Study on carvacrol and cinnamaldehyde polymeric films: mechanical properties, release kinetics and antibacterial and antibiofilm activities A. Nostro, R. Scaffaro, M. D’Arrigo, L. Botta, A. Filocamo, A. Marino & G. Bisignano Applied Microbiology and Biotechnology ISSN 0175-7598 Volume 96 Number 4 Appl Microbiol Biotechnol (2012) 96:1029-1038 DOI 10.1007/s00253-012-4091-3
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Author's personal copy Appl Microbiol Biotechnol (2012) 96:1029–1038 DOI 10.1007/s00253-012-4091-3
APPLIED MICROBIAL AND CELL PHYSIOLOGY
Study on carvacrol and cinnamaldehyde polymeric films: mechanical properties, release kinetics and antibacterial and antibiofilm activities A. Nostro & R. Scaffaro & M. D’Arrigo & L. Botta & A. Filocamo & A. Marino & G. Bisignano
Received: 11 March 2012 / Revised: 4 April 2012 / Accepted: 6 April 2012 / Published online: 4 May 2012 # Springer-Verlag 2012
Abstract Polyethylene-co-vinylacetate (EVA) films with different concentrations (3.5 wt% and 7 wt%) of essential oil constituents, carvacrol or cinnamaldehyde, were prepared and characterized by mechanical, antibacterial and antibiofilm properties. The incorporation of the compounds into copolymer films affected their elastic modulus, tensile stress and elongation at break. Carvacrol and cinnamaldehyde act as plasticizers which reduce the intermolecular forces of polymer chains, thus improving the flexibility and extensibility of the film. The analysis of the surface characteristics demonstrated that essential oil constituents lowered the contact angle values without causing any remarkable variation of the surface roughness. The films allowed progressive diffusion of the bioactive molecules and the kinetic of release was correlated with the damaging effect on bacterial growth. The kill curves proved that the film with essential oil constituents (7 wt%) had a significant bactericidal effect (reduction of 4 and 2 log CFU) against Staphylococcus aureus and Escherichia coli and a bacteriostatic effect against Staphylococcus epidermidis and Listeria monocytogenes (reduction of about 1 log CFU). With regard A. Nostro : M. D’Arrigo : A. Filocamo : A. Marino : G. Bisignano Dipartimento Farmaco-Biologico, University of Messina, Viale Annunziata, 98168 Messina, Italy R. Scaffaro : L. Botta Dipartimento di Ingegneria Civile, Ambientale, Aerospaziale, dei Materiali, University of Palermo, Viale delle Scienze, Ed. 6, 90128 Palermo, Italy A. Nostro (*) Dipartimento Farmaco-Biologico, University of Messina, Viale Annunziata, 98168 Messina, Italy e-mail:
[email protected]
to biofilm formation the biomass formed on polymeric films surface was significantly reduced if compared with the pure copolymer control. The results were confirmed by fluorescence microscopy images by Live/dead staining. The reduction in the surface tension coupled to an inherent bactericidal property of carvacrol and cinnamaldehyde could in turn affect the initial attachment phase of bacteria and compromise the normal biofilm development. Keywords Carvacrol . Cinnamaldehyde . Polymeric film . Mechanical properties . Release kinetic . Biofilm
Introduction Carvacrol (2-methyl-5-(1-methylethyl)-phenol) and cinnamaldehyde (3-phenyl-2-propenal) are aromatic essential oil constituents with a wide-spectrum antimicrobial activity, extended to food spoilage or pathogenic microorganisms, including drug-resistant and biofilm forming bacteria (Burt 2004; Dorman and Deans 2000; Elgayyar et al. 2001; Inouye et al. 2001; Nostro et al. 2004, 2007, 2009). Their antibacterial activity have been attributed to the considerable effects on the structural and functional properties of cytoplasmatic membrane (Gill and Holley 2004; Lambert et al. 2001). Concerning the literature in the food field, carvacrol and cinnamaldehyde have been studied in the preservation of a wide range of foods, including vegetables, fruit, dairy products, fish and meat (Burt 2004). Carvacrol is a predominant monoterpenic phenol which occurs in aromatic plants and in many essential oils of the family Labiatae including Origanum, Satureja, Thymbra, Thymus and Corydothymus species. It is categorized as Generally Recognized as Safe (GRAS) by the U.S. Food and Drug Administration (FDA; http://www.cfsan.fda.gov/∼dms/
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eafus.html, Center for Food Safety and Applied Nutrition 2006; Commission of the European Communities 1999) and it is used as a flavoring agent in sweets, beverages and chewing gum (Fenaroli 2002). Cinnamaldehyde is the main component in cassia oil as well as cinnamon bark oil. It is categorized as GRAS by the Flavoring Extract Manufacturers’ Association and it is approved for food use by the FDA. The industry utilizes food grade cinnamaldehyde in non-alcoholic beverages, ice cream, candy, baked goods, chewing gum, condiments and meats at levels ranging from 9 to 4,900 ppm (Fenaroli 2002). Carvacrol and cinnamaldehyde are molecules with interesting applicative prospects. This has stimulated researchers to study different antimicrobial formulations and polymeric materials for biomedical and food packaging applications. In this context, the essential oils incorporated into different type of