《气味科学中的健康密码》课程教学资源（阅读材料）Oregano and lavender essential oils as antioxidant and antimicrobial additives of biogenic gelatin films

Industrial Crops and Products 71(2015)205-213 Contents lists available at ScienceDirect Industrial Crops and Products ELSEVIER journal homepage:www.elsevier.com/locate/indcrop Oregano and lavender essential oils as antioxidant and antimicrobial CrossMark additives of biogenic gelatin films J.F.Martuccia.*,L.B.Gendeb,L.M.Neira,R.A.Ruseckaitea Instituto de Investigaciones en Ciencia y Tecnologia de Materiales(INTEMA).Av.Juan B.Justo 4302.7600 Mar del Plata,Buenos Aires,Argentina bResearch Center in Social Bees(Arthropods Laboratory).CONICET-Natural and Exact Sciences Faculty,Mar del Plata University.Funes 3350.7600 Mar del Plata,Buenos Aires,Argentina ARTICLE INFO ABSTRACT Article history: The chemical composition of the essential oils obtained by hydrodistillation from fully-formed,dried Received 4 November 2014 oregano leaves (Origanum vulgareOriganum vulgare)and lavender leaves and flowers (Lavandula offic- Received in revised form 24 March 2015 inalis)were analyzed by GC/MS.The effectiveness of oregano (OEO)and lavender (LEO)essential Accepted 26 March 2015 oils and a mixture LEO:OEO (50:50)in inhibiting Escherichia coli and Staphylococcus aureus growth were determined.Both essential oils inhibited the growth of the microorganisms tested,being more Keywords: Active films sensitive to gram-positive bacteria.OEO yielded the lowest values of minimum inhibitory con- Essential oils centration(MICoEo=1600-1800ppm vs.MICLEo=2000 ppm against E.coli:MICoEo=800-900ppm vs. Gelatin MICLEo =1000-1200 ppm against S.aureus),due to the higher content of phenolic compound,which also Antimicrobial provides antioxidant capacity(IC50oEo=297+89 ppm vs.IC50LEo *6000 ppm).Mixture results indicated Antioxidant an antagonist antimicrobial effect between OEO and LEO.Gelatin-based films added with OEO or LEO. were prepared by casting(2000-6000 ppm).Mechanical,optical and water vapor barrier properties were determined to observe film functionality.OEO effect on the functional properties of gelatin films was not significant.LEO,in the highest concentration analyzed,promotes a slight change in water vapor perme- ability of Ge-based films(1.46 x 10-13 to 6.8 x 10-14 Kg.m/Pa.s.m2).due to its high hydrophobic nature. Oregano containing gelatin films exhibited the highest antimicrobial and antioxidant properties. 2015 Elsevier B.V.All rights reserved. 1.Introduction well as materials intended for food packaging applications(Gomez- Guillen et al.,2007;Martucci and Ruseckaite,2009;Ahmad et al. The main driving force for the growth of worldwide food indus- 2012:Martucci et al.,2012:Teixeira et al.,2014).Biodegradabil- try is the scope and range of food preservation and shelf life ity.excellent biocompatibility.plasticity.adhesiveness.abundance, extension technology(Sadaka et al..2013).Active packaging is gain- and low cost are the main reasons for the wide range of applications ing increasing attention from researchers and the industry due to of this biopolymer. its potential to provide quality and safety benefits.Active packaging The consumer's desire for natural ingredients and for chemi- is a type of packaging that changes its conditions as a way to extend cal preservative-free foods has increased the popularity of natural life or enhance safety or sensory properties while maintaining food antimicrobial agents (Sadaka et al..2013).In this framework. quality (Vermeiren et al.,1999).In view of the health concerns the addition of essential oils to biopolymer films as natural bac- expressed by consumers and current environmental problems, teriostatics could be an interesting election.Essential oils have research is now focusing on the development of sustainable pack- well-recognized properties,such as antimicrobial (Kulevanova and aging materials based on annually renewable natural biopolymers Panovska,2001:Gende et al.,2010,b:Teixeira et al.,2013a,b). such as polysaccharides and proteins (Gomez-Estaca et al.,2010) antibacterial (Canillac and Mourey,2001:Min and Oh,2009,b: As a multifunctional protein,gelatin is an heteropolymer derived Teixeira et al..2013a,b)and antioxidant properties(Burt,2004: from collagen.Gelatin-based systems are applied in numerous Kacaniova et al.,2012:Danh et al.,2012.b:Teixeira et al.,2013a,b). fields,including food,pharmaceuticals,photographic industry.as These properties can be attributed to the high content of terpenic compounds(o-pinene,B-pinene,1,8-cineol,menthol,linalool)or phenolic compounds such as carvacrol,eugenol and thymol (Burt, 2004).It is common knowledge that essential oils are characterized Corresponding author.Tel.:+542234816600x249 by changes in their chemical composition,depending on the state of E-mail address:jmartucci@fi.mdp.edu.ar (J.F.Martucci). http://dx.doi.org/10.1016/j.indcrop.2015.03.079 0926-6690/2015 Elsevier B.V.All rights reserved

Industrial Crops and Products 71 (2015) 205–213 Contents lists available at ScienceDirect Industrial Crops and Products j ou rnal homepage: www.elsevier.com/locate/indcrop Oregano and lavender essential oils as antioxidant and antimicrobial additives of biogenic gelatin films J.F. Martucci a,∗, L.B. Gende b, L.M. Neiraa, R.A. Ruseckaitea a Instituto de Investigaciones en Ciencia y Tecnología de Materiales (INTEMA), Av. Juan B. Justo 4302, 7600 Mar del Plata, Buenos Aires, Argentina b Research Center in Social Bees (Arthropods Laboratory). CONICET- Natural and Exact Sciences Faculty, Mar del Plata University, Funes 3350, 7600 Mar del Plata, Buenos Aires, Argentina a r t i c l e i n f o Article history: Received 4 November 2014 Received in revised form 24 March 2015 Accepted 26 March 2015 Keywords: Active films Essential oils Gelatin Antimicrobial Antioxidant a b s t r a c t The chemical composition of the essential oils obtained by hydrodistillation from fully-formed, dried oregano leaves (Origanum vulgareOriganum vulgare) and lavender leaves and flowers (Lavandula offic￾inalis) were analyzed by GC/MS. The effectiveness of oregano (OEO) and lavender (LEO) essential oils and a mixture LEO:OEO (50:50) in inhibiting Escherichia coli and Staphylococcus aureus growth were determined. Both essential oils inhibited the growth of the microorganisms tested, being more sensitive to gram-positive bacteria. OEO yielded the lowest values of minimum inhibitory con￾centration (MICOEO = 1600–1800 ppm vs. MICLEO = 2000 ppm against E. coli; MICOEO = 800–900 ppm vs. MICLEO = 1000–1200 ppm against S. aureus), due to the higher content of phenolic compound, which also provides antioxidant capacity (IC50OEO = 297 ± 89 ppm vs. IC50LEO » 6000 ppm). Mixture results indicated an antagonist antimicrobial effect between OEO and LEO. Gelatin-based films added with OEO or LEO, were prepared by casting (2000–6000 ppm). Mechanical, optical and water vapor barrier properties were determined to observe film functionality. OEO effect on the functional properties of gelatin films was not significant. LEO, in the highest concentration analyzed, promotes a slight change in water vapor perme￾ability of Ge-based films (1.46 × 10−13 to 6.8 × 10−14 Kg.m/Pa.s.m2), due to its high hydrophobic nature. Oregano containing gelatin films exhibited the highest antimicrobial and antioxidant properties. © 2015 Elsevier B.V. All rights reserved. 1. Introduction The main driving force for the growth of worldwide food indus￾try is the scope and range of food preservation and shelf life extensiontechnology (Sadaka et al., 2013).Active packaging is gain￾ing increasing attention from researchers and the industry due to its potentialto provide quality and safety benefits.Active packaging is a type of packaging that changes its conditions as a way to extend life or enhance safety or sensory properties while maintaining food quality (Vermeiren et al., 1999). In view of the health concerns expressed by consumers and current environmental problems, research is now focusing on the development of sustainable pack￾aging materials based on annually renewable natural biopolymers such as polysaccharides and proteins (Gomez-Estaca et al., 2010). As a multifunctional protein, gelatin is an heteropolymer derived from collagen. Gelatin-based systems are applied in numerous fields, including food, pharmaceuticals, photographic industry, as ∗ Corresponding author. Tel.: +542234816600x249. E-mail address: jmartucci@fi.mdp.edu.ar (J.F. Martucci). well as materials intended for food packaging applications (Gómez￾Guillén et al., 2007; Martucci and Ruseckaite, 2009; Ahmad et al., 2012; Martucci et al., 2012; Teixeira et al., 2014). Biodegradabil￾ity, excellent biocompatibility, plasticity, adhesiveness, abundance, and low cost are the main reasons for the wide range of applications of this biopolymer. The consumer’s desire for natural ingredients and for chemi￾cal preservative-free foods has increased the popularity of natural antimicrobial agents (Sadaka et al., 2013). In this framework, the addition of essential oils to biopolymer films as natural bac￾teriostatics could be an interesting election. Essential oils have well-recognized properties, such as antimicrobial (Kulevanova and Panovska, 2001; Gende et al., 2010,b; Teixeira et al., 2013a,b), antibacterial (Canillac and Mourey, 2001; Min and Oh, 2009,b; Teixeira et al., 2013a,b) and antioxidant properties (Burt, 2004; Kacániová ˇ et al., 2012; Danh et al., 2012,b; Teixeira et al., 2013a,b). These properties can be attributed to the high content of terpenic compounds (-pinene, -pinene, 1,8-cineol, menthol, linalool) or phenolic compounds such as carvacrol, eugenol and thymol (Burt, 2004). Itis common knowledge that essential oils are characterized by changes in their chemical composition, depending on the state of http://dx.doi.org/10.1016/j.indcrop.2015.03.079 0926-6690/© 2015 Elsevier B.V. All rights reserved.

