Growth inhibition of the stored fish (Ethmalosa fimbriata) fungus Aspergillus flavus, exposed to extracted essential oils from Callistemon citrinus and Ocimum canum

The aim of this study was to evaluate the antifungal effects of essential oils from Callistemon citrinus and Ocimum canum against Aspergillus flavus. Major components in the oil of C. citrinus were 1,8cineole (60.6%), α-pinene (18.5%), limonene (5.0%) and α-terpineol (5.0%). The oil of O. canum was mainly composed of 1.8-cineole (20.8%), linalol (14.3%), eugenol (11.9%), terpinen-4-ol (7.4%) and germacrene D (4.9%). Inhibition of the mycelia growth of A flavus increased significantly (p < 0.05) with the essential oils concentrations. Positives correlations were observed between inhibition percentages and the concentration of C. citrinus (p < 0.001; r = 0.873) and O. canum oils (p< 0.001; r = 0.768). O. canum oil was fungicide at 325 ppm while C. citrinus was fungistatic at all the tested concentrations with the highest inhibition percentage means of 89.74 %. Antifungal activity of the essential oil of O. canum was significantly (p < 0.001) higher than that of C. citrinus. Antioxidant activity of O. canum (49.3 mol/l) was significantly (p < 0.001) higher than that from C. citrinus (5.0 mol/l). These observations suggest the possible exploitation of the oils from O.canum and C.citrinus as potential approach for smoked Ethmalosa fimbriata preservation against A. flavus.


INTRODUCTION
Ethmalosa fimbriata, a fish commonly known as "Bonga", belongs to the family Clupeidae.In Cameroon, the farming of this fish is a common occupation of people living in the coastal areas and along major river banks.It is one of the best sources of proteins, vitamins, essential fatty acids and minerals.It contains essential nutrients required for supplementing both infant and adult diets (Stansby, 1987;Abdullahi et al., 2001).Various brands of oils are also extracted from the flesh and the fluids of this fish.Due to its high susceptibility to microbial spoilage, autolysis, oxidation and hydrolysis of fats, fish is a highly perishable product (Frazier and Westhoff, 1978).Care is therefore required in handling as well as its preservation for food.The commonly used post-harvest method of preserving E. fimbrata in Cameroon is smoke drying.This process can increases the shelf life of fish by eliminating microorganisms or prevent their growth (Falioye et al., 2002;Ali et al., 2011).However, despite this treatment, smoke dried fish is frequently altered by many fungi such as members of the genus Aspergillus especially when stored in an unsuitable environment (Falioye et al., 2002;Gram, 2010).Our preliminary survey in some local markets in Cameroon showed that Aspergillus flavus was the most commonly occurring fungus on stored E. fimbriata.This fungus is well known to decrease the commercial and nutritive value of the fish and produce a number of toxic metabolites including aflatoxin (Edema and Agbon, 2010).Additional control methods of this spoiling mold are essential.In this respect, natural products could be developed for reducing losses during storage.Selected plants and their essential oils have been evaluated as natural sources of compounds for food preservatives due to their antimicrobial and antioxidant effects (Tasadjieu et al., 2009;Prakash et al., 2015).Their main components show antioxidant activities and antimicrobial activity against a wide range of microorganisms including filamentous fungi (Hyldgaard et al., 2012;Sameza et al., 2014).Variable results have been observed depending on the origin of biological substances; testing conditions and target microorganisms (Delaquis et al., 2002;Hyldgaard et al., 2012).Cameroon flora is very rich with aromatic plants which have various biological activities (Amvam et al., 1998).Among these, Callistemon citrinus and Ocimum canum are used as spice, condiment, ornamental plants and African medicine.Previous works showed that, essential oils extracted from Ocimum sp and Callistemon sp. had many biological activies including antifungal and antioxidant.These activities were related to the main component 1,8 cineole (Kumar et al., 2011;Shukla et al., 2012;Alves Silva et al., 2013).In certain households in Cameroon, leaves of these plants are used to protect stored food products.The aim of this study was to evaluate the antioxidant potential and antifungal activities of essential oils from C. citrinus and O. canum against Aspergillus flavus, one of the most occurring spoiling fungi of stored smoke dried E. fimbriata in Cameroon.

