This article was downloaded by: [McMaster University] On: 07 May 2014, At: 10:26 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Archives Of Phytopathology And Plant Protection Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gapp20 Antifungal activity of essential oils and their combinations in in vitro and in vivo conditions Mysore Gopal Tejeswini a , Hebbale Vasanth Sowmya a , Shimoga Prabhakar Swarnalatha a & Pradeep Singh Negi a a Fruit and Vegetable Technology Department, Council of Scientific and Industrial Research-Central Food Technological Research Institute, Mysore, India. Published online: 11 Jul 2013. To cite this article: Mysore Gopal Tejeswini, Hebbale Vasanth Sowmya, Shimoga Prabhakar Swarnalatha & Pradeep Singh Negi (2014) Antifungal activity of essential oils and their combinations in in vitro and in vivo conditions, Archives Of Phytopathology And Plant Protection, 47:5, 564-570, DOI: 10.1080/03235408.2013.814235 To link to this article: http://dx.doi.org/10.1080/03235408.2013.814235 PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
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This article was downloaded by: [McMaster University]On: 07 May 2014, At: 10:26Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registeredoffice: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Archives Of Phytopathology And PlantProtectionPublication details, including instructions for authors andsubscription information:http://www.tandfonline.com/loi/gapp20
Antifungal activity of essential oils andtheir combinations in in vitro and invivo conditionsMysore Gopal Tejeswinia, Hebbale Vasanth Sowmyaa, ShimogaPrabhakar Swarnalathaa & Pradeep Singh Negiaa Fruit and Vegetable Technology Department, Council ofScientific and Industrial Research-Central Food TechnologicalResearch Institute, Mysore, India.Published online: 11 Jul 2013.
To cite this article: Mysore Gopal Tejeswini, Hebbale Vasanth Sowmya, Shimoga PrabhakarSwarnalatha & Pradeep Singh Negi (2014) Antifungal activity of essential oils and their combinationsin in vitro and in vivo conditions, Archives Of Phytopathology And Plant Protection, 47:5, 564-570,DOI: 10.1080/03235408.2013.814235
To link to this article: http://dx.doi.org/10.1080/03235408.2013.814235
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the“Content”) contained in the publications on our platform. However, Taylor & Francis,our agents, and our licensors make no representations or warranties whatsoever as tothe accuracy, completeness, or suitability for any purpose of the Content. Any opinionsand views expressed in this publication are the opinions and views of the authors,and are not the views of or endorsed by Taylor & Francis. The accuracy of the Contentshould not be relied upon and should be independently verified with primary sourcesof information. Taylor and Francis shall not be liable for any losses, actions, claims,proceedings, demands, costs, expenses, damages, and other liabilities whatsoever orhowsoever caused arising directly or indirectly in connection with, in relation to or arisingout of the use of the Content.
This article may be used for research, teaching, and private study purposes. Anysubstantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
Fruit and Vegetable Technology Department, Council of Scientific and Industrial Research-Central Food Technological Research Institute, Mysore, India
(Received 8 June 2013; final version received 9 June 2013)
Natural additives are in demand for the control of microbial growth in foods. Severalnatural compounds including essential oils (EOs) are being explored for food uses.In the present investigation, the antifungal activity of cinnamaldehyde, eugenol, pep-permint and clove EOs and their combinations was evaluated against 12 species ofAspergillus, Fusarium, Penicillium and Rhizopus in in vitro and tomato fruit system(in-vivo). The EOs were able to inhibit complete growth of tested fungi at or below0.6% level and 80 μL of EOs (except peppermint oil) in in vitro condition andtomato system, respectively. The fractional inhibitory studies showed either additiveor indifferent effect by combining eugenol and peppermint, and indifferent or antag-onist effect by combining the cinnamaldehyde and clove in both in vitro and in vivostudies. The findings may be useful for application of these EOs in foods, but theireffects on organoleptic quality of foods need to be investigated.
Food-borne diseases are a growing public health problem worldwide. It is estimated thateach year in the USA, 31 species of pathogens cause 9.4million cases of food-borneillnesses (Scallan et al. 2011). Therefore, food safety has become an important publicconcern as microbial contamination not only increases the risk of food-borne diseasebut also reduces the shelf life of foods. Contamination by fungi such as Rhizopusspecies, Aspergillus species and Penicillium species is the major cause of rapid deterio-ration of fresh commodities, which affects their quality and shortens the shelf life(Lichter et al. 2002). For many years, synthetic fungicides have been used to solve thisproblem; however, the association of synthetic chemicals with the adverse effect onhuman health and the development of fungicide-resistant strains have stimulated thesearch for new strategies for controlling postharvest decay. In recent years, manyresearchers have focused on the search of natural antimicrobial agents, and the plantextracts (Negi 2012; Ravikumar & Garampalli 2013), and essential oils (EOs) (Burt2004; Mohammadifar et al. 2012) are gaining more importance.
