1. Introduction
The overuse of chemicals in food preservation poses health risks to consumers. For this reason, it is important to prioritize research and the use of bio-pesticides during crop storage. Essential oils are increasingly studied for their ability to inhibit microorganisms and extend food shelf life due to their antimicrobial properties, including antibacterial, antiviral, and antifungal activities. They exhibit significant antibacterial activity against foodborne pathogens such as Salmonella and Escherichia coli, with oils like thyme (Thymus vulgaris) and cinnamon (Cinnamomum zeylanicum) reducing bacterial loads in meats and dairy products [1]. Certain essential oils, such as lemon oil (Citrus limon) and eucalyptus oil (Eucalyptus globulus), have demonstrated antiviral properties, with lemon oil inhibiting specific foodborne viruses [2]. The incorporation of essential oils like garlic (Allium sativum) and rosemary into food packaging can prolong shelf life by slowing bacterial growth [3]. The control of fungal growth in food using local plant extracts, particularly EOs, could be an effective and healthy solution. Indeed, several EOs have the capacity to produce a wide range of antifungal metabolites [4], [5] that can inhibit fungal growth, preserve food quality and safety and extend food shelf life [6]. Due to their natural origin, EOs are more prized by consumers than chemical agents. These oils are also recognized by the Food and Drugs Administration (FDA) as non-dangerous [7]. EOs of Lippia multiflora tested against strains of Aspergillus flavus isolated from wheat bran showed a 100% inhibition rate at a concentration of 2.5% [8].
In addition, Tiendrebeogo et al. [9] reported that Lippia multiflora EO had the ability to inhibit the mycelial growth of Bipolaris oryzae, Pyricularia oryzae and Fusarium moliniformes strains isolated from rice by up to 100% at concentration ranged between 100 to 600 ppm. Etienne et al. [10] showed that the essential oil of Lippia multiflora was able to completely inhibit the growth of strains of Aspergillus sp., Rhizopus sp. and Fusarium sp. isolated from potatoes at a concentration of 0.666 µL/mL after 7 days incubation. EOs obtained from Ocimum species, such as Ocimum gratissimum, inhibited the growth of several fungi, including the plant pathogens Botryosphaeria rhodina, Rhizoctonia sp. and two strains of Alternaria sp. EOs of Ocimum americanum inhibited the growth of strains of Candida albicans, Candida glabrata, Candida tropicalis, Candida krusei and Candida parapsilosis [11]. The work of Gakuubi et al. [12] showed that Eucalyptus camaldulensis EO was able to completely inhibit the mycelial growth of several Fusarium species, including F. oxysporum, F. solani, F. verticillioides, F. proliferatum and F. subglutinans at concentrations ranged between 7 and 8 μL/mL after five days of incubation. Several studies have shown that the EO of Eucalyptus cameldulensis has the ability to inhibit the growth of a wide range of fungi, such as Aspergillus niger, Chaetomium globosum, Rhizopus oryzae, Thanatephorus cucumeris and F. oxysporum [13]. Somda et al. [14] also reported that the EO of Eucalyptus camaldulensis had antifungal activity against strains of Glomerella graminicola, Phoma sorghina and F. moniliforme isolated from the soil.
EOs are natural sources of biomolecules that deserve to be explored because, as well as having the ability to inhibit fungal growth, they are also able to reduce the synthesis of mycotoxins in foods [12] [13] [15] [16]. Recent studies demonstrated the inhibitory effect of Cymbopogon citratus and Cymbopogon schoenanthus EOs on the synthesis of aflatoxin B1 [17] and, the possibility of controlling Aspergillus flavus and Aspergillus parasiticus on corn, using two formulations based on EOs of Cymbopogon giganteus and Eucalyptus camaldulensis [18].
Ouili et al. [19] recently isolated two mycotoxin-producing strains of Aspergillus flavus from Bambara groundnut seeds. The objective of this study is to evaluate the antifungal activity of essential oils from Lippia multiflora, Eucalyptus camaldulensis, and Ocimum americanum against these two aflatoxin-producing fungal strains.
2. Material and Methods
2.1. Essential Oils
The EOs of Lippia multiflora, Ocimum americanum and Eucalyptus cameldulensis used for the tests in our study were obtained from the Institut de Recherche en Sciences Appliquées et Technologies (IRSAT) of Burkina Faso (Figures 1-3). These EOs were extracted for 3 h using the hydrodistillation method in a clenvenger-type apparatus. Anhydrous sodium sulphate was used to dry the EO, which was then stored in an airtight, glass container at 4˚C in a refrigerator.
2.2. Antifungal Screening of EOs
Antifungal activity was tested using the two strains of Aspergillus flavus (AVBF26, AVBF66) isolated from Bambara groundnut. Previous studies have shown that these two strains are able to produce aflatoxins B1 and B2 when inoculated onto rice [19].
