Characterisation and antimicrobial activity of active polypropylene films containing oregano essential oil and Allium extract to be used in packaging for meat products

1 Characterization and antimicrobial activity of active polypropylene films containing Oregano essential oil and Allium extract to be used in packaging for meat products. María Llana-Ruiz-Cabello1, Silvia Pichardo1*, José María Bermudez3, Alberto Baños2, Juan José Ariza2, Enrique Guillamón2, Susana Aucejo3, Ana M. Cameán1 1Area of Toxicology, Faculty of Pharmacy, Universidad de Sevilla, Profesor García González n°2, 41012 Seville. Spain. 2DOMCA S.A. Camino de Jayena s/n, 18620 Alhendín, Granada, Spain. 3Area of Packaging materials and Systems, ITENE, C/ Albert Einstein 1, 46980 Paterna, Valencia, Spain. *Corresponding author: Silvia Pichardo Sánchez Area of Toxicology, Faculty of Pharmacy, Universidad de Sevilla, Profesor García González n°2, 41012 Seville. Spain. E-mail address: [email protected] Tel: +34 954 556762 Fax: +34 954 556422 2 Abstract Cooked ham is more prone to spoilage than other meat products due to its properties, making the preservation techniques a key step in its commercialization. One of the most promising preservation strategies is the use of active packaging. Oregano essential oil (OEO) and Proallium® (an Allium extract) have previously shown to be useful in polylactic acid (PLA) active films for ready to eat salads. The present work aims to study the suitability of polypropylene (PP) films containing OEO and Proallium® in the preservation of cooked ham. Concerning the technological features of the studied material, no remarkable change in the mechanical or optical properties of PP films containing the active substances were recorded in comparison to the PP film without extracts. Although films containing both active substances were more flexible than the control film and less strong, highlighting the plasticization effect of the natural extracts. Moreover, physical properties changed when active substances were added to the film. The incorporation of 4% Proallium® affected in a higher extend the transparency of the film than in the case of 8% OEO, undergoing a decrease in the transparency of 40 and 45%, respectively. Moreover, only the film containing the highest amount of OEO (8%) decreased significantly the thickness. Both active substances showed antibacterial properties; however, Proallium® active films seemed to be more effective against Brochotrix thermosphacta than PP films containing OEO, with all percentages of Proallium® killing the bacterial population present in the ham after 60 days. In addition, materials containing the lowest Proallium® content exhibited the higher acceptability by the consumers in the sensory analyses with a willing purchase of 63-100%, even better than the control package (56-89%). In fact, 2% of Proallium® obtained the best results in the smell study performed by the panellists. Keywords: polypropylene; oregano essential oil; Allium extract; active packaging 3 Abbreviations: Analysis of variance multifactor (ANOVA); Brochothrix thermosphacta (B. thermosphacta); colony forming units (CFU); crystallisation temperature (Tc); elastic modulus (E); elongation at break (EAB); enthalpy of crystallization (ΔHc); enthalpy of melting (ΔHm); essential oils (EOs); ethylene vinyl alcohol (EVOH); least significant difference (LSD); melting temperature (Tm); oregano essential oil (OEO); polylactic acid (PLA); polypropylene (PP); propyl thiosulphinate oxide (PTSO); Spanish Type Culture Collection (CECT); tensile strength (TS); tetrahydrofuran (THF); thermogravimetric analysis (TGA). 