Effect of glycerol and Ca+2 addition on physicochemicalproperties of edible carrageenan/porphyran-based filmsobtained from the red alga, Pyropia columbina

Raúl E. Cian & Pablo R. Salgado & Silvina R. Drago &

Adriana N. Mauri

Received: 12 August 2014 /Revised and accepted: 26 October 2014# Springer Science+Business Media Dordrecht 2014

Abstract Our aim was to study the effect of glycerol and Ca+2 addition on physicochemical properties of phycocolloid(carrageenans/porphyrans)-based films obtained fromPyropia columbina aqueous fraction. Films were formed bycasting from aqueous dispersions of phycocolloid-enrichedfraction (P) with different levels of glycerol (0, 12.5, and25 g (100 g)−1 dry solid) and the optional addition of Ca+2

(16 g (100 g)−1 dry solid), yielding six formulations accordingto a multi-factorial design (P, PG1, PG2, PCa, PCaG1, andPCaG2). All films were homogeneous, flexible, and slightlygreenish. Moisture content, water solubility, and water vaporpermeability of films were significantly increased by addingglycerol to the formulation (11, 12, and 6 %). Increasingglycerol content in formulations (12.5 to 25 g (100 g)−1 drysolid) allows obtaining more stretchable but less resistantfilms (with less tensile strength and elastic modulus) thanthose produced without plasticizer (P). Ca+2 addition maskedglycerol effect in some properties (water solubility and watervapor permeability), but in others, the effect of Ca+2 wasadded to the plasticizer glycerol effect (mechanical proper-ties). Ca+2 could stabilize the three-dimensional structure of Pphycocolloids by interactions between sulfate groups,

promote water retention, and open film structure. Use of theseadditives yielded films from P. columbina with a wide rangeof mechanical properties, thus extending the scope of thesematerials.

Keywords Edible films . Red seaweeds . Phycocolloids .

Calcium . Plasticizers

Introduction

Marine microorganisms such as bacteria, microalgae, andseaweeds represent a large source of valuable materials (LeiriaCampo et al. 2009). Three commercially exploited carbohy-drate polymers (phycocolloids) from marine microorganismsare (1) alginates from brown seaweeds, (2) carrageenans, and(3) agar both isolated from red seaweeds (Souza et al. 2012).

Carrageenans are water-soluble polymers with a linearchain of partially sulfated galactans, which have high poten-tiality as a film-forming material. The number and position ofsulfate groups on the disaccharide repeating unit determine theclassification in three major types: lamda (λ), kappa (κ), andiota (ι) (Al-Alawi et al. 2011), which have sulfate contents of41, 33, and 20 % (w/w), respectively, resulting from one, two,and three sulfate ester groups per dimeric unit (Karbowiaket al. 2006). The main difference between the highly sulfatedcarrageenans from the less sulfated agars is the presence of D-galactose and anhydro-D-galactose in carrageenans and D-galactose, L-galactose, or anhydro-L-galactose in agars(Gómez-Ordóñez and Rupérez 2011). Noteworthy thatporphyrans are the main agar from the Pyropia species. Thesesulfated polysaccharides comprise the hot-water-soluble por-tion of the cell wall and intercellular region (Zhang et al.2005).

These phycocolloids have been used for the developmentof biodegradable films with good mechanical and barrier

R. E. Cian : S. R. DragoInstituto de Tecnología de Alimentos, Facultad de IngenieríaQuímica, Universidad Nacional del Litoral (UNL), 1° deMayo 3250,(3000) Santa Fe, Argentina

R. E. Cian : P. R. Salgado : S. R. Drago :A. N. MauriConsejo Nacional de Investigaciones Científicas y Técnicas(CONICET), Av. Rivadavia 1917, (C1033AAJ) Buenos Aires,Argentina

P. R. Salgado (*) :A. N. MauriCentro de Investigación y Desarrollo en Criotecnología deAlimentos(CIDCA) CONICET CCT La Plata y Facultad de Ciencias Exactasde Universidad Nacional de La Plata (UNLP), 47 y 116 S/N°,(B1900JJ) La Plata, Argentinae-mail: psalgado78@gmail.com

J Appl PhycolDOI 10.1007/s10811-014-0449-5

properties (Blanco Pascual et al. 2014; Giménez et al. 2013;Karbowiak et al. 2006; Rhim 2012; Sousa et al. 2010). In thissense, these films can inhibit/regulate the migration of mois-ture, oxygen, carbon dioxide, and lipids and also carry foodingredients to improve food properties when they are used aspackaging (Martins et al. 2012).

In a previous study, edible films were prepared from aque-ous fractions extracted from the red alga Pyropia columbina(found on hard substratum in the coasts of Patagonia, Argen-tina) without ulterior purification steps, which implies alower-cost process than the use of purified phycocolloid frac-tions (carrageenans and agars). These films—processed with-out the addition of any plasticizer—showed high tensilestrength, low elongation at break, and interesting antioxidantproperties (Cian et al. 2014).