For the antifungal screening, various concentrations (112.5, 225, 450, 900, 1800 et 3600, 9000, 10,800, 12,600 μg∙mL−1) of EOs from each plant (Lippia multiflora,
Figure 1. Plant of Lippia multiflora.
Figure 2. Plant of Ocimum americanum.
Figure 3. Plant of Eucalyptus camaldulensis.
Ocimum americanum and Eucalyptus cameldulensis) were incorporated into molten Potato Dextrose Agar (PDA) (approximately 44˚C) containing 0.1% Tween-20. Approximately 20 ml of each mixture (PDA agar + EO) obtained was distributed in Petri dishes (90 mm), and after solidification of the culture medium, plugs of a 7-day fungal culture (4 mm), taken from the actively grown region of the colonies, were placed in the center of the Petri dishes and incubated at room temperature. Fungal colony diameters were measured after seven days of incubation. Each test was carried out in triplicate. PDA agar without EO was used as a negative control and the antifungal agent Calthio C infused into the PDA agar (final concentration 2.5 mg/l) was used as a positive control. The inhibition rates of fungi using EOs were calculated using the following formula:
where dc = Mean mycelial growth diameter in the negative control plates (PDA without EOs).
dt = Mean mycelial growth diameter in the treatment.
The MIC was defined as the lowest concentration of EO that caused 100% inhibition. For the minimum fungicidal concentration (MFC) determination, fungal discs whose growth had been completely inhibited in the MIC experiment were transferred to new PDA plates previously prepared without EO. These plates were then incubated at room temperature for 5 days. The MFC is the lowest EO concentration at which fungal inhibition is irreversible in the absence of the inhibitor (EO).
2.3. Statistical Analysis
The data were analyzed using XLSTAT software and the mean separation was done by LSD at P = 0.05.
3. Results and Discussion
Antifungal Activity of EOs
The inhibition rates of the three EOs (Lippia multiflora, Ocimum americanum and Eucalyptus cameldulensis) tested against the strains of Aspergillus flavus (AVBF26 and AVBF66) are presented in Table 1.
Table 1. Growth inhibition (%), MIC and MFC of EOs.
EOs (μg∙mL−1) |
Growth inhibition (GI) |
A. flavus (AVBF26) |
A. flavus (AVBF66) |
Ocimum americanum |
112.5 |
5.46 ± 3.9gg |
2.29 ± 0.81eh |
225 |
16.94 ± 2.4ff |
8.62 ± 3.72eg |
450 |
36.06 ± 2.68ee |
28.73 ± 3.25df |
900 |
38.79 ± 2.79ee |
36.78 ± 2.15de |
1800 |
54.09 ± 1.34dd |
48.85 ± 0.81cd |
3600 |
63.38 ± 0.77cc |
55.74 ± 2.15cc |
9000 |
83.06 ± 2.79bb |
82.75 ± 4.22bb |
10,800 |
100.00 ± 00aa* |
100.00 ± 00aa*˚ |
12,600 |
100.00 ± 00aa˚ |
100.00 ± 00aa |
Eucalyptus camaldulensis |
112.5 |
17.48 ± 1.55ff |
10.34 ± 1.41ef |
225 |
18.03 ± 1.34ff |
12.06 ± 1.41ef |
450 |
48.63 ± 2.4ee |
39.08 ± 1.61de |
900 |
60.1 ± 2.4dd |
41.37 ± 1.41dd |
1800 |
61.20 ± 2.4dd |
57.47 ± 0.81cc |
3600 |
67.21 ± 1.34cc |
59.19 ± 0.81cc |
9000 |
74.86 ± 0.77bb |
75.28 ± 0.81bb |
10,800 |
100.00 ± 0.00aa* |
100.00 ± 00aa*˚ |
12,600 |
100.00 ± 0.00aa˚ |
100.00 ± 00aa |
Lippia multiflora |
112.5 |
34.42 ± 2.32fg |
28.16 ± 1.63gg |
225 |
51.36 ± 1.55ef |
47.12 ± 0.81ff |
450 |
67.76 ± 2.79de |
63.21 ± 2.15ee |
900 |
73.22 ± 5.41cdd |
68.96 ± 1.41dd |
1800 |
81.42 ± 2.04bcc |
74.13 ± 1.41cc |
3600 |
87.97 ± 0.77bb |
89.08 ± 0.81bb |
9000 |
100.00 ± 00aa*˚ |
100.00 ± 00aa*˚ |
10,800 |
100.00 ± 00aa |
100.00 ± 00aa |
12,600 |
100.00 ± 00aa |
100.00 ± 00aa |
Means with the same letter(s) show no significant difference (multivariate analysis, Fisher's protected LSD at 𝑝 ≤ 0.05). ˚MFC; *MIC.
It can be seen that the sensitivity of fungi (AVBF26, AVBF66) increases when the concentrations of EOs increase (Figure 4, Figure 5).
