ANTIFUNGAL ACTIVITY OF THE ESSENTIAL OIL OF CINNAMON (CINNAMOMUM ZEYLANICUM), OREGANO (ORIGANUM VULGARE) AND LAURAMIDE ARGINE ETHYL ESTER (LAE) AGAINST THE MOLD ASPERGILLUS FLAVUS CECT 2949

Abstract

Minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) for oregano EO, cinnamon EO and LAE were determined using a direct-contact antifungical assay by macrodilution method. Cinnamon gave the strongest inhibition followed by oregano and LAE with similar results. LAE showed no antifungal activity in vapor phase, so growth and kill-kinetics were studied by direct contact. Colony diameter of Aspergillus flavus was measured by agar dilution method at different concentrations of cinnamon and oregano EOs, resulting cinnamon EO much more active than oregano EO. In the vapor phase test, the influence of the time of exposure to the cinnamon EO and oregano EO was evaluated, showing relevant differences in their behavior. Kill kinetics showed a different behavior among the three agents. [PUBLICATION ABSTRACT]

Headnote

ABSTRACT

Minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) for oregano EO, cinnamon EO and LAE were determined using a direct-contact antifungical assay by macrodilution method. Cinnamon gave the strongest inhibition followed by oregano and LAE with similar results. LAE showed no antifungal activity in vapor phase, so growth and kill-kinetics were studied by direct contact.

Colony diameter of Aspergillus flavus was measured by agar dilution method at different concentrations of cinnamon and oregano EOs, resulting cinnamon EO much more active than oregano EO. In the vapor phase test, the influence of the time of exposure to the cinnamon EO and oregano EO was evaluated, showing relevant differences in their behavior. Kill kinetics showed a different behavior among the three agents.

Keywords: Aspergillus flavus CECT 2949, cinnamon essential oil, colony radial growth, lauramide argine ethyl ester, orégano essential oil, volatile effect.

INTRODUCTION

During the last decades, prevention of foodborne spoilage has increased the research of new alternatives leaving behind the traditional ones (Quiroga et al., 2009). Many studies in the scientific literature demonstrated the powerful of essential oils as antimicrobial agents (Burt, 2004); (Kalemba and Kunicka, 2003). Their advantages have been tested as antimicrobial packaging where the agents can either interact directly with the spoiled organisms or interact with the environment inside the package (Lopez and Sanchez et al., 2007); (Rodriguez and Nerin et al., 2008); (Nielsen and Rios, 2000).

Antifungal activity of the essential oils of Cinnamomum zeylanicum and Origanum vulgare and the non volatile agent Lauramide argine ethyl ester (LAE) was evaluated against the mold Aspergillus ßavus CECT 2949. Many previous works concerning the antimicrobial activity of essential oils have been reported, however, LAE is a recent substance still in investigation (Luchansky and Call et al., 2005) (Porto-Fett and Campano et al., 2010). Due to the scarce knowledge reported in fungi compared to bacteria, different methodologies have been applied to increase information about these shelf-life extenders.

MATERIALS AND METHODS

Microbial culture and growth conditions

Strain of Aspergillus ßavus CECT 2949 from the Spanish Collection CECT of the University of Valencia, Spain, was used. The fungus was stored at - 18°C in sterilized skimmed milk. Fungal conidia were harvested after inoculation on Malt Extract Agar (MEA) for 1 0 days at 25°C and transferred to a test tube with physiological saline solution (NaCl 0,9%). A final concentration of 106cfu/mL was reached and confirmed by plate counting in culture media.

Active agents

The essential oils of Cinnamon [Cinnamomum zeylanicum, CAS 805-91-6) and orégano [Origanum vulgare, CAS 8007-11-2) were supplied by Argolide (Spain) and the agent Lauramide arginine ethyl ester (LAE, CAS 60372-77-2) by Lamirsa (Spain).

Minimal inhibitory concentration (MIC) and minimalfiingicidal concentration (MFC)

MIC was determined by macrodilution method in yeast extract broth. Serial dilutions of each active agent were prepared (Pinto and Salgueiro et al., 2007). Both essential oils were diluted into etanol (v/v) and LAE into sterilized water (w/v) and mixed into yeast extract broth to reach a range of 1 ,600-100 ppm final concentration. A 106cfu/mL of Aspergillus ßavus suspension was added to each sample. Test tubes with a 2 mL of total volume were incubated for 48h at 25°C in continuous shaking. The minimal inhibitory concentration was determined by visual method, as the lowest concentration where no growth was observed. Then, 100 µL of these non-growth concentrations were seeded with a Drigalsky sterile loop into different Petri dishes containing 15 mL of Malt Extract Agar (MEA). After 3-5 days of incubation period at 25° C, minimal fungicidal concentration was determined as the lowest concentration where no colony was developed. This test was carried out three times by duplicate.