It is known that physicochemical properties of biopolymerfilms can be modified by the addition of plasticizers or cross-linking agents (Han and Gennadios 2005; Baldwin et al.2012). Glycerol, a low molecular weight and non-volatilepolyol, is widely used as a plasticizer, and its addition to thesepolysaccharide films could significantly improve their flexi-bility and guarantee their processability (Vieira et al. 2011).Calcium, being a divalent cation, is capable of forming intra-molecular bridges between the sulfate groups of adjacentanhydro-D-galactose and D-galactose residues, and it has beenreported that its presence favored carrageenan gelation(Thrimawithana et al. 2010). The effect of calcium on alginateedible films has been studied (Olivasa and Barbosa-Cánovas2008; Sartori et al. 1997; Rhim 2004), but not the carrageenan/porphyran films from P. columbina. As far as we know, thereare no published works related to the production ofcarrageenan/porphyran-based films with plasticizers from thisseaweed and even less with addition of calcium.

The aim of this work was to study the effect of glycerol andCa+2 additions on physicochemical properties of phycocolloid(carrageenans/porphyrans)-based films obtained fromP. columbina aqueous fraction.

Materials and methods

Preparation of phycocolloid (carrageenans/porphyrans)fraction from P. columbina

One kilogram of different specimens of P. columbina washand-picked in Punta Maqueda (46° 00′ S, 67° 34′ W) inspring. Punta Maqueda, a pristine beach far away from an-thropogenic activities, is located within San Jorge Gulf at30 km to the south of Comodoro Rivadavia, Argentina. Sam-ples were taken to the laboratory at 4 °C inside plastic bags. Toremove adherent seawater, sediment, organic debris, macrofauna, and epibiota, they were scraped and rinsed with dis-tilled water. P. columbina samples were dried to environment

temperature (22–25 °C) and ground to obtain a powder with aparticle size lower than 1 mm, using a laboratory hammer mill(Retsch, Germany). Then, samples were passed through a 20-mesh sieve (0.85 mm) and stored at 4 °C in plastic bags untilanalysis.

Phycocolloid-enriched fraction (P) from P. columbina wasprepared as follows.P. columbina algae powder was dispersedat 50 g kg−1 in cold distilled water (at 4 °C) for 2 h and filteredthrough a 50-mesh sieve (0.297 mm). The residue of thefiltration (particle size >0.297 mm) was subjected to twowashings with cold distilled water (4 °C), dispersed at50 g kg−1 in hot distilled water (at 95 °C) for 2 h, and thencentrifuged at 3000×g for 45 min at 45 °C. The supernatantobtained from hot distilled water extraction was thephycocolloid-enriched fraction and had 1 g (100 g)−1 drysolid. P composition on a dry basis was the following: protein,14.3 %; total carbohydrates, 72 %; sulfate content, 12.6 %;and ash, 6.4 %. The chlorophyll a and total chlorophyllcontent from P was determinate spectrophotometrically ac-cording to Wolfenden et al. (1988).

Film preparation

Films were prepared by casting from aqueous dispersions ofphycocolloid-enriched fraction (P) (1 g dry solid g (100 g)−1

dispersion) with different amounts of glycerol (0, 12.5, and25 g (100 g)−1 dry solid) and the optional addition of Ca+2

(16 g (100 g)−1 dry solid), yielding six formulations accordingto a multi-factorial design. Formulations were named as fol-lows: P, PGl (P+12.5 g glycerol g (100 g)−1 dry solid), PG2

(P+25 g glycerol g (100 g)−1 dry solid), PCa (P+16 g Ca+2 g(100 g)−1 dry solid), PCaG1 (P+12.5 g glycerol g (100 g)−1

dry solid+16 g Ca+2 g (100 g)−1 dry solid), and PCaG2 (P+25 g glycerol g (100 g)−1 dry solid+16 g Ca+2 g (100 g)−1 drysolid).

The film-forming dispersions were agitated in a magneticstirrer for 1 h at room temperature. Twenty milliliters of eachfilm-forming dispersion was poured on polystyrene Petridishes (64 cm2) and then dehydrated at 60 °C for 5 h in anoven with air flow circulation (Yamato, DKN600, USA).Resulting films were conditioned 48 h at 20 °C and 58 %relative humidity (RH) in desiccators with saturated solutionsof NaBr before being peeled from the casting surface forcharacterization.

Rheology of film-forming dispersions

Rotational analyses of P, PG1, PG2, PCa, PCaG1, andPCaG2 film-forming dispersions were performed at 25 °C ina Haake ReoStress 600 (Thermo Haake, Germany) with a 1-mm gap serrated plate–plate sensor system PP35. Dispersionswere maintained at 25 °C by a circulating water bath connect-ed to the jacket surrounding the sensor system during testing.

J Appl Phycol

Shear stress (τ) was determined as a function of shear rate (D).The shear rate was increased from 0 to 500 s−1 in 2 min,maintained for 1 min, and then decreased from 500 to 0 s−1 in2 min. Flow (n) and consistency (K) indexes were determinedadjusting experimental results with the Ostwald de Waelemodel (Eq. 1).

