FILM PROPERTIES OF κ/ι-hybrid CARRAGEENAN NATURAL POLYMER.

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1 FILM PROPERTIES OF κ/ι-hybrid CARRAGEENAN NATURAL POLYMER. F.D.S. Larotonda 1, L. Hilliou 2, M.P. Gonçalves 3, A.M. Sereno 4 * 1 FEUP - larotond@fe.up.pt; 2 FEUP - hilliou@fe.up.pt; 3 FEUP - pilarg@fe.up.pt; 4* REQUIMTE-CEQUP, Departamento de Engenharia Química, Faculdade de Engenharia da Universidade do Porto,Rua Dr. Roberto Frias, s/n Porto-Portugal - sereno@fe.up.pt The main renewable and natural biopolymers are obtained from polysaccharides, lipids and proteins. A polysaccharide that have been widely studied and utilized in pharmaceutical and biotechnological products is carrageenan, a biopolymer extracted from red seaweeds. The use of a κ/ι-hybrid carrageenan obtained from Mastocarpus stellatus (an underexploited Portuguese seaweed) for the production of films was studied. Films were produced spreading hot aqueous κ/ι-carrageenan solutions over an acrylic plate by an automatic film applicator, and let to dry at room temperature to form the film. Two different plasticizers were tested as an alternative to improve the film properties, a hydrophilic (glycerol) and a hydrophobic (triethylcitrate). The addition of plasticizers improves the film properties: glycerol was the one with best results in terms of mechanical properties, but the triethylcitrate also improves the films hygroscopic properties. The results indicate that the κ/ι-hybrid carrageenan extracted from Mastocarpus stellatus is a good source for the production of biodegradable films. Introduction Chemically synthesized polymeric films are widely used for packaging in food industry, because they are easily and inexpensively produced from uniform raw materials and are flexible as well as durable. A serious disadvantage of these films is that they are not biodegradable. On the contrary, films constituted by edible components are biodegradable and can be potentially used for enhancing the stability and quality of foods. Moreover, edible films may be applied also by dipping or spraying with the result to significantly reduce the packaging waste [1, 2, 3]. Edible and/or biodegradable films are not meant to totally replace synthetic packaging films, however they do have the potential to replace the conventional packaging in some applications. An edible film coating, acting as an efficient moisture, oxygen, or aroma barrier, can reduce the amount of packaging [2]. Edible films are made of various materials, are formed by various processes, and have various properties. The main renewable and natural biopolymers are obtained from polysaccharides, lipids and proteins. Polysaccharides may include cellulose derivative, starches and their derivatives, seaweed extracts such as carrageenan and alginates, pectins, and chitosan. Protein film formers include collagen, gelatin, whey protein, corn zein, soy protein, and wheat gluten. Polysaccharide and protein film materials are characterized by high moisture permeability, low oxygen and lipid permeability at lower relative humidities, and compromised barrier and mechanical properties at high relative humidities [4]. With regard to the polysaccharides, because of their wide variety of chemical structures, various filmforming behaviors may occur during the manufacture. Consequently, films made from different types of polysaccharides may to display a wide range of properties [5]. Biopolymers from marine sources have been studied and utilized in pharmaceutical and biotechnological products for decades. Carrageenans are water-soluble galactose polymers extracted from red seaweed, which are extensively used in food and pharmaceutical industries as gelling and stabilizing agents. Carrageenan is classified in three industrially relevant types, which present ideal chemical structures with associated gelling and viscous enhancer properties. However, carrageenan biopolymers are actually chemically complex [6] and are better described as hybrids of ideal monomers and biological precursors monomers [7]. The arrangement of these monomers and their respective amount in the macromolecule are specific to the type of seaweeds that produce the carrageenan [6]. κ-carrageenan has one negative charge per disaccharide with a tendency to form excellent gel and film forming properties, and exhibit the highest tensile strength when compared with that of λ- and ι-carrageenan films. The gelling power of κ- and ι- carrageenans imparts excellent film forming properties [8]. After an extraction optimization [9] and a viscoelastic characterization [10], the use of a κ/ι-hybrid carrageenan obtained from Mastocarpus stellatus (an underexploited Portuguese seaweed) for the production of films was studied. The effect of different plasticizers, a hydrophilic (glycerol) and a hydrophobic (triethylcitrate), was evaluated in order to improve films functional properties (hygroscopicity, mechanical, water barrier and optical).

