Investigation of the antifungal activity of different essential oils and their nanofibers

There has been an increased tendency to usage of natural antimicrobials and preservatives for food products in recent years. Essential oils (EOs) which are natural, antimicrobial and liquid secondary plant metabolite are getting importance to protect food with increasing awareness against synthetics and health problems occurring as a result of food borne diseases. They are composed of about sixty different components. Not only major components but also minor components play critical role for antimicrobial activity. There are several extraction methods of essential oils. However, the extraction method, extraction place of plant organ, season, soil properties, climate, oxygen and age of plant vary the properties of essential oil. The components included in the composition of essential oil affect microbial cells by various mechanisms including degradation of cell membrane, changing the equilibrium inside the cell by influencing permeability and disrupting enzyme functions. The main reason of these results is due to the hydrophobic characteristics. On the other hand, stability of essential oils is extremely low under environmental stresses like temperature and light. Therefore, they require some methods to preserve these properties. In this study, fungal strains were isolated from the packaged bread products (organic, whole wheat and sliced bread) which are about to expire date. After the molecular identification, P. carneum, A. flavus and A. niger were selected to perform following experiments. The antifungal effect of ginger, cumin, cinnamon, black pepper, origanum and clove essential oils and their volatile compounds were investigated by disc diffusion and disc volatilisation method, respectively. The petri dishes were incubated at 25ºC for 6 days and the diameter of inhibition zones were measured after 3 and 6 days. Among EOs, while ginger and black pepper affected tested fungal strains slightly, origanum was the most effective EO against all tested fungal strains during 6 days incubation. The second most effective EO was cumin at the end of the 3 days but its effect did not be preserved after 3 more days. The other remain EOs clove and cinnamon showed moderate antifungal impact. In the second part of this study, first four EOs (cumin, cinnamon, origanum and clove) found effective against fungal strains were selected to determine minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) values by poisoned culture medium method and the used concentration of EOs were 500, 750, 1000, 1500, 2000, 3000 and 5000 µg/mL. The highest MIC value was obtained in case of cumin EO at 3000 µg/mL concentration for P. carneum and the highest MFC value was obtained from the results of cumin and origanum as concentration of 3000 µg/mL. While some of EOs had same MIC and MFC values, some of them gave next concentration of MIC as an MFC value. Pursuant to consequences of MIC and MFC values, clove and cinnamon EOs were found more effective at lower concentrations. Thus, these two EOs were used to produce nanofibers for checking the antifungal activity of nanofiber films of EOs. The electrospinning solution was prepared as a mixture of polyvinyl alcohol (PVA) solution (6 %, 3 hours mixing, 80ºC, 700 rpm), β-cyclodextrin (2 %, continued 1-hour mixing, 55ºC, 700 rpm) and EO (2 %, continued 2.5 hours mixing, 55ºC, 700 rpm). Viscosity, surface tension and electrical conductivity analysis were performed for electrospinning solution. After production of nanofibers (procedure driven by 20-gauge steel needle, in distance 12 cm, at 0.4 mL/s flow rate by voltage in range of 14-16 kV), the characterization of EO nanofilms were examined by Fourier Transform Infrared (FTIR). Besides, raw materials (EO, PVA and β-CD) were also examined for comparison. The characteristic bands of PVA and β-CD were detected on the spectrum line; however, most of specific bands of EOs disappeared in consequence of accomplished encapsulation process. The produced nanofiber films were tested against both P. carneum and A. niger. In the result of analysis, cinnamon EO film showed higher inhibition activity than clove EO film. At the end of the 1-day incubation of films inside fungal suspension, these suspensions were inoculated to medium and fungal colonies were counted. The results of cinnamon film which were 2.64 – 2.51 log (CFU/mg) were considerably lower than clove film which were 3.18 – 3.24 log(CFU/mg). In the final part of study, organic bread sample collected from market was packed with nanofiber films and stored at 25°C. The prepared samples were waited until observing fungal growth on control bread sample. After three days, although the bread sample without film moulded, there was no fungal growth on bread packed with films. In conclusion, the higher antifungal activity of cinnamon and clove EOs among others and an alternative, natural and open to improvement option for bread packaging technology were showed and discussed by this study.