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LWT - Food Science and Technology 59 (2014) 533e539

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LWT - Food Science and Technology

journal homepage: www.elsevier .com/locate/ lwt

Characterization of fish protein films incorporated with essential oilsof clove, garlic and origanum: Physical, antioxidant and antibacterialproperties

Bárbara Teixeira a,b,c, António Marques a,c,*, Carla Pires a,c, Cristina Ramos a,Irineu Batista a,c, Jorge Alexandre Saraiva b, Maria Leonor Nunes a,c

aDivision of Aquaculture and Upgrading (DivAV), Portuguese Institute for the Sea and Atmosphere (IPMA, I.P.), Av. Brasília, 1449-006 Lisbon, PortugalbResearch Group of Organic Chemistry, Natural and Agro-food Products (QOPNA), Chemistry Department, Aveiro University, Campus Universitáriode Santiago, 3810-193 Aveiro, Portugalc Interdisciplinary Center of Marine and Environmental Research (CIIMAR), University of Porto, Rua das Bragas, 289, 4050-123 Porto, Portugal

a r t i c l e i n f o

Article history:Received 9 February 2012Received in revised form16 May 2012Accepted 12 April 2014Available online 25 April 2014

Keywords:Hake muscle proteinsBiodegradable filmsActive packagingBioactive propertiesPhysical properties

* Corresponding author. Division of AquaculturPortuguese Institute for the Sea and Atmosphere (IPMLisbon, Portugal. Tel.: þ351 213 027 025; fax: þ351 2

E-mail addresses: amarques@ipma.pt, marques_amURL: http://www.ipma.pt/en/investigacao/index.js

http://dx.doi.org/10.1016/j.lwt.2014.04.0240023-6438/� 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

Consumers are demanding high quality foods using environmental-friendly packaging systems andnatural preservatives, like edible/biodegradable films or coatings with bioactive properties. In thiscontext, films prepared with Cape hake by-products proteins were combined with three essential oils(garlic, clove, and origanum) and characterized in terms of physical, mechanical, antioxidant, and anti-bacterial properties. Control films, without essential oils, were homogeneous, transparent, slightly yel-low, and mechanically resistant. The incorporation of garlic, clove, and origanum essential oils in this filmsignificantly decreased thickness, water solubility, breaking force and elongation, whereas increased thefree radical scavenging activity of films. Particularly, clove films showed lower water vapour permeabilitythan control films, and the highest antibacterial activity (against Shewanella putrefaciens). Garlic filmswere the most yellowish and had the highest antioxidant activity. Finally, origanum films were rathersimilar to the control, particularly in colour, transparency and reducing power. In conclusion, proteinsrecovered from Cape hake by-products combined with essential oils have adequate properties withapplicability in new preservation food packaging systems.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

In recent years, edible and biodegradable films and coatingsprepared with proteins, polysaccharides, and lipids have receivedincreasing attention. Some examples of their commercial applica-tions are: collagen casings for sausages, confectioner’s glaze madefrom shellac, corn zein and gelatin-based coatings for pharma-ceuticals, and waxes on various fruits (Gennadios, Hanna, & Kurth,1997). Factors contributing to the interest in films and coatingsdevelopment include: consumers demand for high quality foodsand natural preservatives; food processors’ needs for new storagetechniques; environmental concerns over disposal of nonrenew-able food packaging materials; and opportunities for creating new

e and Upgrading (DivAV),A, I.P.), Av. Brasília, 1449-00613 015 948.@yahoo.com (A. Marques).p

market outlets for under-utilized film-forming ingredients(Gennadios et al., 1997).

Edible and biodegradable films must meet a number of specificfunctional requirements, including colour, appearance, barrierproperties, mechanical, and rheological characteristics, which aredependent on the type of material used and type of application(Guilbert, Gontard, & Gorris, 1996). Films primarily composed ofproteins usually have suitable mechanical and optical properties,but show poor water vapour barrier properties because of theirhydrophilic nature (Guilbert et al., 1996). Active compounds likeessential oils can be added to films to improve their functionalproperties, such as water vapour permeability, as well as antimi-crobial and antioxidant properties (García, Martino, & Zaritzky,2000; Oussalah, Caillet, Salmiéri, Saucier, & Lacroix, 2004; Seydim& Sarikus, 2006).

