Comparative antimicrobial activity of clove and fennel essential oils against food borne pathogenic fungi and food spoilage bacteria

Antifungal and antibacterial activities of essential oils obtained from fennel seeds (Feoniculum vulgare Mill) and clove buds (Syzygium aromaticum) were studied by agar well dilution technique. Both essential oils (EOs) from fennel and clove exhibited pronounced and varying degrees of growth inhibition against fungal (86 to 39%) and bacterial pathogens (42 to 20%). Fennel oil depicted significant and greater fungitoxicity in case of three fungal strains Alternaria alternate (7.7, 3.8 cm) Fusarium oxysporum (5.9, 4.1 cm) and Aspergillus flavus (4.5, 3.7 cm) except two Aspergillus strains, Aspergillus acculeatus and Apergillus fumigatus where clove oil showed greater inhibition zone (5.5, 5.9 cm) (3.5, 3.7 cm) respectively. A. alternate was found to be most sensitive strain, which growth was suppressed up to 86% by fennel seeds oil. Bactericidal activity of culinary spices was evaluated against five food spoilage bacteria namely: Pseudomonas syringae, Bacillus subtilis, Escherichia coli, Staphylococcus sp., and Aeromicrobium erythreum. Fennel oil was found fairly active against bacterial strains as compared to clove oil with highest antibacterial activity against Gram positive bacteria Bacillus subtilis (3.8 cm) and least against Gram negative bacteria E. coli (2.2 cm). The summarizing results from the present investigation showed that fennel seeds oil is a relatively stronger antimicrobial agent against broad range of pathogens as compared to clove oil, except in case of certain Aspergillus strains and E. coli.


INTRODUCTION
Food-borne diseases are still a major problem in the world, in spite of the modern improvements in food hygiene, even in well-developed countries (WHO, 2002).A variety of microorganisms lead to food spoilage and food borne diseases.In the advance stage preservation, preservatives must be used to prevent the growth of spoiling microbes in the food industry (Sagdıc and Ozcan, 2003).In foods for decades, synthetic preservatives have been used that may lead to negative health consequences, such as convert ingested materials into toxic substances, increasing cost, handling hazards, residues on food that threat to human environment (Namiki, 1990;Paster and Bullerman, 1988;Farag et al., 1989).Consumer awareness of natural food products and a growing concern of microbial resistance toward conventional preservatives have led to exploring naturally-occurring antimicrobials for food preservation (Gould, 1995).Essential oils (EOs) of spices and herbs at first place are safe and stable as natural foodstuffs have been added to food since ancient times.Spices essential oils appeal to all who question safety of synthetic food additives and at the same time demand high-quality (Nakatani 1994;Cutler et al., 1995).Kitchen spices used in daily life also used in traditional medicines to treat infectious diseases seem to be a potent source of new bioactive secondary metabolites.The antimicrobial effects of spices are mostly due to the essential oils present in their composition (Arora-Dlijit and Kaur, 1999).
Fennel (Foeniculum vulgare Miller), with a sweet, earthy flavor which belongs to the family Apiaceae has long been used as herbal remedy.Medicinally, fennel is used as analgesic, antioxidant, antispasmodic, antiinflammatory, carminative, and diuretic (Oktay et al., 2003;Mimica-Dukic et al., 2003).Recently, antimicrobial activities of fennel seed extracts and essential oils has been investigated and explored value of this commonly used kitchen spice (Ozcan et al., 2006;Mata et al., 2007) .Essential oil of Fennel (EOF) can be used as possible bio fungicides, alternative to synthetic fungicides against phytopathogenic fungi (Soylu et al., 2007).The essential oil and extracts of clove (EOC) are used as a topical application to relieve pain and to promote healing, antiaging, cardiovascular disease, arthritis , infections (skin, flu, bacterial, viral and fungal, hepatitis, parasitic), digestive problems (nausea, vomiting, diarrhea), skin cancer, thyroid dysfunction and also finds use in the fragrance and flavouring industries (Chaieb et al., 2007;Liu et al., 1997;Kim et al., 1998;Zheng et al., 1992;Cai and Wu, 1996).Strong antimicrobial activity of in clove essential oil is referred to the rich amount of eugenol.This phenolic compound is capable to denature proteins and reacts phospholipids of cell membrane alternating their permeability (Briozzo, 1989;Deans and Ritchie, 1987).Gulfraz et al. (2008) showed inhibition of fennel oil against Bacillus cereus, Bacillus magaterium, Bacillus pumilus, Bacillus substilis, Eschericha coli, Klebsiella pneumonia, Micrococcus lutus, Pseudomonos pupida, P. syringae, and Candida albicans as compared to its ethanolic and methanolic extracts.Eugénia et al. (2009) evaluated the minimum fungicidal concentration of the clove oil and its main component, eugenol, against Candida, Aspergillus and dermatophyte clinical and American Type Culture Collection strains.It has been proved that clove oil and eugenol have considerable antifungal activity against clinically relevant fungi, including fluconazole-resistant strains.
Regarding the safety of food and human health, there has been increasing interest to replace synthetic preser-vatives with natural, effective and nontoxic compounds.The purpose of the present study was to evaluate the antimicrobial effectiveness of the essential oils of Fennel and clove functioning both as flavoring and additive.

