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The potential effect of clove, cinnamon, mustard, peppermint, eucalyptus, citronella, camphor, rose, lemon and lemon grass essential oils against fungi identified from areca palm leaves and palmyra palm leaf sheath were investigated. The fungi were identified by 18s rRNA sequencing method. An agar dilution method was employed to determine the minimum inhibitory concentration (MIC) of essential oils. Zone inhibition tests and the inhibitory effect of the leaf and sheath dip treated and vapour treated with essential oils against those fungi were examined. With an MIC of 0.02 µl ml-1Musturd essential oil had the strongest inhibitory effect. The efficacy of mustard oil vapour on the fungal inhibition was relatively higher when compared to the liquid phase. It is also comparatively more potential than chemical fungicides.
In southeast Asia and India, areca palm (Areca catechu) leaf sheath dropped naturally and Palmyra palm (Borassus flabellifer) leaf have been traditionally used as an environmentally friendly food serving and packaging material for centuries1. Now a days with modern technology, this natural and rigid material is compressed into different shapes and used to serve and pack food. Over the past 20 years, the use of bio-based packaging materials to prolong the shelf-life and improve the quality of fresh food products has been receiving increased attention2. Palmyra palm leaves are employed in making utilitarian, aesthetic, artistic, creative, culturally attached, decorative, functional, traditional, religiously and socially symbolic and significant handicraft products too.
The presence and growth of fungi in the above materials may cause food spoilage and also results in a reduction in quality and quantity3. Aflatoxins produced by Aspergillus species are known to be potent hepatocarcinogens in animals and humans. Therefore, the presence of toxigenic fungi and mycotoxins in foods packing and serving materials and handicraft products present a potential hazard to human and animal health.
Some chemical compounds are used as fungicides and they are commonly made up of 90% sulfur and are very toxic. Copper sulfate (bluestone) is one of the commonly used fungicides among copper forms. Bordeaux mixture a combination of copper sulfate and lime is an oldest fungicide that successfully used for more than 150 years on ornamentals, fruits and vegetables. Phosphorous acid another effective fungicide that acts over the fungi either by inhibiting a particular process or by inducing a defence response in the agent to show inhibiting activity. Biphenyl is also a fungicide that has the ability to inhibit the sporulation process of fungi.
As the chemical preservatives are considered responsible for many carcinogenic and teratogenic attributes as well as residual toxicity, consumers tend to be mistrustful of chemical additives and thus there is demand for natural and socially more acceptable preservatives4. Considerable interest has developed on the use of essential oils to effectively retard growth and mycotoxin production. A renewed interest in natural preservation appears to be stimulated by present food safety concerns, growing problems with microbial resistance, and a rise in production of minimally processed food, together with green image policies of food industries. Numerous studies have documented the antifungal5, 6 effects of plant essential oils. Natural products may regulate the cellular effects of aflatoxins and evidence suggests that aromatic organic compounds of spices can control the production of aflatoxins7.
The use of the polymerase chain reaction (PCR) offers great advantage compared with conventional microbiological testing8, 9, 10. Fungal primers specific for the conserved sequence of 18s rRNA gene common to all fungi have been used to detect fungal specimens11.
The objective of this work is to evaluate the antifungal activity of ten essential oils namely clove oil, cinnamon oil, mustard oil, peppermint oil, eucalyptus oil, citronella oil, camphor oil, rose oil, lemon oil and lemon grass oil on fungi commonly found on the areca palm leaf sheath and palmyra palm leaf surface and compare them with the effect of chemical fungicides.
2. Materials and methods
2.1 Essential oil and leaf sheath
Food grade essential oils (clove oil, cinnamon oil, mustard oil, peppermint oil, eucalyptus oil, citronella oil, camphor oil, rose oil, lemon oil, and lemongrass oil) derived from steam distillation were derived from Eastern Distributors, Coimbatore. The Areca palm leaf sheath and palmyra leaves used for making handicraft products were obtained from Natural Fibre Handicraft centre, Punnayadi village, Kanyakumari district, Tamil Nadu, India.
Three fungal strains were separated from areca palm leaf sheath and Palmyra Palm leaf surfaces using Agar Plate technique in Potato Dextrose Agar (PDA) medium and morphological identification was carried out. The isolated fungi were the major causes of deterioration the leaf sheath, leaf and intermediate moisture food wrapped in them.