polymeric matrices, such as polypropylene and polyethylene/ethylene vinyl alcohol copolymer or lowdensity polyethylene have been presented (Gutiérrez et al. 2009; López et al. 2007; Persico et al. 2009). At the same time, the essential oils or their constituents could be used at concentrations that are low enough to minimally alter the impact on the organoleptic properties of the product (McClements 1999). However, polymeric films are susceptible to bacterial colonization and biofilm formation (Hogt et al. 1986). Therefore, the incorporation of natural antimicrobial molecules that also prevent the bacterial adhesion appears to be a suitable strategy in order to control microbial contamination and biofilm related adverse effects in the biomedical, food and industrial fields. Currently, only a few studies have reported the effect of natural molecules incorporated into polymeric materials on biofilm formation. Poly(ethylene-covinyl acetate) copolymer (EVA) containing nisin have proved effective in controlling bacterial growth and biofilms production of some food borne pathogens (Nostro et al. 2010; Scaffaro et al. 2011, 2012). EVA has several industrial applications. It is typically used to produce film not only for producing film for greenhouses covering (Scaffaro et al. 2009) and for food packaging applications (Kirwan and Strawbridge 2003), but also in systems for controlled drug release (Arnold et al. 2008). EVA is characterized by low friction coefficients and high adhesivity. Because of these properties, it is usually used in multilayer film as outer layer, to promote self-sealing and welding (Kirwan and Strawbridge 2003). The incorporation of antimicrobials or other molecules by melt processing allows the use of common processing equipment to prepare films or sheets thus making it possible to produce large amounts of materials in a single step without using solvents. This has obvious positive implications for environmental and economic factors.
Appl Microbiol Biotechnol (2012) 96:1029–1038
This study focuses on the preparation-by melt processing-and on the characterization of copolymeric films provided with antibacterial and antibiofilm properties coming from carvacrol and cinamaldehyde in order to find potential uses in different biomedical and food applications.
Materials and methods Polymeric films preparation The polymer used in the frame of this work is a sample of a poly(ethylene-co-vinylacetate) (EVA14, Greenflex FC45, Polimeri Europa, vinylacetate content 14 %). Carvacrol (2methyl-5-(1-methylethyl)-phenol, purity ≥97 %) and cinnamaldehyde (3-phenyl-2-propenal, purity ≥98 %) were supplied by Sigma Aldrich and used as received without further purification. Blends containing 3.5 wt% and 7 wt% of both essential oils have been prepared by melt mixing using a batch mixer (Brabender PLE330) at the temperature of 120 °C and a rotational speed of 64 rpm. EVA14 was first fed to the mixer. After melting (about 3 min), the oil was added and the blend was processed for no longer than 1 min in order to minimize the evaporation of the additive. Once completed this phase, the material was immediately fed out the mixer and immersed in liquid nitrogen in order to inhibit any further volatilization process of the oils. The materials were then stored at 0 °C in vacuum packed plastic bags until their final use. Films were prepared by compression moulding using a Carver Laboratory press. The material was preventively ground, placed in a mould between two Teflon sheets and pressed at 120 °C and 100 bar for about 2 min to obtain a film 200 μm thick. Mechanical properties and contact angle measurements Tensile mechanical measurements were carried out by using a dynamometer (Instron model 3365, Instron, UK) on rectangular shaped specimens (10×90 mm) cut off from films prepared as described above. The grip distance was in all the cases 30 mm and the crosshead speed 1 mm/min for the first 2 min (in order to measure the elastic modulus) and 100 mm/ min thereafter. Static contact angles were measured on all the samples using deionized water as a fluid by an FTA 1000 (First Ten Ångstroms, UK) instrument. Scanning electron microscopy The morphology of the films surface was analysed by scanning electron microscopy (SEM; FEI Quanta 200 ESEM). The specimens were obtained by cutting them off directly from the films prepared as described above and thereafter
Author's personal copy Appl Microbiol Biotechnol (2012) 96:1029–1038
