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Comparative Study of Antifungal Activities of Six Selected Essential Oils against Fungal Isolates from Cheese Wagashi in Benin



P. Sessou, S. Farougou, S. Ahounou, Y. Hounnankpon, P. Azokpota, I. Youssao and D. Sohounhloue
 
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ABSTRACT

The study has compared the antifungal efficacy of six essential oils, Cinnamomum zeylanicum, Cymbopogon citratus, Ocimum gratissimum, Pimenta racemosa, Syzygium aromaticum and Zingiber officinale, tested in culture medium and in traditional cheese wagashi system against moulds belonging to Aspergillus, Penicillium, Fusarium and Scopulariopsis genera in perspective to select the most actives as substitutes of chemical preservatives for wagashi preservation. Results obtained from this work indicated that Syzygium aromaticum, Pimenta racemosa, Ocimum gratissimum and Cymbopogon citratus essentials oils were the most actives extracts at in vitro assay in decreasing order with strong fungistatic activity against the isolates tested; the pronounced activity was provided by S. aromaticum essential oil. The effectiveness of these actives oils on the less sensitive moulds common to these oils showed that, among these extracts that of Syzygium aromaticum in particular exerted high sporale reduction against all the strains tested. In sum, Syzygium aromaticum essential oil possessed the highest antifungal activity both in culture medium and in wagashi system. Essential oils of C. citratus, O. gratissimum, P. racemosa and above all that of S. aromaticum, among the six extracts investigated, were the most promising oils as wagashi additives in substitution of synthetic chemicals ones to extend shelf life time of this by-product of milk for its valorization. Further studies are needed to be performed on the safety of oils for human, the shelf life time of this cheese and its acceptability when treated with essential oils to reduce and control pathogen contamination or native microflora.

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P. Sessou, S. Farougou, S. Ahounou, Y. Hounnankpon, P. Azokpota, I. Youssao and D. Sohounhloue, 2013. Comparative Study of Antifungal Activities of Six Selected Essential Oils against Fungal Isolates from Cheese Wagashi in Benin. Pakistan Journal of Biological Sciences, 16: 1751-1757.

DOI: 10.3923/pjbs.2013.1751.1757

URL: https://scialert.net/abstract/?doi=pjbs.2013.1751.1757
 
Received: January 13, 2013; Accepted: March 03, 2013; Published: May 16, 2013



INTRODUCTION

In Benin, a local cheese wagashi obtained by traditional process is a nutritional food containing proteins which allows its use as substitutes of eggs and meat in many dishes of rural than citizens populations. This product could help to fight against nutsritional problems due to animals’ proteins deficiency (Keke et al., 2008; Sessou et al., 2012a, b). However, the unsatisfactory conditions of process and preservation of this foodstuff may lead to its contamination by adulterated and pathogenic microorganisms particularly moulds (Aissi et al., 2009; ICMSF, 2005). Moulds contamination of wagashi may result in qualitative and quantitative losses of this product. Also, moulds may produced toxic metabolites such as mycotoxins inside wagashi contaminated (Singh et al., 2010). Thus, a better control to prevent contamination and spoilage of wagashi during the production, sale and distribution in order to extend its shelf life time and minimize public health hazards is necessary. Synthetic chemicals could be used to control these pathogens. However, these chemical preservatives have been considered as sources of many diseases such as carcinogenic, nephritic and teratogenic threats (Hsouna et al., 2011; Barkat and Bouguerra, 2012). The same time, consumers are looking for natural products which have less impact on human health and environment and even containing less synthetic preservatives. In the meantime, a lower food salt content is recommended by the World Health Organization, in order to reduce the incidence of cardiovascular disease (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, secondary metabolites of plants, have recently been recognized as bioactive agents possessing antimicrobial activities and classified as Generally Recognized as Safe substances (ESO GRAS-182.20) by the Food and Drug Administration (Food and Drug Administration, 2005; Hsouna et al., 2011; Varona et al., 2013). Therefore they could be used to control many pathogenic and spoilage microorganisms in foods (Velazquez-Nunez et al., 2013). Several studies have reported Cinnamomum zeylanicum, Cymbopogon citratus, Ocimum gratissimum, Pimenta racemosa, Syzygium aromaticum and Zingiber officinale essential oils to be some of the best agents having broad spectrum potentials to inhibit foodborne pathogens and spoilage organisms (Burt, 2004; Pinto et al., 2009; Sessou et al., 2012a-c; Yehouenou et al., 2012a, b). Their applications as control agents of fungal isolates from cheese wagashi in Benin are widely studies (Sessou et al., 2012a-f, 2013). The objective of the present work is to make a comparative study on the antifungal activities of these oils in order to select the most actives as substitutes of chemicals synthetic preservatives for the valorization of this product.