206 J.F.Martucci et aL.Industrial Crops and Products 71(2015)205-213 development of the plant,the part used for the extraction,the geo- The quantitative and qualitative analysis of lavender and graphical location,and the physical and chemical characteristics of oregano oils were carried out by gas chromatography(GC)coupled the soil and climate(Gende et al..2010). to mass spectrometry (GC/MS).The experiments were performed The use of gelatin based films with aqueous plant extracts using an Agilent gas chromatograph(GC)model 7890 A(Agilent, appears to be a promising technology in food packaging materi- Palo Alto,USA)equipped with an auto-sampler ALS and coupled to als.These films can reduce surface microbial populations,enhance an Agilent single quadrupole mass spectrometer(MS)model 5975C oxygen barrier,and reduce the use of synthetic packaging mate- (Agilent,Palo Alto,USA).The GC was equipped with an Agilent 5MS rials since gelatin and essential oils are derived from renewable column (100m x 0.25 mm internal diameter and 0.25 um thick- resources (Gomez-Estaca et al.,2009,2010:Perez-Mateos et al. ness).Helium was used as carrier (1.5 mL/min)in constant flow 2009:Min and Oh,2009:Ahmad et al.,2012;Teixeira et al.,2014) mode,with a total GC run time of 30 min.The injector temperature The inclusion of antimicrobial and/or antioxidant compounds into was kept at 280C in a split less mode and using an injection vol- edible films provides a novel way to improve the safety and shelf ume of 1 uL.The oven temperature was programmed to increase life of ready-to-eat foods.Some plant EOs and their components are from 50C,hold 2 min,increased to 260 at 10C/min and then hold compatible with the sensory characteristics of fruits and vegetables for 2 min.The mass spectrometer was operated in electron impact and have been shown to prevent bacterial growth,as reported by (El)mode at 70eV with anion source temperature at 230C and several reviews about this subject(Burt,2004;Sanchez-Gonzalez quadrupole temperature 190C.A scan rate of 0.6s (cycle time: et al.,2011;Eca et al.,2014) 0.2s)was applied,covering m/z range from 29 to 500.The identi- The objectives of this study were to determine the antimi- fication of EOis components was achieved by matching their mass crobial activity of oregano (Origanum vulgare L)and lavender spectra to that reported in the literature(Adams,2007).Quantita- (Lavandula officinalis L)essential oils and their main components tive data were derived by integration of FID area percentages with against Escherichia coli and Staphylococcus aureus by a serial dilution no use of collection factors. method.It was also to investigate the antibacterial efficacy of both oils incorporated into mammalian gelatin-based films using the 2.4.Preparation of control and EO-added gelatin films agar diffusion method.Optical and mechanical properties,water vapor permeability and the ability of the films to provide microor- Gelatin power (5g)was dissolved in 100 mL of buffer phos- ganisms'protection and lipid oxidation were analyzed. phate (pH 7)at room temperature.After dissolution for 30 min under continuous stirring.propylene glycol (8%v/v of solution) 2.Experimental was added as plasticizer and emulsifier agent.An adequate mixing of the plasticizer,lavender essential oil (LEO)or oregano essential 2.1.Materials oil(OEO)was incorporated to obtain final concentrations between 2000 and 6000 ppm.Control formulation was prepared in the Bovine hide gelatin(Ge)type Bwas kindly supplied by Rousselot same way,replacing EOs by buffer phosphate.Afterwards mixtures (Argentina).Bloom 150,isoionic point (Ip)5.3.Buffer Phos- were homogenized at 20000 rpm for 5 min by using a homoge- phate pH 7(Ciccarelli,Argentina).Propylene Glycol (PG,molar nizer (UltraturaxT25 basic,IKA-Werke GMBH Co.,KG Staufen, mass:76.09 g/mol;HLB:7.4-9.3)(Bolivar chemicals,Argentina). Germany).Films were obtained by casting and dried at 35C in 1,1-Diphenyl-2-picrylhydrazyl (DPPH)(Sigma-Aldrich,EEUU) a forced-air oven(Memmert UFE550,Germany)for 20h until con- potassium hexacyanoferrate lll and trichloroacetic acid(Ciccarelli, stant weight.Dried film samples were manually peeled off from the Argentina)were analytical grade and used as received mold and conditioned in a laboratory humidity chamber at 25+2C and 65+2%relative humidity(RH)prior to analysis.Resultant films 2.2.Source of Bacteria were designated as Ge(control gelatin films).OEO-Ge (oregano essential oil -added gelatin film)and LEO-Ge (lavender essential oil-added gelatin film).respectively. Food-borne pathogens were used to assess the antimicro- bial proprieties,which includes the gram-negative bacteria 2.5.Analysis Escherichia coli 0157:H7 ATCC 32158(ATCC,American Type Culture Collection)and Gram-positive Staphylococcus aureus ATCC 25923. 2.5.1.Thickness These strains were obtained in Eosin-methylene blue (EMB)and Film thickness was measured using a 0-25 mm manual microm- Baird Parker agar respectively.Vegetative cells of each microor- eter,with a resolution of 0.01 mm.The reported values are the ganism were streaked on Mueller Hinton agar and incubated at average of four readings taken randomly on each film sample. 37+0.5C for 24h.Microbial broth was then suspended in dou- ble distilled sterile water.The density of bacteria suspension was adjusted until the visible turbidity was equal to 0.5 Mc Farland 2.5.2.Optical properties standard before testing. Color was measured by a CIE L'ab*system using a LoviBond Colorimeter RT500(Neu-Isenberg,Germany)with an 8 mm diam- eter measuring area.Total color difference (AE).hue angle (hab) 2.3.Isolation and characterization of oregano and lavender and chroma(C'ab)were calculated as the average of six samples essential oils using the following equations: Falciform,fully-formed oregano leaves (Origanum vulgare L) C*ab=(a*2+(b*2 (1) and lavender leaves and flowers (Lavandula officinalis L)were col- lected in the geographical area of Mar del Plata(3800'24.17"S-57 33'55.89"W)during July 2011.Plant specimens were classified h*ab arctg(as b米 (2) and stored in the herbarium of vascular plants(AL 17 and PV 97. Arthropods laboratory,Faculty of Sciences,Universidad Nacional △E=V(Aa*)+(△b*2+(△L*2 (3) de Mar del Plata).Essential oils were extracted by hydrodistillation using a Clevenger type apparatus according to the method reported where△L',△a'and△b'referred to differences between the white elsewhere(Gende et al.,2010)from freshly dried plant material standard (used as the film background)and sample color values

206 J.F. Martucci et al. / Industrial Crops and Products 71 (2015) 205–213 development of the plant, the part used for the extraction, the geo￾graphical location, and the physical and chemical characteristics of the soil and climate (Gende et al., 2010). The use of gelatin based films with aqueous plant extracts appears to be a promising technology in food packaging materi￾als. These films can reduce surface microbial populations, enhance oxygen barrier, and reduce the use of synthetic packaging mate￾rials since gelatin and essential oils are derived from renewable resources (Gómez-Estaca et al., 2009, 2010; Perez-Mateos et al., 2009; Min and Oh, 2009; Ahmad et al., 2012; Teixeira et al., 2014). The inclusion of antimicrobial and/or antioxidant compounds into edible films provides a novel way to improve the safety and shelf life of ready-to-eatfoods. Some plant EOs and their components are compatible with the sensory characteristics of fruits and vegetables and have been shown to prevent bacterial growth, as reported by several reviews about this subject (Burt, 2004; Sánchez-González et al., 2011; Ec¸ a et al., 2014) The objectives of this study were to determine the antimi￾crobial activity of oregano (Origanum vulgare L.) and lavender (Lavandula officinalis L.) essential oils and their main components against Escherichia coli and Staphylococcus aureus by a serial dilution method. It was also to investigate the antibacterial efficacy of both oils incorporated into mammalian gelatin-based films using the agar diffusion method. Optical and mechanical properties, water vapor permeability and the ability of the films to provide microor￾ganisms’ protection and lipid oxidation were analyzed. 2. Experimental 2.1. Materials Bovine hide gelatin (Ge)type B was kindly supplied by Rousselot (Argentina), Bloom 150, isoionic point (Ip) 5.3. Buffer Phos￾phate pH 7 (Ciccarelli, Argentina), Propylene Glycol (PG, molar mass: 76.09 g/mol; HLB: 7.4–9.3) (Bolivar chemicals, Argentina), 1,1-Diphenyl-2-picrylhydrazyl (DPPH) (Sigma–Aldrich, EEUU), potassium hexacyanoferrate III and trichloroacetic acid (Ciccarelli, Argentina) were analytical grade and used as received. 2.2. Source of Bacteria Food-borne pathogens were used to assess the antimicro￾bial proprieties, which includes the gram-negative bacteria Escherichia coli O157:H7 ATCC 32158 (ATCC, American Type Culture Collection) and Gram-positive Staphylococcus aureus ATCC 25923. These strains were obtained in Eosin-methylene blue (EMB) and Baird Parker agar respectively. Vegetative cells of each microor￾ganism were streaked on Mueller Hinton agar and incubated at 37 ± 0.5 ◦C for 24 h. Microbial broth was then suspended in dou￾ble distilled sterile water. The density of bacteria suspension was adjusted until the visible turbidity was equal to 0.5 Mc Farland standard before testing. 2.3. Isolation and characterization of oregano and lavender essential oils Falciform, fully-formed oregano leaves (Origanum vulgare L.) and lavender leaves and flowers (Lavandula officinalis L.) were col￾lected in the geographical area of Mar del Plata (38◦ 00 24.17” S-57◦ 33 55.89” W) during July 2011. Plant specimens were classified and stored in the herbarium of vascular plants (AL 17 and PV 97, Arthropods laboratory, Faculty of Sciences, Universidad Nacional de Mar del Plata). Essential oils were extracted by hydrodistillation using a Clevenger type apparatus according to the method reported elsewhere (Gende et al., 2010) from freshly dried plant material The quantitative and qualitative analysis of lavender and oregano oils were carried out by gas chromatography (GC) coupled to mass spectrometry (GC/MS). The experiments were performed using an Agilent gas chromatograph (GC) model 7890 A (Agilent, Palo Alto, USA) equipped with an auto-sampler ALS and coupled to an Agilent single quadrupole mass spectrometer (MS) model 5975C (Agilent, Palo Alto, USA). The GC was equipped with an Agilent 5MS column (100 m × 0.25 mm internal diameter and 0.25 m thick￾ness). Helium was used as carrier (1.5 mL/min) in constant flow mode, with a total GC run time of 30 min. The injector temperature was kept at 280 ◦C in a split less mode and using an injection vol￾ume of 1 L. The oven temperature was programmed to increase from 50 ◦C, hold 2 min, increased to 260 at 10 ◦C/min and then hold for 2 min. The mass spectrometer was operated in electron impact (EI) mode at 70 eV with anion source temperature at 230 ◦C and quadrupole temperature 190 ◦C. A scan rate of 0.6 s (cycle time: 0.2 s) was applied, covering m/z range from 29 to 500. The identi- fication of EOıs´ components was achieved by matching their mass spectra to that reported in the literature (Adams, 2007). Quantita￾tive data were derived by integration of FID area percentages with no use of collection factors. 2.4. Preparation of control and EO-added gelatin films Gelatin power (5 g) was dissolved in 100 mL of buffer phos￾phate (pH 7) at room temperature. After dissolution for 30 min under continuous stirring, propylene glycol (8% v/v of solution) was added as plasticizer and emulsifier agent. An adequate mixing of the plasticizer, lavender essential oil (LEO) or oregano essential oil (OEO) was incorporated to obtain final concentrations between 2000 and 6000 ppm. Control formulation was prepared in the same way, replacing EOs by buffer phosphate. Afterwards mixtures were homogenized at 20000 rpm for 5 min by using a homoge￾nizer (UltraturaxT25 basic, IKA-Werke GMBH & Co., KG Staufen, Germany). Films were obtained by casting and dried at 35 ◦C in a forced-air oven (Memmert UFE550, Germany) for 20 h until con￾stant weight. Dried film samples were manually peeled off from the moldandconditionedina laboratoryhumidity chamber at 25 ± 2 ◦C and 65 ± 2% relative humidity (RH) prior to analysis. Resultant films were designated as Ge (control gelatin films), OEO-Ge (oregano essential oil -added gelatin film) and LEO-Ge (lavender essential oil-added gelatin film), respectively. 2.5. Analysis 2.5.1. Thickness Film thickness was measured using a 0–25 mm manual microm￾eter, with a resolution of 0.01 mm. The reported values are the average of four readings taken randomly on each film sample. 2.5.2. Optical properties Color was measured by a CIE L*a*b* system using a LoviBond Colorimeter RT500 (Neu-Isenberg, Germany) with an 8 mm diam￾eter measuring area. Total color difference (E), hue angle (h*ab) and chroma (C*ab) were calculated as the average of six samples using the following equations: C ∗ ab = (a∗) 2 + (b∗) 2 (1) h ∗ ab = arctg( b∗ a∗ ) (2) E = (a∗) 2 + (b∗) 2 + (L∗) 2 (3) where L*, a* and b* referred to differences between the white standard (used as the film background) and sample color values.

