds present in gliadins.

amino acids.19 Water solubility is an important factor regarding film end use because water insolubility of cast films is a required property when applied in contact with high-moisture content foods. In contrast to glutenin films which maintained their integrity after immersion in water, gliadin films broke apart upon contact with water. Since gliadin amino acid composition is similar to that of the glutenins,20 this behavior may be attributable to the absence of intermolecular covalent bonds between gliadins. Treatment of gliadins with cysteine greatly improved the water resistance of films since they did not suffer visual loss of integrity upon immersion. Thus, cysteine acts as a reducing agent promoting intra- and/or intermolecular sulfydryl/disulfide interchange reactions and hence polymerization. Consequently, the new protein network becomes more compact with an increase in the cohesiveness between polypeptide chains, which makes it more difficult for water to penetrate the film. Figure 2 is a representation of how polymerization of gliadins can take place. It can be expected that film matter solubilized in water be mainly the glycerol added to the film since gluten proteins are highly insoluble in water. For cysteine-treated gliadin films, water solubility was determined at glycerol concentrations ranging from 0 to 66% (g/100 g dry film). Relationship between water solubility and glycerol content can be

1506


described by a first-order linear regression, i.e., WS ) 0.95 ( 0.03 × [glycerol content] + 5.44 ( 0.82, R2 ) 0.998; as can be seen, the experimental values of film dry matter solubilized in water are similar to the amount of glycerol added to the film. Water solubility for unplasticized films was 6.4 ( 1 (g/100 g of dry film), which is attributable to residual starch in the film and/or soluble low molecular weight polypeptides. Water solubility could not be evaluated for films made from untreated gliadins since films broke apart after immersion in water. It is important to point out that the water solubility values for cysteine-treated gliadin films were similar to those reported for glutenin films11 (WS ) 0.91 ( 0.03 × [glycerol content] + 6.5 ( 0.6, R2) 0.998). In agreement with these findings, Kunte et al.21 found that films made from the soy globulin 11S fraction had lower water solubility values than films made from the soy globulin 7S fraction. This was attributed to both the lower molecular weight of 7S proteins and the greater tendency of 11S proteins to form disulfide bonds which stabilize the film network. Several authors have also reported a decrease in the water solubility of films made from several protein sources after cross-linking with aldehydes. Formaldehyde, for instance, substantially reduced the water solubility of soy protein films22,23 and cottonseed protein films.24 Dialdehyde starch improved the water resistance of egg white protein films,25 soy protein films,26 and zein films,27 whereas enzymatic treatment of whey protein with transglutaminase also decreased film solubility in water.28 Thermal treatment, UV, and γ-irradiation of proteins have also been employed to improve the water resistance of protein films.29-32 Opacity and Color. Transparency is a requirement for films that are to be directly applied over the food surface as a coating, and it is often desirable when they are used as a primary package. In this context, the nature of the hydrocolloid and the solvents used for film casting as well as the additives included in the film formulation directly affect the film’s final appearance. The apparent opacities of films made from glutenins, gliadins, and gliadins treated with cysteine were not significantly influenced by glycerol content across the range of concentrations studied. Cysteine-treated gliadin films presented greater apparent opacity values than untreated films (79 ( 3 and 35 ( 2 A nm, respectively) but lower values than glutenin films (102 ( 3 A nm). The greater transparency of gliadin films could be related to the high solubility of these proteins in 70% (v/v) aqueous ethanol. Addition of cysteine to the filmforming solution could cause the association of subunits via disulfide bonds and new noncovalent interactions thus exposing nonpolar groups of amino acid side chains to the aqueous surface environment. Rearrangement of proteins could modify the interactions of amino acid side chains with the solvent, decreasing their solubility and therefore affecting the final transparency of the cast films. Formation of protein aggregates through cysteine treatment could also decrease solubility of proteins in ethanol and therefore increase opacity of films.


Figure 3. Moisture sorption isotherms for unplasticized and glycerol plasticized films at 23 °C. Table 1. Effect of Glycerol on L*, H*, and C* Coordinates of Films %glycerol

gliadin film

11 22 33 44 55 66

97.8 ( 0.3 97.9 ( 0.2 97.3 ( 0.3

11 22 33 44 55 66

4.1 ( 0.3 3.9 ( 0.2 4.5 ( 0.1

11 22 33 44 55 66

92.4 ( 0.1 92.3 ( 0.5 92.4 ( 0.7

CYS-gliadin film L* 97.0 ( 0.2 97.2 ( 0.3 96.8 ( 0.5 96.9 ( 0.2 97.0 ( 0.2 96.5 ( 0.3

