Biological control of spoilage and pathogens moulds in culture medium and Beninese traditional cheese wagashi by Syzygium aromaticum essential oil

The investigation highlighted the antifungal effectiveness in culture medium and traditional cheese wagashi foodsystem of Syzygium aromaticum ( Eugenia caryophyllata ) essential oil (EO) against spoilage and toxinogenic moulds isolated from wagashi produced in Benin. The chemical composition of the EO obtained by hydrodistillation, characterized through GC-FID and GC-MS analysis, revealed eugenol (75.2%) and trans- -caryophyllene (12.0%) as major components. The evaluation of in vitro antifungal activity of this oil showed a significant fungistatic activity against Aspergillus ( flavus , tamarii , niger , aculeatus , ustus , terreus ), Penicillium ( brevicompactum , citrinum , griseofulvum ), Fusarium ( poae , verticillioides ) and Scopulariopsis brevicaulis with MIC ranged from 200 to 600 mg/L due probably to its richness in eugenol. Moreover, this EO had fungicidal activity against Aspergillus terreus and S. brevicaulis which were the most sensitive respectively at 600 and 400 mg/L. The assessment of antifungal activity of the oil studied in wagashi foodsystem against the less sensible isolates at in vitro assay revealed high sporale reduction rate (55% at least at 1000 mg/L) on all species investigated above all on Penicillium citrinum and Aspergillus aculeatus , two harmful mycotoxins producers in cheese. Results obtained indicate the possibility of exploiting S. aromaticum EO to preserve wagashi against moulds contamination and probably mycotoxins inhibition during wagashi storage


INTRODUCTION
Cheese is highly nutritious food with many diverse flavor and texture and it can be used as a snack or as a part of dish or prepackaged conveniences food (Elkhider et al., 2011).Soft unripened traditional cheese locally called wagashi is an indigenous cheese produced in Benin.Due to its proteins content, it could efficaciously contribute to the resolution of nutritional problems due to the deficiency of proteins (Kèkè et al., 2008;Sessou et al., 2012a, b).However, this foodstuff is produced and preserved using rudimentary methods under unsanitary conditions which may lead to its contamination by spoilage and pathogenic microorganisms especially fungi (Aissi et al., 2009;Sessou et al., 2012a, b, c, d, e).The fungal growth in this product may result in several kinds of cheese spoilage: off-flavours, toxins, discolouration, mycolytic enzymes, rotting and formation of pathogenic or allergenic propagules.The deterioration of sensorial properties is often due to the production of exoenzymes during growth.Filamentous fungi can produce a vast number of enzymes: lipases, proteases and carbohydrase.Once inside the food, these enzymes may continue their activities independent of the destruction or removal of the mycelium (Filtenborg et al., 1996, Angelini et al., 2006).The production of mycotoxins, in particular, has a major negative impact of fungal growth in cheese (Nasser, 2001;ICSMF, 2005).Thus, a better control to prevent contamination and spoilage during the production, sale and distribution and to extend the shelf life of wagashi is necessary to avoid its contamination by mycoflora and minimize public health hazards.Synthetic chemicals could be used to control these pathogens.However, there is a strong debate about the safety aspects of these chemical preservatives since they are considered responsible for many carcinogenic and teratogenic attributes as well as residual toxicity (Hsouna et al., 2011, Barkat andBouguerra, 2012).Concurrently, modern society is looking for natural products that have less impact on the environment and that contain less synthetic antimicrobial food additives.In the meantime, a lower food salt content is also being promoted by the World Health Organization, in an attempt to reduce the incidence of cardiovascular diseases (WHO, 2002;Angelini et al., 2006).For these reasons, alternative methods to control cheese-borne fungi of wagashi and consequently to improve the safety of the product are needed to be performed (Goni et al., 2009;Lv et al., 2011).Essential oils (EO's) obtained from many plants have recently gained a great popularity and scientific interest.Attention is being paid to these compounds as a new alternative to prevent the proliferation of microorganisms and protect food from oxidation (Hsouna et al., 2011;Varona et al., 2013).They have been recognized as bioactive components with antimicrobial activity and classified as generally recognized as safe substances (ESO GRAS -182.20) by the Food and Drug Administration (FDA, 2005).Therefore they could be used to prevent growth of many pathogenic and spoilage microorganisms in foods (Velázquez-Nuñez et al., 2013).Several studies have reported results on their preservative action (Nielsen and Rios, 2000;Burt, 2004;Sessou et al., 2012g).Essential oil obtained from spice Syzygium aromaticum also called Eugenia caryophyllata is effective against many strains (Lopez et al., 2005;Pawar and Thaker, 2006;Pinto et al., 2009).Based on our knowledge, its use as cheese preservative has been few studied.The efficacy essential oil on fungal isolates from wagashi must be verified in order to measure its potential biopreservation for the valorization of this product.The objective of this study was to investigate antifungal activity of S. aromaticum flowers buds oil against spoilage and toxinogenic moulds isolated from wagashi in culture medium and in this food stuff for its potential use as preservative.