Aromatic spices
Fennel seeds and Clove buds were purchased from Local market in Lahore city (Pakistan) during March, 2010.The spices were identified by The Flora of Pakistan (Nasir and Ali, 1978).The voucher specimen PU.IAGS.HHC.635 and PU.IAGS.HHC.636 was given to sweet fennel seeds (Foeniculum vulgare Mill.) and clove [Syzygium aromaticum (L.)Merrill and Perry], respectively and deposited in the Herbarium of Institute of Agricultural Sciences, IAGS, University of the Punjab (PU) Lahore, Pakistan.The seeds and buds were ground using pestle and mortar and packed in polythene bags and placed in a dried place for further oil extractions.

Essential oil (EO) extraction
For oil extraction, 1 kg ground of each spices material was subjected to hydrodistillation for 4 h to obtain essential oil.The residue was removed by filtration through filter paper.The essential oils were dried over anhydrous sodium sulfate and stored in black vials at 5°C.Essential oils yield was calculated as follows: Weight of EO recovered Yield (%) = × 100 Weight of spices Each essential oil dilution (60 mg/ml) was prepared in dimethylsulphoxide (DMSO), followed by sterilization using a 0.45 μm membrane filter.

Microbial strains
The EOs of clove and fennel was tested against a range of 10 microorganisms, collected from First Fungal Culture Bank of Pakistan, (FCBP), Institute of Mycology and Plant Pathology, University of The Punjab Lahore Pakistan (Table 1).These fungal and bacterial cultures were maintained on Malt Extract Agar (MEA 2%) and nutrient agar (NA) at 4°C respectively and revived on fresh medium for further study.Fungal inoculum was prepared from 7 days old culture in sterilized distilled water with help of hematocyotmeter (10 8 CFU/ml).Each bacterial inoculum was prepared from 24 days old culture (10 5 CFU/mL; 0.5 Mac-Farland).

Agar-well diffusion assay
Standardized inoculum of fungi and bacteria (100 µL) each was spread onto a malt extract Agar (MEA) and nutrient Agar (NA) plates respectively with the help of sterile spreader.The inoculated plates were allowed to dry and sterile cork borer of diameter 8.0 mm was used to bore wells in center of inoculated agar plates.Subsequently, a 60 μL volume of oil of test spices were introduced in wells.Sterile DMSO served as the control.The plates were allowed to stand for 1 h to diffuse and then incubated at 37°C for 24 h for bacteria and for 5 days for fungi.The zone of inhibition was recorded to the nearest size in cm.Percentage inhibition and index of antifungal activity was calculated with the following formulas

Statistical analysis
The antimicrobial activity was determined by measuring the diameter of zone of inhibition which was statistically analyzed by applying Duncan's Multiple Range Test (Steel and Torrie, 1980) using computer software COSTAT.