C*

H*

glutenin film 95.7 ( 0.2 96.0 ( 0.4 95.5 ( 0.2 95.5 ( 0.3 95.8 ( 0.3 95.8 ( 0.4

4.1 ( 0.2 3.9 ( 0.1 4.2 ( 0.2 4.1 ( 0.2 4.2 ( 0.1 4.2 ( 0.2

6.4 ( 0.3 6.4 ( 0.4 6.8 ( 0.1 6.1 ( 0.2 6.5 ( 0.3 6.6 ( 0.2

92.5 ( 0.4 93.0 ( 0.2 92.3 ( 0.4 92.2 ( 0.7 92.5 ( 0.6 92.8 ( 0.2

91.9 ( 0.2 92.0 ( 0.3 91.8 ( 0.3 92.1 ( 0.3 92.3( 0.4 92.1 ( 0.3

Along with film transparency, film color is another important factor concerning the final consumer acceptance of the packaged product. Color coordinates of films are shown in Table 1. In general, films made from wheat gluten proteins had a yellowish coloration more marked in glutenin films which presented lower average hue angle values (H*gliadin ) 92.4 ( 0.1, H*gliadin+CYS ) 92.5 ( 0.4, H*glutenin ) 91.9 ( 0.2). Gliadin and cysteine-treated gliadin films had similar chroma values (C* ) 4.1 ( 0.3 and C* ) 4.1 ( 0.2 respectively), whereas the color intensity of glutenin films was greater as indicated by higher chroma values (C* ) 6.4 ( 0.3). Gliadin and cysteine-treated gliadin films were lighter in color than glutenin as determined by highest L* values (L*gliadin ) 97.7 ( 0.3, L*gliadin+CYS ) 97.0 ( 0.2, L*glutenin ) 95.7 ( 0.2). These results are in agreement with higher opacity values found for glutenin films. Glycerol had no effect on the color coordinates of films. Moisture Sorption Isotherms. Equilibrium moisture sorption isotherms were measured at 23 °C for gliadin and cysteine-treated gliadin films at 0% and 33% glycerol content and are represented in Figure 3. The overall effect of glycerol on the moisture sorption of control and cysteine-treated films was similar, increasing the moisture content of both. Equilibrium moisture sorption values of treated and untreated



gliadin films were similar to those of glutenin films (results not shown). The amount of moisture bound by a protein depends primarily on its amino acid composition, the number of exposed polar groups, protein conformation, and surface polarity.33 Polymerization of gliadins did not modify the water uptake of the films. Interestingly, the moisture sorption capacities of ellipsoid-like gliadins34 and long-extended glutenins have been found to be similar35 although the proteins have different conformations. This suggests that water uptake is governed by the amino acid composition of the protein rather than the conformation adopted and, hence, that analogous hydrophilic groups are capable of binding water through hydrogen bond formation. However, it is worth remarking that even though gliadin films have similar moisture holding capacity, they loose their integrity at high water activity values (>0.75), whereas glutenin or cysteinetreated gliadin films could easily be handled above 0.9aw. Intermolecular cross-linking of cysteine-treated gliadins and partial unfolding exposing hydrophobic groups to a high aw environment could explain this observation. All isotherms had a type II sigmoid shape in the BET isotherm classification36 corresponding to a multilayer sorption model typical of biological and food materials. The first region of the curve corresponds to a hydration monolayer where water molecules are strongly linked to the film by hydrogen bonds. The linear part of the sorption isotherm indicates that water molecules are adsorbed as a multilayer, this relation extending up to about 55% or 65% relative humidity depending on the film glycerol content. The convex upper part of the curve is characterized by a sharp upswing of the water gained as the water activity increases, suggesting self-association of water molecules. The moisture isotherms of films were fit to the Guggenheim-Anderson-deBoër (GAB) equation which has shown a good fit to moisture sorption isotherms of many food products over a wide range of aw, up to 0.937 EMC ) WmCkaw/[(1 - kaw)(1 - kaW + Ckaw)]

(3)

where EMC is the equilibrium moisture content of the films on a dry basis; aw is the water activity, Wm is the BrunauerEmmett-Teller (BET) monolayer moisture content and represents the water content corresponding to saturation of all primary adsorption sites by one water molecule, C is the Guggenheim constant and represents the energy difference between the water molecules attached to primary sorption sides and those absorbed to successive sorption layers, and k is a factor correcting properties of the multilayer molecules with respect to the bulk liquid. When k ) 0, the GAB equation reduces to the BET equation. To evaluate the goodness of the fit, the root-mean-square (RMS, %) was calculated

%RMS )

[x

[

∑

M

-M Mexp N

exp

]

calc

]

2

× 100

(4)

where N is the number of experimental points, Mexp is the experimental equilibrium moisture content value, Mcalc is the calculated equilibrium moisture content value. The lower the RMS value, the better the fit.