Archives of Phytopathology and Plant Protection, 2014Vol. 47, No. 5, 564–570, http://dx.doi.org/10.1080/03235408.2013.814235
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Cinnamaldehyde oil, the major compound in Cinnamomum osmophloeum leaf EOs,possessed stronger antifungal activity when compared to the other components (Wanget al. 2005). Several reports have shown the antimicrobial effect of eugenol (Davidson1997), and cinnamaldehyde and eugenol applied above 200 ppm concentration com-pletely inhibited mould growth and aflatoxin production (Wang et al. 2005). Peppermintoil is also reported to possess antifungal activity (Davidson 1997; Edris & Farrag2003).
Usually, combination of EOs or artificial mixtures of purified main componentsaffect multiple biochemical processes in the micro-organisms and inhibits their growth.In recent years, there has been an increased interest in the use of natural antimicrobialagents in combinations (either with processing techniques or among themselves) to con-trol food-borne pathogenic micro-organisms. However, to our knowledge, there is nostudy describing in vivo antifungal efficacy of a combination of EOs. Therefore, theaim of the present investigation is to study the antifungal activity of cinnamaldehyde,eugenol, peppermint and clove EOs and their combinations against 12 species ofAspergillus, Fusarium, Penicillium and Rhizopus in in vitro and in in vivo (tomato fruitsystem) for their potential application in control of postharvest spoilage of storedtomatoes.
Materials and methods
EOs and chemicals
The cinnamaldehyde (99.75% purity) and clove oil (eugenol content of 85.08%, v/v)were purchased from Loba Chemie Pvt. Ltd. (Mumbai, India), and eugenol (98% purity)and peppermint oil (4% Methyl acetate and 44% menthol) were purchased from HiMediaLaboratories Pvt. Ltd. (Mumbai, India). The oil was dissolved in the sterile distilledwater with the help of tween 80 and then used for the antifungal activity assays inin vitro and tomato systems. All the microbiological media were also purchased fromHiMedia and all solvents and chemicals used in the study were of AR grade.
Test organisms
The strains used were comprised of 12 fungal species belonging to four different gen-era. Six species of Aspergillus, four species of Fusarium and one species each of Rhizo-pus and Penicillum were used as test organisms. The fungal strains were isolated fromdifferent food or plant materials by plating onto potato dextrose agar (PDA) and identi-fied up to the species level by microscopic examination after staining with lacto-phenolcotton blue dye.
The fungal strains were cultured on the PDA slants and grown for five days at roomtemperature (28 ± 2 °C). The spore suspension was prepared from freshly growing cul-tures by using the saline solution (0.85% NaCl, w/v). To the 10ml of saline solution, 2loops of spores were added and mixed properly. Then, the spore count was done usinghaemocytometer and the suspension was diluted to 104 spores/ml for use in the presentstudy.
In vitro assay
The minimum inhibitory concentration (MIC) of EOs against various fungal strains wasdetermined using the method described by Xing et al. (2012) with slight modification.
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PDA was dispensed into conical flasks (50ml each) and autoclaved. Before pouringmedia on to the Petri plates, different concentrations of oil were added into 50ml PDAunder aseptic conditions. The emulsion without EOs (water and tween 80 in same quan-tity) served as control. After solidification of media, the plates were spot inoculatedwith 10 μL of spore suspension (104 spores/ml) and incubated for 3–5 days under roomtemperature conditions (28 ± 2 °C). The plates were observed regularly for growth of thefungus and the MIC of EO was defined as the lowest concentration of the EO capableof inhibiting the complete growth of fungal strain in question (Xing et al. 2012). EachMIC value was ascertained in minimum of three experiments.
In vivo assay
The in vivo antifungal assay was done using tomato fruit according to the proceduregiven by Badawy and Rabea (2009) with slight modification. Before treatment, thefruits were surface disinfected with 2% sodium hypochlorite for 3min, rinsed with tapwater and then air dried in laminar hood. The fruits were surface wounded (threewounds/fruit) with a sterile cork borer and 20 μL of EO emulsion was added to theindividual wounds. The emulsion without EO served as control. After 1 h of EO treat-ment, 10 μL of spore suspension (104 spores/ml) of each of the 12 fungal strains wasadded to different wounds. The control and treated fruits were put in ethanol-sanitisedthermocol boxes with two layers of handmade filter paper. Approximately, 25ml of ster-ile water was sprayed on filter paper before placing tomatoes in the box to maintainhumidity. After placing tomatoes, the box was covered with transparent sheets and incu-bated for 3–5 days at room temperature (28 ± 2 °C). The tomatoes were regularlyobserved for fungal growth during incubation period and MIC was defined as the low-est concentration of the particular EO at which no growth of the fungal strain in ques-tion was observed even after five days. Each MIC value was ascertained in minimum ofthree experiments.