In the seafood processing industry, a substantial amount of by-products are generated that can be used to recover proteins toprepare films and restructured seafood products. Protein films havebeen successfully prepared using fish proteins, including

B. Teixeira et al. / LWT - Food Science and Technology 59 (2014) 533e539534

myofibrillar and sarcoplasmic proteins (e.g. Benjakul, Artharn, &Prodpran, 2008; Cuq, Gontard, & Guilbert, 1997). Essential oils ofaromatic plants like clove, garlic, and origanum show strong anti-microbial and antioxidant properties (e.g. Benkeblia, 2004;Bounatirou et al., 2007; Misharina & Samusenko, 2008). There-fore, incorporation of essential oils in films can improve function-alities of films. In this context, the aim of the current work was tostudy the physical, mechanical, antioxidant, and antibacterialproperties of films prepared with fish proteins recovered from Capehake by-products and essential oils from aromatic plants (clove,garlic and origanum).

2. Material and methods

2.1. Chemicals

Essential oils of clove from Eugenia spp. (C8392; lot 116K1861;buds distillation; origin: Indonesia; SigmaeAldrich), garlic (bio-logical source not specified; W250317; lot 04712 EE-148; bulbssynthetic organic material; origin: Mexico; SigmaeAldrich), andoriganum from Thymus capitatus (W282812; lot 21417CL-214; driedflowering herb steam distillation; origin: Spain; SigmaeAldrich),glycerol, a,a-diphenyl-b-picrylhydrazyl (DPPH), sodium bromide,trizma hydrochloride (TriseHCl), sodium azide, and potassiumhexacyanoferrate III were obtained from SigmaeAldrich (SigmaAldrich Chemie GmbH, Steinheim, Germany); phosphate buffer,ferric chloride, trichloroacetic acid, and ascorbic acid were pur-chased from Fluka (Buchs, Germany); tryptic soy agar and platecount agar fromMerck (Darmstadt, Germany); brain heart infusionbroth and maximum recovery diluent from Oxoid (Basingstoke,Hampshire, England); ethylene diamine tetracetic acid (EDTA cali-bration sample) from LECO (LECO corporation, St. Joseph, USA);ethanol had a purity grade of 99% and the water used was Milli-Qpurified and distilled.

2.2. Film preparation

Fish proteins were recovered from frozen by-products resultingfrom the portioning (fish ‘sawdust’ and cut offs) of Cape hake(Merluccius capensis) by alkaline solubilisation following a meth-odology previously described (Batista, Pires, Nelhas, & Godinho,2006). Recovered proteins (90 g/100 g protein content) werefreeze-dried, packed under vacuum conditions, and stored at�30 �C until utilisation.

Hake protein powder (30 g) was added to water (2 L) and ho-mogenized (5000 rpm, 1 min) using a Polytron homogenizer. ThepH was adjusted to 11 with 1 mol/L sodium hydroxide and me-chanically stirred, followed by centrifugation (10,000 g, 15 min,5 �C) to remove insoluble material. The protein concentration of thesoluble fractionwas determined (see methodology below), glycerolwas added at 59 g/100 g of protein, and the mixture was gentlystirred (30 min). Afterwards, the essential oils of clove, garlic, andoriganumwere added to protein film forming solutions, emulsifiedin a Polytron homogenizer (13,500 rpm, 2 min) and the emulsionwas degassed under vacuum (20 min) and casted on plates toobtain films with 4 mg of protein and 1 mL of essential oil per cm2

when present. Control films had the same amount of protein persurface area. The plates were placed on levelled surfaces to obtainfilms with homogeneous thickness, dried in a ventilated dryingchamber (30 �C, 50% relative humidity, 20 h), peeled off, and storedat room temperature at 57% relative humidity in desiccators withsaturated solutions of sodium bromide. Four types of films wereprepared (treatments): a) without essential oils; b) with cloveessential oil; c) with garlic essential oil; and d) with origanumessential oil.

The protein content of the soluble fraction was determined us-ing a FP-528 LECO nitrogen analyser (LECO, St. Joseph, USA), cali-brated with EDTA (carbon e 41.07 � 0.17, hydrogen e 5.55 � 0.02,nitrogen e 9.57 � 0.03), according to the Dumas method (Saint-Denis & Goupy, 2004). All determinations were performed intriplicate.