4 1. Introduction Meat and meat products are good media for the growth of microorganism (Van Haute et al. 2016). Cooked ham is more prone to spoilage than other meat products due to its low salt content, pH near 6 and water activity higher than 0.945. In addition, cooked ham is usually sliced and packaged before being sold and consequently its shelf-life could decrease (Baños et al. 2012). Therefore, the preservation strategies for these foods are of great importance. New trends in consumer demands, regarding minimally processed food or ready-to-eat fresh food, are driving a search for innovative ways to maintain food quality, freshness and safety (Appendini and Hotchkiss 2002). One of these new developments is active packaging, which was defined in Regulation (EC) No 450/2009. These materials include antimicrobial active films that can be classified in two categories: those made by using polymers inherently antimicrobial; and those that involve the direct incorporation of the antimicrobial additive into the packaging films (Suppakul, et al. 2003). Active food packaging systems containing antimicrobial additives are polymeric materials developed to release these active substances into the food in order to prevent microbial growth and to extend foodstuff shelf-life (Del Nobile et al. 2009). Polyolefins have been used as food packaging matrices in the development of active films due to their mechanical, barrier, optical and thermal properties (Ramos et al. 2012; Gigli et al. 2014). Polypropylene (PP) is a polyolefin with low water vapor transmission, medium gas permeability, good resistance to greases and chemicals, good abrasion resistance, high temperature stability and good gloss and high clarity. All of these features make PP suitable for food packaging (Robertson 2012; Ramos et al. 2012; Ramos et al. 2014; Muriel- Galet et al. 2013). Regarding active substances, several studies have shown that consumers have recently become more informed about food additives and tend to choose those with additives of 5 natural origin rather than their synthetic analogues (Carocho et al. 2015). In this regard, essential oils (EOs) and plant extracts have been the target of extensive research because these substances have demonstrated biological properties that exhibit benefits in food (Llana- Ruiz-Cabello et al. 2015; Maisanaba et al. 2017; Ribeiro-Santos et al. 2017). Therefore, new alternatives based on aromatic plants, such as EOs, have been studied (Ramos et al. 2014). The advantages observed for active packaging containing EOs compared with direct addition of these additives to the food, such as lower amount of compounds required or extended effects due to controlled migration from the film to the food are increasing the demand of these materials (Sanchez-Silva et al. 2014). Besides, when the additive is sprayed directly to food, the active substances could interact with food constituents causing their denaturalization. This phenomenon can be avoid also by using active packaging containing EOs (Sung et al. 2013). Among EOs, oregano essential oil (OEO) and its major compounds, carvacrol and thymol, have shown its usefulness as active substance in food packaging. A previous experimental study performed in our laboratory have proved the suitability of OEO when incorporated to a matrix of polylactic acid (PLA) as antioxidant agent in ready-to-eat salads (Llana-Ruiz-Cabello et al. 2016). Muriel-Galet et al. (2013) also showed the ability of ethylene vinyl alcohol (EVOH)- coated PP films containing OEO to improve the shelf life of packaged salad. Similarly, Ramos et al. (2012) reported the antimicrobial activity of PP films containing carvacrol and thymol against bacterial strains present in foods such as Escherichia coli or Staphylococcus aureus. Moreover, another plant extract, Proallium®, is a commercial product based on Allium extract that is composed of organosulphur compounds. Garlic EO and its major organosulphur compounds have shown antimicrobial activity when they are included in active packaging (Sung et al. 2014; Teixeira et al. 2014). Specifically, we have previously observed that films of PLA containing Proallium® showed antimicrobial activity against bacteria, yeast and molds 6 (Llana-Ruiz-Cabello et al. 2015b). However, this is the first work dealing the use of active packaging containing Proallium® in the preservation of ham. The aim of the present work was to develop new active films based on PP containing OEO and Proallium® and further characterize them though optical, mechanical and thermal properties. Moreover, the effectiveness of these films in the preservation of cooked ham was also evaluated. 