τ ¼ K :Dn ð1Þ

where τ is the shear stress (Pa), K is the consistency index(Pa sn), D is the shear rate (s−1), and n is the flow index(dimensionless).

Apparent viscosities (ηapp, mPa s) were calculated in theupwards curves at 60, 300, and 500 s−1 for each film-formingdispersion. Determinations were carried out in triplicate.

Film characterization

Thickness Film thickness was measured by a digital coatingthickness gauge (Check Line DCN-900, USA). Measure-ments were done at five positions along the rectangular stripsfor the tensile test, and at the center and at eight positionsround the perimeter for the water vapor permeability (WVP)determinations. The mechanical properties and WVP werecalculated using the average thickness for each film replicate.

Color Film color was determined with a Konica MinoltaChroma Meter CR-400 (Konica Minolta Chroma Co., Japan)set to C illuminant/2° observer. A CIE-Lab color scale wasused to measure the degree of lightness (L*), redness (+a*) orgreenness (−a*), and yellowness (+b*) or blueness (−b*) ofthe films. The instrument was calibrated using a white stan-dard plate with color coordinates of L*standard=97.55, a-*standard=−0.03 and b*standard=1.73 provided by Minolta.Film color was measured on the surface of this standard plate,and total color difference (ΔE*) was calculated as follows:

ΔE� ¼L�film– L�standardð Þ2 þ a�film– a�standardð Þ2 þ b�film– b�standardð Þ2

h i0:5

ð2Þ

Color can also be expressed by use of polar (or cylindrical)coordinates L*, C*, and h*, where L* is the same as previ-ously, C* is chroma or saturation index, and H* is hue. Thefollowing equations were used to convert L*a*b* coordinatesto L*C*h* coordinates.

C � ¼ a�2 þ b�2� �0:5 ð3Þ

h � ¼ arc tan b �.a�

� �ð4Þ

Values were expressed as the means of nine measurementson different areas of each film.

Opacity Each film specimen was cut into a rectangular pieceand placed directly in a spectrophotometer test cell, and mea-surements were performed using air as the reference. A spec-trum of each film was obtained in an UV-vis spectrophotom-eter (Beckman DU650, Germany). The area under the absorp-tion curve from 400 to 800 nmwas recorded (absorbance units(AU)×nm) and divided by film thickness (nm) (García et al.2006). The opacity of films was expressed as AU. Fivereplications for each formulation were performed.

Moisture content (MC) Small specimens of films collectedafter conditioning were cut and placed on Petri dishes thatwere weighed before and after oven drying at 105 °C for 24 h(ASTM D644-99 2004). MC values were determined in trip-licate for each film and calculated as the percentage of weightloss relative to the initial weight.

Water solubility (WS) WS was determined following themethod described by Gontard et al. (1994) with slight modi-fications. Film portions were weighed (diameter=2 cm; P0~0.03–0.05 g) and placed in an Erlenmeyer flask (250mL) with50 mL of distilled water (containing 0.02 %w/v sodium azide)and then sealed and shaken at 100 rpm for 24 h at 20 °C(Ferca, TT400 model, Argentina). The solution was thenfiltered through Whatman no. 1 filter paper (previously driedand weighed) to recover the remaining undissolved film,which was desiccated at 105 °C for 24 h (Pf). WS wascalculated as follows:

WS ¼ P0⋅ 100− MCð Þð Þ−P f½ �⋅100.

P0⋅ 100−MCð Þ½ � ð5Þ

where P0 is the initial filmweight (g),Pf is the final dry filmweight (g), andMC is themoisture content (%).WS tests werecarried out in triplicate.

Water vapor permeability (WVP) Water vapor permeabilitytests were conducted according to ASTM method E96-00(ASTM 2004) with some modifications. Each film samplewas sealed over a circular opening of 0.00185 m2 in apermeation cell that was stored at 20 °C in desiccators.To maintain a 75 % relative humidity (RH) gradientacross the film, anhydrous silica (0 % RHc) was placedinside the cell and a saturated NaCl solution (75 %RHd) was used in the desiccators. The RH inside thecell was always lower than that outside, and watervapor transport was determined from the weight gainof the permeation cell. When steady-state conditionswere reached (about 1 h), eight weight measurementswere made over 5 h. Changes in the weight of the cellwere recorded and plotted as a function of time. Theslope of each curve (Δm/Δt, g H2O s−1) was obtainedby linear regression, and the water vapor transmissionrate was calculated from the slope divided by the

J Appl Phycol

permeation cell area (A, m2). WVP (g H2O Pa−1 s−1 m−1) wascalculated as follows:

WVP ¼ WVTR.

PVH2O⋅ RHd– RHcð Þ� �h i

⋅d ð6Þ

where WVTR is the water vapor transmission rate(g H2O s−1 m−2); PV

H2O is the saturation water vapor pressureat test temperature (2339.27 Pa at 20 °C); (RHd−RHc) is therelative humidity gradient across the film, expressed as afraction (0.75); A is the permeation (m2) area; and d is thefilm thickness (m). Each WVP value represents the meanvalue of at least three samples taken from different films.