Plant material and essential oil extraction
C. citrinus and O. canum plants were harvested in March 2011 in Douala, Cameroon and dried during 3 days at room temperature (28 ± 2°C).Herbarium/plants were identified at the National Herbarium of Cameroon.Leaves of C. citrinus and O. canum were steam-distilled for 4 h using a Clevenger apparatus.Each oil recovered was dried over anhydrous Na2SO4, stored in an ambercolored flask and kept at 4°C until use.

Essential oils analysis
Essential oils obtained were analyzed by Gas chromatography (GC).It was performed on a Varian-HP 5890 with flame ionization detector fitted with a fused silica capillary column (30 m x 0.25 mm coated with DB-1, film thickness 0.25 mm).The temperature program was set to 60°C-246°C at 3°C/min, with injector temperature set at 200°C, detector temperature set at 200°C carrier with N2 gas set to 1 ml/min.The linear retention indices of the components were determined relatively to the retention times of series of n-alkanes and the percentage composition were obtained from electronic integration measurements without taking into account relative response factors.Gas chromatography coupled with mass spectrometry analyses were performed using a Hewlett-Packard GC 5890 equipped with an HP-1 (cross-linked methyl siloxane) fused column (30 m x 0.25 mm, film thickness 0.25 μm) and interfaced with a quadrupole detector (model 5970); temperature programmed at 70°-200°C (10°C/min); injector temperature, 220°C; temperature of connection parts, 180°C; carrier gas, helium at a flow rate of 0.6 ml/min; injection type, split, 1:10 (1μl of a 10:100 pentane solution); ionization voltage, 70 eV; electron multiplier, 1400 eV; mass range, 35-300; scan rate, 2.96 scan/seconde.The identification of the constituents was assigned based on comparison of their retention indices and their mass spectra with those published by Adams (2007).

Aspergillus flavus
The Aspergillus flavus used in this study was isolated from fresh smoked fish samples of E. fimbriata purchased from a local market (Douala Cameroon).Fish tissue segments (2-4 mm) from flesh and gills were cut using a sterile scalpel and seeded aseptically on A. flavus selective medium (Griffin and Garren, 1974).After 3-4 days of incubation at 30°C, the mycelia emerging from the tissues were transferred to fresh medium.Colonies of A. flavus developing from tissue segments were transferred to Czapez-Dox Agar for confirmation of identity according to the criteria of Raper and Fennell (1965).The cultures were stored at 4°C on Sabouraud Dextrose Agar (SDA)-chloranphenicol slants and sub-cultured once a month.

Antifungal assay
The agar incorporation method (Lahlou, 2004) was used to evaluate the antifungal activity of the essential oils.The test was carried out in 90 mm Petri dishes containing SDA-chloramphenicol medium.The oils were first diluted with Di Methyl Sulphur Oxide (DMSO) (ratio1: 9).These essential oils were added aseptically into the medium at an appropriate volume to produce various concentrations ranging from 100 to 800 ppm.SDA-chloramphenicol medium supplemented only with DMSO was used as negative control.After solidification, the media were inoculated with 5 mm discs obtained from the edge of 3-days old mycelia culture of A. flavus.Each treatment consisted of triplicate plates incubated at 30°C in the dark.Mycelia growth was monitored by measuring the growth diameter following two perpendicular lines going through the centre of the dish.These measurements were made daily for 7 days.The inhibition percentage of mycelia growth was calculated by comparing them with those in the blank dish without essential oil using the formula below: %I = (Dc -Dt) / Dc, where Dc is the diameter of microbial colony in the control and Dt the diameter of the colony in the treated plate.The fungicidal or fungistatic activity was determined by transferring the discs from the Petri dishes with no apparent growth into non-supplemented medium.

Antioxidant activity
The antioxidant potential of the essential oils was determined using the FRAP (Ferric Reducing Antioxidant Power) method adapted from Benzie and Strain (1996).FRAP reagent contained 4 ml of 10 mM TPTZ (tripyridyltriazine) in 40 mM HCl plus 4 ml of 10 mM FeCl3,6H2O and 40 ml of 300 mM acetate buffer (pH 3.6).Tenμl of essential oil was diluted in methanol at different concentrations then 75 μl of essential oil-methanol were mixed with 2 ml of FRAP reagent daily prepared.The absorbance was recorded after 12 min incubation at the room temperature with a spectrophotometer UV V-1100 at 593 ηm.FRAP values were obtained by comparing with standard curves created by ascorbic acid solutions concentrations ranging from 50-800 μM.The Equivalent Concentration (EC) was defined as the concentration of antioxidant having a ferric-TPTZ reducing ability equivalent to that of 1 mM FeCl3, 6H2O.EC was calculated as the concentration of antioxidant giving an increase in the FRAP assay equivalent to the theoretical absorbance value of a 1 mM of ascorbic acid solution, determined by using the corresponding regression equation.