2.3. Colour

Films colour parameters (L*, a* and b*) weremeasured (n¼ 6 foreach film) using a colorimeter (CR-410, Konica Minolta Camera, Co,Ozaka, Japan) with a measure cell opening of 50 mm. For mea-surements, films were placed on a white standard plate. Chroma(C*), hue (h*) and whiteness (W) were estimated using thefollowing equations, accordingly to Atarés, Bonilla, and Chiralt(2010):

C* ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiða*Þ2 þ ðb*Þ2

q

h* ¼ arctgb*a*

W ¼ 100�ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffið100� L*Þ2 þ ða*Þ2 þ ðb*Þ2

q

Colour was expressed as the difference of colour (DE*) in thedifferent parameters, accordingly to the equation (García & Sobral,2005):

DE* ¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiðDL*Þ2 þ ðDa*Þ2 þ ðDb*Þ2

q

where DL*, Da* and Db* correspond to the variation between thecolour parameter of film and that of the white standard plate usedas background.

2.4. Transparency

The absorbance of films (600 nm; spectrophotometer UNICAMUv/Vis UV2; ATI-UNICAM, Cambridge, United Kingdom) and filmthickness (mm; in the same location of absorbance readings) weremeasured (n¼ 5 for each film) in order to address film transparencyusing the following equation:

Transparency ¼ A600

x

where A600 is the absorbance of films and x is the film thickness(mm) (Shiku, Hamaguchi, & Tanaka, 2003). Films less transparentshow higher transparency values.

2.5. Opacity

Opacity was measured accordingly to the Hunterlab method(Anonymous, 2008) using the same equipment of colour mea-surement. The opacity (%) of films was calculated with reflectancemeasurements of each film (n ¼ 6) with standard black and whitebacking plates, accordingly to the following equation:

Opacity ¼ Yblack backing

Ywhite backing� 100

where Y is the CIE tristimulus value of the filmwith the black (Yblackbacking) or white (Ywhite backing) backing plates.

B. Teixeira et al. / LWT - Food Science and Technology 59 (2014) 533e539 535

2.6. Thickness

Film thickness was measured in nine different locations of eachfilm, using a Digimatic tube micrometer model BMD-25D (MITU-TOYO MFG Co. Ltd., Kawasaki, Japan).

2.7. Water vapour permeability

Water vapour permeability (WVP) was measured in duplicatefor each film using the method described by Gontard, Guilbert, andCuq (1992). The films were sealed in cells containing silica gel (0%RH) and then stored at 30 �C in desiccators with water. The cellswere weighed at regular intervals for a period of 30 h, and theWVPestimated as follows:

WVP ¼ w� xA� t � ðP2 � P1Þ

where w is the weight gain (g), x is the film thickness (m), A is theexposed area of film (m2), t is the time of weight gain (s), and P2eP1is the vapour pressure differential across the film (Pa).

2.8. Film and protein solubility

Film solubility in water (n ¼ 5) was determined according to themethod of Hoque, Benjakul, and Prodpran (2011). Films (3 � 2 cm2)were weighted and immersed in water (10 mL) with sodium azide(0.1 g/100 mL). The mixture was continuously shaken (24 h; roomtemperature) and centrifuged (3000 g; 10 min; 25 �C). Unsolubi-lised films were dried (24 h; 105 �C) and film solubility wasdetermined by subtracting the weight of unsolubilised dry matterfrom the initial weight of dry matter and expressed as a percentageof the total weight.

The protein concentration in the water (n ¼ 5) was determinedusing the Bradford method (Bio-Rad Protein Assay Kit, Bio-Rad,Hercules, California, USA). Protein solubility was expressed aspercentage of total solubilised film protein at 20 �C for 24 h.

2.9. Mechanical properties

Prior to measuring the mechanical properties, films wereconditioned in desiccators with saturated solutions of sodiumbromide (57% relative humidity) at room temperature during 72 h.The puncture and tensile tests were performed using an Instrontexture analyser (Instron 4301, Bucks, United Kingdom) and dataobtained with the Instron Corporation software (version 5.02,1985e1990).

The force and deformation at the films breaking point weredetermined using a puncture test. Each film (10� 10 cm2) was fixedbetween two cells with a circular hole (16 mm Ø) in the centre ofthe cells, where a 3 mm diameter plunger moving at 6 cm min�1

perforated the films. The puncture deformation (PF) was calculatedconsidering that the stress was homogeneously distributed alongthe film at the breaking point (Sobral, Menegalli, Hubinger, &Roques, 2001), with the following equation:

PF ¼ Dll0

¼ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiD2 þ l20

q� l0

l0

whereD is the stretch distance of films at the breaking point, l is thefinal length of the film and l0 is the initial length of the film (equal tothe radius of the cell hole). Five films were used per treatment, withnine measurements recorded per film.