RESULTS
The yield obtained of clove and fennel seed oils were 17.46 and 0.72 % respectively.Both spice EOs showed considerable inhibitory potential against fungi (39 to 86%) as well as bacteria (24 to 42%) against all the test food borne pathogens (Tables 2 and 3).The results from the well diffusion method indicated that A. alternate (86%) and B. subtilis (42%) were the most sensitive microorganisms tested, showing the largest inhibition zones 7.7 and 3.8 cm respectively.However, fennel seeds overall depicted far greater antimicrobial potential than clove oil where (7.7, 3.8 cm) (5.9, 4.1 cm) (4.5, 3.7 cm) (3.8, 2.2 cm) (3.3, 2.4 cm) (2.3, 2.1 cm) (2.5, 2.4 cm) zone of inhibition were calculated against A. alternata, A. flavus, B. subtilis, P. syringae, Staphylococcus sp. and Aeromicrobium erythreum respectively.In case of fungi fennel oil maximum suppressed the growth of A. alternate (86%); A. fumigatus was found to be least inhibited (39%).Bactericidal activity was maximum in case of B. subtilis (42%) and minimum in E. coli (24%).Clove oil significantly inhibited the food borne pathogens comparable with that of fennel oil in case of A. acculeatus (65, 61%), Aspergillus fumigatus (42, 39%) and E. coli (29, 24%). A. acculeatus was found to be most sensitive with 5.9 cm inhibition zone and E. coli (2.6 cm) fungal and bacteria strain respectively, in case of clove oil.

DISCUSSION
Food borne pathogenic fungi and food spoilage bacteria cause damage to human health reveal drug resistance due to inadequate use of antibiotics.The main advantage of natural sources is that they do not enhance the antibiotic resistance, commonly encountered with the longterm use of synthetic antibiotics (Nenad et al., 2007).Thus, there is a need for the discovery of novel substances from natural sources, including spices.In the present study, the antimicrobial activity of the two spices fennel and clove was evaluated against a panel of food borne pathogens.Fennel oil and clove oil significantly suppressed the microbial growth of all tested five fungal strains with inhibition zone (3.5 to 7.7 cm) and five bacterial strains (2.1 to 3.8 cm) (Tables 2 and 3)..As far as the antifungal results are concerned, fennel oil showed higher inhibition against Alternaria alternate (86, 42%) Fusarium oxysporum (65, 42%) and Aspergillus flavus (51, 41%) as compared to clove oil whereas, clove oil displayed greater pathogenicity in case of A. fumigatus (42, 39%) and Aspergillus acculeatus (65, 61%) than fennel oil.Other workers also reported fennel oil as week antifungal for Aspergillus species than clove oil (Sunita and Rai, 2008).Overall fennel oil displayed highest antifungal potential against A. alternate while clove oil inhibited the A. acculeatus (Table 2).
The antifungal activity of the clove oil and its main component, eugenol (85.3%) were investigated by Eugénia et al. (2009) against Candida, Aspergillus and dermatophyte clinical and American Type Culture Collection strains.The EO and eugenol showed inhibitory activity against all the tested strains.Mainly, phenolic components of essential oils are considered responsible for the antimicrobial activity, followed by aldehydes, ketones, and alcohols (Azzouz and Bullerman, 1982: Shelef et al., 1983: Akgul, 1989).It is difficult to attribute the activity of natural essential oils which are complex mixtures to a particular constituent, it is reasonable to assume that the activity of clove oil can be related to the presence of a high concentration (85.3%) of eugenol.Different modes of action are involved in the antimicrobial activity of EOs (Burt, 2004).The activity may, in part, be due to their hydrophobicity, responsible for their partition into the lipid bilayer of the cell membrane, leading to permeability alteration and a consequent leakage of cell contents.As typical lipophiles, essential oils can travel through the cell wall and cytoplasmic membrane, disrupt the structure of the different layers of polysaccharides, fatty acids and phospholipids, and permeabilize them (Burt, 2004).Many reports Bakkali et al., 2008;Pasqua et al., 2007;(Hammer et al., 2004) suggest that antimicrobial mechanism is because of membrane damage.Bacteriostatic action involving permeabilization of the membranes is associated with ion loss, loss of proton, depletion of ATP and reduction of membrane potential.Clove oil and eugenol also considerably reduce the quantity of ergosterol which is a specific fungal cell membrane component responsible for maintaining cell function and integrity (Rodriguez et al., 1985).Germ tube formation by microbes albicans was completely or almost completely inhibited by clove oil and eugenol concentrations below the MIC values (Eugénia et al., 2009).

Conclusion
Fennel (F.vulgare) and cloves (S. aromaticum) were found to have important antimicrobial activity against the food borne pathogenic fungi and food spoilage bacteria.In this regard the use of culinary spices fennel seeds and clove buds as natural preservatives in food products are valuable not only for increasing shelf life of foodstuffs but it could be a future target for replacing chemical additives and synthetic antimicrobial agents.

Table 1 .
List of fungal and bacterial species.

Table 2 .
Effect of Fennel (F.vulgare)and cloves (S. aromaticum) essential oils on food born pathogenic fungal growth.