2. Materials and Methods 2.1 Supplies and Chemicals Polypropylene ISPLEN® PP 050 G2M was purchased from Repsol YPF S.A. (Madrid, Spain). Oregano essential oil was acquired from El Jarpil (Almería, Spain). Proallium® with 14.5% of PTSO (Llana-Ruiz-Cabello et al. 2015b) as active agent was provided by Domca S.L. (Alhendin, Granada, Spain). Chemicals for the different assays were purchase from Sigma-Aldrich (Spain) and VWR International Eurolab (Spain). Ham for the in vivo assays was purchase in a local market (Valencia, Spain). 2.2 Film preparation Two types of active PP films were obtained, one of them containing nominal OEO contents of 2, 5 and 8 % (w/w) and the other containing nominal Proallium® contents of 2, 3 and 4 % (w/w). Films were prepared by melt blending in a twin-screw extruder (DSE 20-40D, Brabender, Germany). Extrusion temperatures were set at 200-205 ºC working at a screw sped of 70 min-1. Active substances were introduced in the mixer once the polymer was melted and were fed into the barrel through the lateral liquid port at L/D 10 to reduce possible volatility 7 and degradation losses. The average width and thickness of the films were around 10 cm and 80 μm, respectively. 2.3 Quantification of the overall content of OEO and Proallium® incorporated into PP films The amount of OEO and Proallium® contained in PP films was evaluated using Thermogravimetric Analysis (TGA). Briefly, samples were heated to 900ºC and the loss of mass was measured in a thermal analyser Q5000IR (TA Instruments, New Castle, DE, USA) as described in Llana-Ruiz-Cabello et al. (2015b). 2.4 Quantification of carvacrol, thymol and propyl thiosulphinate oxide incorporated into PP films The main constituents of OEO (i.e. carvacrol and thymol) and Proallium® (i.e. propyl thiosulphinate oxide, PTSO) incorporated into the PP films were extracted using tetrahydrofuran (THF) as solvent. Then, Thermo Finnigan gas chromatograph (GC) equipped with a DB-5MS column (Agilent Technologies, USA) (30 m, 0.25 mm, film thickness 0.25 μm) using helium as a carrier gas (flow rate of 1 ml min−1) was used for the analyses of active substance. The quantification of carvacrol and thymol was performed following the method described by Llana-Ruiz-Cabello et al. (2016) and the quantification of PTSO was carried out according to Llana-Ruiz-Cabello et al. (2015b). 2.5 Physical properties The light transmission and transparency of films were measuredat selected wavelengths between 200 and 800 nm, using a Jasco V-630 UVeVisible recording spectrophotometer (Madrid, Spain) according to the method described in Llana-Ruiz-Cabello et al. (2015b). The transparency of the films was calculated according to Han and Floros (1997), as follows: 8 Transparency values = (-logT600)/x; where T600 is the fractional transmittance at 600 nm; and x: is the film thickness in mm. Thickness were measured was measured in at least six different locations on each film using a digital micrometre Mitutoyo 547-400 Absolute (Kawasaki, Japan). The results are expressed as the median ± standard deviation. 2.6 Mechanical properties Films were conditioned for 48 h at 25 ºC and 50±5 % RH before testing their mechanical properties. Tensile strength (TS), elongation at break (EAB) and elastic modulus (E) were determined at room temperature using an M350-20CT Universal Testing Machine (Testometric, Rochdale, England) according to UNE-EN-ISO 527-1, with slight modifications. 