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 (P<0.05)(Table 4).A decrease conditions and/or by the gelatin matrix hindering the migration in the breaking force was previously recorded in gelatin films of EO components into the agar medium (Gomez-Estaca et al., 2009Gomez-Estaca et al.,2009 Hosseini et al.,2009:Pereda et al.. incorporated with antioxidant extracts (Tongnuanchan et al.,2012: Kavoosi et al.,2013:Li et al.,2014).The addition of EOs possibly 2011:Teixeira et al,2014).Nonetheless,based on the values of resulted in the lowered interaction between gelatin monomers, the vapor pressure and boiling points of the major components and probably hindered the polymer chain-to-chain interactions of the essential oils used (Table 6)and the processing temper- and,consequently,led to a decrease in TS.Other authors assigned ature(35C during drying operations).it was assumed that the this behavior to the presence of the structural discontinuities pro- content of the active agents in the final films was almost invari- voked by oil incorporation.The reduction in the stretching capacity able and the essential oil components were retained by the protein has also been reported by other authors in protein-oil added films matrix. (Atares et al.,2010b:Li et al.,2014). The incorporation of OEO resulted in a significant decrease of Table6 TS(P<0.05),though no significant effect(P>0.05)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) (Table 4). A decrease in the breaking force was previously recorded in gelatin films incorporated with antioxidant extracts (Tongnuanchan et al., 2012; Kavoosi et al., 2013; Li et al., 2014). The addition of EOs possibly resulted in the lowered interaction between gelatin monomers, and probably hindered the polymer chain-to-chain interactions and, consequently, led to a decrease in TS. Other authors assigned this behavior to the presence of the structural discontinuities pro￾voked by oil incorporation. The reduction in the stretching capacity has also been reported by other authors in protein-oil added films (Atarés et al., 2010b; Li et al., 2014). The incorporation of OEO resulted in a significant decrease of TS (P < 0.05), though no significant effect (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.