Functional properties and antimicrobial activity of gelatin-based films containing propolis

Transcription

1 Functional properties and antimicrobial activity of gelatin-based films containing propolis R.B Bodini a, C. S. Favaro-Trindade a, P.J.A. Sobral a, R. A. Carvalho a a Food Engineering Department, FZEA University of São Paulo. AV. Duque de Caxias Norte, , Pirassununga/SP (rosecarvalho@usp.br) ABSTRACT The interest in the use of active packaging for food conservation has increased, along with a higher demand for natural antimicrobial substances, due to higher consumer awareness of the potential health risks caused by synthetic compounds consumption. Among natural additives, propolis, has awaken the interest of the researchers for its antibacterial and antioxidant properties. Thus, the aim of this work was to investigate the effect of the addition of an ethanolic propolis extract (EPE) to gelatin-based films plasticized with acethyltributhyl citrate (AC) on the mechanical properties, solubility and the antimicrobial activity against Staphylococcus aureus. For the EPE production, 30g of propolis resin (type 12) were mixed with 100mL ethyl alcohol (80%), and the solution was kept under mechanical stirring (500rpm, 30minutes) at 50 C, and filtered after 24hours under refrigeration. The films were produced by casting, using 2g of gelatin/100g of film forming solution, 30g of AC/100 g of gelatin, 35g of lecithin/100g of plasticizer and EPE in the concentrations of 0, 5, 40 or 200g/100g of gelatin, and evaluated for their mechanical properties and water solubility (Sol), after storage (5 days, relative humidity = 58%, temperature = 25 C). The antimicrobial activity of the films against Staphylococcus aureus was analyzed by agar diffusion method. The EPE addition caused variation of the tensile strength (T) of the films (58.7 to 66.4MPa), which also presented variations for the elongation (%). The water solubility of the films presented significant increase (Sol = 18,6%) for 200% of EPE. The activity against S. aureus was observed for the films containing 40 and 200% of EPE, with significant increase of inhibition diameter with increase of EPE concentration. The films kept their antimicrobial activities during 177 days of storage. These results demonstrated the antimicrobial capacity of gelatin-based films added of propolis, and their stability over time. Keywords: film; protein; bioactive; acethyltributhyl citrate; caracterization. INTRODUCTION In recent years, research on packaging has given greater attention to biodegradable films, produced from natural biopolymers. This makes sense for the environment because these materials are fully compatibles, from renewable sources, highly recyclable, compostable and biodegradable. Among the natural macromolecules used, the gelatin has been has been extensively used in several studies for production of edible or biodegradable films, due to its functional properties, large-scale production and low cost [1,2,3]. However, the gelatin films are very sensitive to environmental conditions, due to the hydrophilic character of the biopolymers and also the usual plasticizer. Then, the use of a hydrophobic plasticizer, such as the acethyltributhyl citrate (hydrophobic plasticizer) could avoid this sensitivity. This alternative plasticizer shows no toxicity and can be used in contact with food [4]. On the other hand, due to constant concern on preventing chemical and especially microbiological deterioration in food [5], the interest in active packaging has increased. Active packaging deliberately incorporates active components intended to release or to absorb substances into, onto or from the packaged food or the environment surrounding the food, to prolong food shelf-life [6]. Active antimicrobial active films are mainly designed to act by direct contact with the foods by incorporating antimicrobial functionalities. In this sense, many natural substances such as enzymes, bacteriocins and essential oils [7,8,9,10,11] have been studied for their functional properties as active additives for edible films. However, propolis, a natural substance widely found and used by man since ancient times because of its pharmacological properties [12], does not have major role as an additive in edible films. But, many scientific studies have suggested that propolis has a great therapeutic potential, especially regarding inflammatory, antimicrobial, anticancer and antioxidant activities [13,14,15,16], which reinforces the possibility of using this compound as an additive in polymeric materials.