MATERIALS AND METHODS

Essential oils studied: Six plants, Cinnamomum zeylanicum, Cymbopogon citratus, Ocimum gratissimum, Pimenta racemosa, Syzygium aromaticum and Zingiber officinale collected in Benin and identified by Doctor YEDOMOHAN of National Herbarium of Benin were hydrodistilled and the essential oils obtained were analyzed by GC/MS and GC/FID. The major compounds in these oils are reported in Table 1.

Strains of filamentous fungi tested: The strains investigated were constituted of adulterated and pathogenic moulds A. flavus, A. niger, A. tamarii, A. terreus, Fusarium poae, F. verticillioides, P. citrinum, P. griseofulvum, Aspergillus aculeatus, A. ustus, Penicillium brevicompactum and Scopulariopsis brevicaulis identified by morphological and microscopic characteristics (Sessou et al., 2012a).

Table 1: Major components of essential oils studies

Preparation of conidial suspension: Conidial suspension concentration of mould was determined by a haemocytometer and the suspension was diluted with 0.05% Tween 80 solution to give a final concentration of 108 spores mL-1 approximatively (Gandomi et al., 2009).

Antifungal assay in culture medium: The test was performed by the agar medium assay described by Tatsadjieu et al. (2009) with different concentrations of essential oil, 200, 400, 600, 800 or 1000 mg L-1. The MGI (Mycelia Growth Inhibition) was determined on each species for each oil. The Minimal Fungicidal Concentration (MFC) values were determined by the method described by Angelini et al. (2006).

Antifungal assay in cheese wagashi foodsystem: The procedure performed is that of Sessou et al. (2012f). About 10 g of sterile cheese wagashi was added to 90 mL of 0.1% peptone in stomacher bags and homogenized for 2 min in a stomacher. Essential oil was added to the cheese mixture to achieve final concentrations wished. 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. The assay was conducted on moulds which were less sensitive at in vitro assay and common to actives essential oils in order to compare their activities.

Statistical analysis: Statistical analysis was carried out with SAS software (SAS, 1989) and several procedures were used. The procedure of generalized linear models (Proc-GLM) was used for the analysis of the variance and the means of inhibition percentage of three independent replicate trials were then calculated and compared by a Z-test using Statistica version 6.0 (StatSoft, 2010).

RESULTS

Six essential oils obtained from Cinnamomum zeylanicum, Cymbopogon citratus, Ocimum gratissimum, Pimenta racemosa, Syzygium aromaticum and Zingiber officinale were compared based on their ability to control foodborne pathogens and spoilage moulds. Among the essential oils tested, those of Syzygium aromaticum, Pimenta racemosa, Ocimum gratissimum, Cymbopogon citratus exhibited broad-spectrum antifungal activities in culture medium against the species of moulds investigated; while those of Zingiber officinale and Cinnamomum zeylanicum showed less antifungal activity, they inhibited the mycelial growth in a few fungal strains (Table 2 and 3). Syzygium aromaticum essential oil was the most effective as antifungal agents, followed by Pimenta racemosa, Cymbopogon citratus and Ocimum gratissimum essential oils in decreasing order at in vitro test (Table 3). In all cases the inhibitory effects of these oils were linked to dose tested (p<0.05). Based on minimal inhibitory concentrations of these extracts, those of Syzygium aromaticum and Pimenta racemosa were the most actives to fight against Aspergillus and Penicillium species. To fight against Fusarium species, S. aromaticum oil is the most recommended followed accessory by Pimenta oil whereas Ocimum gratissimum, Cinnamomum zeylanicum, Cymbopogon citratus and Syzygium aromaticum constituted the most actives control agents of Scopulariopsis brevicaulis specie. Aspergillus tamarii was the less sensitive common specie to essential oils of Syzygium aromaticum, Ocimum gratissimum and Cymbopogon citratus while Penicillium citrinum was the less sensible common specie to Syzygium aromaticum, Pimenta racemosa and Cymbopogon citratus essential oils at in vitro test. Penicillium brevicompactum and Fusarium verticillioides were the less sensitive common species to Pimenta racemosa and Ocimum gratissimum essential oils in culture medium test.