LF.Martucci et aL Industrial Crops and Products 71 (2015)205-213 207 2.5.3.Scanning electronic microscopy (SEM) 2.5.7.2.Determination of DPPH radical scavenging activity.DPPH Fracture surfaces were observed on a Phillips 505 microscope radical scavenging activity(RSA)was determined on the basis of (Eindhoven,The Netherlands)at 10 kV.All specimens were sputter- the method by Yen and Hsieh (1995)with slight modifications.A coated with gold. volume of 400 uL of each essential oil solution(0-6000 ppm)was mixed with 2 mL of a 0.06 mmol/L solution of DPPH in methanol. 2.5.4.Water uptake Mixtures were shaken vigorously and allowed to stand in the dark Water uptake test was performed gravimetrically.Samples were for 30 min at room temperature.The absorbance of the resulting placed in pre-weighed aluminum cups and dried at 45C in a vac- solutions was monitored at 517nm using an UV-visible spec- uum oven(Gallenkamp,UK)up to constant weight(mo).Specimens trophotometer(Agilent 8453.China).The control was prepared were then kept at 25+2C in a humidity chamber at 65+2%RH in the same manner except that methanol was used instead of following the procedure described in ASTM E104-95.Samples were the sample.DPPH radical scavenging activity(RSA)was calculated removed at specific time intervals and weighed with a precision of according to the following equation(Yen and Hsieh,1995): +0.0001g until reaching constant weight(mf).The water uptake at equilibrium(WUeg)was calculated as the weight gain according RSA A517sample 100 to: (6) A517contro wUg(%)=100m-mo) (4) mo where A517sample and A517control correspond to the absorbances at 517 nm of the radical (DPPH.)in the absence and presence of This experiment was carried out on four specimens of each sample antioxidant respectively.IC50 is defined as the efficient concen- to ensure results reproducibility. tration required to decrease the initial DPPH radical concentration by50% 2.5.5.Mechanical properties In the case of films,samples were immersed in liquid nitrogen Tensile strength at breaking(TS)and percentage of elongation and subsequently crushed and grounded with a pestle.A precise (e%)were measured according to the ASTM638 94 D standard using amount of crushed film (0.4g)was mixed with 4mL of methanol an Instron 4467 Universal Testing Machine(Buckinghamshire,Eng- in a caped tube,stirred vigorously and allowed to stand overnight land)with a 5 kN load cell and at a crosshead speed of 10 mm/min (about 12h).Afterwards,tubes were centrifuged at 5000 rpm for Samples were stored at 65+2%RH and 25+2C prior to measure- 10 min(Sartorius type 4-15.Germany)and the supernatant was ments.Reported results were obtained from at least 10 samples for recovered and reserved for DPPH radical scavenging activity and each type of film. reducing power as outlined above for EOs.In the case of reducing power,final results were expressed per g of film. 2.5.6.Water vapor permeability (WVP) Water vapour permeability (WVP)was performed gravimet- rically at 25C,using the ASTM E96-95 desiccant method.All 2.5.8.In vitro antibacterial assay of essential oils and films specimens were equilibrated at 65+2%RH at 25+2C for 48h. 2.5.8.1.Determination of Minimum Inhibitory Concentration (MIC). Afterwards,test films were fixed onto opening cells containing sil- A broth microdilution method was used to determine MIC(Gende ica gel (0%RH)and the cells were placed in an controlled humidity et al.,2010).Appropriate amounts of essential oil were mixed in chamber at65±2%RH and25±2°C.The air gap inside the cell was water and emulsified with 8%(v/v)propylene glycol,thus obtain- ~1.2 cm and the film area exposed for water vapor transmission ing a mother solution (MS).For broth microdilution,100 uL of was 13.8 cm2.The cells were weighed hourly over a 10h period. Muller Hilton medium was placed in each of the 96-well microtiter WVP was calculated from the following equation: plates and then diluted with MS to obtain serial dilutions.Micro- WVP (kg ms-1 Pa-m2)=ArApe bial biomass suspension of E.coli and S.aureus (approximately (5) 10>-10 CFU/mL)was individually added to each serial dilution. yielding final value concentrations ranging from 12.5 to 2000 ppm. where w is the weight gain of the cup(Kg)at time t(s):e is the All microtiter plates(with positive and negative controls)were film thickness(m):A is the exposed area of the film(m2):APis the incubated at 35+0.5C for 48h in order to determine MIC val- vapor pressure difference across the film(Pa).All measurements ues.Experiments were conducted in triplicate.Chloramphenicol were made in triplicate. (30 wg)was used as a reference standard. 2.5.7.7 In vitro antioxidant assay 2.5.7.1.Ferric reducing antioxidant power(FRAP)assay.FRAP assay 2.5.8.2.Antimicrobial activity of films by disc diffusion method. was carried out according to the method described by Oyaizu Antibacterial activity on films was assessed using the agar diffusion (1986).Basically,1 mL of the essential oils (2000-6000 ppm)was method described by Pereda et al.(2011).Films were aseptically mixed with phosphate buffer(2.5 mL,0.2 mol/L,pH 6.6)and 2.5 ml cut into a 10mm diameter disc using a circular knife and then of potassium hexacyanoferrate Ill(K3Fe(CN)6.1%(w/v)).The mix- placed on agar plates,previously seeded with 100 uL of inoculum ture was incubated at 50C for 20min.Afterwards 2.5 mL of a containing approximately 105-106 CFU/mL of each tested bacteria. 10%(w/v)trichloroacetic acid solution was added to the mixture. The plates were then incubated at 37C for 24h.The diameter of which was then centrifuged at 5000rpm for 10 min(Sartorius type the growth inhibition zone surrounding the film discs was accu- 4-15,Germany).A 2.5mL aliquot of the upper layer was mixed rately measured with a manual caliper from the center of the film with 2.5 mL of deionized water and 0.5 mL of a 0.1%(w/v)FeCl3 (Mitutoyo,Japan).Each assay was performed by triplicate on two solution.Absorbance was measured at 700 nm in a UV-visible spec- separate experimental runs. trophotometer(Agilent 8453,China).The increased absorbance of the reaction mixture indicated increased reducing power.Ascorbic acid(AA)was used as reference material and the final results were 2.5.9.Statistical analysis expressed as ppm of ascorbic acid equivalents(AAE).All tests were Experimental data were statistically analyzed by one-way anal- performed in triplicate and the graph was plotted with the average ysis of variance (ANOVA)using the OriginPro 8 software and of the three determinations. Tukey's test for comparison of means at a 5%of significance level

J.F. Martucci et al. / Industrial Crops and Products 71 (2015) 205–213 207 2.5.3. Scanning electronic microscopy (SEM) Fracture surfaces were observed on a Phillips 505 microscope (Eindhoven, The Netherlands) at 10 kV.All specimens were sputter￾coated with gold. 2.5.4. Water uptake Wateruptake test wasperformed gravimetrically. Samples were placed in pre- weighed aluminum cups and dried at 45 ◦C in a vac￾uumoven (Gallenkamp, UK) up to constant weight(m0). Specimens were then kept at 25 ± 2 ◦C in a humidity chamber at 65 ± 2% RH following the procedure described in ASTM E104-95. Samples were removed at specific time intervals and weighed with a precision of ±0.0001 g until reaching constant weight (mf). The water uptake at equilibrium (WUeq) was calculated as the weight gain according to: WUeq(%) = 100(mf − m0) m0 (4) This experiment was carried out on four specimens of each sample to ensure results reproducibility. 2.5.5. Mechanical properties Tensile strength at breaking (TS) and percentage of elongation (%) were measured according to theASTM 638 94 D standard using an Instron 4467 Universal Testing Machine (Buckinghamshire, Eng￾land) with a 5 kN load cell and at a crosshead speed of 10 mm/min. Samples were stored at 65 ± 2% RH and 25 ± 2 ◦C prior to measure￾ments. Reported results were obtained from at least 10 samples for each type of film. 2.5.6. Water vapor permeability (WVP) Water vapour permeability (WVP) was performed gravimet￾rically at 25 ◦C, using the ASTM E96-95 desiccant method. All specimens were equilibrated at 65 ± 2% RH at 25 ± 2 ◦C for 48 h. Afterwards, test films were fixed onto opening cells containing sil￾ica gel (0%RH) and the cells were placed in an controlled humidity chamber at 65 ± 2% RH and 25 ± 2 ◦C. The air gap inside the cell was ∼1.2 cm and the film area exposed for water vapor transmission was 13.8 cm2. The cells were weighed hourly over a 10 h period. WVP was calculated from the following equation: WVP kg ms−1 Pa−1m−2  = w AtP e (5) where w is the weight gain of the cup (Kg) at time t (s); e is the film thickness (m); A is the exposed area of the film (m2); P is the vapor pressure difference across the film (Pa). All measurements were made in triplicate. 2.5.7. 7 In vitro antioxidant assay 2.5.7.1. Ferric reducing antioxidant power (FRAP) assay. FRAP assay was carried out according to the method described by Oyaizu (1986). Basically, 1 mL of the essential oils (2000–6000 ppm) was mixed with phosphate buffer (2.5 mL, 0.2 mol/L, pH 6.6) and 2.5 mL of potassium hexacyanoferrate III (K3Fe(CN)6, 1% (w/v)). The mix￾ture was incubated at 50 ◦C for 20 min. Afterwards 2.5 mL of a 10% (w/v) trichloroacetic acid solution was added to the mixture, which was then centrifuged at 5000 rpm for 10 min (Sartorius type 4-15, Germany). A 2.5 mL aliquot of the upper layer was mixed with 2.5 mL of deionized water and 0.5 mL of a 0.1% (w/v) FeCl3 solution.Absorbance was measured at 700 nm in a UV–visible spec￾trophotometer (Agilent 8453, China). The increased absorbance of the reaction mixture indicated increased reducing power. Ascorbic acid (AA) was used as reference material and the final results were expressed as ppm of ascorbic acid equivalents (AAE). All tests were performed in triplicate and the graph was plotted with the average of the three determinations. 2.5.7.2. Determination of DPPH radical scavenging activity. DPPH radical scavenging activity (RSA) was determined on the basis of the method by Yen and Hsieh (1995) with slight modifications. A volume of 400 L of each essential oil solution (0–6000 ppm) was mixed with 2 mL of a 0.06 mmol/L solution of DPPH in methanol. Mixtures were shaken vigorously and allowed to stand in the dark for 30 min at room temperature. The absorbance of the resulting solutions was monitored at 517 nm using an UV–visible spec￾trophotometer (Agilent 8453, China). The control was prepared in the same manner except that methanol was used instead of the sample. DPPH radical scavenging activity (RSA) was calculated according to the following equation (Yen and Hsieh, 1995): RSA(%) =  1 − A517sample A517control  100 (6) where A517sample and A517control correspond to the absorbances at 517 nm of the radical (DPPH•) in the absence and presence of antioxidant respectively. IC50 is defined as the efficient concen￾tration required to decrease the initial DPPH radical concentration by 50%. In the case of films, samples were immersed in liquid nitrogen and subsequently crushed and grounded with a pestle. A precise amount of crushed film (0.4 g) was mixed with 4 mL of methanol in a caped tube, stirred vigorously and allowed to stand overnight (about 12 h). Afterwards, tubes were centrifuged at 5000 rpm for 10 min (Sartorius type 4-15, Germany) and the supernatant was recovered and reserved for DPPH radical scavenging activity and reducing power as outlined above for EOs. In the case of reducing power, final results were expressed per g of film. 2.5.8. In vitro antibacterial assay of essential oils and films 2.5.8.1. Determination of Minimum Inhibitory Concentration (MIC). A broth microdilution method was used to determine MIC (Gende et al., 2010). Appropriate amounts of essential oil were mixed in water and emulsified with 8% (v/v) propylene glycol, thus obtain￾ing a mother solution (MS). For broth microdilution, 100 L of Muller Hilton medium was placed in each of the 96-well microtiter plates and then diluted with MS to obtain serial dilutions. Micro￾bial biomass suspension of E. coli and S. aureus (approximately 105–106 CFU/mL) was individually added to each serial dilution, yielding final value concentrations ranging from 12.5 to 2000 ppm. All microtiter plates (with positive and negative controls) were incubated at 35 ± 0.5 ◦C for 48 h in order to determine MIC val￾ues. Experiments were conducted in triplicate. Chloramphenicol (30 g) was used as a reference standard. 2.5.8.2. Antimicrobial activity of films by disc diffusion method. Antibacterial activity on films was assessed using the agar diffusion method described by Pereda et al. (2011). Films were aseptically cut into a 10 mm diameter disc using a circular knife and then placed on agar plates, previously seeded with 100 L of inoculum containing approximately 105–106 CFU/mL of each tested bacteria. The plates were then incubated at 37 ◦C for 24 h. The diameter of the growth inhibition zone surrounding the film discs was accu￾rately measured with a manual caliper from the center of the film (Mitutoyo, Japan). Each assay was performed by triplicate on two separate experimental runs. 2.5.9. Statistical analysis Experimental data were statistically analyzed by one-way anal￾ysis of variance (ANOVA) using the OriginPro 8 software and Tukey’s test for comparison of means at a 5% of significance level.