2.7 Thermal analysis A Differential Scanning Calorimeter DSC Q2000 (TA Instruments) was used to develop the thermal analysis of films. Crystallisation temperature (Tc), and melting temperature (Tm) of the different PP films were measured using a Differential Scanning Calorimeter DSC Q2000 (TA Instruments). Specimens weighing 6 mg were heated at the rate of 10 ºC min-1 from 20 ºC to 250 ºC. Then, the samples were cooled at 10 ºC min-1 from 250 ºC to -50 ºC. Finally, the samples were heated again at 10 ºC min-1 up to 250 ºC. The crystallinity was calculated as the ratio between the enthalpy of the melting point to the corresponding theoretical enthalpy of 100 % crystalline PP (estimated as 165 J/g for PP according to Zhu (2002) in percentage basis. 2.8 Antimicrobial activity in cooked ham 2.8.1 Microbial strain 9 Brochothrix thermosphacta, supplied by the Spanish Type Culture Collection (CECT), was grown overnight on brain heart infusion (BHI, Sharlau, Barcelona, Spain) at 28 ºC in aerobic atmosphere. 2.8.2 Experimental design Three slides of ham were placed in polystyrene trays (135 x 180 mm, BANDESUR, Alcalá la Real, Spain) for each treatment and inoculated with a suspension of B. thermophacta to yield a final bacterial level of 105 colony forming units (CFU)/cm2 using a Digralsky spreader. In order to separate the slides the developed films were used as interlayer. Different trays were prepared using PP and PP containing OEO (2, 5 and 8 %) or PP containing Proallium® (2, 3 and 4 %). Samples packaged were stored in PP/polyamide plastic bags under vacuum (Technotrip EVT- 10-2-CD-SC, Barcelona, Spain) and stored at 5 ºC. The antimicrobial activity of PP films was evaluated after 1, 6, 12, 21, 27 and 60 days of packaging by studying the evolution of B. thermophacta population in the cooked ham along the storage time. 2.8.3 Microbial analysis Samples of the surface of each slide of ham were taken at selected times to measure viable counts of bacteria. Each slide (aprox. 14 g) was aseptically removed and homogenized with 125 ml of peptone water (0.1 % peptone) using a Masticator blender (IUR, Barcelona, Spain) for 1 min. Then, a decimal dilution series was prepared for plating on STAA agar (Oxoid LTD, Basingstoke, Hampshire, England). Finally, plates were incubated aerobically at 28 ºC for 48-72 h before bacterial counts were determined. Results were expressed as CFU/cm2. 16 decreased the microbial counts of this microorganism. Therefore, the use of these interlayer films suppose a novel tool that in combination with other hurdles, such as refrigeration or vacuum packaging, could extend the shelf life of meat products. The ability of OEO, and its major compounds, incorporated in different film matrices to reduce microbial counts in food has been reported by several authors. Ramos et al. (2012) reported that PP films containing 8 % of carvacrol and thymol and films containing an 8 % mixture of both substances showed antimicrobial activity against Staphylococcus aureus and Escherichia coli. Antimicrobial activity was observed in salad packaged in EVOH-coated PP films containing OEO (Muriel-Galet et al. 2013) and polylactic acid (PLA) films containing OEO (Llana-Ruiz- Cabello et al. 2016). 