Table 2: Mycelial growth inhibition, fungistatic and fungicidal activities of six essential oils investigated
FS: Fungistatic activity, FC: Fungicidal activity, data in the line followed by different letters are significantly different (p<0.001), The values are means of the repetitions±standard deciation, Key: A. Acu: Aspergillus aculeats, A. Fla: Aspergillus flavus, A. Nig: Asperillus niger, A. Tam: Aspergillus tamarii, A. Tecitrinum, P. Gri: Penicillium griseofulvum, S. Brev: Scopulariopisi brevicaulis, +++: Significative (p<0.001), ns: Non significative. The results presented in this table are those significant for comparative activites of oil studied

Table 3: Percentage of studied fungal strains inhibited at in vitro test

Fig. 1: Inhibition of Aspergillus tamarii in traditional cheese wagashi by S. aromaticum, C. citratus and O. gratissimum essential oils at concentration of 1000 mg L-1

Fig. 2: Inhibition of Penicillium citrinum in traditional cheese wagashi by S. aromaticum, C. citratus and P. racemosa essential oils at concentration of 1000 mg L-1

The active oils (S. aromaticum, Pimenta racemosa, Ocimum and Cymbopogon citratus) at in vitro assay were tested against their common less sensitive moulds in cheese wagashi and showed high sporale reduction of Aspergillus tamarii and Penicillium citrinum (Fig. 1 and 2) with Syzygium aromaticum oil. In fact, quantum of Aspergillus tamarii was reduced to 1.02 log 10 CFU g-1 of its load at fourteenth day of storage at 1000 mg L-1 with Syzygium aromaticum essential oil whereas the spore load of the same strain was less reduced, respectively to 2.79 log 10 CFU g-1 and 2.4 log 10 CFU g-1 by essential oils of Cymbopogon citratus and Pimenta racemosa at the same concentration the day 14.

Fig. 3: Inhibition of P. brevicompactum and F. verticillioides in traditional cheese wagashi by P. racemosa and O. gratissimum essential oils at concentration of 1000 mg L-1

At the same time, the spore load of the control of Aspergillus tamarii without essential oil has increased of 6 log 10 CFU g-1 to 8 log 10 CFU g-1 on the fourteenth day of storage. The same remarks were observed with these oils on Penicillium citrinum specie. Figure 3 showed that essential of Ocimum gratissimum were more active on Fusarium verticillioides than that of Pimenta racemosa in cheese wagashi foodsystem. The same remark was observed for these oils on Penicillium brevicompactum. In fact, microbial load of Fusarium verticillioides was reduced to 2.83 log 10 CFU g-1 of its load at fourteenth day of storage at 1000 mg L-1 with Ocimum gratissimum whereas in presence of Pimenta racemosa essential oil at the same concentration, the quantum of Fusarium verticillioides increased until to 6.99 log 10 CFU g-1 the day 14. The same observations were noted for these oils against Penicillium brevicompactum where the reduction of spore load of this mould is pronounced with Ocimum gratissimum oil than that of Pimenta extract. In sum, these experimentations in wagashi food system revealed that the activity of S. aromaticum is higher than that of P. racemosa essential oil and this last than that of Cymbopogon citratus on Penicillium citrinum. On Aspergillus tamarii, Syzygium aromaticum activity is higher than that of Cymbopogon citratus essential oil and this last than that of Ocimum gratissimum. The activity of O. gratissimum was significative on Fusarium verticillioides than that of P. racemosa essential (Table 4).