2 Thus, the aim of this work was the production and characterization (mechanical properties, solubility and antimicrobial activity) of gelatine-based films added with different concentrations of ethanolic propolis extract, including the evaluation of the effect of the storage time on the stability of the of propolis antimicrobial activity inside of gelatine matrix. MATERIALS & METHODS Materials For the production of films, the following products were used: commercial type A gelatin provided by GELITA of Brazil Ltda. (São Paulo, Brazil), acethyltributhyl citrate (AC) (Aldrich), lecithin (E 322, Caramuru, São Simão - GO) and ethyl alcohol (Synth). A Brazilian propolis (Star Rigel, Raffard - SP) was used for the production of ethanolic propolis extract type 12. To evaluate the antimicrobial activity BHI broth (BD), bacteriological peptone (BD) and nutrient agar (Merck) were used. The Staphylococcus aureus bacteria used in the microbiological analysis was obtained from Andre Tosello Foundation (Campinas). Propolis extraction The ethanolic propolis extract (EPE) was prepared [17] using 30g of crushed Brazilian propolis resin (type 12) in 100mL of ethyl alcohol (80%), and kept at 50ºC under mechanical stirring (500rpm, 30minutes). After this step, the solution was rapidly cooled, stored refrigerated for 24 hours, and then filtered using Whatman nº1 filter. Film production For film production, acethyltributhyl citrate (AC), used as plasticizer (C AC = 30g/100g of gelatin), and lecithin, used as emulsifier (C Lecitina = 35g/100g of plasticizer) were added in ethyl alcohol (20g/100g of film forming solution) and kept under magnetic stirring for 2h, at room temperature. Gelatin (2g/100g of film forming solution) was hydrated (at room temperature, 30minutes) and solubilized (T = 55ºC, 30minutes). After those periods, the solutions (gelatin + water and ethyl alcohol + lecithin + plasticizer) were mixed and homogenized magnetically. The ethanolic propolis extract was then added at the concentrations of 5, 40 or 200g/100g of gelatin. The final solution was homogenized at 6000rpm (10min) using an ultra-turrax (IKA, T25) and poured in acrylic plates (65g of solution/plate) and dried in oven with forced ventilation at 30ºC for 24h. Mechanical properties The mechanical properties of the films were evaluated by tensile tests [18], using a texture TA.XT2 (TA Instruments), where the film samples (15x100mm) were attached on a specific probe (tensile grips). The initial separation distance was 80mm and the test speed was 0,9mm/s. The tensile strength (T, MPa) and the elongation at break (E, %) were obtained directly of the tensile strength versus elongation curves, while the elastic modulus (ME, MPa) was determined calculating the slope of the linear segment of the curve. Water solubility For the solubility analysis [19,20], the film samples, cut as discs (diameter = 2cm) were immersed in distilled water (50mL) and kept under mechanical stirring (shaker Marconi-MA141) for 24h, at 25ºC. After this time, the samples were dried (105ºC, 24h), and weighed determining then the final dry mass of the samples (m f ). The initial mass (m i ) was determined knowing the sample moisture. The solubility (Sol), expressed in terms of dissolved dry mass, was calculated according to Equation 1. mi mf Sol = 100 m (1) i Antimicrobial activity The determination of the inhibition diameter of films [21], stored at room temperature and relative humidity (22-25ºC, RH = 50-58%) was evaluated for 177 days. Initially, the S. Aureus bacteria (Andre Tosello Foundation) were reactivated in BHI broth at 37ºC for 24 hours. Nutrient agar plates, previously prepared, were then inoculated on the surface with 0.1mL of S. aureus (10 4 CFU/mL) and discs of films,