Tensile strength (i.e. force at breaking point per initial cross-sectional area) and elongation percentage were measured using

film strips (10 � 2 cm2) fixed in the extremities of a self-aligninggrip. The initial grip separation was 5 cm and crosshead speedwas 6 cm min�1. Tensile tests were carried out in 16 film strips pertreatment.

2.10. Antioxidant activity

The antioxidant activity of films was evaluated using filmpowders. Films were ground with a pestle in a mortar, using liquidnitrogen.

2.10.1. Free radical scavengingThe scavenging effect of DPPH free radical was determined in

triplicate according to the method of Weng, Osako, and Tanaka(2009). Film powders (20 mg) in 2 mL TriseHCl buffer (0.1 mol/L,pH 7.4) were added to 2 mL of 0.2 mmol/L DPPH in ethanol (95 mL/100 mL). The mixture was shaken vigorously and allowed to standat room temperature in the dark for 30 min. The mixture wascentrifuged (4500 g, 10 min) and the absorbance of supernatantwas recorded at 517 nm in a spectrophotometer (UNICAM UV/VisUV2; ATI-UNICAM, Cambridge, United Kingdom). A negative con-trol was prepared using all reagents, except the film powder. DPPHradical-scavenging activity was calculated with the followingformula:

Percentage of inhibition ð%Þ ¼ Abscontrol � Abssample

Abscontrol� 100

where Abssample and Abscontrol correspond to the absorbance ofsample and control, respectively.

2.10.2. Reducing powerThe reducing power of films was determined in triplicate ac-

cording to the modified method of Oyaizu (1986) as described byWeng et al. (2009). Film powders (20 mg) with 2 mL water, 2 mLphosphate buffer (0.2 mol/L, pH 6.6) and 2 mL potassium hex-acyanoferrate III (1 g/100 mL) were incubated at 50 �C for 20 min.Afterwards, 2 mL trichloroacetic acid (10 g/100mL)were added andthe mixture was centrifuged (1500 g, 10 min, room temperature). A2 mL aliquot of the supernatant was mixed with 2 mL water and0.4 mL ferric chloride (0.1 g/100 mL). The absorbance was recordedafter 10 min at 700 nm in a spectrophotometer (UNICAM UV/VISUV2; ATI-UNICAM, Cambridge, United Kingdom). A negative con-trol was prepared using all reagents except the film powder,whereas a positive control was also prepared (10e70 mg mL�1

range) in order to plot the absorbance of ascorbic acid againstconcentration. The results were expressed as mg of ascorbic acidper g of film.

2.11. Antibacterial activity

The antibacterial activity tests included foodborne spoilage andpathogenic bacteria acquired from the American Type CultureCollection (ATCC) or the Spanish Type Culture Collection (CECT):Brochothrix thermosphacta (CECT 847), Escherichia coli (ATCC25922), Listeria innocua (CECT 910), Listeria monocytogenes (CECT5873), Pseudomonas putida (CECT 7005), Salmonella typhimurium(ATCC 14028), and Shewanella putrefaciens (CECT 5346). Thesestrains, kept at �70 �C in a cryopreservative solution (Microbank,Pro-lab Diagnostics, Richmond Hill, ON, Canada), were inoculatedin tryptic soy agar (TSA) and incubated overnight at 30 �C, exceptL. monocytogenes that was inoculated in plate count agar (PCA).Subsequently, one colony from each culture was inoculated in brainhearth infusion broth (BHI) and incubated at 30 �C for 18e24 hwith

Table 1Colour parameters of fish protein films (n ¼ 6). In each line, different italized lettersdenote significant differences (p < 0.05) within films. Values are presented asaverage�standard deviation. Abbreviations: L*, a* and b*e colour parameters of theCIE L*a*b* system; C*e chroma; h*e hue;Wewhiteness; DE*e difference of colourbetween colour parameters of film and that of the white standard plate used asbackground.

No essential oil Garlic Clove Origanum

L* 93.6 � 0.6a 91.9 � 0.3b 91.9 � 1.0b 93.4 � 0.3a

a* �1.7 � 0.1b �1.9 � 0.1c �1.7 � 0.1b �1.4 � 0.0a

B* 5.9 � 0.6c 12.0 � 1.0a 8.9 � 2.6b 5.8 � 0.2c

C* 6.1 � 0.6c 12.2 � 1.0a 9.1 � 2.6b 5.9 � 0.2c

H* 106.6 � 0.9a 99.2 � 0.6b 101.2 � 2.4b 104.0 � 0.5a

W 91.1 � 0.8a 85.4 � 0.9b 87.8 � 2.6b 91.1 � 0.4a

DE* 4.7 � 0.6b 11.0 � 1.0a 8.0 � 2.8a 4.6 � 0.2b

B. Teixeira et al. / LWT - Food Science and Technology 59 (2014) 533e539536

shaking (75 rpm), in order to obtain freshly cultured microbialsuspensions (108e109 CFU mL�1) for tests.