Mechanical properties Tensile strength (TS), elastic modulus(EM), and elongation at break (EAB) of the films were deter-mined using a texture analyzer TA.XT2i (Stable Micro Sys-tems, Surrey, England) equipped with a tension grip systemA/TG, according the procedures outlined in the ASTM meth-od D882-02 (ASTM 2004). The measurements were made at20 °C and 65 % RH in a controlled room. Film probes of70 mm long and 6 mmwide were used, and a minimum of sixprobes were prepared from each film. The initial grip separa-tion was set at 50 mm and the crosshead speed at 0.5 mm s−1.The curves of force (N) as a function of distance (mm) wererecorded by the Texture Expert V.1.15 software (Stable MicroSystems, Surrey, England). Tensile properties were calculatedfrom the plot of stress (tensile force/initial cross-sectionalarea) versus strain (extension as a percentile of the originallength). TS and EABwere determined directly from the stress-strain curves, and EM was determined as the slope of theinitial linear portion of this curve. Twelve replications for eachformulation were performed.

Scanning electron microscopy (SEM) of films SEM of filmcross section (cryofractured by immersion in liquid nitrogen)and film surface were performed. The pieces of films weremounted on aluminum stubs using a double-sided tape and

were coated with a thin gold layer using a cool sputter system(SCD 005, Bal-Tec, Switzerland). SEM images were acquiredwith a scanning electron microscope (XL-20, Philips, Nether-lands), using an acceleration voltage of 20 kV for all thesamples.

Fourier transform infrared spectroscopy (FTIR)analysis Infrared spectra of studied films were acquired usinga Nicolet iS 10 Infrared Spectrometer (Thermo Scientific,USA). Measurements were performed at room temperature.The spectra were obtained in the 4000–400 cm−1 range byaccumulation of 60 scans at 4 cm−1 resolution. FTIR spectrawere recorded using the OMNIC software version 7.3 (Ther-mo Electron Corporation, USA). Prior to sample collection, abackground spectrum was recorded and subtracted from thesample spectra. The spectra were baseline corrected at1812 cm−1 and normalized for comparison purposes. Tworeplications for each formulation were performed.

Statistical analysis

Results are expressed as mean±standard deviation and wereanalyzed by analysis of variance (ANOVA). Means weretested with the Fisher least significant difference test for pairedcomparison, with a significance level α=0.05, using theStatgraphics Plus version 5.1 software (Statgraphics, USA).

Results and discussion

Rheology of film-forming dispersions

Rheological behavior of film-forming dispersions obtainedfrom phycocolloid-enriched fractions (P) added with glyceroland/or calcium is shown in Table 1. All dispersions exhibited anon-Newtonian shear-thinning behavior (n<1). The powerlaw (or the Ostwald de Waele model) fitted satisfactorily with

Table 1 Rheological behavior of film-forming dispersions obtained from phycocolloid-enriched fractions (P) added with glycerol and/or calcium

Formulations Ostwald de Waele parameters Apparent viscosity (mPa s)

K (Pa sn) n D=60 s−1 D=300 s−1 D=500 s−1

P 2.15±0.28b 0.47±0.01a 233±28b 104±12ab 78±9a

PG1 2.16±0.03b 0.47±0.01a 233±1b 107±2b 79±2a

PG2 2.05±0.13b 0.47±0.01a 235±16b 104±7ab 79±8a

PCa 1.59±0.05a 0.50±0.01b 205±5a 96±1ab 72±1a

PCaG1 1.57±0.04a 0.50±0.01b 199±1a 93±1ab 69±1a

PCaG2 1.50±0.06a 0.51±0.01b 195±8a 91±3a 69±2a

G1, 12.5 g glycerol/100 g dry solid; G2, 25 g glycerol/100 g dry solid; and Ca, 16 g Ca+2 /100 g dry solid. Results are expressed as mean value±standard

deviation. Different letters in the same column mean significant differences between samples (p<0.05), according to Fisher’s least significant differencetest

J Appl Phycol

the experimental data (r2>0.98) for P, PG1, PG2, PCa, PCaG1,and PCaG2 film-forming dispersions. The corresponding fittingparameters, n and K, are shown in Table 1. These results agreewith those of Marcotte et al. (2001) and Lizarraga et al. (2006)who worked with carrageenan aqueous dispersions at similarconcentration and temperature. The incorporation of glycerol tofilm-forming dispersions—with or without Ca2+—did notmodify its rheological properties. P, PG1, and PG2 dispersions,which differed in its glycerol concentrations, showed similarconsistency and flow indexes (K and n) and similar apparentviscosities (calculated at 60, 300, and 500 s−1) (p>0.05). Thesame effect was observed for PCa, PCaG1, and PCaG2 disper-sions (p>0.05). The presence of Ca2+ in film-forming disper-sions produced an important decrease in K and a slight increasein n (p<0.05), which only was reflected in the apparent viscos-ities at low deformations, as P, PG1, and PG2 film-formingdispersions have higher values of apparent viscosity than PCa,PCaG1, and PCaG2 (p<0.05) (only at 60 s−1) (Table 1). Thissuggests that the addition of calcium hindered the polymer–polymer interactions in P, PG1, and PG2 dispersions. Plasticizerand cross-linking effects expected by adding glycerol and Ca2+

were not reflected in the rheology of the film-formingdispersions.