- Preparation of Inocula
Spores of the test fungi were obtained from mycelia grown on Potato Dextrose Agar (PDA) at 30° C for 14 days, and were collected by flooding the surface of the plates with ~5ml sterile saline solution (NaCl,8.5 g/l water) containing Tween 80 (0.1 % v/v). After the spores were counted using a haemocytometer, the suspension was standardized to concentrations of 107 spores/ml by dilution made using sterile water before use. The viability of all strains were checked using quantitative colony counts at 107 CFU/ml.
- Fungal identification
DNA was extracted from the fungal pellet and purified using PureFast® Fungal Genomic DNA purification kit. Fungal ITS forward and reverse primers were used in the PCR reaction Each 50 ml PCR reaction consisted of 2U of Taq DNA polymerase, 10X Taq reaction buffer, 2mM MgCl2, 1μl of 10mM dNTPs mix and PCR additives. PCR reactions were run using the following parameters: (1) 94º C for 3 min, (2) 30cycles of 94ºC for 1 min, 58º C for 1 min, and 72 º C for 1min, and (3) 72º C for 5 min. Clean PCR products were sequenced using ITS primers, assembled and edited. Nucleotide sequences were compared to those in the Gene Bank Database with the Basic Local Alignment Search Tool (BLAST) algorithm to identify known closely related sequences. The 18s rDNA sequences were aligned and phylogenic tree was constructed by neighbour joining method.
- Inhibition of molds by Essential oils
Determination of minimal inhibitory concentration (MIC) of the essential oils on each test fungus was performed by the agar dilution method. The essential oil was added aseptically to sterile PDA to make an agar solution with essential oil the concentration used was 50 µl. The resulting agar solutions were immediately poured into petriplate dishes after vortex. The plates were spot inoculated with 100µl (107 spore/ml) of each fungus. The vegetable oil (olive oil) was used as a control. The inoculated plates were incubated at 25°c for 3 days. At the end of the incubation period, the plates were evaluated for the presence or absence of microbial growth. The MIC value was determined as the lowest concentration of the essential oil at which absence of growth was recorded12.
- Zone of Inhibition of leaf sheath Dip-Treated with Essential oils
The disc diffusion method was employed to determine a zone of inhibition (ZI) of areca palm leaf sheath and palmyra palm leaf discs treated with essential oils. One hundred microlitres of a suspension containing 107 (CFU)/ml of individual fungi was spread on the PDA plate. Sets of 3 random triplicate specimens of the leaf sheath disc of 6mm in diameter were dip-treated according to ASTM test method D4445-91 (American Society Testing Materials 1998) for 15s with each essential oil at its MIC obtained from inhibition of fungi tests. Vegetable oil (olive oil) was used as a control. Different dilutions of essential oils were made with methanol. The disc was placed in sterile beaker that was covered tightly with a plastic sheet to prevent drying of the essential oil and stored in an aseptic cabinet for 24h. This allowed the draining of excess oil and time for the essential oil to penetrate into the leaf sheath before inoculation of the PDA. The inoculated plates were then incubated at 25°c for 3 days. The antifungal activity of the treated leaf sheath was evaluated by measuring the zone of inhibition (the width in mm, of the clear zone outside the disc) against the test organisms12.
2.6 Mold Test on Areca palm leaf sheath and palmyra palm leaf using Mustard oil
Sets of 3 triplicate specimens of all the 3 leaf sheath plate of 10mm in width and 70mm in length were dip-treated with mustard oil over the range of 1-10 µg/ml. Vegetable oil (olive oil) was used as a control. Essential oils and vegetable oil were diluted with methanol. Dip-treated specimens were held in a closed container overnight at room temperature before inoculation with spores of the test fungus.
The dip-treated specimens were inoculated with 1ml of each spore inoculums (107 spores/ml) and were incubated at 25°c with 100%RH chamber for 45 days. The specimens were then individually rated for fungus growth cover. The specimens were then individually rated for fungus growth cover on a 0-5 scale (0, no growth; 1, 20 % cover; 2, 40 % cover; 3, 60 % cover; 4, 80 % cover; 5, 100 % cover) according to ASTM test method D4445-91 (American Society for Testing and Materials 13). The percentage area of stain and fungus (based on a control) for each essential oil concentration was calculated as (A/B) x100, where A is the total score for each fungus at each concentration of essential oil, and B is the total score for each fungus in the controls12.
2.7 Zone of inhibition using vapour phase of essential oil
For investigation of the effect of essential oil vapour 1ml of each fungal spore suspension of 107 spores/ml was inoculated on a potato dextrose agar and incubated at 25° C for 12 hrs for getting the exponential growth phase of the fungi. Then 20µl of each essential oil was put on the sterile discs (6mm) in upper lid of the inverted petriplates where it gets converted to essential oil vapour. The petriplate was sealed with parafilm and further incubated for another 3 days at 25° C. Vegetable oil was used as the control. This allows the essential oil vapours to act against the fungi. Zone of inhibition was observed.