2 Experimental Sample preparation Carrageenan extracted from Mastocarpus stellatus [9] was utilized to prepare films, using as plasticizers glycerol (GLY) and triethylcitrate (TEC), at different amounts (0, 10, 20 and 40%) in relation to the amount of carrageenan. The total concentration of polymers in water was 4%, which leads to optimal solution viscosity with respect to the knife coating technique used. The solutions containing carrageenan and plasticizers were submitted to heating to 70 C under stirring during 30 minutes for total solubilization of carrageenan. The solution was put over an acrylic plate and spread over the plate by an Automatic Film Applicator model 1132N (Sheen, UK) and let to dry at room temperature using a fan to form the film. The speed of application was 300 mm/s. The thickness of film samples was measured at four different points using a thicknesses comparator Digimatic Indicator ID-H (Mitutoyo Co., Japan) giving a mean value of 15 ± 1 µm. The samples were codified in accordance with the following: κ/ι-hybrid carrageenan (M) and plasticizers (P), glycerol (G) and triethylcitrate (T), and the respective formulations, for example M:P(G)-90:10 was the formulation with 90% of κ/ι-hybrid carrageenan and 10% of plasticizer, in this case glycerol. Water sorption isotherms Water sorption isotherms were determined by the gravimetric method. Samples with dimensions of 30 x 30 mm were previously dried at 50 C during 24 h in a vacuum oven. The samples were then placed in desiccators with different relative humidities, imposed by the use of saturated saline solutions. The experiment was carried out at 25 C. The samples were weighed periodically until they reached constant weight, after which the sample moistures were determined by the gravimetric method. The Guggenheim-Anderson-de Boer (GAB) model (Eq. 1) was used to represent the experimental sorption data [11]. X = [( 1 ka )( 1 ka + Cka ] w CkX a 0 w (1) w w ) where X is the equilibrium moisture content at the water activity a w, X 0 is the 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 sites and those absorbed to successive sorption layers, and k is the corrective constant taking into account properties of multilayer molecules with respect to the bulk liquid. GAB equation parameters were calculated from STATISTICA software (version 6.0). Mechanical properties The mechanical properties were studied using a TAXT2 (Stable Micro Systems, England) in accordance with ASTM D [12]. Five sample strips (25x100mm) of each formulation were cut and clamped between grips. Force (N) and deformation (mm) were recorded during extension at 0.2 mm/s and with an initial distance between the grips of 60 mm. The parameters determined were: stress at break (MPa), strain at break (%) and Young s modulus (GPa). The samples were conditioned in a dessicator with a relative humidity of 53% for a week before performing the mechanical assays. Water Vapor Permeability (WVP) WVP tests were conducted using ASTM method E96-95 [13] with some modifications. Each film sample was sealed over a circular opening 0.003m 2 in a permeation cell that was stored at 25ºC in a dessicator. The driving force, expressed as water vapor partial pressure, was Pa. To maintain this driving force corresponding to a 98% relative humidity (RH) gradient across the film, anhydrous calcium chloride (2% RH) was used inside the cell and distilled water (100% RH) was used in the dessicator. A fan was operated within the chamber to avoid the stagnant air. The test cell was periodically weighted after steady state conditions were reached (after about 2h, a constant weight variation rate was observed). WVP (g m -1 s -1 Pa -1 ) was calculated using the Eq. 2: Δm x WVP = A Δt Δp (2) Δm where is the weight gain (g) of the test cell, x is the film thickness (m), and A is exposed area (0.003m 2 ) during duration (s) under partial water vapor pressure (Pa). Δt Δp Optical properties Film color was determined by a Minolta CR300 series (Tokio, Japan) using the CIELab color parameters, lightness (L*) and chromacity parameters a* (red green) and b* (yellow blue) were measured. The color of the films was expressed (Eq. 3) as the difference of color (ΔE*).