3.6 Sensory analyses Figure 2 shows the median scores for each sensory descriptor at the different storage times and packaging material as well as the box and whisker plot for the scores obtained along storage time for each packaging material. Smell was in general the lowest scored descriptor in comparison to the others and thus the most critical one (Figure 2A). The panellists detected in most of the cases the inherent smell of the oregano and Allium extracts and they evaluated the smell as something unexpected. In fact, general acceptability and purchase intention were highly correlated to the smell. Multifactor ANOVA showed a significant impact of both, packaging materials and days of storage (p < 0.05) on the sensory perception of the smell. Ham in the control package was above the threshold value of 5 from day 1 to day 11. From that point, smell was slightly below the threshold up to day 60. The active materials containing OEO were significantly below the scores given to the control package. Materials containing 3% and 4% Proallium® were below the control package although non-significant differences were found. The best results for the 17 smell were obtained for materials containing 2% of Proallium®. This material was scored above 5 for the entire storage time. Figure 2B shows the median scores for visual appearance. In this case, this sensory attribute was above or very close to the threshold value for the different materials and storage time. ANOVA identified the storage time as non-significant (p > 0.05). In the case of the packaging materials, just the material containing 2% OEO was significantly different from the other materials, scoring the worst. Surprisingly, visual appearance was better evaluated than smell, but scarcely influenced in the purchase intention of consumers. General acceptability showed a similar trend to the smell (Figure 2C). ANOVA showed a significant impact of the storage time and packaging material (p < 0.05). Acceptability of the ham packaged in the control material decreased from day 1 to day 11 and remained close or above the threshold up to day 60. The active materials containing OEO and Proallium® showed values in general below the control package although with very few significant differences. The exemption was PP containing 2% Proallium®, which scored the best and above the threshold for the entire storage. Finally, Table 5 shows the percentage of panellists willing to purchase or consume the ham packaged into the different materials and storage time. The best results were obtained for the control material and the PP containing 2% Proallium®. Purchase intention was higher than 50% for both materials and along the entire storage time. Materials containing OEO were in general below 50% including 0% purchase intention at some specific days. 4. Conclusions Polypropylene (PP) films containing Proallium® or OEO showed similar thermical and mechanical properties as PP. Moreover, Proallium® active films seemed to be more effective against B. thermosphacta than PP films containing OEO. Regarding the sensorial analyses, 18 materials containing the lowest Proallium® percentage was highly accepted by the consumers, even better than the control package. However, oregano flavour was noticed as strange to consumers, and had a negative impact in purchase intention. Acknowledgements: the authors wish to thank the Spanish Ministry of Science and Innovation (AGL2012-38357-C02-01) co-financed by FEDER funds and Junta de Andalucía (AGR-7252) for the financial support for this study and the Microscopy Service of CITIUS. María Llana-Ruiz- Cabello also gratefully acknowledges Junta de Andalucía for her predoctoral grant associated to the AGR7252 project. 19 References Ahmad M, Benjakul S, Prodpran T, Agustini TW. 2012. Physico-mechanical and antimicrobial properties of gelatin film from the skin of unicorn leatherjacket incorporated with essential oils. Food Hydrocoll. 28: 189–199. DOI: 10.1016/j.foodhyd.2011.12.003 Altiok D, Altiok E, Tihminlioglu F. 2010. Physical, antibacterial and antioxidant properties of chitosan films incorporated with thyme oil for potential wound healing applications. J. Mater Sci: Mater Med. 21: 2227-2236. DOI: 10.1007/s10856-010-4065-x Appendini P, Hotchkiss JH. 2002. Review of antimicrobial food packaging. Innov. Food Sci. Emer. Technol. 