Film characterization

Appearance and microstructure All phycocolloid-basedfilms, with or without glycerol and Ca2+, were found to behomogeneous and flexible. Table 2 shows their thickness,color parameters, and opacity. P, PG1, and PG2 films hadsimilar thickness values (~17 μm) (p>0.05), indicating thatglycerol addition not affect this property. In contrast, additionof Ca2+ significantly increased film thickness (~21 μm)(p<0.05). This result agrees with those of Rhim (2004), whoworked with alginate films with different levels of calciumchloride (0.01, 0.02, and 0.03 g CaCl2g

−1 alginate). In thiswork, the authors found that thickness of alginate films madewith 0.03 g CaCl2g

−1 alginate was significantly higher than

those obtained for alginate alone. This effect on film thicknesscould be attributed to the binding of Ca2+ to phycocolloidmatrix. Janaswamy and Chandrasekaran (2002) found thatcalcium ions and -carrageenan forms a threefold, right-hand-ed, half-staggered, parallel, double helix of 26.42 Å pitch,stabilized by hydrogen bonds. In this context, the stronginteractions between the sulfate groups of neighboring helices,mediated by calcium ions and water molecules, could beresponsible for stabilizing these three-dimensional structuresof higher thickness.

CIE-Lab color parameters (L*, a*, and b*), total colordifference (ΔE*), chroma (C*), hue (h), and opacity of filmsgive an idea of the visual aspect of the developed materials.All films present a greenish coloration, which was character-ized by negative a* values, given mainly by chlorophyllpigments that are present in P. columbina. In this regard,chlorophyll a and total chlorophyll content from P was 3.4±0.2 and 3.6±0.1 μg/g, respectively. The film’s color wasmodified only by the addition of Ca2+. PCa, PCaG1, andPCaG2 films had lower L*, a*, and b* values than thoseobtained from P, PG1, and PG2 films (p<0.05). However,no significant differences were obtained for ΔE*, C*, and hvalues (p>0.05) from different films. The reduction of L*, a*,and b* also was observed by Rhim (2004) for alginate filmswith 0.12 g CaCl2/100 g polysaccharide.

Film opacity is a critical property if the film will be used as asurface food coating. Transparent films are characterized by lowvalues of the area below the absorption curve. As shown inTable 2, glycerol addition decreased the opacity of the resultingfilms (p<0.05), while the presence of Ca2+ did not affect it(p>0.05). In this sense, PG1, PG2, PCaG1, and PCaG2 filmswere less opaque than the P and PCa ones. These results agreewith those reported by García et al. (2006) for corn starch andchitosan films plasticized by glycerol. It is noteworthy that Ca2+

addition did not increase film opacity as happens in otherpolysaccharide-based films (Fang et al. 2002; Fabra et al. 2010).

Undoubtedly, intense color limits some potential applica-tions of these materials in food packaging. For example, they

Table 2 Thickness, CIE-Lab color parameters (L*, a*, and b*), total color difference (ΔE*), chroma (C*), hue (h), and opacity of films obtained fromphycocolloid-enriched fractions (P) with glycerol and/or calcium

Formulations Thickness (μm) Color parameters Opacity×10−4 (UA)

L* a* b* ΔE* C* h

P 16.1±0.2a 79.4±1.0b −3.1±0.1b 20.6±0.5b 26.3±0.5a 20.9±0.7a 98.6±0.3a 14.3±0.0b

PG1 17.1±0.2a 80.0±0.6b −3.2±0.2b 20.6±0.9b 25.9±0.4a 20.8±1.3a 98.8±0.6a 11.7±0.1a

PG2 18.0±2.3a 79.6±0.8b −3.2±0.1b 20.5±0.3b 26.1±0.5a 20.7±0.3a 98.7±0.2a 10.4±1.2a

PCa 21.3±0.9b 77.9±0.6a −2.9±0.0a 19.5±0.3a 26.5±0.4a 19.7±0.4a 98.4±0.3a 14.1±0.2b

PCaG1 21.6±0.4b 77.9±1.1a −2.9±0.0a 19.5±0.5a 26.5±0.7a 19.7±0.7a 98.5±0.3a 10.5±0.4a

PCaG2 21.9±0.7b 77.6±1.0a −2.9±0.0a 19.3±0.2a 26.7±0.8a 19.6±0.3a 98.6±0.2a 10.4±1.7a

G1, 12.5 g glycerol/100 g dry solid; G2, 25 g glycerol/100 g dry solid; and Ca, 16 g Ca+2 /100 g dry solid. Results are expressed as mean value±standard

deviation. Different letters in the same columnmean significant differences between samples (p<0.05), according to Fisher’s least significant difference test

J Appl Phycol

could not be used for products that should be easily visiblethrough the package (such as minimally processed vegetables)because the impaired visualization may reduce the acceptabil-ity of potential consumers. In contrast, such films could beused, if their properties are adequate, for applications in wherecolor is irrelevant or in those where color may have anadditional usefulness, as in the case of packaging productsthat are sensitive to visible light radiations (i.e., that can bealtered or degraded by 400–800 nm light), such as dairyproducts, baby foods, soy-based sauces, and nutritional ormedicinal products (Salgado et al. 2010; Krikor et al. 2011).