Plants material and extraction of the essential oil
The dried flowers buds of clove (Eugenia caryophyllata) were collected at Malanville in North of Republic of Benin at November 2011 and were identified by Doctor YEDOMOHAN of National Herbarium of Benin.They were hydrodistilled for about 3 h, using a Clevenger apparatus.Oil recovered in a dark sterile glass was dried over anhydrous sodium sulfate and stored at +4°C until it was used (Yehouenou et al., 2012a, b).

Chemical analysis of S. aromaticum essential oil
Quantitative and qualitative analyses of the essential oil of S. aromaticum were carried out by gas chromatography/flame ionization detection (GC/FID) and gas chromatography/mass spectrometry (GC/MS).GC/FID analyses were performed using a Varian CP-3380 GC equipped with a DB5 (100% dimethylpolysiloxane) fitted with a fused silica capillary column (30 m x 0.25 mm, film thickness 0.25 µm) and Supelcowax 10 (polyethylene glycol) fused capillary column (30 m x 0.25 mm, film thickness 0.25 µm); temperature program 50°-200°C at 5°C/min, injector temperature 220°C, detector temperature 250°C, carrier gas N2 at a flow rate of 0.5 mL.min-1.Diluted samples (10/100, v/v, in methylene chloride) of 2.0 µL were injected manually in a split mode (1/100).The percentage compositions were obtained from electronic integration measurements without taking into account relative response factors.The linear retention indices of the components were determined relatively to the retention times of a series of n-alkanes (C9-C20).GC/MS analyses were performed using a Hewlett Packard apparatus equipped with a HP1 fused silica column (30 m x 0.25 mm, film thickness 0.25 µm) and interfaced with a quadruple detector (Model 5970).Column temperature was programmed from 70 to 200°C at 10°C/min; injector temperature was 220°C.Helium was used as carrier gas at a flow rat of 0.6 mL.min-1, the mass spectrometer was operated at 70 eV.2.0 µL of diluted samples (10/100, v/v, in methylene chloride) were injected manually in the split mode (1/100).The identification of individual compounds was based on the comparison of their relative retention times with those of authentic samples on the DB5 column and by matching the linear retention indices and mass spectra of peaks with those obtained from authentic samples and/or the NBS75K.L and NIST98.L libraries and published data (Adams, 2007).

Strains of filamentous fungi tested
The strains used in this study were constituted of spoilage and pathogens moulds Aspergillus flavus, Aspergillus niger, Aspergillus tamarii, Aspergillus terreus, Fusarium poae, Fusarium verticillioides, Penicillium citrinum, Penicillium griseofulvum, Aspergillus aculeatus, Aspergillus ustus, Penicillium brevicompactum, and Scopulariopsis brevicaulis.They have been isolated and identified from a traditional cheese wagashi collected near its vendors.Colonies of these moulds isolated from Dichloran Rose Bengal Chloramphenicol medium by dilution method (ISO 21527-1: 2008) were purified by streaking onto malt extract agar and then three point inoculated onto malt extract agar and Czapeck yeast autolysate agar before identification based both on macroscopic characters (colony growth, colony diameter) and microscopic characters using the identification schema of Samson et al. (1995) and Pitt and Hocking (2009).