3 Δ E (3) * * 2 * * 2 * * 2 * = ( L Ls ) + ( a as ) + ( b bs ) * L s * a s * b s where (97,10), (0,05) e (1,76) are the CIELab standards for the white standard, used as the film background. The ultraviolet (UV) and visible light barrier properties of the films were measured at selected wavelengths from 200 to 600nm using a UV-visible Unicam spectrometer model Helios Alpha. Results and Discussion Films characteristics Homogeneous, thin, flexible and transparent films were obtained from κ/ι-hybrid carrageenan by the knife coating technique (semi-continuous process). All the films were easily removed from the acrylic plate and showed smooth surfaces. The films were very sensible to water, being easily dissolved in distilled water. The films plasticized with hydrophobic plasticizer (triethylcitrate) presented a little water exudation. Water sorption isotherms Moisture sorption isotherms of the film samples obtained from carrageenan, varying the plasticizer type and relative content are presented in Figure 1, together with the GAB model fitted for each sample. The GAB equation parameters and the correlation coefficients are presented in Table 1. The values of k (<1) and the correlation coefficient (r > 0.98) show that GAB equation gives a good fit to experimental values, except for the data sets corresponding to films plasticized with triethylcitrate. All the samples presented a sigmoidal shape indicating that the equilibrium moisture content increases slowly with increasing environmental a w up to 0.7, beyond which a steep rise in moisture content in film samples was observed. The isotherms corresponding to the films containing glycerol exhibit a more hygroscopic behavior (X 0 increases and C decreases with increasing glycerol content), as expected because of the high hydrophilicity of this plasticizer. The films with hydrophobic plasticizer showed a similar behavior in terms of water sorption, but the moisture content absorbed was lower than the film of pure carrageenan and the carrageenan-glycerol films. Mechanical properties The stress-strain curve of κ/ι-hybrid carrageenan without plasticizer shows the typical pattern of brittle materials, because they exhibited high values of tensile stress at break, breaking force and elastic modulus and low values of strain at maximum breaking force. Plasticizers addition in the films exhibits the expected effect on the mechanical properties, since strain at break increased and stress decreased compared with the unplasticized films. Films plasticized with glycerol and triethylcitrate presented similar values of mechanical properties for the concentrations of 10 and 20%. For the concentration of 40%, films with glycerol present better results than the films with triethylcitrate. Figure 2 shows the stress-strain curves for all the samples studied. Water Vapor Permeability (WVP) Figure 3 shows WVP of κ/ι-hybrid carrageenan films varying the amount and type of plasticizer. WVP values ranged between 0.72 and 1.49 x g m -1 s -1 Pa -1. WVP increases with the addition of plasticizer, when compared to pure κ/ιhybrid carrageenan. At lower concentrations of κ/ι-hybrid carrageenan in the formulation WVP decreases, despite the increase of plasticizer. This result indicates that carrageenan exhibits a great influence on WVP. The films with hydrophobic plasticizer showed lower values of WVP compared with the hydrophilic plasticizer. Optical properties Visually, all the films had a slightly yellow appearance. Instrumental color determination performed on films showed no-significant (P>0.05) differences in the color parameters (L*, a* and b*) values for all assayed films (see Table 2). Color difference (ΔE*) increases with the addition of plasticizer, decreasing with higher values of plasticizer concentration, in the case of glycerol, possibly due to the effect of dilution of glycerol, which is a colorless substance [14]. For the triethylcitrate this does not occur. Table 3 lists the light transmission at selected wavelengths for the κ/ι-hybrid carrageenan films plasticized with glycerol and triethylcitrate. All the films showed good barrier properties to UV light in the 280nm region, decreasing the light transmission when compared with the initial wavelength of 200nm. The increase of the hydrophilic plasticizer (glycerol) increases the values of light transmission in all the wavelength range measured, while the increase of the hydrophobic plasticizer (triethylcitrate) decreases the values of light transmission.