Volatile effect of cinnamon and orégano versus time

Lauramlde arginine ethyl ester (LAE) did not show any activity in vapor phase, so, cinnamon and orégano were employed as active agents. 100 µ?^ of 106cfu/mL of Aspergillus flavus suspension was inoculated into the petri dishes. Then, 5 \XL of each essential oil was applied into the cover lid, and the Petri dishes were incubated with the lid upside down (Lopez and Sanchez et al., 2005). Four exposure times were evaluated (24h, 48h, 72h, 96h) and 3 replicates were prepared for each time and each essential oil. So, a total of 24 Petri dishes were carried out in the same way. Two controls without active agent were performed to assure the correct growth of the fungus. Every 24h the covers of 3 replicates of each sample were removed. After 12 days of incubation period at 25° C, inhibition area (cm) was measured.

Colony radial growth

The experiment was carried out in direct contact with the culture media (MEA). The three active substances were used in a range of 1-4% of concentration. Dilutions of essential oils (v/v) were made into ethanol and LAE (w/v) into sterilized water. Three replicates of a total volume of 100 [iL were prepared into eppendorf tubes and poured into 15 mL of malt extract agar (MEA) previously liquefied and maintained at 45°C of temperature. The mixture was homogeneously shaken in all directions. Once the medium containing the active agent was solidified, a 10 µ? drop of 106cfu/mL fungal suspension was inoculated into the middle of each Petri dish. Controls were performed with addition of ethanol in the culture media instead of active agent. The incubation period at 25°C stopped at 9 days, point where the control reached the border of the dish. Antifungal activity was measured in relation to the colony diameter (cm) developed (Boyraz and Ozcan, 2006).

Kill curves

An initial concentration of 105 cfu/mL of fungal suspension was mixed with the minimal fungicidal concentration (MFC) for orégano and LAE. Cinnamon needed a higher concentration, so MFC and two times MFC were tested. The mixture was diluted into yeast extract, placed into 2 mL test tube of total volume and incubated at 25°C in continuous shaking. Different time points were established depending on the active agent evaluated, then, 100 µL of each mixture were inoculated into 15 mL MEA Petri dishes. After 3-5 days of incubation period, fungal colony was counted (cfu/mL).

RESULTS

Minimal inhibitory concentration (MIC) and minimalfiingicidal concentration (MFC)

As shown in tab. 1, the lowest concentrations were obtained for cinnamon essential oil, while LAE and Oregano gave the same results.

Volatile effect of cinnamon and orégano versus time

On one hand (fig. la), oregano's activity manifested a dependence of time of exposure. As we can see, replicates exposed for only 24h showed a faster decrease of the inhibition area, reaching the complete inhibition at sixth day. Not significance differences were obtained between replicates exposed for 48h, 72h and 96h, that is, oregano's antifungal activity against Aspergillus ßavus in vapor phase needs at least 24h of exposure.

On the other hand (fig. lb), cinnamon did not show differences between replicates exposed for different times. This behavior could be explained by a faster release of volatile compounds in the first 24h.

Colony radial growth

Colony diameter were used as a parameter of antifungal activity in direct contact with culture media. LAE did not show any activity (fig. 2a), non differences were obtained between control and any concentration. Oregano showed low activity at 1% concentration, however, colony diameter were strongly reduced with higher concentrations, obtaining total inhibition at 4% (fig. 2b). As we can observed, cinnamon was the most active agent in this test since non growth was found at any concentration (fig. 2c).

Kill curves

An initial kinetic study was performed in direct contact with yeast extract broth. Oregano (fig. 3a) evidenced the fastest activity where non colony was counted after 20 min of assay and LAE showed the same result after 3h (fig. 3a). However, cinnamon (fig. 3b) needed longer contact, in about 16h MFC produced a growth reduction of 2 logaritmic units while two times the MFC gave a reduction of 3 units.

CONCLUSIONS

Although both essential oils gave antifungal activity in all experiments, differences between them were found. In particular, the results in vapor phase test showed signs of a faster release of volatile compounds in the case of cinnamon. Stronger activity was also found for cinnamon in MIC and MFC determinations and in the test performed in direct contact with culture media. These results are in correlation with other authors (Lopez and Sanchez et al., 2005; 2007) and are essential to perform the antimicrobial packaging, considering cinnamon essential oil more active than orégano essential oil in the case of fungi.

The advantage of LAE in relation to the essential oils is the fact that organoleptic modifications of products are minimized. However, LAE has not any activity in vapor phase, needing the presence of water to be dissociated and to be able to act, medium where it has a strong activity (Kawamura, 2008). This fact means that the substance requires direct contact with the product and the presence of water. In conclusion, choice of antifungal agent will depend on the kind of food product and package performed.

ACKNOWLEDGMENTS

This work has been financed by the project AGL-2008-04363 from the Spanish Ministry of Science and Innovation and by the European Project Nafispack.

References

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