Antibacterial activity of the films was evaluated by the agardiffusion method. Freshly bacterial suspensions were adjusted to1 � 107 CFU mL�1 in BHI and spread on the surface of TSA or PCAusing a sterile cotton swab. Subsequently, edible film discs (1.5 cmØ) were placed on the agar surface. After staying at 4 �C for 2 h, Petridishes were incubated at 30 �C for 24 h, except L. monocytogenesthat was incubated during 48 h. Antibacterial activity was evalu-ated by measuring the total diameter of the inhibition zone,including the film disc, to the nearest millimetre.

The antibacterial properties of films were also evaluated withthe macrodilution method (NCCLS, 1999). Films (5 � 5 cm2) wereimmersed in 10 mL of the bacterial suspensions (1 �107 CFUmL�1)in BHI. After staying 4 h at 30 �C with shaking (75 rpm) ten-folddilutions of the bacterial suspensions were performed inmaximum recovery diluent and spread in TSA or PCA for thedetermination of the bacterial concentration.

In both methods, bacterial suspensions without films were usedas positive controls, whereas films without bacterial strains wereused as negative controls. All determinations were performed intriplicate.

2.12. Statistical analysis

Differences between films were tested with analysis of variance(ANOVA), followed by multiple comparisons tests (Tukey HSD). Ifdata could not meet ANOVA assumptions, non-parametric analysisof variance (Kruskall-Wallis) was performed, followed by non-parametric multiple comparisons test (Dunn). All statistical ana-lyses were tested at 0.05 level of probability with the softwareSTATISTICA� 6.1 (Statsoft, Inc., Tulsa, OK, USA).

Table 2Physical and mechanical properties of fish protein films tested. In each line, differentpresented as average �standard deviation.

n No essential oil

Transparency 5 1.8 � 0.1c

Opacity 6 15.9 � 0.9a

Thickness (mm) 9 28.3 � 4.0a

Water vapour permeability( � 10�11 g m�1 s�1 Pa�1)

2 5.9 � 0.1ab

Puncture tests 9Force (N) 6.1 � 2.0a

Deformation (mm) 7.0 � 1.1b

Puncture deformation (%) 33.5 � 9.7b

Tensile tests 16Force (N) 3.5 � 0.9a

Elongation (%) 147.9 � 31.8a

Tensile strength (Mpa) 6.1 � 1.7a

3. Results and discussion

3.1. Physical properties

Colour parameters of films are shown in Table 1. Fish proteinfilms showed high L* values and were slightly yellowish. Similarresults were previously reported for fish muscle proteins-basedfilms (e.g. Artharn, Benjakul, Prodpran, & Tanaka, 2007;Chinabhark, Benjakul, & Prodpran, 2007; Limpan, Prodpran,Benjakul, & Prasarpran, 2010). The addition of essential oilsaffected some colour parameters depending on the essential oilused, i.e. darker, yellowish, higher chroma, lower hue and lesswhiteness films (garlic and clove), slightly less green (origanum)and slightly more green (garlic). In previous studies, the addition ofessential oils also affected film colour parameters, being influencedby the amount and type of essential oil used (e.g. Atarés, Bonilla,et al., 2010; Atarés, Jesús, Talens, & Chiralt, 2010).

Fish protein films without essential oil and with origanumweremore transparent than clove and garlic films (Table 2). Similartransparency values of fish protein films were reported in previousstudies (e.g. Benjakul et al., 2008), whereas lower transparency hasalso been referred (e.g. Chinabhark et al., 2007), which could beattributed to different protein content in films. The change intransparency of films with different essential oils might be relatedwith the type and amount of essential oil, as well as film thickness.

Opacity did not reveal statistical differences among films, withvalues ranging between 15.9 and 16.4 (Table 2). Previous studiesreported lower opacity values in fish protein films subjected todifferent thermal treatments (García & Sobral, 2005).