4 Table 1 GAB equation parameters for moisture sorption isotherms (r is the correlation coefficient) Film X 0 C k r M:P-100: M:P(G)-90: M:P(G)-80: M:P(G)-60: M:P(T)-90: M:P(T)-80: M:P(T)-60: Table 2 Color standards of κ/ι-hybrid carrageenan films plasticized with glycerol and triethylcitrate. Film Δa* (a* s = 0.05) Δb* (b* s =1.76) ΔL* (L* s =97.1) ΔE* M:P-100: M:P(G)-90: M:P(G)-80: M:P(G)-60: M:P(T)-90: M:P(T)-80: M:P(T)-60: Table 3 Light transmission (%) of κ/ι-hybrid carrageenan films plasticized with glycerol and triethylcitrate. Wavelength (nm) Film M:P-100: M:P(G)-90: M:P(G)-80: M:P(G)-60: M:P(T)-90: M:P(T)-80: M:P(T)-60:

5 moisture content (g H 2 O/g dry solids) M:P - 100:0 M:P(G) - 90:10 M:P(G) - 80:20 M:P(G) - 60:40 M:P(T) - 90:10 M:P(T) - 80:20 M:P(T) - 60: a w Figure 1 Experimental data (symbols) for moisture sorption isotherms of the films obtained from κ/ι-hybrid carrageenan varying the amount (0, 10, 20 and 40%) and type of plasticizer (glycerol and triethylcitrate), and the respective fitted GAB curves (lines). stress (MPa) M:P - 100:0 M:P(G) - 90:10 M:P(G) - 80:20 M:P(G) - 60:40 M:P(T) - 90:10 M:P(T) - 80:20 M:P(T) - 60: strain (%) Figure 2 Tensile mechanical behavior (stress vs. strain curves) of films formulated with: κ/ι-hybrid carrageenan (M:P-100:0), κ/ι-hybrid carrageenan plasticized with glycerol (M:P(G)) and with triethylcitrate (M:P(T)).The concentration of plasticizers varying from 0 to 40%. Films were formulated with 4% of components in solution.

6 WVP (g m -1 s -1 Pa -1 ) 1.6x x x x x x x x M:P-100:0 M:P-90:10 M:P-80:20 M:P-60:40 formulation P(G) - glycerol P(T) - triethylcitrate Figure 3 Water vapor permeability of films formulated with: κ/ι-hybrid carrageenan (M), κ/ι-hybrid carrageenan plasticized with glycerol (G) and with triethylcitrate (T). Films were formulated with 4% of components in solution. Conclusion Homogeneous, flexible and transparent films were obtained from κ/ι-hybrid carrageenan by the knife coating technique plasticized with a hydrophilic and a hydrophobic plasticizers. Practically, all studied properties were affected by the addition and concentration of the plasticizers, although the carrageenan concentration also affects some properties because of its hygroscopic behavior. Film hygroscopicity increases with the addition of glycerol (hydrophilic plasticizer) and decrease with the addition of triethylcitrate (hydrophobic plasticizer). The tests of mechanical properties showed that unplasticized carrageenan film is a good packing material with high tensile strength, whereas plasticized carrageenan film is a good material for producing flexible films. The plasticizer addition on the film formulation causes an increase in elongation at break, a decrease in the tensile strength and the elasticity modulus, as it was expected. At a plasticizer concentration of 40%, glycerol had best results, while for other studied concentrations both plasticizers perform similarly in terms of mechanical properties. Water vapor permeability increases with the addition of plasticizer, but is very influenced by the carrageenan concentration in the film formulation. Comparing the plasticizers, triethylcitrate had lower WVP values than glycerol, as it was expected because of its hydrophobic behavior. As far as optical properties are concerned glycerol performed better than triethylcitrate. These results indicate that κ/ιhybrid carrageenan extracted from Mastocarpus stellatus domestic red seaweed from the Portuguese coast, can be used to produce edible films with interesting mechanical and optical properties. Acknowledgements The authors wish to thanks the financial support from FCT (Project POCTI/EQU/45595/2002), the support from CYTED (Project XI.20) and the scholarship of F.D.S. Larotonda, supported by the Programme Alβan, the European Union Programme of High Level Scholarships for Latin America, scholarship nº E04D027282BR.. References 1. J.J. Kester; O.R. Fennema, Food Technol. 1986, 40, M. Krochta; C.D. Johnston, Food Technol. 1997, 51, K.S. Miller; M. Krochta, Trends Food Sci. Tech. 1997, 8, A.L. Brody, Food Technol. 2005, 59, 65.

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