208 J.F.Martucci et aL Industrial Crops and Products 71(2015)205-213 Table 1 (Inouye et al.,2001).Gram-positive bacteria,in turn,lack the rigid Main compounds,expressed as percentage of chromatographic area of L officinalis and O.vulgare essential oils. outer membrane being more susceptible to the action of EOs. The effectiveness of pure constituents of OEO and LEO against Components() the studied bacteria was statistically lower than that of EOs Lavander (Lavandula officinalis) Oregano (Origanum vulgare) (Table 2)indicating that minor ingredients are critical to the activ- Linalool 53.50 ity,as previously observed by others (Kulevanova and Panovska Carvacrol 26.70 Camphor 8.40 p-Cimene 1520 2001:Canillac and Mourey,2001:Gende et al.,2010:Danh et al. Terpinen-4-ol 7.60 y-Terpinene 15.10 2012:Teixeira et al.,2013a).Furthermore,both pathogens were 18-cineol 6.80 Terpinene 7.50 less susceptible to the action of lavender oil and to its major compo- Borneol 4.70 o-Pinene 5.60 nent,linalool (Table 2),suggesting that phenolic compounds seem Linalyl acetate 420 Iso borneol 3.80 080 3.40 to govern the antibacterial ability of Eos(Burt,2004:Tassou et al. Lavandulvl acetate Terpinolene Hexilacetate 0.60 B-Myrcene 3.40 2000).Similar trend was previously observed for thyme and basil 1-0cten-3-0 055 c-Thujene 3.40 essential oils(and their major compounds:thymol,estragol,car- 3-octanone 0.40 o-Terpineol 2.30 vacrol,linalool,and p-cymene)being carvacrol the one exerting Myrcene 030 Methyl carvacrol 220 the strongest antibacterial activity against E.coli followed by thy- Caryophyllene 1.40 Sabinene 1.40 mol.Estragol and linalool,in turn,exhibited limited antibacterial Endo borneol 120 activity (Bagamboula et al.,2004). Thymol 1.10 The potential antagonistic or synergic effect among components Terpinen-4-ol 1.10 of both essentials oils was experimentally analyzed on mixtures B-Phellandrene 0.60 Camphene 0.40 of LEO:OEO 50:50 The obtained results (Table 2)reflected that 1.8-cineol 0.30 the tested microorganisms were less susceptible to the action of Total 87.85 Total 96.1 the mixture,resulting in a lower antimicrobial effect than the sum of the single effects produced by each essential oil individu- ally,thereby indicating a possible antagonistic effect between their 3.Results and discussion components(Canillac and Mourey,2001:Sadaka et al.,2013). The antioxidant capacity of EOs was revealed through their 3.1.Characterization of oregano and lavender essential oils reducing power and DPPH radical scavenging efficiency,respec- tively.In Fig.1 a,the FRAP value points reflect a ten-fold higher Active components in oregano and lavender essential oils iden- reducing power of OEO than that of lavender oil,in accordance with tified by GC/MS are summarized in Table 1.The quantitative the presence of electron donor chemicals such as carvacrol and thy- analysis of lavender essential oil revealed a prevalence of linalool mol,which can react with free radicals and turn them into more (53.50%),followed by camphor (8.40%).terpinen-4-ol (7.60%)and stable products,and so terminate radical chain reactions (Burt 1.8-cineol(6.80%).The results of the chemical profile of LEO com- 2004).Moreover,OEO exhibited a strong dose-dependent reducing pare favorably with those of earlier studies (Inouye et al.,2001 power rising from 11 ppm of AA for 200 ppm up to 114 ppm of AA Dahn et al.,2012 Teixeira et al.,2013a).The prevailing compounds for 3000 ppm.Higher OEO concentrations did not induce signifi- of oregano essential oil were aromatic monoterpenes:carvacrol cant changes in the reducing power,indicating a saturation level (26.70%)and thymol (1.10%)and aliphatic monoterpenes being p- of around 3000ppm.By contrast,no significant variations were cymene(15.20%).y-terpinene(15.10%)and terpinene(7.50%)the evidenced for LEO activity which exhibited a lag phase of up to major constituents of this group.Plants that produce carvacrol and 2000 ppm followed by a minor increment of up to 17 ppm for a thymol almost always have relatively high amounts of p-cymene concentration of 6000 ppm.These results are in agreement with and y-terpinene,which are biosynthetic precursors for both phe- OEO composition constituted by phenolic compounds(Table 1). nolic monoterpenes (Poulose and Croteau,1978).Differences in The phenolic compounds are free radical acceptors that delay or essential oil compounds may be influenced by geographical vari- inhibit the autoxidation initiation step or interrupt the autoxida- ables,time of plant harvesting.preparation process extraction tion propagation step (Kacaniova et al..2012:Eca et al..2014) method and quantification conditions (Burt,2004:Gende et al. Fig.1 b clearly shows that the radical scavenging activity of OEO is 2010).Several studies on O.vulgare ssp.cultivated in different significantly higher as compared to that of LEO for the same concen- regions worldwide have centered their attention on the variabil- trations.As it can be observed in Fig.1 b,OEO free radical scavenging ity of chemical composition.Different percentages of thymol and effect also exhibited a dose-dependent increase having a 74%RSA carvacrol have been reported as majority compounds in this vegetal for 6000 ppm,in line with the results reported by Kacaniova et al. specie(D'Antuono et al.,2000;Oussalah et al.,2004:Gomez-Estaca (2012)for the same concentration of oregano essential oil.LEO did etal,2009). not show significant activity for all concentrations tested,i.e.,from EOs antibacterial efficiency was quantified by determining the 2000 to 6000 ppm.The weakest activity of LEO could be associated minimum inhibitory concentration (MIC).Oregano and lavender with the absence of phenolic compounds in its composition. essential oils,together with their major components.c.a.carvacrol and linalool,were evaluated against E.coli and S.aureus.MIC values Table 2 are summarized in Table 2.It is noteworthy that greater inhibition Antimicrobial activity of essential oils against E coli and S.aureus strains. of oils and pure compounds was observed against S.aureus.This finding is in line with early studies focused on the action of whole Minimum inhibitory concentration(MIC) E.coli S.aureus EOs against food spoilage organisms and food-borne pathogens Lavender EO (LEO) 2000 1000-1200 (Inouye et al.,2001:Burt,2004)and it is thought to arise as a Oregano Eo(oEO 1600-1800 800-900 LE0:0E050-50 >2000 1600-1800 result of the differences in their cell membrane structure and Thymol 1000 800-900 the hydrophobic character of essential oils and their components Carvacrol 600 500-600 (Vaara,1992:Inouye et al.,2001 Inouye et al.,2001).Gram nega- Linalool 2000 2000 tive bacteria possess an outer membrane surrounding the cell wall Chloramphenicol 8 2 composed by hydrophilic polysaccharides which restricts diffusion Notes:Data are MIC (ug/mL)range values.The antimicrobial activity was deter- of hydrophobic compounds such as OEs and their main constituents mined by triplicate analyses for oil and strains

208 J.F. Martucci et al. / Industrial Crops and Products 71 (2015) 205–213 Table 1 Main compounds, expressed as percentage of chromatographic area of L. officinalis and O. vulgare essential oils. Components(%) Lavander (Lavandula officinalis) Oregano (Origanum vulgare) Linalool 53.50 Carvacrol 26.70 Camphor 8.40 p-Cimene 15.20 Terpinen-4-ol 7.60 −Terpinene 15.10 1,8- cineol 6.80 Terpinene 7.50 Borneol 4.70 − Pinene 5.60 Linalyl acetate 4.20 Iso borneol 3.80 Lavandulyl acetate 0.80 Terpinolene 3.40 Hexilacetate 0.60 - Myrcene 3.40 1-octen-3-ol 0.55 − Thujene 3.40 3- octanone 0.40 - Terpineol 2.30 Myrcene 0.30 Methyl carvacrol 2.20 Caryophyllene 1.40 Sabinene 1.40 Endo borneol 1.20 Thymol 1.10 Terpinen-4-ol 1.10 - Phellandrene 0.60 Camphene 0.40 1,8- cineol 0.30 Total 87.85 Total 96.1 3. Results and discussion 3.1. Characterization of oregano and lavender essential oils Active components in oregano and lavender essential oils iden￾tified by GC/MS are summarized in Table 1. The quantitative analysis of lavender essential oil revealed a prevalence of linalool (53.50%), followed by camphor (8.40%), terpinen-4-ol (7.60%) and 1.8-cineol (6.80%). The results of the chemical profile of LEO com￾pare favorably with those of earlier studies (Inouye et al., 2001; Dahn et al., 2012 Teixeira et al., 2013a). The prevailing compounds of oregano essential oil were aromatic monoterpenes: carvacrol (26.70%) and thymol (1.10%) and aliphatic monoterpenes being p￾cymene (15.20%), - terpinene (15.10%) and terpinene (7.50%) the major constituents of this group. Plants that produce carvacrol and thymol almost always have relatively high amounts of p-cymene and -terpinene, which are biosynthetic precursors for both phe￾nolic monoterpenes (Poulose and Croteau, 1978). Differences in essential oil compounds may be influenced by geographical vari￾ables, time of plant harvesting, preparation process extraction method and quantification conditions (Burt, 2004; Gende et al., 2010). Several studies on O. vulgare ssp. cultivated in different regions worldwide have centered their attention on the variabil￾ity of chemical composition. Different percentages of thymol and carvacrol have been reported as majority compounds in this vegetal specie (D’Antuono et al., 2000; Oussalah et al., 2004; Gómez-Estaca et al., 2009). EOs antibacterial efficiency was quantified by determining the minimum inhibitory concentration (MIC). Oregano and lavender essential oils, together with their major components, c.a. carvacrol and linalool, were evaluated against E. coli and S. aureus. MIC values are summarized in Table 2. It is noteworthy that greater inhibition of oils and pure compounds was observed against S. aureus. This finding is in line with early studies focused on the action of whole EOs against food spoilage organisms and food-borne pathogens (Inouye et al., 2001; Burt, 2004) and it is thought to arise as a result of the differences in their cell membrane structure and the hydrophobic character of essential oils and their components (Vaara, 1992; Inouye et al., 2001 Inouye et al., 2001). Gram nega￾tive bacteria possess an outer membrane surrounding the cell wall composed by hydrophilic polysaccharides which restricts diffusion of hydrophobic compounds such as OEs and their main constituents (Inouye et al., 2001). Gram-positive bacteria, in turn, lack the rigid outer membrane being more susceptible to the action of EOs. The effectiveness of pure constituents of OEO and LEO against the studied bacteria was statistically lower than that of EOs (Table 2) indicating that minor ingredients are critical to the activ￾ity, as previously observed by others (Kulevanova and Panovska, 2001; Canillac and Mourey, 2001; Gende et al., 2010; Danh et al., 2012; Teixeira et al., 2013a). Furthermore, both pathogens were less susceptible to the action of lavender oil and to its major compo￾nent, linalool (Table 2), suggesting that phenolic compounds seem to govern the antibacterial ability of Eos (Burt, 2004; Tassou et al., 2000). Similar trend was previously observed for thyme and basil essential oils (and their major compounds: thymol, estragol, car￾vacrol, linalool, and p-cymene) being carvacrol the one exerting the strongest antibacterial activity against E. coli followed by thy￾mol. Estragol and linalool, in turn, exhibited limited antibacterial activity (Bagamboula et al., 2004). The potential antagonistic or synergic effect among components of both essentials oils was experimentally analyzed on mixtures of LEO:OEO 50:50 The obtained results (Table 2) reflected that the tested microorganisms were less susceptible to the action of the mixture, resulting in a lower antimicrobial effect than the sum of the single effects produced by each essential oil individu￾ally, thereby indicating a possible antagonistic effect between their components (Canillac and Mourey, 2001; Sadaka et al., 2013). The antioxidant capacity of EOs was revealed through their reducing power and DPPH radical scavenging efficiency, respec￾tively. In Fig. 1 a, the FRAP value points reflect a ten-fold higher reducing power of OEO than that of lavender oil, in accordance with the presence of electron donor chemicals such as carvacrol and thy￾mol, which can react with free radicals and turn them into more stable products, and so terminate radical chain reactions (Burt, 2004). Moreover, OEO exhibited a strong dose-dependent reducing power rising from 11 ppm of AA for 200 ppm up to 114 ppm of AA for 3000 ppm. Higher OEO concentrations did not induce signifi- cant changes in the reducing power, indicating a saturation level of around 3000 ppm. By contrast, no significant variations were evidenced for LEO activity which exhibited a lag phase of up to 2000 ppm followed by a minor increment of up to 17 ppm for a concentration of 6000 ppm. These results are in agreement with OEO composition constituted by phenolic compounds (Table 1). The phenolic compounds are free radical acceptors that delay or inhibit the autoxidation initiation step or interrupt the autoxida￾tion propagation step (Kacániová ˇ et al., 2012; Ec¸ a et al., 2014). Fig. 1 b clearly shows that the radical scavenging activity of OEO is significantly higher as compared to that of LEO for the same concen￾trations.As it canbe observed inFig. 1 b, OEO free radical scavenging effect also exhibited a dose-dependent increase having a 74% RSA for 6000 ppm, in line with the results reported by Kacániová ˇ et al. (2012) for the same concentration of oregano essential oil. LEO did not show significant activity for all concentrations tested, i.e., from 2000 to 6000 ppm. The weakest activity of LEO could be associated with the absence of phenolic compounds in its composition. Table 2 Antimicrobial activity of essential oils against E. coli and S. aureus strains. Minimum inhibitory concentration (MIC) E. coli S. aureus Lavender EO (LEO) 2000 1000–1200 Oregano EO (OEO) 1600-1800 800–900 LEO:OEO 50:50 >2000 1600–1800 Thymol 1000 800–900 Carvacrol 600 500–600 Linalool 2000 2000 Chloramphenicol 8 2 Notes: Data are MIC (g/mL) range values. The antimicrobial activity was deter￾mined by triplicate analyses for oil and strains.