Water susceptibility of films The main disadvantage of filmsderived from biopolymers is their high water susceptibility,which, in general, is affected by the hydrophilic or hydrophobicnature of the plasticizers and cross-linkers added to formulationto modify their mechanical properties. Table 3 shows the mois-ture content (MC), water solubility (WS), and water vaporpermeability (WVP) of the obtained films. Glycerol additionsignificantly increases the moisture content of films with orwithout Ca2+ (PG1, PG2, PCaG1, and PCaG2). This result wasexpected since glycerol is a highly hygroscopic molecule gen-erally added into film-forming dispersions to prevent filmbrittleness (Shaw et al. 2002). In this sense, Karbowiak et al.(2006) found that water content of carrageenan films was farmore sensitive to relative humidity when glycerol was includ-ed. In that work, the authors showed that glycerol increases thewater sorption of carrageenan film, and this increment becomesmore significant at water activity above 0.7, whereas no signif-icant difference is noticeable for water sorption at lower wateractivities. It is noteworthy that the addition of Ca+2 also in-creased the moisture content of films. Therefore, Ca2+ additionnot only could be stabilizing the three-dimensional structure ofP phycocolloids by interactions between sulfate groups but alsopromoting water retention. This was corroborated, since mois-ture content of PCa was significantly higher than that of P(p<0.05). PCaG1 and PCaG2 exhibited the highest moisturecontent (~19 %), indicating that Ca+2 exerts an additional effectwith glycerol. Film water solubility showed a similar behavior

to moisture content. P films had the lowest WS(~85 %) (Table 2). This property significantly increased inPG1 and PG2 films with glycerol addition (p<0.05) and inPCa with Ca+2 addition (p<0.05) and was the highest inPCaG1 and PCaG2 films (~98 %). This effect is commonlyfound by adding glycerol to formulations based on biopoly-mers and is favored precisely because of the hydrophilicnature of the molecule (Baldwin et al. 2012). In the case ofCa+2, it is clear that any additional interactions that could beformed among phycocolloid chains bridged by these ions areunstable in water, this being facilitated by the greater thicknessof the respective films and its greater water content.

Table 3 also shows the WVP of the studied films. As can beseen, the addition of glycerol to P matrix increases the WVP.This phenomenon is related to the ability of the plasticizer toestablish favorable hydrogen bonds with the polymer, therebydisrupting intermolecular polymer interactions (this mechanismbeing favored by high relative humidities), with consequentincrease of polymer chain mobility and decreasing film barrierproperties (Gontard et al. 1993; Hernández-Muñoz et al. 2004).A similar value of WVP was reported by Martins et al. (2012)for κ-carrageenan film with 30 g glycerol (100 g)−1 polysac-charide. On the other hand, the addition of Ca+2 increases theWVP, regardless of the glycerol presence, suggesting that ahigh Ca2+ level masks the glycerol effect. Additionally, PCa,PCaG1, and PCaG2 films exhibit the greater thickness. Asreported previously for other hydrophilic films (pectin, am-ylose, cellulose ethers, sodium caseinate, and soybean pro-teins), WVP increases with film thickness (Ghorpade et al.1995).

Mechanical properties of films Tensile strength (TS), elonga-tion at break (EAB), and elastic modulus (EM) of the films areshown in subpanels a–c of Fig. 1, respectively. Mechanicalproperties of P films were very interesting. Even without therequirement of a plasticizer, films were flexible and had veryhigh TS (~50 MPa) and EM (~29.5 MPa), but had low EAB(~3 %). Similar values of TS and EM were reported by Rhim(2012) for agar/κ-carrageenan blend films.

Table 3 Moisture content (MC), water solubility (WS), and water vapor permeability (WVP) of films obtained from phycocolloid-enriched fractions (P)from P. columbina added with glycerol and/or calcium

Formulations MC (g 100 g−1) WS (%) WVP×1011 (g H2O Pa−1 s−1 m−1)

P 10.7±0.2a 84.5±2.2a 4.9±0.1a

PG1 11.9±0.4b 95.0±0.2b 5.2±0.1b

PG2 16.0±0.4c 95.1±2.2b 5.2±0.2b

PCa 17.0±0.6d 96.3±1.6bc 6.7±0.1c

PCaG1 18.7±0.4e 98.1±0.8c 6.8±0.1c

PCaG2 18.9±0.3e 98.7±0.7c 6.6±0.2c

G1, 12.5 g glycerol/100 g dry solid; G2, 25 g glycerol/100 g dry solid; and Ca, 16 g Ca+2 /100 g dry solid. Results are expressed as mean value±standard

deviation. Different letters in the same column mean significant differences between samples (p<0.05), according to Fisher’s least significant difference test

J Appl Phycol

As glycerol content increased in the formulation (PG1 andPG2), films progressively decreased TS and EM (p<0.05) andgradually increased EAB (p<0.05). These results were ex-pected because glycerol acts as a plasticizer on P matrixinterfering with the formation of polymer–polymer interac-tions and favored by the additional plasticizing effect ofexcess water absorbed by glycerol. In this sense, a smallincrease in glycerol level results in a large drop in tensilestrength with concomitant increase of EAB and WVP

(Debeaufort and Voilley 1997; Tanaka et al. 2001; Fairleyet al. 1996).