Fish protein films had an average thickness of 28 mm (Table 2),which was similar to values registered in previous studies withdifferent proportions of protein and polyvinyl alcohol per surfacearea (e.g. Limpan et al., 2010). Still, higher thickness values wereobtained in previous studies (e.g. Artharn et al., 2007), and thesedifferences might be related with different protein content in films(Prodpran & Benjakul, 2005). The incorporation of essential oils infilms significantly decreased thickness to 12e17 mm, being garlicand clove films those with lower thickness values. Pires et al. (2011)also found a decrease in films thickness in the presence of essentialoil at the concentrations used in the current study. The interactionof essential oil and the casting surface might contribute to higherdegree of compaction of film matrix, since it was observed anincreasing adhesion of films to the surface as oil level increased.Additionally, the combination of proteins and compounds withhydrophobic interactions might also cause different rearrange-ments in the protein matrix, and consequently affect thickness.Nonetheless, other studies reported that essential oils of oregano,

italized letters denote significant differences (p < 0.05) between films. Values are

Garlic Clove Origanum

22.9 � 2.3a 7.0 � 3.3b 2.3 � 0.2c

16.2 � 0.6a 16.4 � 0.9a 16.2 � 1.0a

12.8 � 7.0c 12.3 � 4.7c 17.2 � 6.0b

4.3 � 1.0bc 3.8 � 0.9c 7.8 � 0.8a

3.7 � 0.4b 3.5 � 1.1b 6.3 � 1.1a

8.9 � 1.3b 15.2 � 2.0a 13.2 � 1.3a

50.4 � 12.1b 115.0 � 22.4a 93.0 � 14.2a

2.1 � 0.9b 1.8 � 0.6b 2.4 � 1.5b

53.3 � 21.1b 55.7 � 31.7b 83.2 � 50.3b

6.6 � 2.7a 7.3 � 2.3a 6.4 � 4.0a

B. Teixeira et al. / LWT - Food Science and Technology 59 (2014) 533e539 537

thyme, clove, and cinnamon increased thickness of alginate andchitosan-based films (Benavides, Villalobos-Carvajal, & Reyes, 2012;Hosseini, Razavi, & Mousavi, 2009), while oregano, lemongrass, andcinnamon essential oils did not affect thickness of alginate-applepuree films (Rojas-Graü et al., 2007). The differences amongstudies might be related with the interaction between essentialoils, casting surface and film forming solutions.

The results of water vapour permeability of films varied be-tween 3.8 and 7.8 � 10�11 g m�1 s�1 Pa�1 (Table 2). Water vapourpermeability of control films was similar to values obtained inprevious studies with fish muscle protein-based films (e.g. Limpanet al., 2010), whereas, few studies revealed one order of magnitudelower values (e.g. Hamaguchi, WuYin, & Tanaka, 2007). Proteinconcentration may be responsible for such differences (Prodpran &Benjakul, 2005). The incorporation of clove essential oil signifi-cantly reduced the permeability of films to water vapour, while nostatistical differences were detected with films containing garlic ororiganum essential oils. In previous studies, the addition of edibleoils (e.g peanut and corn oils) to fish protein films also decreasedwater vapour permeability (Tanaka, Ishizaki, Suzuki, & Takai, 2001),while the incorporation of garlic essential oil in alginate-basedfilms increased the water vapour permeability (Pranoto, Salokhe,& Rakshit, 2005). The distinct composition of essential oils couldbe responsible for the differences observed, as the water vapourtransfer process depends on the hydrophilic-hydrophobic ratio offilm components. Still, it cannot be assumed that the water vapourpermeability of edible films is reduced simply by adding a hydro-phobic component, but the impact of lipid addition on the micro-structure of the emulsified film is also a determining factor (Atarés,Jesús, et al., 2010).

Films and protein solubility results are shown in Fig. 1. Fishprotein films immersed in water become hydrated and thicker,without visually loosing their integrity. Control films showed 34%soluble fraction, which was lower than several other values re-ported earlier (Artharn et al., 2007; Shiku, Yuca Hamaguchi,Benjakul, Visessanguan, & Tanaka, 2004). The protein solubility ofcontrol films was within the values obtained in other studies (e.g.Benjakul et al., 2008). The incorporation of essential oils in filmsreduced films solubility, principally for origanum and clove films(10%), which was similar to the findings of Hosseini et al. (2009) inchitosan-based films with clove and cinnamon, but not with thymeessential oil. In contrast, protein solubility was significantly higherin films incorporated with clove, whereas statistically lower valueswere obtained with films with origanum.

3.2. Mechanical properties

The results from tensile and puncture tests of fish proteins filmsare summarized in Table 2.