Statistical analysis
Data were analyzed using Sigmastat version 2.03 (Synstat Sofware Inc).Results are presented in term of means ± standard deviation.Multiple comparisons of mean values were set up using one-way parametric ANOVA when the normality and equal variance test passed.When these conditions were not matched, we used the non-parametric Krustcall-Wallis test.

Effects of essential oils on fungal growth
Results showed that, the inhibition of the mycelia growth of the fungus increased significantly (p < 0.05) with the essential oils concentrations (Table 2).Total inhibition occurred at 325 ppm with the essential oil from O. canum.For C. citrinus the highest inhibition percentage means was 89.74%.O. canum oil was fungicide at 325 ppm while C. citrinus was fungistatic at all the tested concentrations.Results showed that from 200 ppm, the activity of the essential oil of O. canum was significantly (p < 0.001) higher than that of C. citrinus.There was a positive correlation between inhibition growth of A. flavus and the concentration of C. citrinus oil (p < 0.001; r = 0.873) and O. canum oil (p < 0.001; r = 0.768).Essentials oils from various sources exhibit broad-spectrum antimicrobial activity and their biological properties have been related to their chemical composition (Nguefack et al., 2012;Djenane et al., 2013).Indeed, compounds such as 1.8-cineole, pinene, limonene, terpineol and eugenol are present in both essential oils analyzed and have been shown to exert various biological activities including antifungal (Isman, 2000;Dayan et al., 2009).It has been demonstrated that essential oils inhibit postharvest pathogens mainly due to their direct effect on the mycelia growth by affecting the cellular metabolism of the pathogen (Serrano et al., 2005;Regnier et al., 2010).The hydrophobicity of the oils and their components allows them to partition in the lipid layer of the fungal cell membranes and result in disruption in membrane structure and cell membrane integrity (Beckman 2000).In addition, the inhibiting activities of these essential oils may not only be attributable to their major components but, to a synergistic effect of individual minor and/or major compounds (Nguefack et al., 2012  Bautista-Banos, 2014).

Antioxidant activities of the essential oils
Mean values of equivalent concentration of antioxidant activities showed that the antioxidant potential of O. canum and C. citrinus essential oils were 49.3 and 5.0 mol/l respectively ( be related to the presence of compounds such as eugenol, thymol and 1-8 cineole known for their antioxidant properties (Mishra et al., 2013).We also identified these compounds in O. canum and C. citinus oil in noticeable amount (Table 1).The essential oils from O. canum and C. citrinus exhibited antifungal activity since they were able to kill or inhibit the mycelia growth of A. flavus.They also had antioxidant activities.Through these properties, they could be used as an approach solution to preserve stored smoked E. fimbriata and other foodstuff.Nevertheless, detailed studies are required to study the preservative effect of these oils directly on the dried fish.

Figure 1 .
Figure 1.Chromatogram of chemical analysis of C. Citrinus essential oil.

Table 3
Prakash et al. (2011)the activity of the oil from O. canum is significantly (p < 0.001) higher than that from C. citrinus.Previous studies ofPrakash et al. (2011)reported that the essential oils of Ocimum sp. had antioxidant activities.Antioxidants retard oxidation and are sometimes added to meat and poultry products to prevent or slow oxidative degradation of fats.Antioxidant agents are effective due to different mechanisms such as free radical scavenging, chelating of pro-oxidant metal ions or quenching singlet-oxygen formation(Lopez-Luzt et al., 2008).These activities could Figure 2. Chromatogram of chemical analysis of O. canum essential oil.

Table 1 .
Yields and chemical composition of essential oils of Callistemon citrinus and Ocimum canum from Cameroon.

Table 2 .
Range and mean values of the percentages of growth inhibition of A. flavus at different concentrations of essential oils.

Table 3 .
Variation of equivalent concentration (mol/l) of antioxidant of Callistemon citrinus and Ocimum canum essential oils.