3 20mm in diameter, were placed on the surface of the plates, which were then incubated at 37ºC for 24 hours. The inhibition diameter was measured considering the initial diameter of the films. Previously to the microbiological analysis, the films were exposed to ultraviolet light for 30 minutes to decontaminate. The tests were performed in triplicate for each extract concentration in gelatin-based film (0, 5, 40 and 200%). Statistical analysis Statistical analysis was performed using the SAS software (version 9.2). Significant differences between means were evaluated by Duncan tests (p < 0.05). RESULTS & DISCUSSION Mechanical properties The incorporation of the ethanolic propolis extract affected the tensile strength (T) of the films (Table 1). The tensile strength fo films with the concentration of 200%, of propolis was lower (P<0.05) than the others films. Similar behaviour was observed for the elongation at break (E). Regarding the elastic modulus (EM), in general, the propolis-added films had higher (P<0.05) values of this parameter. As the production of the film plasticized with acetyltributyl citrate involves an emulsifying step, the distribution of propolis in the matrix is extremely complex which may have caused this variability. Table 1. Tensile strength (T), elongation (E) and elastic modulus (EM) of films plasticized with acethyltributhyl citrate (AC) as a function of ethanolic propolis extract concentration (C EPE ). Plasticizer C EPE (g/100g of gelatin) T (MPa) E (%) EM (MPa) AC ± 2.1 b 7.7 ± 1.1 b 14.7 ± 2.9 c ± 3.4 a 14.9 ± 3.1 a 16.3 ± 1.6 ab ± 2.3 b 8.2 ± 1.5 b 15.2 ± 0.8 bc ± 3.2 c 5.0 ± 1.0 c 17.4 ± 0.4 a Different small letters in the same column indicate significant differences (p <0.05). Mean difference obtained by Duncan test, using the SAS software. Pastor et al. [22] found that the addition of bioactive agent did not significantly affect the tensile strength and elastic modulus of the films based on hydroxypropyl methylcellulose (HPMC) containing ethanolic propolis extract in different concentrations (0 to 1.5%). However, the elongation at break of these films decreased with increasing concentration of the ethanolic propolis extract [22]. Regarding another study, the increase in concentration of thyme oil in chitosan films also caused a significant reduction of the tensile strength of the films, and decreased the percentage of elongation, as a consequence of the increasing in film porosity [23]. Other authors [24] found that the addition of cinnamon essential oil in films of chitosan significantly increased the tensile strength of the films, which was attributed to the strong interaction between polymer and oil that had a crosslinking effect, reducing the free volume and molecular mobility of the polymer. Water solubility The results presented in Table 2 show that the concentration of 40% of EPE decreased the solubility of the films in comparison to the control film, while with the concentration of 200% of EPE, the solubility increased approximately 129%. Probably, these results are related to the distribution of the extract in the gelatin matrix and to interactions of polyphenols, present in propolis, with proteins. Gómez-Estaca et al. [9], working on gelatin films with essential oil of clove, observed the the additive also increased film solubility, and this behavior was attributed to the establishment of interactions between protein and polyphenols that weakened the interactions that stabilize the protein network [9]. However, in films based on chitosan, the addition of cinnamon essential oil caused decrease in the water solubility of the films [24]. Regarding moisture (Table 2), no change was observed due to the increased concentration of EPE.

4 Table 2. Solubility (Sol) and moisture (H) of films plasticized with acethyltributhyl citrate (AC) as a function of ethanolic propolis extract concentration (C EPE ). Plasticizer C EPE (g/100g of gelatin) Sol (g/100g of film) U (g/100g of film) AC ± 1.3 b 9.8 ± 1.6 a ± 0.6 b 8.5 ± 0.7 b ± 1.1 c 10.3 ± 0.5 a ± 2.6 a 9.4 ± 0.9 ab Different small letters in the same column indicate significant differences (p <0.05). Mean difference obtained by Duncan test, using the SAS software. Antimicrobial activity The films added of ethanolic propolis extract (0, 5, 40 and 200%) were evaluated qualitatively for their antimicrobial activity against Staphylococcus aureus. The results of this study showed that the films with concentrations of 0 and 5g of EPE/100 of gelatin did not have antimicrobial activity, presenting bacterial growth on the their surfaces (Figure 1). However, for concentrations above 5%, an effective inhibition of the tested bacteria was observed (Figure 1) with significant increase of the inhibition diameter with the increase of extract concentration, around 22.9 and 26.9mm for concentrations of 40 and 200%, respectively. (a) (b) (c) (d) Figure 1. Antimicrobial activity of films plasticized with acethyltributhyl citrate against S. aureus: (a) C EPE = 0g/100g of gelatin, (b) C EPE = 5g/100g of gelatin, (c) C EPE = 40g/100g of gelatin and (d) C EPE = 200g/100g of gelatin. Alginate-based films added with garlic oil [25], films of whey protein containing essential oils of oregano and garlic [26] and films of soy protein fortified with oils oregano or thyme [8] also showed an inhibitory effect on the S. Aureus. The results of the inhibition diameter, in general, showed that films added of propolis (Figure 2) kept their antimicrobial activity during the period studied (177 days), for two concentrations (40 and 200%) tested. However, it was not possible to set a correlation between storage time and antimicrobial activity due to the variation of the inhibition diameter, which may be associated with variations in the distribution of the ethanolic propolis extract in the polymer matrix.