Determination of fractional inhibitory concentration indices
The fractional inhibitory concentration (FIC) index of clove oil and cinnamaldehyde, andeugenol and peppermint oil combinations against all the test fungi were calculated in thein vitro and in vivo conditions. The oils in combination studies were used in 1: 1 ratio(v/v). The FIC indices were calculated as FICclove + FICcinnamaldehyde, where FICclove =(MICclove combination/MICclove alone) and FICcinnamaldehyde = (MICcinnamaldehyde
combination/MICcinnaaldehyde alone); and FICeugenol + FICpeppermint, where FICeugenol =(MICeugenol combination/MICeugenol alone) and FICpeppermint = (MICpeppermint combination/MICpeppermint alone). The results were interpreted as synergy (FIC < 0.5), additive(0.5 < FIC < 1), indifference (1 < FIC < 4) or antagonism (FIC > 4) (Schelz et al. 2006).Each MIC value was ascertained in minimum of three experiments.
Results and discussion
Food products require protection against fungal spoilage during their storage. In thepresent study, the EOs used were able to inhibit complete growth of tested fungi at orbelow 0.6% level in in vitro condition, except for peppermint oil where almost 2% oilwas required for similar inhibition. A similar concentration of 2% of clove oil for com-plete growth inhibition of several mycotoxigenic moulds (Aspergillus flavus, Aspergillusparasiticus, Penicillium patulum and Penicillium citrinum) has been reported earlier
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also (Azzouz & Bullerman 1982). In our study, cinnamaldehyde was found to be themost effective antifungal agent followed by eugenol. Different fungi showed variableresponse to different EOs; however, Fusarium proliferatum was least resistant to pep-permint oil, A. flavus to eugenol, Aspergillus nidulans to clove oil, and Fusarium solaniand Fusarium moniliforme to cinnamaldehyde (Figure 1). Cinnamon oil has beenreported to inhibit the growth of several moulds, yeasts and bacteria (Matan et al.2006). Similarly, antiaflatoxigenic activity of eugenol (Jayashree & Subramanya 1999)and antifungal and antiaflatoxigenic effect of lemon grass EO (Parnagama et al. 2003)are also reported in literature.
Spoilage of fruits and vegetables by fungi is the major concern during storage as itaffects the quality and shortens their shelf life. In the present study, the completegrowth inhibition of artificially inoculated fungi in wounded tomatoes was observed ator below 80 μL of EOs, except for peppermint oil where almost 200 μL of oil wasrequired for similar effect. The inhibition trend was almost similar to in vitro studies,except a higher oil concentration was required in food system (Figure 1). A need ofhigher concentration of antimicrobial compound in in vivo as compare to in vitroconditions is reported earlier also (Gutierrez et al. 2009; Catherine et al. 2012). In the
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present study, among the four EOs, cinnamaldehyde showed highest inhibitory effectagainst all the tested fungi (except A. flavus); however, in a study of efficacy of EOsfrom clove and cinnamon against six fungi (Aspergillus niger, Alternaria alternata,Colletotrichum gloeosporioides, Lasiodiplodia theobromae, Phomopsis viticola andRhizopus stolonifer) causing postharvest decay of grapes, clove oil showed higherinhibition than cinnamon (Sukatta et al. 2008). Aminifard and Mohammadi (2012)reported that ammi and anise EOs inhibited the grey mould growth on tomato fruits at200–800 μLL�1 concentration and increased the shelf life of tomato besides main-taining its postharvest quality.
In order to define the influence of the combinations of the EOs on microbial growth,the FIC indices are widely used. In the present study, the fractional inhibitory studiesshowed either additive or indifferent effect by combining eugenol and peppermint EOs,and indifferent or antagonist effect by combining the cinnamaldehyde and clove EOs inboth in vitro and in vivo studies (Table 1). In general, the eugenol and peppermint EOscombination was found better than the cinnamaldehyde and clove EOs combination asit showed lower FIC indices and produced additive effect against A. flavus in in vitroand against Aspergillus ochraceus in both in vitro and in vivo conditions. Combinationof eugenol and peppermint EOs showed indifferent effect for all other fungi, whereascombination of cinnamaldehyde and clove EOs even produced antagonistic effectagainst several fungi in both in vitro and in vivo conditions. It is reported that thecombination of clove and rosemary show all three (synergistic, additive and antagonis-tic) effects depending on the micro-organism in question (Fu et al. 2007). Nguefacket al. (2012) reported that the fungicidal activity of mixtures of fractions from the sameEOs or from different EOs (Cymbopogon citratus, Ocimum gratissimum and Thymusvulgaris) was synergistic, additive or antagonistic. Similarly, combination of the Cymbo-pogon. giganteus and C. citratus EOs exerted synergistic, additive and indifferent anti-microbial effects depending on the micro-organism and concentration used (Bassoleet al. 2011).
This study showed that the tested EOs are effective against some of the fungi andcinnamaldehyde was the most effective against all the fungi tested in the present study.
Table 1. FIC of clove oil–cinnamaldehyde and peppermint–eugenol combinations against fungiin in vitro and in vivo conditions.
EOs were able to inhibit growth of fungi in tomato system also; however, a slightlyhigher concentration was required. Combination studies showed additive effect for somecombinations, and these combinations can be exploited in order to maximise theantifungal activity of EOs. Use of combinations of Eos will help in reduction in theconcentrations of individual EOs required to produce a desired antifungal effect, andwill reduce their impact on product sensory quality; however, their effect on overallacceptability of products need further investigations.
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