2.8 Inhibition of fungi by Fungicides
Determination of minimal inhibitory concentration (MIC) of the fungicides on each fungi was performed by the agar dilution method. The fungicides were added aseptically to sterile PDA. The resulting agar solution was immediately poured into petriplate dishes after vortex. The plates were inoculated with 100µl (107 spores/ml) of each spore inoculum. The plate that was not inoculated with the test fungi was used as a control. The inoculated plates were incubated at 25° C for 3 days. At the end of the incubation period, the plates were evaluated for the presence or absence of fungus growth. The MIC value was determined as the lowest concentration of the fungicides at which absence of growth of the fungi were observed.
2.9 Zone of inhibition of fungicides against fungi
Spore suspension of each fungus at the concentration of 107 spores/ml was inoculated on PDA plates by spread plate technique. Discs dipped in selected fungicides (CuSO4, OPA and Biphenyl) were prepared and placed onto the inoculated PDA plates, and incubated at 25°C for 3 days. After incubation zone of inhibition was measured and tabulated.
3. Results and Discussion
3.1 Fungal identification
Based on the results of the 18s rRNA sequence analyses (BLAST and phylogenies) and morphological comparisons of the fungal isolates, the sequences of fungus 1 showed maximum percentage of similarity with the sequences of the species Aspergillus niger, while sequence of fungus 2 and 3 had higher percentage of similarity to the sequences of Aspergillus flavus and Aspergillus oryzae respectively (Fig. 1).
3.2. Inhibition of fungi by Essential oils
In the agar dilution method (Table 1) all 10 essential oils exhibited fungistatic effect on the test fungi. Vegetable oil used at 10-500 mg ml_1 as a control showed no inhibition on the test fungi. With an MIC of 0.02 µl ml-1 mustard oil was the most potent inhibitor. Eucalyptus oil and lemon grass oil had the least MIC of 50 µl ml-1 and 30 µl ml-1 respectively. Cinnamon oil, clove oil and lemon oil had MIC of 2 µl ml-1, 4 µl ml-1 and 8 µl ml-1 respectively. Mustard oil, eucalyptus oil, lemon grass oil, cinnamon oil, clove oil and lemon oil had MICs similar in all the three test fungi. Citronella oil camphor oil, rose oil and peppermint oil had different MIC for each test fungi. Citronella oil, camphor oil and peppermint oil had MIC of 6 µl ml-1 for Aspergillus flavus and rose oil had MIC of 8 µl ml-1. Rose oil and peppermint oil had similar MIC for Aspergillus oryzae (8 µl ml-1) , citronella oil and camphor oil had 2 µl ml-1 and 12 µl ml-1 MIC respectively. MIC against Aspergillus niger was 12 µl ml-1 , 10 µl ml-1 , 4 µl ml-1 and 8 µl ml-1 for citronella oil, camphor oil, rose oil and peppermint oil respectively. Strong antifungal activity of these oils has been reported by many authors. 14, 15, 16
3.3 Inhibition of fungi by fungicides
Copper sulphate inhibited all three fungi with an MIC of 600 µg ml-1 in PDA. Orthophosphoric acid was also a strong inhibitor with an MIC of 2 µl ml-1,12 µl ml-1and 14 µl ml-1 against Aspergillus niger, Aspergillus oryzae and Aspergillus flavus respectively. But biphenyl was a weak fungicide, when compared with other fungicides with an MIC of 260 mg ml-1 against all three fungi (Table 2).
3.4. Zone of Inhibition of leaf sheath dipped in Essential oils
Zone of inhibition (ZI) of the essential oils at their MICs obtained via. the disc diffusion method confirmed that all the essential oils at their MICs inhibited the three fungi. Even though all essential oils used exhibited ZI, relatively large inhibition was found in mustard oil (Table 3). It is confirmed that mustard oil was the strongest inhibitor compared to all other essential oils. Cinnamon oil had the next highest zone of inhibition in all the three fungi. Clove oil also acted as a potent inhibitor. Aspergillus oryzae was more sensitive to mustard oil, lemon oil, clove oil, lemongrass oil and cinnamon oil. Aspergillus niger and Aspergillus flavus was highly sensitive to mustard oil and cinnamon oil. Aspergillus flavus was sensitive to lemon oil too. Peppermint oil showed medium inhibition in Aspergillus oryzae and was a weak inhibitor in Aspergillus niger and Aspergillus flavus. Rose oil acted as a medium inhibitor in all the three fungi. Aspergillus oryzae was highly sensitive to lemongrass oil while Aspergillus flavus and Aspergillus niger were moderately sensitive.