In tensile tests, the force needed to break fish protein filmswithout essential oils, as well as its elongation percentage, werestatistically higher than in films with essential oils. The elongation

Fig. 1. Films solubility and protein solubility of fish protein films tested. In eachgraphic, different letters denote significant differences (p < 0.05) between films. Barsrepresent average values, whereas error bars indicate the standard deviation (n ¼ 5).

at the breaking point of films without essential oils was higher thanthose reported in previous studies (ca. 17e125%; Artharn, Benjakul,& Prodpran, 2008; Chinabhark et al., 2007; Prodpran & Benjakul,2005). In contrast, its tensile strength was similar to values ob-tained by Prodpran and Benjakul (2005) and by Sabato,Nakamurakare, and Sobral (2007), whereas higher values wereobtained by Artharn et al. (2008). The differences found in the forceand elongation of films tested in the current study might be relatedwith films thickness, as the results of tensile strength (force atbreaking point per initial cross-sectional area) were not statisticallydifferent. Similarly, Atarés, Bonilla, et al. (2010) concluded that thepresence, type, and content of essential oils does not affect tensilestrength of sodium caseinate-based films.

The force needed to puncture films without essential oil wasstatistically higher than in films incorporated with garlic and clove,whereas no differences were detected with origanum films. Addi-tionally, filmswith clove and origanum significantly increased filmspuncture deformation compared to films without essential oil andgarlic films. In the case of tilapia films, the puncture forcewas lower(below 4 N) for the same amount of plasticizer (García & Sobral,2005), and the differences could be attributed to the thermaltreatments performed in tilapia films forming solutions. In a pre-vious study, puncture force decreased with the incorporation ofessential oil with the same concentration used in the current study(Pires et al., 2011).

3.3. Antioxidant activity

The results of DPPH radical-scavenging activity and reducingpower of fish protein films are shown in Figs. 2 and 3, respectively.Fish protein films showed antioxidant activity that can be due tothe presence of free sulfhydryl groups and other aminoacids such astryptophan, methionine, and tyrosine on hake fish proteins, asmentioned by Taguchi, Iwami, Kawabata, and Ibuki (1988) andFaraji, McClements, and Decker (2004). The incorporation ofessential oils significantly increased the DPPH radical-scavengingactivity of fish protein films. The highest antioxidant activity wasobtained with clove and garlic films (72% of inhibition). The dif-ferences found in the antioxidant activity of films with differentessential oils, are due to different compounds with antioxidantactivity in essential oils composition. The DPPH radical-scavengingactivity of films with origanum and clovewas lower than that of thefree essential oil, whereas garlic films revealed higher activity thanthe essential oil itself. Such decrease could be due to interactionsbetween the components of films and essential oils that could nolonger be available to interact in the antioxidant activity reactions,and to the loss of volatile compounds of essential oils during thefilms drying process. In contrast, the increase in the antioxidantactivity observed with garlic films might be due to the presence of

Fig. 2. Antioxidant activity of essential oils and fish protein films tested, measuredwith the free radical scavenging method. In each graphic, different letters denotesignificant differences (p < 0.05) within films or essential oils. Bars represent averagevalues, whereas error bars indicate the standard deviation (n ¼ 3). Abbreviations:DPPH e a,a-diphenyl-b-picrylhydrazyl.

Fig. 3. Antioxidant activity of essential oils and fish protein films tested, measuredwith the reducing power method. In each graphic, different letters denote significantdifferences (p < 0.05) within films or essential oils. Bars represent average values,whereas error bars indicate the standard deviation (n ¼ 3).

B. Teixeira et al. / LWT - Food Science and Technology 59 (2014) 533e539538

di-2-propenyldisulfide and di-2-propenyltetrasulfide in thisessential oil, both with disulfide bonds that might be cleaved in thefilm forming solutions and able to act as scavenging hydroxylradicals increasing the antioxidant activity in the DPPH method,and not in the ferric reducing power method.

The reducing power of films without essential oil was lowerthan that of surimi films of Alaska pollack (Weng et al., 2009). Theincorporation of clove and garlic essential oils significantlyincreased films antioxidant activity, but not for origanum. Onceagain, the reducing power of films with origanum and clove waslower than that of the free essential oil, whereas garlic filmsrevealed higher activity than the essential oil itself.

3.4. Antibacterial activity

The antibacterial activity of films is shown in Table 3. The agardiffusion method revealed only inhibitionwith both Listeria strains.Inhibition of L. monocytogenes only occurred with garlic and orig-anum films, whereas L. innocuawas only inhibited by films withoutessential oil and films with origanum.