Table 4: Residual spores load of moulds in cheese wagashi treated with 1000 mg L-1 of essential oils at the end of storage time (14th day)

DISCUSSION

Food security is a major preoccupation both for consumers and the food industry, particularly as the number of food-associated infections cases continues to rise (Alzoreky and Nakahara, 2003). According to World Health Organization, about 30% of people in industrialized countries, annually, suffer from foodborne diseases (WHO, 2002). Microorganisms play a major parts in stored food contamination by deterioring them quantitatively and qualitatively. These strains are mainly controlled by synthetic fungicides which can often be problematic when used for treatment because of their high residual toxicity to mammals (Bakkali et al., 2008). The use of synthetic chemicals to fight against microbial strains has been discouraged due to their negative effects on food and human health. In addition to these impacts on food and consumers’ health, microorganisms can also develop resistance to synthetic controlling agents. The use of higher concentrations of synthetic agents to control the microbial resistance increase the risk of high content of toxic residues by the products (Barkat and Bouguerra, 2012). Furthermore, the limitations of some synthetic food additives use by food industry and regulatory agencies have led to great importance to natural antimicrobial components, especially essentials oils which are secondary metabolites of plants (Singh et al., 2010). Essential oils as well as their constituents possess a broad range of activities among which antibacterial and antifungal activities are largely investigated (Nguefack et al., 2007). Food mycoflora has been traditionally few studies contrary to bacterial flora of foodstuffs. Better, essential oils have received great attention concerning their antibacterial activities whereas the effects of these substances on food spoilage and pathogenic moulds were few investigated (Burt, 2004; Angelini et al., 2006). According to our studies essential oils of Syzygium aromaticum, Pimenta racemosa, Ocimum gratissimum and Cymbopogon citratus were the most harmful extracts against species of moulds investigated. The activities of these oils could be linked to their richness in phenolic and aldehydic compounds. In fact, considering Zingiber officinale and Cymbopogon citratus essential oils, both contained geranial, in minor percent in Z. officinale essential oil and in large proportion in Cymbopogon citratus essential oil which was the more active of these two oils. The antifungal activity of each one of these oils may be linked to their content in geranial which may possess a high antifungal activity. Considering essential oils of Pimenta racemosa and Syzygium aromaticum belonging to Myrtaceae family, both contained eugenol in large proportion with that of Syzygium aromaticum in high percentage than that of Pimenta racemosa. From these two oils S. aromaticum extract was the more active. The activity of these two oils may be due to their content in eugenol which is a phenolic compound generally possessing antimicrobial activity (Burt, 2004, Lopez-Reyes et al., 2010). The two oils containing eugenol possessed high antifungal activity than those containing geranial. The antifungal activity of eugenol may be higher than that of geranial. Also, the composition of Ocimum gratissimum in thymol, a phenolic compound may be at the origin of the antifungal activity of this oil. The antifungal activity of this last extract than that of Pimenta on Fusarium verticillioides may be link to the fact that thymol has more negative effect on cellular membrane of Fusarium species than that of eugenol. These hypotheses relative to the antifungal activity of thymol, geranial and eugenol must be confirmed by further studies in which antifungal activity of each molecular will be only tested on each genera of strain Aspergillus, Penicillium and Fusarium. Altogether, essential oils of Syzygium aromaticum, Pimenta racemosa, Ocimum gratissimum and Cymbopogon citratus were the most promising antifungal agents which could be used for traditional cheese wagashi preservation in Benin.

CONCLUSION

The present investigation concluded that Syzygium aromaticum, Pimenta racemosa, Ocimum gratissimum and Cymbopogon citratus possessed differentially high fungistatic activity against Aspergillus, Penicillium, Fusarium and Scopulariopsis brevicaulis genera and could be used as wagashi preservatives in replacement of synthetic chemicals ones for the valorization of this foodstuff. The use of these natural substances as antifungal agents, may be of interest given that essential oils are of natural origin and safer for human health and the environment and there is less chance that the pathogenic microorganisms will develop resistance. Toxicity of these extracts and acceptability of wagashi treated with these oils are need to be investigated.

ACKNOWLEDGMENTS

The authors are grateful to Doctor SEBASTIEN TINDO DJENONTIN in memoriam (He was coldly shot by thugs at his home March 11th, 2013; May he rest in peace) for its scientific contribution. They are also thankful to University Council of Development (CUD) for its financial support.

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