Table 2. Guggenheim-Anderson-de Boër Model Constants and Root Mean Square (RMS) for Glutenin, Gliadin, and CysteineTreated Gliadin Films at 0% and 33% of Glycerol Content film type

Wm

C

k

RMS,%

glutenin gliadin CYS-gliadin gliadin+GLY CYS-gliadin+GLY glutenin+GLY

5.8 6.3 6.0 9.2 9.1 9.0

42.9 110.4 50.2 38.8 17.9 24.5

0.80 0.80 0.80 0.93 0.93 0.93

2.4 1.9 1.5 2.3 2.7 3.1

The constants Wm, k, and C were calculated according to Bizot.38 Table 2 shows the values of the constants for glutenin, gliadin, and cysteine-treated gliadin films. From the table, it can be seen that k and C values were within the ranges 0.24 < k e 1, and 5.67 e C e ∞, respectively. Keeping k and C within these ranges ensures that the BET monolayer values differ by not more than (15.5% from the true monolayer. Constants beyond these regions do not describe the sigmoidal type of the isotherm or yield a much bigger error in estimating the BET monolayer according to the study carry out by Lewicki.39 In agreement with the work of Coupland et al.,40 C values of glutenin, glutenin, and cysteine-treated gliadin films decreased when these were plasticized with glycerol, although it is difficult to give a physical interpretation of this behavior. BET monolayer for all of the films presented values within the range reported by Bull for several proteins.41 All of the films presented similar values of BET monolayer (see Table 2). These results are in agreement with various studies showing that there is a good correlation between the number of water molecules calculated to exist in a BET monolayer and the number of polar protein groups.42 As observed in Table 2, addition of glycerol to the films increased the BET monolayer value since the number of polar groups in the film was increased. The range of water activities in which the self-association of water molecules occurs can be determined from an isotherm sorption model such as the GAB equation by applying the clustering function developed by Zimm and Lundberg.43,44 The clustering function is written as1 ∂(aw/φ1) G11 ) -(1 - φ1) -1 V1 ∂aw

(5)

where Φ1 is the volume fraction of the solute. When G11/V1 is greater than -1, water is expected to cluster. The function G11 V1

1 + φ1

(6)

represents a measure of the cluster dimension. A value higher than 1 indicates the formation of water aggregates. The onset of water auto-association (clustering) in unplasticized glutenin, gliadin, and cysteine-treated gliadin films was observed to occur at water activities of 0.57, 0.64, and 0.63 respectively; addition of glycerol favored water molecules clustering at lower water activity values (0.48, 0.52, and 0.49 respectively) since glycerol attaches to hydrogenbinding amino acids reducing the availability of such sites

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Figure 4. Values of 1 + Φ1G11/V1 shown as a function of water activity at 23 °C.

Figure 5. Moisture sorption isotherm at 23 °C of cysteine-treated films plasticized with 33% glycerol.

for water molecules. These results are in agreement with those found for collagen films plasticized withpolyols.45 In Figure 4, the function 1 + Φ1G11/V1 shows that the dimension of water clusters in gluten protein films increases with water activity. The additivity principle was applied to predict the sorption isotherm of glycerol plasticized cysteine-treated gliadin films on the basis of the sorption behavior of pure components. Calculation was done using the formula EMCi ) xPEMCPi + xGEMCGi

(7)

where EMCi is the equilibrium moisture content for the mixture at aw ) ai; EMCPi is the equilibrium moisture content of protein at aw ) ai; EMCGi is the equilibrium moisture content of glycerol at aw ) ai; xP ) (g protein/g film) and xG ) (g glycerol/g film). The sorption isotherm of pure glycerol was obtained from Bell and Labuza.46 Experimental and calculated isotherms are represented in Figure 5; the experimental isotherm is superimposable with that predicted in the water activity interval ranged 0.11-0.75. At higher water activity values, the predicted isotherm overestimates the moisture content of films compared with experimentally observed values. The protein component of the film domi-