Antifungal assay in culture medium
The test was performed by the agar medium assay (Tatsadjieu et al., 2009).Potato dextrose agar (PDA) medium with different concentrations of essential oil (200, 400, 600, 800 or 1000 mg.L-1) were prepared by adding appropriate quantity of essential oil to melted medium, followed by addition of Tween 80 (100 µL to 100 mL of medium) to disperse the oil in the medium.About 20 ml of the medium were poured into glass Petri-dishes (9 cm x 1.5 cm).Each Petri-dish was inoculated at the centre with a mycelial disc (6 mm diameter) taken at the periphery of a fungus colony grown on PDA for 48 h.Positive Control (without essential oil) plates were inoculated following the same procedure.Plates were incubated at 25°C for 8 days and the colony diameter was recorded each day.Minimal inhibitory concentration (MIC) was defined as the lowest concentration of essential oil in which no growth occurred.The mycelia growth inhibition (MGI) percentage was calculated according to the equation: Where, dc = mean diameter for control -6 mm and dt = mean diameter for treated mycelium -6 mm.
The minimal fungicidal concentration (MFC) values were determined by the method described by Angelini et al. (2006).This was done by subculturing the inhibited fungal discs at MICs on PDA medium without essential oil.Observations were recorded after 7 days of incubation at 25°C.Fungal growth on the seventh day was indicative of a fungistatic nature, while the absence of fungal growth denoted a fungicidal action of the oil.

Preparation of conidial suspension
The strains isolated from cheese wagashi were cultured on potato dextrose agar medium for 10 -14 days at 25 ± 1°C.Conidia were harvested by adding 10 ml of 0.05% Tween 80 solution to culture and gently scraping the mycelia with a sterile inoculating loop to free spores.Conidial concentration was determined by a haemocytometer and the suspension was diluted with 0.05% Tween 80 solution to give a final concentration of 10 8 spores/mL approximatively (Gandomi et al., 2009, Sessou et al., 2012g).

Test in cheese wagashi
The procedure was based on that of Smith-Palmer et al. (2001) and performed by Sessou et al. (2012g).10 g of sterile cheese wagashi was added to 90 ml of 0.1% peptone (CM0009 Oxoid, LTD Basingstoke, Hampshire, England) in stomacher bags and homogenized for 2 min in a stomacher.Essential oil of S. aromaticum was added to the cheese mixture to achieve final concentrations wished (600, 800 and 1000 mg/L).The controls contained peptone but no plant essential oil.The cheese mixture was inoculated with 100 µl of sporale suspension culture, which had been prepared previously.The inoculum was mixed thoroughly with the cheese mixture by gently squeezing the bags by hand and the concentration of mould in the cheese determined at 0 h and 1, 2, 3, 4, 7, 10 and 14 days using the serial dilution and spread plate technique.

Statistical analysis
Data from three independent replicate trials were subjected to statistical analysis using Statistica version 6.0.Differences between means were tested using Z-test.Results were considered statistically significant when p<0.05.

