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.