JF.Martucci et aL Industrial Crops and Products 71(2015)205-213 209 Table3 Thickness and color parameters of obtained films. Thickness(mm) 1 a b C'ab h'ab △E Ge 0.11±0.01m 89.07±0.16 0.07±0.07a 2.22±0.51a 2.23±0.51m 1.54±0.07 10.81±0.53m 0E02000-Ge 0.10±0.01a 88.95±0.159 0.23±0.09a 2.02±0.60 2.03±0.60 1.46±0.09▣ 10.67±0.710 0E03000-Ge 0.13±0.03° 89.01±0.21 0.23±0.11 1.49±1.09 1.50±1.09 1.42±0.11a 10.25±0.99 0E04000-Ge 0.11±0.02° 89.12±0.40° 0.17±0.12 2.17±1.020 2.18±1.02° 1.49±0.120 10.73±1.08 0E05000-Ge 0.11±0.02° 88.96±0.36 -0.23±0.12 5.12±1.11b 5.13±1.11b -1.52±0.12b 13.56±1.01b 0E06000-Ge 0.13±0.02 88.89±0.17 -0.24±0.06b 4.82±0.530 4.82±0.53 -1.52±0.06 13.35±0.52 LFO2000-Ge 0.07±0.02" 89.00±0.28 -0.29±0.21× 496±0.87严 2.68±0.87 -1.23±0.10 12.55±1.28 LE03000-Ge 0.09±0.02y 88.96±0.68 -0.44±0.20 5.18±1.199 520±2.19 -1.49±0.08 13.71±1.09y LE04000-Ge 0.09±0.02y 88.18±0.60 -0.45±0.38x 5.86±1.259 3.89±125w -1.45±0.11y 12.71±1.39y LE05000-Ge 0.11±0.039 88.52±0.49 -0.60±0.23x 5.74±1.750 5.77±1.752 -1.47±0.13y 14.38±1.83y LE06000-Ge 0.11±0.02 88.02±035 -0.56±0.31× 7.23±1.30 7.25±330 -1.49±0.11 15.94±1.38 Any two means in the same column followed by the same letter are not significantly (P>0.05)different according to Tukey test High radical scavenging activity and reducing power has been antioxidant capacity in relation to the content of oregano essen- observed in the main components of oregano essential oil (car- tial oil.The lower antioxidant activity of the mixture denoted a vacrol,thymol,y-terpinene and p-cymene)(Aeschbach et al.,1994: possible antagonistic effect between their components as previ- Oussalah et al.,2004).However,other compounds could contribute ously observed in the antimicrobial activity.Taking into account to the antioxidant properties of oregano oil. the results reported,EOs mixture was not considered for films for- The antioxidant capacity of the LEO:OEO 50:50 mixture was mulation analyzed by DPPH radical scavenging activity and Ferric reduc- ing power assay.The results obtained indicated a reduction in the 3.2.Properties of EO-added gelatin films 3.2.1.Optical properties 140 OEO Gelatin films added with oregano or lavender essential oils,on an individual basis,were prepared by casting using propylene gly- 120 -0…LE0 △LE0:0E050:50 col as plasticizer and emulsifier.The film optical properties are relevant since they play a major role in the appearance of the coated 100 product.In order to compare the differences between control films and those prepared with the essential oils,Table 3 reports the color 80- (wdd) parameters of the films obtained.Visually:Ge films were quite transparent,though they turned yellowish when the essential oil 60 was added.As far as OEO differences are concerned,they were not significant (P=0.05)up to 3000 ppm of OEO(Table 3).which is 40 consistent with the findings reported by Min and Oh(2009)for cat fish gelatin coatings containing oregano essential oil and Teixeira et al.(2014)for fish gelatin films.On the other hand,LEO-Ge films presented higher C"ab and AE*values and lower h'ab.AE*LEO- Ge values were higher than OEO-Ge ones due to the increase in absorption caused by the interaction between the components.In 1000 2000 3000 4000 5000 6000 LEO-Ge films,Cab values increased with the amount oflavender oil EOs concentration (ppm) added,indicating an increase in yellowish coloration.These results suggested that the incorporation of essential oil influenced film color,though changes depended on the type of essential oil.Ahmad OEO et al.(2012)showed that the incorporation of lemongrass oil in o LEO fish skin gelatin films increased its total color difference,yet there 4 LE0:0E050:50 70 was no difference in the color parameters of the films incorporated with bergamot oil.The considerable effects of the incorporation 60 of cinnamon oil on the color of caseinate-based edible films were IC50oeo:697±89ppm reported by Atares et al.(2010a).However,no marked effect on the color parameters of those films was obtained when ginger oil 40、 was added (Atares et al.,2010a).It was suggested that essential oil also influenced the color of gelatin film,depending on the type and 30 concentrations of the essential oil incorporated. 20 3.2.2.Films morphology 10 The cross-section of Ge control film was compact and smooth without pores or cracks(Fig 2a)in agreement with previous reports (Martucci and Ruseckaite,2009:Tongnuanchan et al.2012).With 1000 2000 3000 4000 5000 6000 the addition of essential oils,the cross-section of films became EOs concentration(ppm) slightly rougher as compared to the control(Fig 2b-e):however. no oil droplets were observed in the film thickness even at the Fig.1.(a)Reducing Power and(b)DPPH radical scavenging activity(RSA)oforegano and lavender essential oils.Bars represent the standard deviation from triplicate highest concentration tested.This result indicates that the film- determinations. forming dispersion was a stable so that no collapse of emulsion

J.F. Martucci et al. / Industrial Crops and Products 71 (2015) 205–213 209 Table 3 Thickness and color parameters of obtained films. Thickness (mm) l * a* b* C*ab h*ab E* Ge 0.11 ± 0.01ax 89.07 ± 0.16ax 0.07 ± 0.07 a 2.22 ± 0.51a 2.23 ± 0.51ax 1.54 ± 0.07a 10.81 ± 0.53ax OEO2000-Ge 0.10 ± 0.01a 88.95 ± 0.15a 0.23 ± 0.09a 2.02 ± 0.60a 2.03 ± 0.60a 1.46 ± 0.09a 10.67 ± 0.71a OEO3000-Ge 0.13 ± 0.03a 89.01 ± 0.21a 0.23 ± 0.11a 1.49 ± 1.09a 1.50 ± 1.09a 1.42 ± 0.11a 10.25 ± 0.99a OEO4000-Ge 0.11 ± 0.02a 89.12 ± 0.40a 0.17 ± 0.12a 2.17 ± 1.02a 2.18 ± 1.02a 1.49 ± 0.12a 10.73 ± 1.08a OEO5000-Ge 0.11 ± 0.02a 88.96 ± 0.36a -0.23 ± 0.12b 5.12 ± 1.11b 5.13 ± 1.11b -1.52 ± 0.12b 13.56 ± 1.01b OEO6000-Ge 0.13 ± 0.02a 88.89 ± 0.17a -0.24 ± 0.06b 4.82 ± 0.53b 4.82 ± 0.53b -1.52 ± 0.06b 13.35 ± 0.52b LEO2000-Ge 0.07 ± 0.02y 89.00 ± 0.28x -0.29 ± 0.21x 4.96 ± 0.87x 2.68 ± 0.87x -1.23 ± 0.10x 12.55 ± 1.28x LEO3000-Ge 0.09 ± 0.02xy 88.96 ± 0.68x -0.44 ± 0.20x 5.18 ± 1.19xy 5.20 ± 2.19y -1.49 ± 0.08y 13.71 ± 1.09xy LEO4000-Ge 0.09 ± 0.02xy 88.18 ± 0.60x -0.45 ± 0.38x 5.86 ± 1.25xy 3.89 ± 1.25xy -1.45 ± 0.11y 12.71 ± 1.39xy LEO5000-Ge 0.11 ± 0.03xy 88.52 ± 0.49x -0.60 ± 0.23x 5.74 ± 1.75xy 5.77 ± 1.75yz -1.47 ± 0.13y 14.38 ± 1.83y LEO6000-Ge 0.11 ± 0.02x 88.02 ± 0.35x -0.56 ± 0.31x 7.23 ± 1.30y 7.25 ± 3.30z -1.49 ± 0.11y 15.94 ± 1.38y * Any two means in the same column followed by the same letter are not significantly (P > 0.05) different according to Tukey test. High radical scavenging activity and reducing power has been observed in the main components of oregano essential oil (car￾vacrol,thymol, -terpinene and p-cymene)(Aeschbach et al., 1994; Oussalah et al., 2004). However, other compounds could contribute to the antioxidant properties of oregano oil. The antioxidant capacity of the LEO:OEO 50:50 mixture was analyzed by DPPH radical scavenging activity and Ferric reduc￾ing power assay. The results obtained indicated a reduction in the Fig. 1. (a) Reducing Power and (b) DPPH radical scavenging activity (RSA) of oregano and lavender essential oils. Bars represent the standard deviation from triplicate determinations. antioxidant capacity in relation to the content of oregano essen￾tial oil. The lower antioxidant activity of the mixture denoted a possible antagonistic effect between their components as previ￾ously observed in the antimicrobial activity. Taking into account the results reported, EOs mixture was not considered for films for￾mulation. 3.2. Properties of EO-added gelatin films 3.2.1. Optical properties Gelatin films added with oregano or lavender essential oils, on an individual basis, were prepared by casting using propylene gly￾col as plasticizer and emulsifier. The film optical properties are relevant since they play a major role in the appearance ofthe coated product. In order to compare the differences between control films and those prepared with the essential oils, Table 3 reports the color parameters of the films obtained. Visually; Ge films were quite transparent, though they turned yellowish when the essential oil was added. As far as OEO differences are concerned, they were not significant (P ≥ 0.05) up to 3000 ppm of OEO (Table 3), which is consistent with the findings reported by Min and Oh (2009) for cat fish gelatin coatings containing oregano essential oil and Teixeira et al. (2014) for fish gelatin films. On the other hand, LEO-Ge films presented higher C*ab and E* values and lower h*ab. E* LEO￾Ge values were higher than OEO-Ge ones due to the increase in absorption caused by the interaction between the components. In LEO-Ge films, C*ab values increased with the amount oflavender oil added, indicating an increase in yellowish coloration. These results suggested that the incorporation of essential oil influenced film color,though changes depended on the type of essential oil. Ahmad et al. (2012) showed that the incorporation of lemongrass oil in fish skin gelatin films increased its total color difference, yet there was no difference in the color parameters of the films incorporated with bergamot oil. The considerable effects of the incorporation of cinnamon oil on the color of caseinate-based edible films were reported by Atarés et al. (2010a). However, no marked effect on the color parameters of those films was obtained when ginger oil was added (Atarés et al., 2010a). It was suggested that essential oil also influenced the color of gelatin film, depending on the type and concentrations of the essential oil incorporated. 3.2.2. Films morphology The cross-section of Ge control film was compact and smooth without pores or cracks (Fig 2a)in agreement with previous reports (Martucci and Ruseckaite, 2009; Tongnuanchan et al., 2012). With the addition of essential oils, the cross-section of films became slightly rougher as compared to the control (Fig 2b–e); however, no oil droplets were observed in the film thickness even at the highest concentration tested. This result indicates that the film￾forming dispersion was a stable so that no collapse of emulsion