In vitro antifungal activity of essential oil of S. aromaticum in culture medium
This part of the work concerned the in vitro evaluation of antifungal activity of essential oil of S. aromaticum against twelve fungal isolates of wagashi belonging to Aspergillus, Fusarium, Penicillium and Scopulariopsis.The results obtained from this work indicated that the observed mycelial reduction rate of strains were significantly (p <0.05) influenced by the incubation time and the concentrations of essential oil tested.Indeed, mycelial growth is reduced with the increasing of essential oil concentration while the mycelial growth increased with the duration of incubation (Figure 1).The percentage of mycelial growth inhibition, minimum inhibitory concentrations (MIC) and fungicidal (MFC) of the oil are shown in Table 2. Analysis of this table shows that the essential oil of E. caryophyllata present differentially fungistatic and fungicidal activities against all of strains investigated.The minimum inhibitory concentrations of this oil were ranged from 200 to 600 mg/L and the more sensitive strains in decreasing order were S. brevicaulis (MIC = 200 mg/L; CMF = 400 mg / L) and A. terreus (MIC = 400 mg / L; CMF = 800 mg / L. The least sensitive strains, all entirely inhibited by the oil concentration at 600 mg/L, were A. aculeatus, A. tamarii, F. poae, P. brevicompactum and P. citrinum.The most pronounced activity of the essential oil of S. aromaticum is certainly due to its major component, eugenol, a phenolic compound which is recognized as having potential antimicrobial properties.In fact, according to Burt (2004), essential oils with strong antibacterial and antifungal activities are generally those that are rich in phenolic compounds such as carvacrol, eugenol and thymol.Mechanisms of action of these phenolic compounds and their effects on the cell membrane are known.Their influence on cell permeability in terms of interference on membrane functions (electrons transport, synthesis of amino acids and nucleic acids and enzyme secretion) are certainly at the origin of this character for the observed antimicrobial activity of the oil against the strains investigated.One important characteristic of these phenolic compounds is their hydrophobicity that allows them to bind to the ergosterol, the majority of lipid cell membrane strains, creating a disturbance of the cytoplasmic membrane, disrupting the proton motive force (PMF), electron flow, active transport and coagulation of cell contents (Murray et al., 2003;Burt, 2004;Yèhouénou et al., 2012a and b).In the literature, several studies on the antifungal activity of the essential oil of Syzygium against food pathogens have been reported and their results are in accordance with those of the present work.Indeed, Lopez et al. (2005) showed that the essential oil of S. aromaticum possess antifungal properties on some fungal strains isolated from foods.Pawar and Thaker (2006) showed that the essential oil of clove has an inhibitory activity on A. niger.Studies of Pinto et al. (2009) also showed that the essential oil of S. aromaticum was active against some strains such as A. flavus, Aspergillus fumigatus and Candida.Considering the results obtained in our present studies, the tested essential oil can be used in the fight against pathogens and spoilage moulds of wagashi.

