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.