All the three fungi were resistant to citronella oil. Aspergillus niger and Aspergillus flavus were resistant to eucalyptus oil. In the case of camphor oil inhibitory effect was observed on Aspergillus oryzae and Aspergillus flavus when palmyra leaf was employed while the same oil inhibited the growth of all the three fungi while areca palm leaf sheath was used. Aspergillus niger was resistant to camphor oil, citronella oil and eucalyptus oil. Citronella oil had no inhibitory effect except Aspergillus flavus (areca palm leaf sheath) in which it showed a very mild activity. While the effect of eucalyptus oil was observed Aspergillus oryzae and Aspergillus flavus were sensitive when areca palm leaf sheath was employed while the same oil inhibited the growth of all the three fungi while palmyra leaf was used.
The antimicrobial agents in essential oils may either pass into the agar medium by diffusion or be released through evaporation in the headspace within the plate. This complex mechanism warrants further investigation.
Table 3: Zone of inhibition of leaf sheath dipped in essential oils.
3.5 Zone of inhibition of fungicides against fungi
Aspergillus niger Aspergillus oryzae and Aspergillus flavus were resistance to biphenyl. CuSO4 and orthophosphoric acid were potential fungicides. Aspergillus niger was sensitive to CuSO4 and orthophosphoric acid with a zone of measurement 15.33±0.57 mm and 10±1 mm respectively. In the case of Aspergillus oryzae, the zone of inhibition was 15.33±0.57 mm and 34.66±8.33 mm in CuSO4 and OPA respectively. Whereas, Aspergillus flavus was moderately sensitive with a zone of 23.66±1.52 mm for CuSO4 and 15.33± 0.57 mm for orthophosphoric acid (Table 4).
3.6. Fungi growth on the leaf sheath
Essential-oil-coating on the surface is required for effective prevention of fungi on the surface of the areca palm leaf sheath and Palmyra palm leaf, and so dip-treatment was employed for fungal testing 17. Growth of fungi on leaf sheaths dip-treated with mustard oil and vegetable oil was examined after incubation for 45 days. The average rating for mustard oil showed no growth, while the vegetable oil (control) showed growth in the three fungi within few days. Thus the essential oil showed fungi resistance for up to 45 days.
It should be noted that although oil treatment might have some effects on exclusion of moisture from the test specimens, fungal spores were able to germinate and grow on the control specimens dip-treated with vegetable oil. As a consequence, mechanisms other than moisture exclusion that are caused by components in essential oils should play a key role in inhibition of fungal spore germination.
3.7. Zone of Inhibition using vapour phase of Essential oil
The results tabulated in Table 5 reveals the growth of fungi on the PDA plates showed a zone of inhibition (ZI) at 25°c for 3 days. More effective results were observed against Aspergillus oryzae in mustard oil, clove oil, peppermint oil and lemon oil. Rose oil had no effect on Aspergillus oryzae. Peppermint oil and lemon oil had very low effect on Aspergillus niger, while the mustard oil had the highest effect. Mustard oil and peppermint oil were very effective against Aspergillus flavus than other oils. Vapour phase of mustard oil was efficient against all the three fungal agents. Vegetable oil (control) showed no inhibition. The use of essential oils in vapour phase was reported earlier by Amit and Anushree 18.
Clove oil, cinnamon oil, mustard oil, peppermint oil, eucalyptus oil, citronella oil, camphor oil, rose oil, lemon oil and lemon grass oil were inhibitory to three fungi previously isolated from areca palm leaf sheaths and palmyra leaf. They were the fungal agents mainly responsible for the deterioration of the leaf products and spoilage of food packed in them. These essential oils at their MICs were capable of inhibiting spore germination and growth of these fungi on leaf sheath and leaf discs for at least 45 days in storage at 25 ËšC and 100 % RH. Areca palm leaf sheath and palmyra palm leaf treated with essential oils can therefore be considered to have good potential as an antifungal bio-based material for use in food packaging and other handicraft products. Among the ten essential oils employed for screening, mustard oil was more potential than others. However, consumer sensory tests will be needed to determine concentrations of essential oils suitable for specific products.
The authors gratefully acknowledge the support rentered by Sri Paramakalyani College, Alwarkurichi and Department of Science and Technology, New Delhi.