5 Figure 2. Microbial inhibition diameter (mm) against S. aureus vs storage time (days) of the films plasticized with acethyltributhyl citrate for different concentrations of ethanolic propolis extract of (C EPE ). Different small letters for each concentration of EPE, indicate significant differences (p <0.05). Mean difference was obtained by Duncan test, using the SAS software. CONCLUSION Overall, the gelatin film properties were affected by high concentrations of added EPE. Regarding the antimicrobial activity, the films containing concentrations above 5% of EPE showed effective inhibition against Staphylococcus aureus, and the evaluation of the effect of time of this property showed that the films kept the antibacterial capacity over a period of 177 days. Therefore, in general, the results obtained for the active films were satisfactory, allowing future works aiming the application of these films in the conservation of food products. ACKNOWLEDGE The authors are grateful to FAPESP (Process: 2009/ ) for the financial support. REFERENCES [1] Cao, N.; Yang, X & Fu, Y Effects of various plasticizers on mechanical and water vapor barrier properties of gelatin films. Food Hydrocolloids, 23, [2] Carvalho, R. A.; Grosso & C. R. F Characterization of gelatin based films modified with transglutaminase, glyoxal and formaldehyde. Food Hydrocolloids, 18(5), [3] Thomazine, M.; Carvalho, R.A.; Sobral, P.J.A Physical properties of gelatin films plasticized by blends of glycerol and sorbitol. Journal of Food Science, 70(3), E172-E176. [4] Rahman, M. & Brazel, C. S The plasticizer market: An assessment of traditional plasticizers and research trends to meet new challenges. Progress in Polymer Science, 29, [5] Chen C-P.; Wang, B-J. & Weng Y-M Physiochemical and antimicrobial properties of edible aloe gelatin composite films. International Journal of Food Science and Technology, 45, [6] López-Rubio A., Almenar E., Hernandez-Muñoz P., Lagarón J.M., R., Gavara R Overview of active polymerbased packaging technologies for food applications. Food Reviews International, 20, [7] Del Nobile M. A., Conte A., Incoronato A. L. & Panza O Antimicrobial efficacy and release kinetics of thymol from zein films. Journal of Food Engineering, 89, [8] Emiroğlu Z. K., Yemiş G. P., Coşkun B. K. & Candoğan K Antimicrobial activity of soy edible films incorporated with thyme and oregano essential oils on fresh ground beef patties. Meat Science, 86, [9] Gómez-Estaca J., Lacey A. L., López-Caballero M. E., Gómez-Guillén M. C. & Montero P Biodegradable gelatin-chitosan films incorporated with essential oils as antimicrobial agents for fish preservation. Food Microbiology, 27, [10] Mecitoğlu Ç., Yemenicioğlu A., Arslanoğlu A., Elmaci Z. S., Korel F. & Çetin A. M Incorporation of partially purified hen egg white lysozyme into zein films for antimicrobial food packaging. Food Research International, 39,

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