210 J.F.Martucci et aL Industrial Crops and Products 71(2015)205-213 Table 4 est water uptake(i.e,26.0±6.8‰)，in accordance to its hydrophilic Mechanical properties,water vapor permeability (WVP)and water uptake (WUeg) nature (Martucci and Ruseckaite,2009:Martucci et al.,2012: of Ge films with and without essential oils. Kavoosi et al.,2013).The incorporation of LEO into gelatin matrix Mechanical properties WVP'1013 WUeq reduced the water absorption capacity (14.8+1.0%for 6000 ppm). e(3 TS(MPa) (Kg.m/Pa.s.m2)() It is hypothesized that non polar EOs interact favorably with Ge 10.8±3.0 17.7±2.8m 1.46±0.13 26.0±6.8m hydrophobic domains in gelatin inducing conformational changes 0E02000-Ge 8.3±3.7° 14.0±1.6b 121±0.45 23.8±3.7加 in gelatin chains that cannot promote protein-water interaction 0E03000-Ge 92±3.4 13.9±1.8 0.88士0.019 202±1.90 (Djagny et al.,2001:Kavoosi et al.,2013).Nevertheless,no signifi- 0E04000-Ge 9.7±2.6° 12.2±2.8hc 1.10±0.20° 21.0±2.1a cant differences were noted in OEO incorporated films(P>0.05) 0E05000-Ce 10.1±2.2° 8.9±3.3 0.81±0.039 19.5±1.70 The different behavior could be due to the differences in the 0E06000-Ge 10.0±1.99 113±3.2c 0.84±0.01 18.1±2.3 LE02000-Ge 43±0.8y 88±2.2z 127±0.07r 203±3.1x hydrophobic nature of the oils used,which resulted in a different LEO3000-Ge 7.6±3.09 15.4±2.1w 0.97±0.10 19.6±2.3 ability to attract water to the film network. LE04000-Ge 6.3±2.0￥ 12.1±1.9w 1.05±0.03 17.8±1.69y LE05000-Ge 4.6±0.3 11.7±2.4w 1.02±0.04 17.5±1.4 LE06000-Ge 73±2.09.12.8±2.82w 0.68±0.02W 14.8±1.0 3.2.4.Water vapor permeability WVP values of LEO-Ge films were slightly decreased when Any two means in the same column followed by the same letter are not signifi- cantly different(P>0.05)based on the Turkey test. lavender concentrations remained above 2000 ppm (Table 4, P<0.05).Nevertheless,in OEO-Ge films,no significant differences (Table 4.P20.05)were found among samples with different occurred during dehydration (Hosseini et al..2009;Atares et al., contents of oregano oil phase.Lavender and oregano oil incor- 2010b:Tongnuanchan et al.,2012). poration affected WVP of gelatin film in a different way.due to the hydrophobic nature of essential oils,which affects the 3.2.3.Water uptake hydrophilic/hydrophobic balance of the film(Ojagh et al..2010; Water uptake at the equilibrium of gelatin films incorporated Teixeira et al..2014)based on the water uptake capacity.Sim- with EOs is summarized in Table 4.Gelatin films exhibited the high- ilar results were obtained by Ahmad et al.(2012)in fish skin 15kU X508 5OMm 25/APR/13 15kU X500 58wm 13 31 SEI C D 15k0 X500 58Mm 15 31 SEI 31sE1 E Fig.2.Scanning electron microscopic images of cross sections of Ge(a):LEO3000-Ge(b):LEO6000-Ge(c):OEO3000-Ge(d)and OEO6000-Ge (e)films

210 J.F. Martucci et al. / Industrial Crops and Products 71 (2015) 205–213 Table 4 Mechanical properties, water vapor permeability (WVP) and water uptake (WUeq) of Ge films with and without essential oils. Mechanical properties WVP*1013 WUeq  (%) TS (MPa) (Kg.m/Pa.s.m2) (%) Ge 10.8 ± 3.0ax 17.7 ± 2.8ax 1.46 ± 0.13ax 26.0 ± 6.8ax OEO2000-Ge 8.3 ± 3.7a 14.0 ± 1.6ab 1.21 ± 0.45a 23.8 ± 3.7a OEO3000-Ge 9.2 ± 3.4a 13.9 ± 1.8bc 0.88 ± 0.01a 20.2 ± 1.9a OEO4000-Ge 9.7 ± 2.6a 12.2 ± 2.8bc 1.10 ± 0.20a 21.0 ± 2.1a OEO5000-Ge 10.1 ± 2.2a 8.9 ± 3.3c 0.81 ± 0.03a 19.5 ± 1.7a OEO6000-Ge 10.0 ± 1.9a 11.3 ± 3.2bc 0.84 ± 0.01a 18.1 ± 2.3a LEO2000-Ge 4.3 ± 0.8y 8.8 ± 2.2yz 1.27 ± 0.07x 20.3 ± 3.1x LEO3000-Ge 7.6 ± 3.0xy 15.4 ± 2.1xw 0.97 ± 0.10y 19.6 ± 2.3x LEO4000-Ge 6.3 ± 2.0y 12.1 ± 1.9zw 1.05 ± 0.03y 17.8 ± 1.6xy LEO5000-Ge 4.6 ± 0.3y 11.7 ± 2.4zw 1.02 ± 0.04y 17.5 ± 1.4y LEO6000-Ge 7.3 ± 2.0xy 12.8 ± 2.8zw 0.68 ± 0.02w 14.8 ± 1.0z * Any two means in the same column followed by the same letter are not signifi- cantly different (P > 0.05) based on the Turkey test. occurred during dehydration (Hosseini et al., 2009; Atarés et al., 2010b; Tongnuanchan et al., 2012). 3.2.3. Water uptake Water uptake at the equilibrium of gelatin films incorporated with EOs is summarized in Table 4. Gelatin films exhibited the high￾est water uptake (i.e., 26.0 ± 6.8%), in accordance to its hydrophilic nature (Martucci and Ruseckaite, 2009; Martucci et al., 2012; Kavoosi et al., 2013). The incorporation of LEO into gelatin matrix reduced the water absorption capacity (14.8 ± 1.0% for 6000 ppm). It is hypothesized that non polar EOs interact favorably with hydrophobic domains in gelatin inducing conformational changes in gelatin chains that cannot promote protein-water interaction (Djagny et al., 2001; Kavoosi et al., 2013). Nevertheless, no signifi- cant differences were noted in OEO incorporated films (P ≥ 0.05). The different behavior could be due to the differences in the hydrophobic nature of the oils used, which resulted in a different ability to attract water to the film network. 3.2.4. Water vapor permeability WVP values of LEO-Ge films were slightly decreased when lavender concentrations remained above 2000 ppm (Table 4, P < 0.05). Nevertheless, in OEO-Ge films, no significant differences (Table 4, P ≥ 0.05) were found among samples with different contents of oregano oil phase. Lavender and oregano oil incor￾poration affected WVP of gelatin film in a different way, due to the hydrophobic nature of essential oils, which affects the hydrophilic/hydrophobic balance of the film (Ojagh et al., 2010; Teixeira et al., 2014) based on the water uptake capacity. Sim￾ilar results were obtained by Ahmad et al. (2012) in fish skin Fig. 2. Scanning electron microscopic images of cross sections of Ge (a); LEO3000-Ge (b); LEO6000-Ge (c); OEO3000-Ge (d) and OEO6000-Ge (e) films