In situ effects of S. aromaticum against moulds in wagashi food system
The data presented in Figures 2, 3 and 4 are related to the use of S. aromaticum oil as a substitute of synthetic chemical preservatives in the fight against spoilage and pathogenic moulds A. aculeatus, A. tamarii, F. poae, and P. brevicompactum, P. citrinum in wagashi.These strains were chosen because of their less sensitivity to this extract when tested in vitro in culture medium.As mentioned before, to evaluate the antifungal activity of S. aromaticum essential oil in the food system, three distinct oil concentrations was adopted with respect to the full inhibitory concentrations (minimal and highest concentrations) found for all tested moulds.A blank test, without the essential oil was also conducted.The concentrations of essential oil tested in situ were then 600, 800 and 1000 mg/mL which had totally inhibited all the strains at in vitro experimentation.It should be noted that the effect of oil is reported here for wagashi tenth diluted in peptone water and is not necessarily a real trend in the wagashi solid but nevertheless necessary for its practical importance.Despite this fact, the investigation showed the potential application of the essential oil of S. aromaticum as curator of wagashi.Indeed, after addition of 600, 800 and 1000 mg/L of essential oil in wagashi containing spores of strains, we observed a significant reduction in spore load of strains based on the retention time (storage) and the concentration of essential oil.In all cases, the inhibitory effects were dose-dependent; more, the concentration of the oil is higher, more the sporale reduction rate  increases (Figures 2,3 and 4).The three preparations showed a gradually decrease in the count of spores of these fungi with the increasing of essential oil concentrations.For example, the strain of A. tamarii was reduced to 4.39 log10 cfu/g of its load at fourteenth day of storage at 600 mg/L whereas at 800 and 1000 ppm, the spore load of the same strain was reduced respectively to 4.06 and 2.67 log10 cfu/g.At the same time, the spore load of the control without essential oil has increased to 8.11 log10 cfu/g on the fourteenth day of storage.At 1000 ppm, strains of P. citrinum, A. aculeatus, P. brevicompactum, A. tamarii and F. poae were reduced to 1.02, 1.30, 1.40, 2.14 and 2.67 log10 cfu/g, respectively, of their sporale quantum the fourteenth days of storage.The same trend was obtained on the same strains at the concentration of 800 ppm where the spore loads of these strains were respectively reduced to 2.01, 1.89, 2.40, 2.96 and 4.06 log10 cfu/g at the last day of storage.It is the same observation at 600 mg/L when the same strains were reduced to 2. 21, 2.14, 3.31, 3.33 and 4.39 log10 cfu/g, respectively, in the fourteenth day of storage.At the same time, there was an increase in spore loads 8.11, 8.35, 8.05, 8.27 and 8.11 log10 cfu/g of the respective controls without essential oil of these five strains on the fourteenth day of storage.After all, the essential oil of S. aromaticum is active on all strains but preferably on P. citrinum, P. brevicompactum and A. aculeatus.It is important to notice that no strain has seen its initial quantum reduced to 100% during storage.The percentages of reduction of mold spores in the wagashi are lower than those observed in the culture medium.This fact may be related to more complex matrix (fat and protein) of wagashi than the culture medium.In general, concentrations of essential oils and their compounds necessary to inhibit microbial growth in food are higher than in the culture media (Burt, 2004).This is due to interactions between the compounds and oils from the food matrix (Nuchas and Tassou, 2000;Sessou et al., 2012g).Several authors showed that the fat in the diet could form a protective layer around the micro-organisms, thereby protecting against antimicrobial agents (Sessou et al., 2012g).These researchers also showed that the lipid fraction of food absorbs the antimicrobial agent, thus reducing the oil concentration in the aqueous phase.
The protein content of the diet may also have been a factor in the effectiveness of essential oil (Smith-Palmer et al., 2001;Sessou et al., 2012g).According to Juven et al. (1994), bovine serum albumin neutralized the antimicrobial action of thymol resulting in the formation of complexes between phenolic compounds in oil and protein foods (Omidbeygi et al., 2007;Sessou et al., 2012g).The addition of oil to the mixture of S. aromaticum and mold spores in wagashi exerted a considerable reduction in spore loads of all strains investigated which can lead to spoilage of this foodstuff and produce their metabolites in wagashi and could affect the health of consumers.This study showed that the essential oil of S. aromaticum has antifungal activity and can be used as an alternative of synthetic chemical preservatives to extend shelf life of wagashi.Antimycotoxigenic and toxicity for human tests of this oil are needed to be investigated for practical use of this extract for the valorization of wagashi, a food product much appreciated by the people of Benin and surrounding.

Conclusion
The in vitro and in situ antifungal activities respectively in culture medium and traditional cheese wagashi of S. aromaticum essential oil against twelve fungal isolates of wagashi belonging to Aspergillus, Fusarium, Penicillium and Scopula-      riopsis genera were evaluated in this work.Essential oil of E. caryophyllata constituted of eugenol and trans-caryophyllene as major components showed high effect against all the species tested at in vitro tests and especially high sporale reduction on P. citrinum, A. aculeatus in wagashi foodsystem.Results obtained in the present study allow the use of this oil to fight against moulds contamination of wagashi during storage.Further studies are needed to evaluate the antitoxigenic and toxicity for human of E. caryophyllata essential oil before its practical use as substitute of chemical preservatives of wagashi.

Figure 2 .
Figure 2. Inhibition of moulds investigated in traditional cheese wagashi by S. aromaticum essential oil at concentration of 600 mg/L.

Figure 3 .Figure 4 :
Figure 3. Inhibition of moulds investigated in traditional cheese wagashi by S. aromaticum essential oil at concentration of 800 mg/L.

Figure 4 .
Figure 4. Inhibition of moulds investigated in traditional cheese wagashi by S. aromaticum essential oil at concentration of 1000 mg/L.

Table 2 .
Mycelial growth inhibition, fungistatic and fungicidal activities of essential oil of Syzygium aromaticum.Data in the line followed by different letters are significantly different (p < 0.05).The values are means of three repetitions ± standard deviation.