1312 EVALUATION OF THE SYNERGISTIC EFFECT OF ETHANOL AND LEMONGRASS OIL AGAINST Aspergillus niger Lieu My Dong* 1 , Dang Thi Kim Thuy 2 Address(es): 1 Faculty of Food Technology, University of Food Industry, Ho Chi Minh City, Viet Nam, phone number: +84989961848. 2 Institute of Tropical Biology, Ho Chi Minh City, Viet Nam. *Corresponding author: lieudong289@gmail.com ABSTRACT Keywords: Ethanol, lemongrass oil, antifungal activity, SEM, synergistic effect, tween 20 INTRODUCTION Post-harvest losses are one of the major causes of the loss of fresh vegetables during the supply chain (Nunes et al., 2012). Aspergillus niger is one of the major causes of black rot of plain (Prakash et al., 1988). Therefore, control of A. niger during the preservation is very necessary. The widespread use of synthetic fungicides for preserving agricultural products has significant limitations such as handling of hazards, pesticide residues, and risk to health and the environment (Dharini et al., 2014). Current trends in antimicrobial agent research from the natural origin in which plant-derived essential oils are of great interest. In previous studies, lemongrass oil is considered an essential oil for safe and effective natural preservatives which has an effective antibacterial activity (Vazirian et al., 2012). Essential oils are natural products consisting of a complex mixture of volatile molecules (Mahian et al., 2016), which are liquid, soluble in organic solvents and insoluble in water (Bakkali et al., 2008). The evaluation of the antimicrobial activity, the essential oils are often diluted at different concentrations by emulsifying agents (Burt, 2004). Hilbig et al. (2016) reported that emulsifier has the negative effect on the antimicrobial activity of essential oil (Hilbig, 2016), whereas the combination of ethanol and other antimicrobial agents (chitosan, potassium sorbate …) show the antimicrobial activity better than using in single agent (Romanazzi et al., 2007; Karabulut et al., 2005). Besides essential oil, ethanol also showed antimicrobial effect (Gianfranco et al., 2007). However, to achieve good antibacterial efficacy, ethanol is commonly used at high concentrations leading to increased production costs and the risk of fire safety (Ozgur et al., 2005). Ethanol is made up of hydrophilic (-OH) and hydrophobic (CH3CH2-) radicals. Hydrophilic radicals (- OH) to help dissolve the polarizing elements and ions. Short chain CH3CH2- hydrocarbons can attract non-polar molecules. Therefore, the combination of ethanol and essential oil can both increase the dilution effect of the oil in the media and can synergize with the essential oil to enhance the effectiveness of the antimicrobial. Although the combination of ethanol and the essential oil is very promising, very few studies exist on the synergic effect of ethanol and lemongrass oil on fungi. In this study, the antifungal activity of lemongrass oil and ethanol used alone or in combination against A. niger was evaluated by agar disk diffusion method, mycelial growth inhibition and broth dilution method to determinate MIC and MFC. The ultraviolet (UV) absorption and electrical conductivity of the culture supernatant were used to determine membrane integrity. Scanning electron microscopy (SEM) performed to observe the morphology of Aspergillus niger spores. MATERIAL AND METHODS Materials Aspergillus niger M1 was obtained from strain collection of Faculty of Food Technology, Ho Chi Minh City University of Food Industry. A. niger was grown in PDA (Potato Dextrose Agar) medium at 30 o C for 6 days. Then, the mass was harvested by rinsing plates with PDB (Potato Dextrose Broth). The freshly grown microbial cell at approximately 6 log CFU/mL was used for the evaluation of the antifungal activity. The essential oil in this study was lemongrass oil (Cymbopogon flexuosus) from Tien Giang province, the City is located at 10°25′N 106°10′E in the southern region of Vietnam. Lemongrass was hand-collected and immediately used to obtain lemongrass oil by steam distillation. Lemongrass oil was stored in glass vials in the absence of light until gas chromatography analysis and to test its antifungal activity. The essential oil was directly analyzed by gas chromatography coupled to mass spectrometry (Agilent GC 7890B GC System, 7010 GC/MS Triple Quad). The used column was an HP-5MS (30 m long, 0.25 mm and 0.25 μm film thickness). The operating conditions were as follows: Helium was used as a carrier gas with a back pressure of 0.8 atm; flow rate of 1.0 mL/min; split 1:20; injection volume 0.2 μL; The injector temperature was 250 o C and the oven temperature program started at 60 for 5 min and then increased at the rate of 5 o C/min up to 150 o C at 5 o C/min, and increased from 150 o C to 280 o C at 10 o C/min. The constituents in the essential oils were identified by computer matching of their mass spectral fragmentation patterns with those of compounds in the data bank NIST 98 and Wiley 275 library. Lemongrass oil and ethanol used alone or in combination with or without tween 20 (0.3% v/v) (an emulsifying agent) were used as antifungal agents for the next step. The aim of this work was to evaluate the antifungal efficiency of ethanol and lemongrass oil at the different concentrations, alone and in combination both against Aspergillus niger by agar disk diffusion method, mycelial growth inhibition and broth dilution method to determine MIC and MFC. The ultraviolet (UV) absorption and electrical conductivity of the culture supernatant were used to determine membrane integrity. Scanning electron microscopy (SEM) performed to observe A. niger spore morphology. The results showed that the MIC and MFC values of lemongrass oil (in tween 20 0.3% v/v) was 5 μL/mL and 250 ppm which were 100 and 200 times respectively lower than ethanol. The combination of lemongrass oil in tween 20 with ethanol (20% v/v) show the best result with the inhibition zone and MGI (%) was observed at 2.5 μL/mL and 100% respectively. The UV absorption and electrical conductivity values increased quickly after 16h incubated in the antimicrobial agents whereas there were almost unchanged in the control at the point of time. The SEM results show that, the morphological changes of the A. niger spores due to significant wrinkles and distortion in the samples treated by lemongrass oil or lemongrass oil combining with ethanol while the A. niger spores in ethanol show slight wrinkles. These suggest that ethanol has a synergic effect which enhances the antifungal activity of lemongrass oil. This combination is necessary to ensure the antimicrobial effect as well as reduce the amount of used essential oil. ARTICLE INFO Received 3. 8. 2018 Revised 6. 2. 2019 Accepted 8. 2. 2019 Published 1. 6. 2019 Regular article doi: 10.15414/jmbfs.2019.8.6.1312-1316