In contrast, the macrodilution method used to quantify thebacterial reduction caused by films (5� 5 cm2) revealed inhibitionswith all films tested. None of the films was able to inhibit E. coli andS. typhimurium. In contrast, B. thermosphacta and L. monocytogenes

Table 3Antibacterial activity of fish protein films tested. Results obtained with the agardiffusion method represent the inhibition diameter, including the disc film (1.5 cmØ), whereas the macrodilution method indicates the percentage reduction of bac-terial viable cells obtained with 5 � 5 cm2

films in 10 mL of bacterial suspensions(1 � 107 CFU mL�1). Values are presented as average � standard deviation (n ¼ 3).Abbreviations: N.D. e inhibition not detected.

Noessential oil

Garlic Clove Origanum

Agar diffusion method (cm)Brochothrixthermosphacta

N.D. N.D. N.D. N.D.

Escherichia coli N.D. N.D. N.D. N.D.Listeria innocua 1.6 � 0.0 N.D. N.D. 1.6 � 0.0Listeria monocytogenes N.D. 3.0 � 1.7 N.D. 1.7 � 0.2Pseudomonas putida N.D. N.D. N.D. N.D.Salmonella typhimurium N.D. N.D. N.D. N.D.Shewanella putrefaciens N.D. N.D. N.D. N.D.

Macrodilution method (%)Brochothrixthermosphacta

N.D. 60.2 � 5.3 N.D. 41.5 � 32.3

Escherichia coli N.D. N.D. N.D. N.D.Listeria innocua N.D. 86.9 � 1.8 20.5 � 13.1 66.5 � 8.5Listeria monocytogenes N.D. 68.7 � 22.0 41.4 � 27.6 N.D.Pseudomonas putida 87.2 � 6.7 N.D. N.D. N.D.Salmonella typhimurium N.D. N.D. N.D. N.D.Shewanella putrefaciens 54.0 � 31.7 77.2 � 30.1 92.2 � 3.0 N.D.

were inhibited by garlic and origanum films, L. innocua inhibitionoccurred with all films with essential oils, P. putida inhibition onlyoccurred with films without essential oil, and S. putrefaciens wasinhibited by filmswithout essential oil and by garlic and clove films.The addition of essential oils to films did not reduce the growth ofGram negative bacteria like E. coli, P. putida, and S. typhimuriumwhen comparing with films without essential oil. The resistance ofthese bacterial strains could be due to the complexity of theirdouble layer cell membrane in comparison with the simpler cellmembrane of Gram positive bacteria (Hogg, 2005).

Several studies tested the antibacterial activity of edible filmsincorporated with different natural substances (e.g. Sivarooban,Hettiarachchy, & Johnson, 2008), but fewer focused on filmsincorporated with essential oils. Garlic films showed similar resultsto those obtained by Pranoto et al. (2005), but different than thosereported in the study of Seydim and Sarikus (2006), whereL. monocytogenes was not inhibited by garlic films. Inhibition ofL. monocytogenes by films with clove and Thymus essential oils wasalso obtained by Hosseini et al. (2009). The differences amongstudies could be due to the diversity of active agents in the essentialoils, interactions between essential oil compounds and films con-stituents (alginate, whey protein, and chitosan), and also due todifferences in the amount of essential oil per area of film (not al-ways specified).

4. Conclusions

Hake proteins recovered from by-products of seafood process-ing industries can be used for the preparation of biodegradablefilms. The incorporation of essential oils in these films reduced filmthickness as well as the solubility inwater, affected filmmechanicalproperties, and improved antioxidant activity. Clove films showedthe lowest water vapour permeability and the highest antibacterialactivity (against S. putrefaciens); garlic films presented the highestantioxidant activity; and origanum films were more similar tocontrol films. The results of this study indicate that hake proteinfilms, prepared with by-products, incorporated with clove, garlic,and origanum essential oils, show interesting properties (watervapour barrier and antibacterial and antioxidant activities) to begood candidates to be used in biodegradable food packagingsystems.

Acknowledgements

The first and second authors acknowledge the PortugueseFoundation for Science and Technology for supporting a PhD grant(Ref. SFRH/BD/44254/2008) and a Research contract (ProgramCiência 2008), respectively. The Portuguese Foundation for Scienceand Technology also supported this study through the ResearchProject “FRESHFISH e Preservation of fish products by usingmodified atmosphere packaging and edible coatings with sea bassand sea bream as models” (Ref. PPCDT/DG/MAR/82008/2006). Weare grateful to Dr Rogério Mendes (INRB I.P./L-IPIMAR) for technicalassistance with physical and mechanical properties methodologies.

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