LF.Martucci et aL Industrial Crops and Products 71 (2015)205-213 211 Table 5 Antimicrobial activity measured as inhibition zone expressed as millimeter(mm)and antioxidant activity measured as DPPH radical scavenging activity(RSA)and Ferric reducing power(expressed as ppm of ascorbic acid equivalent,AAEppm/g film)of Ge-based edible films with and without essential oils. EO(ppm) Antimicrobial activity Antioxidant activity E.coli(mm) S.aureus(mm) RSA ( AAE(ppm/g film) OEO-Ge LEO-Ge OEO-Ge LEO-Ge OEO-Ge LEO-Ge 0.0±0.0 0.0±0.0° 0.1±0.1 0.0±0.0 03±0.2 0.1士1.0° 2000 93±1.0 10.8±0.86 12.7±02b 1.5±0.6的 97±18 0.1±2.40 3000 11.7±0.4 113±1.0 17.5±3.1c 3.1±1.1x 129±6c 0.8±230 4000 13.7±0.54 10.6±1.5 36±100 5.4±2.8 135士6 0.3±2.0° 5000 14.1±0.44 13.0±0.8 49±54 6.9±3.5 189±24 0.7±22° 6000 18.0±0.8 15.0±0.5d 60±4 8.6±4.4 241±8 26.0±6.6 Any two means in the same column followed by the same letter are not significantly(P>0.05)different according to Turkey test. Standard antibiotic of chloramphenicol(30 ug/disc)was used as reference or positive control.Clearing zones around the discs were between 21 and 23 mm in diameter for both microorganisms. gelatin films enriched with bergamot and lemongrass essential films,the oils concentrations incorporated into the FFSs were equal oils.Bergamot oil caused an increased in WVP,while lemon- to or above the MIC values of the oils against microorganisms.The grass caused a decrease in WVP of the resulting films.Teixeira inhibitory effect of gelatin films incorporated with 0-6000 ppm et al.(2014)showed a significant reduction in WVP values of fish of oregano and lavender essential oil against E.coli and S.aureus gelatin films by incorporating clove essential oil,while no statisti- is illustrated in Table 5.The results indicate that both microor- cal differences were detected in films containing garlic or oregano ganisms exhibited sensitivity to all the active films.Ge-EOs films essential oils.WVP also decreased significantly with an increase showed an increase in the halo of inhibition with EOs increment. in the concentration of OEO in chitosan-cassava starch,yet this The halos obtained were slightly higher for OEO-Ge films which behavior was observed in films with concentrations greater than are in concordance with the greatest antimicrobial effect of car- 0.5%of OEO (Pelissari et al.,2009).Nevertheless,Atares et al vacrol compared to linalool (the main oil components)(Burt,2004: (2010a)found no significant differences in the WVP of sodium Bagamboula et al..2004).The present study showed that EOs were caseinate films (SC)incorporated with cinnamon or ginger essen- effective against tested bacteria at concentrations less than or equal tial oils owing to the small amount of essential oil used.In short, to 6000 ppm,which is in accordance with most of the reported types and amount of essential oil affected not only the optical works on the antimicrobial effectiveness of protein films contain- properties but also the water vapor permeability of the films ing essential oils,in most cases at concentrations varying between obtained. 1 to 5%w/V(Min and Oh,2009:Gomez-Estaca et al.,2009,2010. 2010 Ahmad et al.,2012). 3.2.5.Mechanical properties Inhibition zones obtained for S.aureus yielded lower values The oil type significantly affected both TS(resistance to elon- than those of E.coli despite the lower MIC value.This could be gation)and e(capacity for stretching)of the films obtained.The explained by the evaporation of volatile compounds involved in addition of LEO in gelatin film caused a significant decrease in TS the antimicrobial activity against S.aureus during films forming and e%if compared to the control (P0.05)was observed on Boiling point (C)and vapor pressure (mmHg)of the main components of oregano deformation at break.A possible explanation therefor lies in the and lavender essential oils. alteration of the plasticizer/gelatin ratio in the EOs added films Boiling point(C) Vapor pressure(mmHg) Water exerted a plasticizing effect on protein films(Martucci and Linalool 199 0.17at25C Ruseckaite,2009).As expressed above,the water uptake of OEO- Camphor 204 0.65at25C Ge film had no significant difference with respect to Ge control film Terpinen-4-ol 212 0.40at200 but was higher than LEO-Ge films (Table 4).Other authors reported 1.8-cineol 176 1.90at25C no difference in the e%and a reduction in TS in biopolymer films carvacrol 236 0.03at25C incorporated with antioxidant extracts (Sanchez-Gonzalez et al. Thymol 232 1.00at64C 177 3.70at38C 2009:Atares et al,2010b:Kavoosi et al.,2013:Wu et al..2014). p-cymene v-Terpinene 182 070at2090 Mechanical properties were also affected by the type and content Terpinene 174 0.80at20C of the essential oil. a-Pinene 156 4.75at25C THE MERCK INDEX,1996.-Encyclopedia of chemicals,drugs,and biologicals. 3.2.6.Antimicrobial activity 12th Ed.-Merck Research Laboratories.Division of Merck CO Inc..Whitehouse Station,NJ.USA. Gelatin films with varying amounts of EOs were tested against b Daubert.T.E R.P.Danner.Physical and thermodynamic propertiesof pure chem- E.coli and S.aureus.To minimize the incorporation of flavor to the icals data compilation.Washington,D.C.:Taylor and Francis,1989

J.F. Martucci et al. / Industrial Crops and Products 71 (2015) 205–213 211 Table 5 Antimicrobial activity measured as inhibition zone expressed as millimeter (mm) and antioxidant activity measured as DPPH radical scavenging activity (RSA,%) and Ferric reducing power (expressed as ppm of ascorbic acid equivalent, AAEppm/g film) of Ge-based edible films with and without essential oils. EO(ppm) Antimicrobial activitya Antioxidant activity E. coli (mm) S. aureus (mm) RSA (%) AAE (ppm/g film) OEO-Ge LEO-Ge OEO-Ge LEO-Ge OEO-Ge LEO-Ge 0 0.0 ± 0.0a 0.0 ± 0.0a 0.1 ± 0.1a 0.0 ± 0.0a 0.3 ± 0.2a 0.1 ± 1.0a 2000 9.3 ± 1.0b 10.8 ± 0.8b 12.7 ± 0.2b 1.5 ± 0.6b 97 ± 18b 0.1 ± 2.4a 3000 11.7 ± 0.4c 11.3 ± 1.0bc 17.5 ± 3.1c 3.1 ± 1.1bc 129 ± 6bc 0.8 ± 2.3a 4000 13.7 ± 0.5d 10.6 ± 1.5bc 36 ± 10d 5.4 ± 2.8c 135 ± 6c 0.3 ± 2.0a 5000 14.1 ± 0.4d 13.0 ± 0.8c 49 ± 5d 6.9 ± 3.5c 189 ± 2d 0.7 ± 2.2a 6000 18.0 ± 0.8e 15.0 ± 0.5d 60 ± 4e 8.6 ± 4.4c 241 ± 8e 26.0 ± 6.6b Any two means in the same column followed by the same letter are not significantly (P > 0.05) different according to Turkey test. a Standard antibiotic of chloramphenicol (30 g/disc) was used as reference or positive control. Clearing zones around the discs were between 21 and 23 mm in diameter for both microorganisms. gelatin films enriched with bergamot and lemongrass essential oils. Bergamot oil caused an increased in WVP, while lemon￾grass caused a decrease in WVP of the resulting films. Teixeira et al. (2014) showed a significant reduction in WVP values of fish gelatin films by incorporating clove essential oil, while no statisti￾cal differences were detected in films containing garlic or oregano essential oils. WVP also decreased significantly with an increase in the concentration of OEO in chitosan-cassava starch, yet this behavior was observed in films with concentrations greater than 0.5% of OEO (Pelissari et al., 2009). Nevertheless, Atarés et al. (2010a) found no significant differences in the WVP of sodium caseinate films (SC) incorporated with cinnamon or ginger essen￾tial oils owing to the small amount of essential oil used. In short, types and amount of essential oil affected not only the optical properties but also the water vapor permeability of the films obtained. 3.2.5. Mechanical properties The oil type significantly affected both TS (resistance to elon￾gation) and % (capacity for stretching) of the films obtained. The addition of LEO in gelatin film caused a significant decrease in TS and % if compared to the control (P 0.05) was observed on deformation at break. A possible explanation therefor lies in the alteration of the plasticizer/gelatin ratio in the EOs added films. Water exerted a plasticizing effect on protein films (Martucci and Ruseckaite, 2009). As expressed above, the water uptake of OEO￾Ge film had no significant difference with respect to Ge control film but was higher than LEO-Ge films (Table 4). Other authors reported no difference in the % and a reduction in TS in biopolymer films incorporated with antioxidant extracts (Sanchez-González et al., 2009; Atarés et al., 2010b; Kavoosi et al., 2013; Wu et al., 2014). Mechanical properties were also affected by the type and content of the essential oil. 3.2.6. Antimicrobial activity Gelatin films with varying amounts of EOs were tested against E. coli and S. aureus. To minimize the incorporation of flavor to the films, the oils concentrations incorporated into the FFSs were equal to or above the MIC values of the oils against microorganisms. The inhibitory effect of gelatin films incorporated with 0–6000 ppm of oregano and lavender essential oil against E. coli and S. aureus is illustrated in Table 5. The results indicate that both microor￾ganisms exhibited sensitivity to all the active films. Ge-EOs films showed an increase in the halo of inhibition with EOs increment. The halos obtained were slightly higher for OEO-Ge films which are in concordance with the greatest antimicrobial effect of car￾vacrol compared to linalool (the main oil components) (Burt, 2004; Bagamboula et al., 2004). The present study showed that EOs were effective againsttested bacteria at concentrations less than or equal to 6000 ppm, which is in accordance with most of the reported works on the antimicrobial effectiveness of protein films contain￾ing essential oils, in most cases at concentrations varying between 1 to 5% w/V (Min and Oh, 2009; Gomez-Estaca et al., 2009, 2010, 2010 Ahmad et al., 2012). Inhibition zones obtained for S. aureus yielded lower values than those of E. coli despite the lower MIC value. This could be explained by the evaporation of volatile compounds involved in the antimicrobial activity against S. aureus during films forming conditions and/or by the gelatin matrix hindering the migration of EO components into the agar medium (Gomez-Estaca et al., 2009Gomez-Estaca et al., 2009 Hosseini et al., 2009; Pereda et al., 2011; Teixeira et al., 2014). Nonetheless, based on the values of the vapor pressure and boiling points of the major components of the essential oils used (Table 6) and the processing temper￾ature (35 ◦C during drying operations), it was assumed that the content of the active agents in the final films was almost invari￾able and the essential oil components were retained by the protein matrix. Table 6 Boiling point (◦C) and vapor pressure (mmHg) of the main components of oregano and lavender essential oils. Boiling pointa(◦C) Vapor pressureb(mmHg) Linalool 199 0.17 at 25 ◦C Camphor 204 0.65 at 25 ◦C Terpinen-4-ol 212 0.40 at 20 ◦C 1,8-cineol 176 1.90 at 25 ◦C carvacrol 236 0.03 at 25 ◦C Thymol 232 1.00 at 64 ◦C p-cymene 177 3.70 at 38 ◦C −Terpinene 182 0.70 at 20 ◦C Terpinene 174 0.80 at 20 ◦C -Pinene 156 4.75 at 25 ◦C a THE MERCK INDEX, 1996.- Encyclopedia of chemicals, drugs, and biologicals. 12th Ed.- Merck Research Laboratories. Division of Merck & CO Inc., Whitehouse Station, NJ, USA. b Daubert, T.E.,R.P. Danner. Physical andthermodynamicproperties ofpure chem￾icals data compilation. Washington, D.C.: Taylor and Francis, 1989.