Lippia multiflora EO, showed the best inhibition rates compared to the rest of the EOs, with a MIC and MFC of 9000 μg∙mL−1 (Table 1). The MIC of Ocimmum americanum and Eucalyptus cameldulensis EOs on the fungi tested was 10,800
Figure 4. Average growth inhibition of the EOs at different concentrations on A. flavus (AVBF26).
Figure 5. Average growth inhibition of the EOs at different concentrations on A. flavus (AVBF66).
μg∙mL−1 and their MFC ranged between 10,800 and 12,600 μg∙mL−1. The lowest concentrations (112.5, 225, 450, 900, 1800 and 3600 μg∙mL−1) of Lippia multiflora EO showed inhibition rates between 34.42% and 87.97% on AVBF26 and 28.16% and 89.08% on AVBF66 (Table 1). Those (112.5, 225, 450, 900, 1800 and 3600, 9000 μg∙mL−1) of Ocimum americanum showed inhibition rates between 5.46 and 83.06% on AVBF26 and 2.29% and 82.75% on AVBF66 (Table 1). The EO of Eucalyptus cameldulensis showed inhibition rates between 17.48 and 74.86% on AVBF26 and 10.34 and 75.28% on AVB66 for the same concentrations (Table 1). These results show that the essential oils of Lippia multiflora, Ocimum americanum and Eucalyptus cameldulensis have fungicidal properties and can be used instead of synthetic fungicides. Numerous studies have shown that EOs are made up of several compounds capable of inhibiting the proliferation of bacteria and fungi in vitro [12] [17] [19] [20]. Antimicrobial and antifungal compounds generally found in EOs are terpenes, alkaloids, lactones, phenolic compounds, flavonoids and naphthoquinones [21]. The main components found in Lippia multiflora EO are c-terpinene, p-cymene, thymyl acetate, thymol and b-caryophyllene; those found in Eucalytus cameldulensis are a-pinene, eucalyptol and limonene. Camphor, 1, 8-cineol, a-pinene camphor, (Z)-methyl cinnamate and trans a-bergamotene are mainly encountered in the EO of Ocimum americanum [8] [21] [22] [23]. The synergistic effect of the antifungal compounds of each EO used in our study could explain the inhibition of the growth of the fungal strains tested. In fact, essential oils (EOs) exhibit inhibitory effects on Aspergillus flavus primarily through several well-established mechanisms. Upon contact, they alter the permeability of fungal cell membranes, leading to leakage of essential internal components and loss of cellular integrity. Recent studies have shown that volatile compounds such as terpenes and phenols present in EOs, including clove oil (Syzygium aromaticum) and thyme oil (Thymus vulgaris), disrupt fungal cell membranes by damaging membrane lipids [24]-[27]. EOs are also effective in inhibiting spore germination and mycelial growth, which are crucial steps for food colonization. For example, cinnamon oil (Cinnamomum verum) and rosemary oil (Rosmarinus officinalis) have been shown to reduce spore germination and inhibit mycelial growth of Aspergillus flavus [28]. Some EOs induce oxidative stress by generating free radicals and reactive oxygen species, thereby damaging fungal cellular structures. Phenolic compounds and EOs such as clove and oregano oil (Origanum vulgare) increase the production of these oxidative agents, leading to cell death in Aspergillus flavus [28]. Additionally, EOs can interfere with fungal metabolic pathways by inhibiting essential growth enzymes. Recent studies have demonstrated that peppermint oil (Mentha piperita) and citronella oil (Cymbopogon citratus) affect protein synthesis and enzymatic activities, disrupting the biological processes of Aspergillus flavus [29]. Some EOs modify the local acidity around the fungi, creating an environment unfavorable for their growth. Lemon oil (Citrus limon) is known to lower the pH in the environment, which limits the growth of Aspergillus flavus [30].
Essential oils offer promising benefits for food preservation due to their antimicrobial and antioxidant properties, providing a natural alternative to synthetic preservatives and contributing to improved food safety. However, their use must be carefully regulated to avoid excessively high concentrations that could affect the taste or quality of the food. The effects of essential oils may vary depending on the type of food and the target microorganism. While their effectiveness has been demonstrated under controlled conditions, further studies are needed to evaluate their performance in large-scale food production environments.
4. Conclusion
The EOs evaluated in this study were all capable of inhibiting the strains of A. flavus tested. Lippia multiflora oil showed the best inhibition rates on fungal strains with a MIC of 9000 μg∙mL−1. It is followed by those of Eucalytus cameldulensis and Ocimum americanum which showed the same MIC (10,800 μg∙mL−1). These EOs are natural sources of biomolecules that can be used for the formulation of new antifungal agents for the preservation of Bambara groundnut seeds. To do this, additional studies are necessary to evaluate the combined effect of these EOs against mycotoxinogenic fungi and their phytotoxicity on Bambara groundnut seeds.
Acknowledgements
This work was supported by the International Foundation for Science (IFS) [I-3-E-6460-1].