3: 113-126. DOI: 10.1016/S1466-8564(02)00012-7 Baños A, Ananou S, Martínez-Bueno M, Gálvez A, Maqueda M, Valdivia E. 2012. Prevention of spoilage by enterocin AS-48 combined with chemical preservatives, under vacuum, or modified atmosphere in cooked ham model. Food Control 24: 15-22. DOI: 10.1016/j.foodcont.2011.08.001 Carocho M, Morales P, Ferreira ICFR. 2015. Natural food additives: Quo vadis? Trends Food Sci. Technol. 45: 284-295. DOI: 10.1016/j.tifs.2015.06.007 Commission Regulation (EC) No 450/2009 of 29 May 2009 on active and intelligent materials and articles intended to come into contact with food. De Nobile MA, Conte A, Buonocore GG, Incoronato AL, Massaro A, Panza O. 2009. Active packaging by extrusion processing of recyclable and biodegradable. J. Food Eng. 93: 1-6. DOI: 10.1016/j.jfoodeng.2008.12.022 Gigli M, Lotti N, Gazzano M, Siracusa V, Finelli L, Munari A, Rosa MD. 2014. Biodegradable aliphatic copolyesters containing PEG-like sequences for sustainable food packaging applications. Polym. Degrad. Stabil. 105: 96-106. DOI: 10.1016/j.polymdegradstab.2014.04.006 Han J H, Floros J D. (1997). Casting antimicrobial packaging films and measuring their physical properties and antimicrobial activity. J. Plastic Film. Sheet. 13: 287-298. 20 Leroy F, Vasilopoulos C, Van Hemelryck S, Falony G, De Vuyst L. 2009. Volatile analysis of spoiled, artisan-type, modified-atmosphere-packed cooked ham stored under different temperatures. Food Microbiol. 26: 94–102. DOI: 10.1016/j.fm.2008.08.005 Llana-Ruiz-Cabello M, Pichardo S, Maisanaba S, Puerto M, Prieto AI, Gutiérrez-Praena D Jos A, Cameán AM. 2015a. In vitro toxicological evaluation of essential oils and their main compounds used in active food packaging: a review. Food and Chemical Toxicology 81: 9-27. DOI: https://doi.org/10.1016/j.fct.2015.03.030 Llana-Ruiz-Cabello M, Pichardo S, Baños A, Núñez C, Bermúdez JM, Guillamón E, Aucejo S, Cameán AM. 2015b. Characterisation and evaluation of PLA films containing an extract of Allium spp. to be used in the packaging of ready-to-eat salads under controlled atmospheres. LWT-Food. Sci. Technol. 64: 1354-1361. DOI: 10.1016/j.lwt.2015.07.057 Llana-Ruiz-Cabello M, Gutiérrez-Praena D, Puerto M, Pichardo S, Moreno F.J, Baños A, Nuñez C, Guillamón E, Cameán AM. 2015c. Acute toxicological studies of the main organosulfur compound derived from Allium sp. intended to be used in active food packaging. Food Chem. Toxicol. 82: 1–11. DOI: 10.1016/j.fct.2015.04.027 Llana-Ruiz-Cabello M, Pichardo S, Bermúdez JM, Baños A, Núñez C, Guillamón E, Aucejo S, Cameán AM. 2016. Development of PLA films containing oregano essential oil (Origanum vulgare L virens) intended to be use in food packaging. Food Addit. Contam. Part A 33: 1374- 1386. DOI: 10.1080/19440049.2016.1204666 Llana-Ruiz-Cabello M, Maisanaba S, Puerto M, Pichardo S, Jos A, Moyano R, Cameán AM. 2017. A subchronic 90-day oral toxicity study of Origanum vulgare essential oil in rats. Food Chem. Toxicol. 101: 36-47. DOI: 10.1016/j.fct.2017.01.001 Maisanaba S, Llana-Ruiz-Cabello M, Gutiérrez-Praena D, Pichardo S, Puerto M, Prieto AI, Jos A, Cameán AM. 2017. New advances in active packaging incorporated with essential oils or their 21 main components for food preservation. Food Reviews International 33: 447-515. DOI: 10.1080/87559129.2016.1175010 Marcos B, Sárraga C, Castellari M, Kappen F, Schennink G, Arnau J. 2014. Development of biodegradable films with antioxidant properties based on polyesters containing α-tocopherol and olive leaf extract for food packaging applications. Food Pack. Shelf Life 1: 140–150. DOI: 10.1016/j.fpsl.2014.04.002 Muriel-Galet V, Cerisuelo JP, López-Carballo G, Aucejo S, Gavara R, Hernández-Muñoz P. 2013. Evaluation of EVOH-coated PP films with oregano essential oil and citral to imrpove the shelflife of packaged salad. Food Control 30: 137-143. DOI: 10.1016/j.foodcont.2012.06.032 Pin C, García de Fernando GD, Ordóñez JA. 2002. Effect of Modified Atmosphere Composition on the Metabolism of Glucose by Brochothrix thermosphacta. Appl. Environ. Microb. 