212 J.F.Martucci et aL.Industrial Crops and Products 71(2015)205-213 3.2.7.Antioxidative capacity Aeschbach.R..Loliger,I.Scott,B.C.,Murcia.A,Butler,I.,HalliwelL,B..Aruoma.O.I.. The DPPH radical-scavenging activity and reducing power val- 1994.Antioxidant actions thymol,carvacrol,6-gingerol,zingerone and ues of gelatin based films are displayed in Table 5.The results are in hydroxy tyrosol.Food Chem.Toxicol.32.31-36. Atares,L.Bonilla,J..Chiralt,A.2010a.Characterization of sodium caseinate-based concordance with the antioxidant capacity of each essential oil.The edible films incorporated with cinnamon or ginger essential oils.J.Food Eng. control film had no DPPH radical scavenging and FRAP activities in 100678687 accordance with other works on gelatin(Tongnuanchan et al.,2012: Atares,L..De Jesus.C..Talens,P..Chiralt,A.,2010b.Characterization of SPl-based edible films incorporated with cinnamon or ginger essential oils.J.Food Eng. Li et al.,2014). 99(3).,384-391 The addition of EOs significantly increased(P<0.05)DPPH rad- Bagamboula,C.F.,Uyttendaele,M.,Debevere,J.,2004.Inhibitory effect of thyme ical scavenging activity (Table 5).The results suggested that the and basil essential oils.carvacrol.thymol,estragol,linalool and p-cymene towards Shigellasonnei and S.flex.Food Microbiol.21.33-42. addition of 6000 ppm of OEO made the film more active against Burt,S 2004.Essential oils:their antibacterial properties and potential DPPH radical.Regarding the gelatin-based film mixed with LEO. applications in foods e a review.Int.J.Food Microbiol.94.223-253. the DPPH radical scavenging capacity could be negligible compared Canillac,N.Mourey.A.,2001.Antibacterial activity of the essential oil of to OEO films.The differences observed between both essential oils Piceaexcelsa on Listeria Staphylococcus aureus and coliform bacteria.Food Microbiol.18,261-268. was previously explained by the higher concentration of phenolic D'Antuono,L.F..Galetti.G.C..Bocchini,P.,2000.Variability of essentials oil contents compounds present in oregano essential oil (Pelissari et al.,2009: and composition of Origanum vulgare L populations from North Mediterranean Area (Liguria Region,Northern Italy).Ann.Bot.86.471-478. Kacaniova et al.,2012:Teixeira et al.,2013b;Li et al.,2014).Along Danh,L.T.Triet,N.D.A..Han,LT.N..Zhao.J..Mammucari,R..Foster.N.2012. these lines,the films mixed with OEO had higher reducing power Antioxidant activity,yield and chemical composition of lavender essential oil as compared to the control and to LEO-Ge films (Table 5).In com- extracted by supercritical CO2.I.Supercrit.Fluids 70.27-34. parison with EOs(Fig.1 a and b),the films presented lower DPPH Djagny.K.B..Wang.Z,Xu,S.,2001.Conformational changes and some functional characteristics of gelatin esterified with fatty acid.J.Agric.Food Chem.49, and FRAP values in the same concentration,probably owing to the 2987-2991 interactions between gelatin matrix and EO components.The inter- Eca,K.S.,Sartori,T.,Menegalli,F.C..2014.Films and edible coatings containing action between antioxidant and gelatin molecules in proteins films antioxidants-a review.Braz.J.Food Technol., http://dx.doi.org/10.1590/bjft.2014.017 has been already suggested by different authors (Gomez-Estaca Gende,L.B..Maggi,M..van Baren,C..di Leo Lira.A.,Bandoni.A..Fritz.R..Eguaras,M.. et al.,2009;Pereda et al.,2011:Teixeira et al.,2014). 2010.Antimicrobial and miticide activities of Eucalyptus globulus essential oils obtained from different Argentine regions.Spanish J.Agric.Res.8(3).642-650. Gomez-Estaca,J Bravo,L Gomez-Guillen,M.C..Aleman,A.,Montero,P..2009. 4.Conclusion Antioxidant properties of tuna-skin and bovine-hide gelatin films induced by the addition of oregano and rosemary extracts.Food Chem.112.18-25. Oregano and lavender essential oils exhibited good antimicro- Gomez-Estaca,J.Lopez de Lacey.A..Lopez-Caballero.M.E.Gomez-Guillen.M.C.. Montero,P..2010.Biodegradable gelatine-chitosan films incorporated with bial properties against E.coli and S.aureus in concentrations above essential oils as antimicrobial agents for fish preservation.Food MicrobioL 27 2000 ppm.Yet oregano proved to be better due to its phenolic com- 889-896. pounds content which also provided antioxidant capacity.Oregano G6mez-Guillen.M.C.Ihl,M..Bifani,V..Silva,A..Montero,P..2007.Edible films made from tuna-fish gelatin with antioxidant extracts of two different murta and lavender essential oils incorporated into gelatin films at low ecotypes leaves(UgnimolinaeTurcz)Food Hydrocolloids 21(7).1133-1143. content(2000-6000 ppm)led to an increase in their antimicrobial Hosseini.M.H..Razavi,S.H.,Mousavi,M.A..2009.Antimicrobial physical and and antioxidant efficacy with little effect on water vapor permeabil- mechanical properties of chitosan-based flms incorporated with thyme,clove and cinnamon essential oils.I.Food Process.Preserv.33(6).727-743. ity and mechanical properties of the resultant films.Ge-based films Inouye,S..Takizawa.T.Yamaguchi,H..2001.Antibacterial activity of essential oils can be used in a wide range of food products,not being harmful for and their major constituents against respiratory tract pathogens by gaseous packaging food material.Oregano-based films exhibited the most contact.J.Antimicrob.Chemother.47.565-573 Kacaniova,M..Vukovic,N.,Hleba.L,Bobkova,A_,Pavelkova,A..Rovna.K.. effective antimicrobial and antioxidant properties.Due to its par- Arpasova,H..2012.Antimicrobial and antiradicals activity of OriganumVulgare ticularly intense color,the incorporation of OEO in concentrations L.and Thymus Vulgaris essential oils.J.Microbiol.Biotechnol.Food Sci.2(1). above 4000 ppm considerably increased the yellowish coloration 263-271. Kavoosi,G..Dadfar.S.M.M.,Purfard,A.M.,2013.Mechanical,physical,antioxidant of gelatin films.OEO4000-Ge film could be a good alternative to and antimicrobial properties of gelatin hilms incorporated with thymol for natural food preservatives as a microbial growth inhibitor and/or potential use as nano wound dressing..Food Sci.78(2).E244-E250. oxidizer.Advances in active packaging materials based on renew- Kulevanova.S.,Panovska,T.K..2001.Antioxidant activity of essential oils of different wild Thymus L species.BulL.Chem.Technol.Maced.20(1).61-66. able sources,such us,gelatin and essential oils will open new lines Li.J.-H.Miao.J..Wu.J.-L,Chen.S.-F..Zhang.Q-Q.2014.Preparation and of work for the development of improved ecofriendly materials. characterization of active gelatin-based films incorporated with natural antioxidants.Food Hydrocolloids 37,166-173. Martucci.J.F,Ruseckaite.R.A.,2009.Tensile properties,barrier properties and Acknowledgments biodegradation in soil of compression-molded gelatin-starch dialdehyde films.J.AppL Polym.Sci.112.2166-2178 This research was financed by the Consejo Nacional de Inves- Martucci,J.F,Accareddu.A.M.E..Ruseckaite,R.A.,2012.Preparation and characterization of plasticized gelatin films cross-linked with low tigaciones Cientificas y Tecnicas (CONICET,grant number PIP concentrations of glutaraldehyde.J.Mater.Sci.47(7).3282-3292. 112-201101-00637)and the Agencia Nacional de Promocion Cien- Min,B.J Oh,J.-H.,2009.Antimicrobial activity of catfish gelatin coating containing tifica y Tecnologica(ANPCyT,grant number PICT 2010-1791)of origanum (Thymus capitarus)oil against gram-negative pathogenic bacteria.J. o0d5C.744】.143-148. Argentina.Authors would like to thank Dr.A.Folabella(Biology Ojagh,S.M..Rezaei.M..Razavi,S.H..Hosseini,S.M.H.,2010.Effect of chitosan Department,FCEyN,UNMDP)and Dr.C.Studdert (IIIB,FCEyN, coatings enriched with cinnamon oil on the quality of refrigerated rainbow UNMDP)for bacterial strains,to A.Coppola and M.Zorzabal for trout Food Chem.120(11 193-198 oregano plant material and Rouselot(Argentina)for kindly supply Oussalah,M.,Caillet,S..Ri.S.S.Saucier.L.Lacroix,M.2004.Antimicrobial and antioxidant effects of milk protein-based film containing essential oils for the gelatin material.Special thanks to Ing.Juan P.Espinosa for kindly preservation of whole beef muscle.J.Agric.Food Chem.52(18).5598-5605. perform GC/MS experiments. Oyaizu,M.1986.Studies on product of browning reaction prepared from glucose amine.Ipn.I.Nutr.44.307-331. Pelissari,F.M.,Grossmann,M.V.E..Yamashita,F,Pineda,E.A.G..2009 References Anumicrobial mechanical and barrer propertes of cassava starch-chitosan films incorporated with oregano essential oiL.J.Agric.Food Chem.57(16). Adams,R.P.,2007.Identification of Essential Oil Components by Gas 7499-7504. Cromatography/Mass Spectroscopy,4th ed.Allured Publishing Corporation. Pereda,M..Ponce.A.G..Marcovich.N.E..Ruseckaite,R.A.,Martucci,J.F.,2011. Carol Stream.Ilinois Chitosan-gelatin composites and bi-layer films with potential antimicrobial Ahmad,M.,Benjakul,S..Prodpran.T..Agustini,T.W.,2012.Physico-mechanical and activity.Food Hydrocolloids 25,1372-1381. antimicrobial properties of gelatin film from the skin of unicorn leatherjacket incorporated with essential oils.Food Hydrocolloids 28(1).189-199

212 J.F. Martucci et al. / Industrial Crops and Products 71 (2015) 205–213 3.2.7. Antioxidative capacity The DPPH radical-scavenging activity and reducing power val￾ues of gelatin based films are displayed in Table 5. The results are in concordance with the antioxidant capacity of each essential oil. The control film had no DPPH radical scavenging and FRAP activities in accordance withother works ongelatin(Tongnuanchanet al., 2012; Li et al., 2014). The addition of EOs significantly increased (P < 0.05) DPPH rad￾ical scavenging activity (Table 5). The results suggested that the addition of 6000 ppm of OEO made the film more active against DPPH radical. Regarding the gelatin-based film mixed with LEO, the DPPH radical scavenging capacity could be negligible compared to OEO films. The differences observed between both essential oils was previously explained by the higher concentration of phenolic compounds present in oregano essential oil (Pelissari et al., 2009; Kacániová ˇ et al., 2012; Teixeira et al., 2013b; Li et al., 2014). Along these lines, the films mixed with OEO had higher reducing power as compared to the control and to LEO-Ge films (Table 5). In com￾parison with EOs (Fig. 1 a and b), the films presented lower DPPH and FRAP values in the same concentration, probably owing to the interactions between gelatin matrix and EO components. The inter￾action between antioxidant and gelatin molecules in proteins films has been already suggested by different authors (Gomez-Estaca et al., 2009; Pereda et al., 2011; Teixeira et al., 2014). 4. Conclusion Oregano and lavender essential oils exhibited good antimicro￾bial properties against E. coli and S. aureus in concentrations above 2000 ppm. Yet oregano proved to be better due to its phenolic com￾pounds content which also provided antioxidant capacity. Oregano and lavender essential oils incorporated into gelatin films at low content (2000-6000 ppm) led to an increase in their antimicrobial and antioxidant efficacy withlittle effect on water vaporpermeabil￾ity and mechanical properties of the resultant films. Ge-based films can be used in a wide range of food products, not being harmful for packaging food material. Oregano-based films exhibited the most effective antimicrobial and antioxidant properties. Due to its par￾ticularly intense color, the incorporation of OEO in concentrations above 4000 ppm considerably increased the yellowish coloration of gelatin films. OEO4000-Ge film could be a good alternative to natural food preservatives as a microbial growth inhibitor and/or oxidizer. Advances in active packaging materials based on renew￾able sources, such us, gelatin and essential oils will open new lines of work for the development of improved ecofriendly materials. Acknowledgments This research was financed by the Consejo Nacional de Inves￾tigaciones Científicas y Técnicas (CONICET, grant number PIP 112-201101-00637) and the Agencia Nacional de Promoción Cien￾tífica y Tecnológica (ANPCyT, grant number PICT 2010-1791) of Argentina. Authors would like to thank Dr. A. Folabella (Biology Department, FCEyN, UNMDP) and Dr. C. Studdert (IIIB, FCEyN, UNMDP) for bacterial strains, to A. Coppola and M. Zorzabal for oregano plant material and Rouselot (Argentina) for kindly supply gelatin material. Special thanks to Ing. Juan P. Espinosa for kindly perform GC/MS experiments. References Adams, R.P., 2007. Identification of Essential Oil Components by Gas Cromatography/Mass Spectroscopy, 4th ed. Allured Publishing Corporation, Carol Stream, Illinois. 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