sputter-coated with a thin layer of gold to avoid electrostatic charging under the electron beam. Kinetic of release of the essential oils constituents from the polymeric films A series of carvacrol and cinnamaldehyde solutions of distilled water containing 2 thru 20 mg/dl of carvacrol and 1 thru 10 mg/dl of cinnamaldehyde were used to obtain a calibration curve correlating the absorbance peak intensity and the essential oil concentration using a UV/vis spectrophotometer (model UVPC 2401, Shimadzu Italia s.r.l., Milan, Italy). In the concentration range here investigated, the calibration curves were found to be a line. The maximum absorbance peaks were detected at 273 nm for carvacrol and at 220 nm for cinnamaldehyde. The release of carvacrol and cinnamaldehyde from the films was investigated by immersing a pre-weighed sample (a square of 5× 5 mm, approximately 4.5 mg) in 10 ml of distilled water. At specific time intervals, the absorbance peak intensity at 273 nm for carvacrol and at 220 nm for cinnamaldehyde of the storage solutions was measured and converted to the quantities of essential oil released based on the calibration line. After each measurement, the samples were immersed in 10 ml of fresh distilled water and the cumulative release of oil here reported was calculated by sequentially adding the oil released after each step. Bacterial strains The bacteria used in this study were Staphylococcus epidermidis ATCC 35984 (positive control for biofilm production), Staphylococcus aureus 815, clinical isolated and previously characterized for many biofilm-related properties (Blanco et al. 2005; Di Stefano et al. 2009), Listeria monocytogenes ATCC 7644 and Escherichia coli ATCC 10536. The strains were stored at −70 °C in Microbanks™; a single bead was removed from the cryovials and directly inoculated in Tryptic Soy Broth (TSB, Oxoid). All reagents were purchased from Sigma Aldrich unless otherwise specified in the text. Measurement of antibacterial activity
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24 h at 37 °C. All tests were performed in duplicate and the antibacterial activity was expressed as the mean of inhibition diameters (mm) produced by polymeric films. Bactericidal activity The bactericidal activity of polymeric films (1 cm2) with or without carvacrol or cinnamaldehyde was evaluated in tubes containing overnight broth cultures adjusted to a concentration of 5×106 CFU/ml (3 ml). These tubes were incubated at 37 °C and after 1, 3, 6, 24 and 48 h samples were serially diluted in sterile PBS (pH 7.4) and were incorporated into melted TSA to determine total cell number. All plates were then incubated at 37 °C for 18–24 h up to 48 h, CFU were counted and time kill plots were constructed. All determinations were performed in duplicate including the growth controls. Antibiofilm activity Cultures were grown overnight in 10 ml of TSB (L. monocytogenes and E. coli) or TSB+1 % glucose (S. aureus and S. epidermidis) and diluted in growth medium to a final concentration of 5×105 CFU/ml as previously described (Nostro et al. 2010). Aliquotes of 3 ml were dispensed into glass test tubes containing either the EVA14 or the EVA14/ oil film (1 cm2). After incubation at 37 °C for 24–48 h, the biofilm formed on the polymeric films was evaluated by (a) biofilm biomass measurement and by (b) Live/Dead BacLight viability kit: (a) For biofilm biomass measurement, the polymeric films were washed twice with sterile PBS (pH 7.4), dried, stained for 1 min with 0.1 % safranin and washed with water. The stained biofilms were resuspended in acetic acid 30 % (v/v) and biofilm biomass was quantified by measuring the optical density at 492 nm using a spectrofotometer EIA reader (Bio-Rad Model 2550). Each assay was performed in triplicate and repeated at least three times. The relative inhibition of biofilm formation was calculated as: 100 ½ðOD492 EVA14 with oil component= OD492 EVA14 without oil componentÞ 100
Disc diffusion test A preliminary screening on the antibacterial activity of polymeric films was carried out using the disc diffusion assay. Overnight broth cultures, adjusted to yield approximately 1×108 CFU/ml were streaked with a calibrated loop on plates containing Tryptic Soya Agar (TSA; Oxoid). Polymeric film discs (1 cm2) were cut and were placed on the inoculated agar surfaces. The plates were observed after
(b) For viability measurements, the polymeric films were rinsed once with PBS and stained by using a Live/Dead BacLight viability kit (Molecular Probes). The solution (1 ml), containing SYTO 9 and propidium iodide mixed in a ratio of 1:1, was added to the film. The films were then incubated at room temperature for 15 min in the dark. After incubation, residual stain was removed. The images were observed using a
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the modified hydrophilicity of the surface is responsible for the change of the contact angle.
fluorescent microscope (Reichert) equipped with halogen lamp, Neoplan 100/1.25 (oil) objective, and 1713 filter cube (Fluorescein) (490/510/520).
Kinetic of release of essential oils components from the polymeric films Statistical analysis The results related to the release kinetics of essential oils components from the polymeric film are reported in Table 2. It can be observed that the amount of oil released was different for the two oils: the carvacrol released after 48 h is about as twice as the cinnamaldehyde. Moreover, the released amount depends strongly on the initial nominal concentration of additive in the polymeric film. In both cases, in fact, the highest amount of oil was released by the 7 wt% containing EVA14 films. In particular, for both materials, the 60–80 % of the whole cumulative oil released after 48 h is released within the first 16 h with a rapid levelling off up to the end of the experiment.
ANOVA test was employed to evaluate any significant differences between the values obtained in EVA and EVA with essential oil components. A p value of