68: 4441– 4447. DOI: 10.1128/AEM.68.9.4441-4447.2002 Ramos M, Jiménez A, Peltzer M, Garrigós MC. 2012. Characterization and antimicrobial activity studies of polypropylene films with carvacrol and thymol for active packaging. J. Food Eng. 109: 513-519. DOI: 10.1016/j.jfoodeng.2011.10.031 Ramos M, Beltrán A, Peltzer M, Valente AJM, Garrigós MC. 2014. Release and antioxidant activity of carvacrol and thymol from polypropylene packaging films. LWT-Food Sci. Tech. 58: 470-477. DOI: 10.1016/j.lwt.2014.04.019 Remenant B, Jaffrès E, Dousset X, Pilet MF, Zagorec M. 2015. Bacterial spoilers of food : Behaviour, fitness and functional properties. Food Microbiol. 45: 45-53. https://doi.org/10.1016/j.fm.2014.03.009 Ribeiro-Santos R, Andrade M, Ramos de Melo N, Sanches-Silva A. 2017. Use of essential oils in active food packaging: Recent advances and future trends. Trend. Food Sci. Tech. 61: 132-140. https://doi.org/10.1016/j.tifs.2016.11.021 22 Robertson GL. 2012. Food Packaging: Principles and practice. Boca Raton FL, CRC Press Taylor and Francis Group. Sanches-Silva A, Costa D, Albuquerque TG, Buonocore GG, Ramos F, Castilho MC, Machado AV, Costa HS. 2014. Trends in the use of natural antioxidants in active food packaging: a review. Food Addit. Contam. Part A 31(3): 374-395. DOI: 10.1080/19440049.2013.879215 Sehrawet M, Kundu SC. 2007. Buying behavior of rural and urban consumers in India: the impact of packaging. Int. J. Consum. Stud. 31: 630–638. DOI: 10.1111/j.1470- 6431.2007.00629.x Sung SY, Sin LT, Tee TT, Bee ST, Rahmat AR, Rahman WAWA, Tan AC, Vikhraman M. 2013. Antimicrobial agents for food packaging applications. Trend. Food Sci. Technol. 33: 110-123. https://doi.org/10.1016/j.tifs.2013.08.001 Sung SY, Sin LT, Tee TT, Bee ST, Rahmat AR, Rahmat WAWA. 2014. Control of bacteria growth on ready-to-eat beef loaves by antimicrobial plastic packaging incorporated with garlic oil. Food Control 39: 214-221. https://doi.org/10.1016/j.foodcont.2013.11.020 Suppakul P, Miltz M, Sonneveld K, Bigger SW. 2003. Active packaging technologies with an emphasis on antimicrobial packaging and its application. Concise Reviews and Hypotheses in Food Science, 68, 408-420. DOI: 10.1111/j.1365-2621.2003.tb05687.x Teixeira B, Marques A, Pires C, Ramos C, Batista I, Saraiva JA, Nunes ML. 2014. Characterization of fish protein films incorporated with essential oils of clove, garlic and origanum: physical, antioxidant and antibacterial properties. Food Sci. Technol. 59: 533-539. DOI: 10.1016/j.lwt.2014.04.024 Van Haute S, Raes K, Van der Meeren P, Sampers I. 2016. The effect of cinnamon, oregano and thyme essential oils in marinade on the microbial shelf life of fish and meat products. Food Control 68: 30-39. DOI: 10.1016/j.foodcont.2016.03.025 23 Zhu L. 2002. Chapter 10 - Thermal analysis of polymer films. In: S.Z.D. Chen (Ed.), Handbook of Thermal Analysis and Calorimetry (Vol. Volume 3, pp. 355-407): Elsevier Science B.V. http://pyrotechnics.net.ru/library/thermo/Handbook_of_Thermal_Analysis_and_Calorimetry_ Volume_3.pdf 24 Figure caption Figure 1. TGA curves obtained for OEO, Proallium, PP film and PP containing OEO and Proallium®. Figure 2. Sensory analysis measured as smell (A), visual appearance (B) and general acceptability (C) of cooked ham in control and active packaging containing OEO (2, 5, 8 %) or Proallium® (2, 3 4 %). Pro: Proallium®. Film sample Overall content (%) Carvacrol content (%) Thymol content (%) PTSO content (%) PP 2% OEO 1.6 ± 0.3 1.10 ± 0.10 0.132 ± 0.011 PP 5% OEO 3.9 ± 0.2 2.9 ± 0.3 0.33 ± 0.02 PP 8% OEO 5.84 ± 0.11 4.2 ± 0.3 0.420 ± 0.011 PP 2% Proallium 1.680 ± 0.014 0.183 ± 0.011 PP 3% Proallium 2.56 ± 0.06 0.196 ± 0.015 PP 4% Proallium 2.82 ± 0.10 0.25 ± 0.06 Table 1. Content of OEO, Proallium® and their main compounds incorporated into the PP films.