nates sorption at low water activities and the soluble component, glycerol, exerts its effect at greater water activities; the difference between the predicted and experimental isotherms is assume to depend on the glycerol concentration in the mixture. This behavior has also been observed for food mixtures of biopolymers and small solutes.47 When water acquires solvent properties in the film (observed in the last segment of the isotherm), the additivity of sorption isotherms no longer applies since glycerol interacts strongly with the protein. Effect of Glycerol and Relative Humidity on WVP. The water vapor permeability of protein films was evaluated at 23 °C and 0-50%, 0-75%, and 0-90% relative humidity across the film as a function of glycerol content which ranged from 11% to 66% depending on the film type and relative humidity conditions. The results are presented in Figure 6. The WVP of gliadin films could not be evaluated either at glycerol concentrations higher than 33% or for glycerol concentrations higher than 22% at 0-90% relative humidity gradient because the films became very sticky and could not be handled. The resistance of gliadin films to moisture permeation improved after cross-linking with cysteine. However, new developed films still have low water barrier properties compared to polymer synthetic films.2 The differences between the WVP of the gliadin control and films made from polymerized gliadins became more acute as the relative humidity gradient and glycerol content of the film increased. Figure 6 shows that the WVP of cysteine-treated gliadin films behaved similarly to that of glutenin films across the whole range of glycerol concentrations and humidity gradient studied. The development of intermolecular disulfide bonds in gliadins plays a more crucial role in the final moisture barrier characteristics of films than hydrophobic or hydrogen bond interactions. Permeation is governed by the thermodynamic dissolution of the permeant in the polymer matrix and its kinetic diffusion through the film. Cross-linking of gliadins is expected to decrease the free volume and segment mobility of polypeptide chains for the water molecules traversing the film. Since cross-linking did not modify water sorption capacity of films, lower WVP values of cysteinetreated films are related to the decrease in the diffusivity of water molecules through the film. Reduction of water vapor permeability has also been achieved in films made from soy and whey protein through covalent cross-linking by γ-irradiation or aldehydes.48,22 It is well-known that reticulation also decreases permeability in conventional polymers used in food packaging.49 Glycerol strongly affects the WVP of protein films. This plasticizer probably acts by establishing hydrogen bonds with proteins thereby reducing protein-protein interactions which diminishes the cohesiveness of the protein network promoting segmental chain mobility. This increase in segmental polymer mobility favors transport of permeant molecules in activated diffusion and consequently enhances the WVP. In addition, glycerol is a very hygroscopic plasticizer which enhances water vapor sorption by films upon exposure to humidity as shown by the sorption isotherms thus permitting a greater degree of molecular mobility in the protein caused by water



Table 3. Empirical Parameters for WVP vs Glycerol Content Curves of Glutenin, Gliadin, and Cysteine-Treated Gliadin Films as Related to Relative Humidity Gradient film type

WVP0 × 1011 [(g m)/(m2 s Pa)]

k × 102

R2

gliadin CYS-gliadin glutenin

50% RH 1.31 1.20 1.01

5.2 4.4 4.5

0.982 0.997 0.946

gliadin CYS-gliadin glutenin

75% RH 4.3 3.4 2.9

4.2 4 4.3

0.995 0.974 0.988

2.1 1.6

0.977 0.909

90% RH gliadin CYS-gliadin glutenin

Figure 6. Water vapor permeability values of films measured at 50% (A), 75% (B), and 90% (C) relative humidity gradient and 23 °C, as related to glycerol content.

molecules. An exponential equation adequately described the variation of the WVP experimental data as a function of glycerol content at a given relative humidity gradient WVP ) WVP0 exp(kx)

(8)

where x is the glycerol concentration (g/100 g dry protein), k is the relative efficiency of glycerol in plasticizing the film, and WVP0 represents WVP for unplasticized films. The values of the parameters for each type of film are given in Table 3, and the corresponding curves are plotted in Figure 6, parts A-C, for 0-50%, 0-75%, and 0-90% relative humidity gradient, respectively. This exponential relationship

9 10.2

for glycerol has also been reported for water vapor and oxygen permeability of several protein and polysaccharidebased films.50,51 As can be expected, increasing the environmental relative humidity favored the loss of barrier properties of these films, a phenomenon that also has been observed in both biobased52-54 and synthetic polar films such as EVOH and polyamides.55,56 The efficiency of glycerol in plasticizing proteins decreased with increasing relative humidity as shown by parameter k in Table 3. As can be observed in Figure 6C, at 90% relative humidity gradient the films are highly plasticized by water and exist above the glass transition temperature (Tg < 0 °C)35 throughout the glycerol concentration tested which makes the change in the slope of the curve less acute. Water vapor permeability values for unplasticized films were obtained from the fitted exponential relationship. It is noteworthy that the gliadin control, polymerized gliadin and glutenin films had similar WVP0 values at 50% relative humidity, however differences among the WVP0 of films were more marked as the surrounding relative humidity increased. This behavior could be related to a change in films from a glassy to a rubbery state. At 50% relative humidity, unplasticized films remain in a glassy state in which molecular segmental motion is restricted in both covalently cross-linked (cysteine-treated) and un-cross-linked (untreated) film networks. As relative humidity increases, films tend to draw in greater quantities of water which disrupts hydrogen bonding between polypeptide chains shifting the glass transition to lower temperatures. At this point, the crosslinked protein network of cysteine-treated gliadins or glutenins could be more effective than monomeric gliadins restricting molecular segmental motion and thus reduce the diffusion of water molecules through the film. Conclusions Cysteine effectively induced polymerization in gliadins through the formation of intermolecular disulfide bonds as evidenced by SDS-PAGE analysis. The water resistance of the derived films was greatly improved compared to untreated films. However, the moisture sorption capacity of