As shown in Fig. 1, PCa, PCaG1, and PCaG2 filmsexhibited higher EAB and lower TS and EM values than P,PG1, and PG2, respectively (p<0.05). It seems that Ca2+ alsoexerts a plasticizing effect on P matrix with and withoutglycerol. These results can be related to Ca2+ that increaseswater content of P matrix. And in this regard, as was previ-ously mention, water molecules could act as strong plasti-cizers because of their increased molecular mobility and elas-ticity in the rubbery state (Karbowiak et al. 2006). Accordingto Sothornvit and Krochta (2000), there are two main types ofplasticizers: (1) molecules capable of forming hydrogenbonds, thus interacting with polymers by interrupting poly-mer–polymer bonding and maintaining the furthest distancebetween polymer chains and (2) molecules capable ofinteracting with large amounts of water to retain more watermolecules, thus resulting in higher moisture content and largerhydrodynamic radius. In this case, it seems that the plasticiz-ing effect of both glycerol and Ca+2 could be associated toboth described mechanisms.

Film microstructure Figure 2 shows SEM micrographs ofsurface and cross section of films. P film was characterizedby a compact, uniform, dense structure and homogenousappearance, indicating a good compatibility betweencarrageenans/porphyrans from P. columbina aqueous fraction.P, PG1, and PG2 film micrographs showed slightly roughsurfaces without any pores. The roughness becomes lessnoticeable by increasing the proportion of glycerol in theformulation. As can be seen, Ca+2 addition increased the filmroughness, being more evident inPCa. As glycerol proportionincreased in these formulations, rough surfaces also graduallydecreased (PCaG1 and PCaG2 films). These results are inagreement with those reported by Bosquez-Molina et al.(2010) for mesquite gum-based edible films containing differ-ent concentrations of CaCl2 (0.0, 0.1, 0.2, 0.3, 0.4, or0.5 g (100 g)−1) plus glycerol (1.5 g (100 g)−1).

SEM micrographs of cross-section films (Fig. 2) confirmthat Ca+2 addition significantly increased film thickness, thiseffect being clearly seen in PCaG1 film. Therefore, Ca2+

would interact with sulfate groups by opening film structure,promoting water retention, and increasing film thickness,causing a net plasticizer effect on the film network that wasreflected in their mechanical properties and watersusceptibility.

Although Ca+2 addition did not exert the expected cross-linking effect, improving the mechanical and/or barrier prop-erties of films, their interactions with the sulfate groups wereverified by FTIR. Infrared spectroscopy is a rapid and non-destructive technique that has been widely used to character-ize different polysaccharides (Martins et al. 2012). Figure 3shows FTIR spectrum (1400–700 cm−1) of P and PCa films.

e

c

d

a

b

a

0

10

20

30

40

50

60

P PCa PG1 PCaG1 PG2 PCaG2

Ten

sile

Str

engt

h (M

Pa)

a

cb

e

d

f

0

10

20

30

40

50

P PCa PG1 PCaG1 PG2 PCaG2

Elo

ngat

ion

at b

reak

(%

)

e

c

d

a

b

a

0

5

10

15

20

25

30

35

P PCa PG1 PCaG1 PG2 PCaG2

Ela

stic

Mod

ulus

(M

Pa)

c

b

a

Fig. 1 Mechanical properties measured in tensile tests: a Tensile strength(TS), b elongation at break (EAB), and c elastic modulus (EM) of filmsobtained from phycocolloid-enriched fractions (P) from P. columbinaadded with glycerol and/or calcium. G1, 12.5 g glycerol (100 g)−1 drysolid; G2, 25 g glycerol (100 g)

−1 dry solid; and Ca, 16 g Ca+2 (100 g)−1

dry solid. Results are expressed as mean value±standard deviation.Different letters in the barsmean significant differences between samples(p<0.05), according to Fisher’s least significant difference test