1510


films was not affected by cross-linking, and the moisture sorption isotherm behaved similarly to those for gliadin and glutenin films fitting the GAB model and with similar BET monolayer values. Cysteine-treated gliadin films improved their water barrier properties compared to control films. This improvement was more acute with increasing humidity and glycerol content in the films, showing water vapor permeability values close to those for naturally polymerized glutenins. Although cysteine is not a cross-linker, it has been shown that it can promote sulfydryl-disulfide interchange reactions leading to the polymerization of gliadins. Thus, cysteine offers a promising alternative to other harmful cross-linkers such as aldehydes for the processing of proteins as bioderived films for food packaging applications. Acknowledgment. We would like to thank Dr. Perry Ng for assistance with SDS-Page analysis and Dr. A. P. MacCabe for a critical reading of the manuscript. P. H.-M. was supported by a fellowship from Generalitat Valenciana. This paper is dedicated to the late Professor Ruben J. Hernandez in whose laboratory part of this work was conducted. References and Notes (1) Gennadios, A.; McHugh, T. H.; Weller, C. L.; Krochta J. M. Edible coatings and films based on proteins. In Edible Coatings and Films to ImproVe Food Quality; Krochta, J. M., Baldwin, E. A., NisperosCarriedo, M. O., Eds.; Technomic Publishing Co.: Lancaster, PA, 1994; pp 201-277. (2) Cuq, B.; Gontard, N.; Guilbert, S. Cereal Chem. 1998, 75, 1-9. (3) Gennadios, A. Protein-based films and coatings; CRC Press: Boca Raton, FL, 2000. (4) Osborne, T. B. The Proteins of the Wheat Kernel; Carnegie Inst.: Washington, DC, 1907. (5) Sanchez, A. C.; Popineau, Y.; Mangavel, C.; Larre, C.; Gueguen, J. J. Agric. Food Chem. 1998, 46, 4539-4544. (6) Mangavel, C.; Barbot, J.; Bervast, E.; Linossier, L.; Feys, M.; Gueguen, J.; Popineau, Y. J. Cereal Sci. 2002, 36, 157-166. (7) Jones, R. W.; Taylor, N. W.; Senti, F. R. Arch. Biochem. Biophys. 1959, 84, 363-376. (8) Woychik, J. H.; Boundy, J. A.; Dimler, R. J. Arch. Biochem. Biophys. 1961, 94, 477-482. (9) Shewry, P. R.; Tatham, A. S.; Forde, J.; Kreis, M.; Miflin, B. J. J. Cereal Sci. 1986, 4, 97-106. (10) Belton, P. S. J. Cereal Sci. 1999, 29, 103-107. (11) Hernańdez-Munõz, P.; Kanavouras, A.; Ng, P. K. W.; Gavara, R. J. Agric. Food Chem. 2003, 51, 7647-7654. (12) AACC ApproVed Methods; American Association of Cereal Chemist: St. Paul, MN, 1992. (13) Laemmli, U. K. Nature 1970, 227, 680-686. (14) Ng, P. K. W.; Bushuk, W. Cereal Chem. 1987, 64, 324-327. (15) Standard Practice for Maintaining Constant Relative Humidity by Means of Aqueous Solutions (E 104-85). Annual Book of ASTM Standards; American Society for Testing and Materials: Philadelphia, PA, 1985; pp 912-916. (16) Karmas, E. Food Technol. 1980, 34, 52-62. (17) Gontard, N.; Guilbert, S.; Cuq J. L. J. Food Sci. 1992, 57, 190195, 199. (18) Optical Methods for Measuring Brightness, Whiteness, Reflectance and Opacity for Paper. B. S. 4432; British Standards Institution: London, 1968. (19) Veraverbeke, W. S.; Delcour, J. A. Crit. ReV. Food Sci. Nutr. 2002, 42, 179-208.

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Gliadins Polymerized with Cysteine: Effects on the ... - ACS Publications

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