J Appl Phycol

Usually, this region is used to obtain information on thestructure of phycocolloids (agars and carrageenans). P spectrashow the characteristic bands of agarocolloids like porphyran(1372, 1250, 1150, 1076, 933, 890, 820, and 771 cm−1). Theseresults agree with those of Zhang et al. (2005) for P. capensis

phycocolloids. The bands at 1370 and 1250 cm−1 can beattributed to the S=O vibration of the sulfate groups. Theimportant band at 1076 cm−1 corresponds to the skeleton ofgalactans, while the band at 890 cm−1 is assigned specificallyto agar-specific bands. Bands at 930 cm−1 in the spectracorrespond to the C–O–C group of 3,6-anhydro-α-L-galactose(Souza et al. 2012). Additionally, the bands at 845, 830, and820 cm−1 are used to infer the position of sulfate group inagarocolloids and are assigned to the 4-sulfate, 2-sulfate, and6-sulfate of D-galactose units, respectively (Maciel et al. 2008;Gómez-Ordóñez and Rupérez 2011). The FTIR spectrum of Pshows a low intensity band at 845 cm−1, attributed to sulfatesubstitution at the C-4 of galactose. The presence of a smallpeak at 820 cm−1 could indicate a low degree of substitutionon C-6. The absence of bands at 830 cm−1 indicates that 2-sulfate galactose and sulfate on the C-2 of 3,6-anhydro-α-L-galactose are not present. These results are in agreement withthose reported by Melo et al. (2002) and Maciel et al. (2008)who worked with two different red seaweeds. Also, the littleband at 890 cm−1 could be attributed to anomeric CH of β-galactopyranosyl residues. Finally, the bands at 1150 and775 cm−1 can be assigned to C–O stretching vibrations ofpyranose ring, in agreement with the FTIR spectrum of dif-ferent red seaweeds reported by Gómez-Ordóñez and Rupérez(2011). On the other hand, only for PCa spectrum, lower

Surface Cross-section Surface Cross-section

without calcium with calciumG

lyce

rol c

onte

nt (

g/10

0 g

dry

solid

s) 0

12.5

25

P

PG1

P

PG2PG2

PG1

PCa

PCaG1

PCa

PCaG2PCaG2

PCaG1

Fig. 2 Scanning electron microscopy (SEM) of films obtained fromphycocolloid-enriched fractions (P) from P. columbina added with glyc-erol and/or calcium. G1, 12.5 g glycerol (100 g)−1 dry solid; G2, 25 g

glycerol (100 g)−1 dry solid; and Ca, 16 g Ca+2 (100 g)−1 drysolid. SEM image magnifications: ×50 for film surface and ×100 for filmcross-section

1400 1300 1200 1100 1000 900 800 700

0.6

1.2

1.8

2.4

3.0

Abs

orba

nce

Wavenumber (cm-1)

PCa

1400 1300 1200 1100 1000 900 800 700

0.6

1.2

1.8

2.4

3.0

1150

890820 7751372

1250

1076

Abs

orba

nce

933

P

Fig. 3 FTIR spectrum (1400 to 700 cm−1) of films obtained fromphycocolloid-enriched fractions (P) and phycocolloid-enriched fractionsadded with calcium at 16 g Ca+2 (100 g)−1 dry solid (PCa)

J Appl Phycol

absorbance at 1372, 1250, and 820 cm−1 than Pwas observed.Therefore, the addition of Ca+2 could affect the spectroscopicbehavior of sulfate groups present in phycocolloids. In thissense, Sartori et al. (1997) found that the addition of CaCl2 toalginate films produced a large shift at 1420 cm−1 (COO-symmetrical stretch peak), reducing band intensity. In thatwork, the absorbance reduction was associated to the replace-ment of sodium by calcium, which changes charge density, theradius, and the atomic weight of the cation associate to thehydrocolloid, creating a new environment around the carbonylgroups (Sartori et al. 1997). This phenomenon could alsooccur in PCa films where calcium would replace sodiumassociated with sulfate group. Furthermore, rates of absor-bance at 1250 cm−1/2920 cm−1 and 1370 cm−1/2920 cm−1

are indexes which relate total sulfate to sugar content (Meloet al. 2002; Rochas et al. 1986). These ratios were 1.5 and 0.9for P films, and 1.1 and 0.6 for PCa films. These results are inconcordance with those mentioned before and suggest a pos-sible effect of Ca+2 on sulfate group from agars and/orcarrageenans.

Conclusions

It was possible to produce biodegradable films based onphycocolloids (carrageenans/porphyrans) extracted fromP. columbina with a wide range of mechanical properties byincorporation of glycerol and/or Ca+2 into film-forming dis-persions. The addition of glycerol to film-forming dispersionswithout Ca2+ allowed producing less resistant but morestretchable and water-susceptible films (with higher watersolubility and water vapor permeability) than those producedin the absence of a plasticizer. Regarding calcium addition, insome properties, Ca2+ masks glycerol effect (water solubilityand water vapor permeability), but in others, the effect ofcalcium is added to a plasticizer glycerol effect (mechanicalproperties). Calcium could stabilize the three-dimensionalstructure of P phycocolloids by interactions between sulfategroups, promote water retention, and open film structure.

Acknowledgments The authors are thankful to the National Agency ofScientific and Technological Support (SECyT, PICT-2010-1837), theUniversidad Nacional de La Plata (11-X494), and the UniversidadNacional del Litoral (CAI+D 2011 PI 0292 LI) for their financial support.

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