Introduction

Safe food enhances individual and population health. Challenges in food safety include chemical, biological, physical, and environmental risks (Fallahzadeh et al. 2018; Fung et al. 2018; Fakhri et al. 2018; Zuccarello et al. 2019; Keramati et al. 2018; Fiore et al. 2019).

Several studies examined the antimicrobial effects of some oils to search for natural fungicides (Mohajeri et al. 2018). The presence and growth of fungi in food may cause spoilage and resulting in a reduction in quality and quantity, besides causing severe medical and economic implications, especially for exporting countries. Some Aspergillus species, such as Aspergillus flavus and Aspergillus parasiticus, are able to produce aflatoxins (AFB1, AFB2, AFG1, AFG2) (Egbuta et al. 2017; Kumar et al. 2017). However, only aflatoxin B1 (AFB1) is considered a carcinogen by the International Agency for Research on Cancer (IARC) having produced sufficient evidence of liver carcinogenicity in experimental animals. In fact IARC (2012) report that transgenic mice exposed to AFB1 showed liver carcinomas (rats showed hepatocellular and kidney carcinomas, tree shrews showed hepatocellular carcinomas, and finally, trouts showed liver tumors) (IARC 2012, and the references therein). Dietary intake is the primary source of human exposure to aflatoxins (Egbuta et al. 2017; Perrone and Gallo 2017; IARC 2012). The occurrence of aflatoxins is influenced by some specific environmental factors (Abrar et al. 2013); hence, the extent of contamination varies with geographic location (involving both tropics and temperate zones), agricultural and agronomic practices, and the susceptibility of commodities to fungal invasion during preharvest, storage, and/or processing phases (Kumar et al. 2017; Umesha et al. 2017). Since the absolute safety is never achieved, many countries have reduced the exposure to aflatoxins by imposing regulatory limits on commodities intended for food and feed use (FAO 2004; Van Egmond and Jonker 2004).

A review of cost-effectiveness analysis on the topic shows that prevention is the most effective method for controlling fungi and mycotoxin contamination and, finally, for preventing health outcomes due to ingestion of contaminated foods (Khlangwiset and Wu 2010). Postharvest treatment (through dispersion) with antifungal agents has been examined to ensure that control can be achieved (Reynoso et al. 2002), although nowadays it is emphasized the need to prevent fungal development and mycotoxin accumulation using eco-friendly natural substances with fungicidal effects (Mie et al. 2017). Essential oils (EOs) so, as natural preservatives, can be of interest for food industry (Mohajeri et al. 2018). The International Organization for Standardization (ISO) (ISO/D1S9235.2) defines an essential oil as a product made by distillation with either water or steam or by mechanical processing or by dry distillation of natural materials. Natural substances are safer to consumers, to animals, and to the whole environment.

Applied research about natural antifungal use in agriculture is supported by growing worldwide demand of reduction in use of pesticides for their carcinogenic, neurotoxic, and mutagenic effects in exposed humans through food chain (EFSA 2014; Fiore et al. 2019; Ferrante and Conti 2017, 2018; Sciacca et al. 2012; Vinceti et al. 2017). A new study highlighted the natural antioxidant activity of some natural products (Derakhshan et al. 2018), and some studies show the efficacy of essential oils in food preservation (Morcia et al. 2017; Prakash et al. 2015a).

EOs are complex mixtures of volatile constituents synthesized by plants, which mainly include terpenes, terpenoids, and aromatic and aliphatic constituents (Morcia et al. 2017). The main categories of EO compounds are terpenes and terpenoids. Terpenes are a large class of naturally occurring hydrocarbons with various chemical and biological properties. Examples of terpenes include p-cymene, limonene, terpinene, sabinene, and α- and β-pinene. EOs and their components have a variety of targets, particularly the membrane and cytoplasm, and in certain situations, they completely alter the morphology of the cells (Kumari et al. 2014; Salas et al. 2016).

They are a valuable natural source of antioxidants and biologically active compounds, and the most attractive aspect that make them and/or their constituents suitable to be used as food preservative is their non-toxicity (Prakash et al. 2015a). In the last years, EOs extracted from different plants were used in the prevention of fungal growth in cereals (Sumalan et al. 2013), table grapes (Kocevski et al. 2013), and dates (Aloui et al. 2014).

Some studies evaluated the effects of citrus EOs against fungal growth. In fact, EOs prepared from kaffir lime (Citrus hystrix DC) and acid lime (Citrus aurantifolia Swingle) epicarps exhibited fungicidal activity against Aspergillus spp. (Rammanee and Hongpattarakere 2011); the EOs of lemon (Citrus lemon L.), mandarin (Citrus reticulata L.), grapefruit (Citrus paradisi L.), and orange (Citrus sinensis L.) showed antifungal activity against A. flavus, Aspergillus niger, Penicillium chrysogenum, and Penicillium verrucosum (Aloui et al. 2014; Morcia et al. 2017; Viuda-Martos et al. 2008); the Citrus sinensis (L.) Osbeck EO significantly reduced the growth of A. niger in a dosage-response manner (Sharma and Tripathi 2008). The antifungal bioactive components in citrus EOs, their potential mechanism of action, and the role of citrus EOs in improvement of food safety have been exhaustively reviewed by Jing et al. (2014). EOs and their flavonoid components have a variety of targets, particularly the membrane and cytoplasm, and in certain situations, they completely alter the morphology of the cells (Nazzaro et al. 2017).

Few articles about the effects of citrus EOs on aflatoxin production are available. The EOs from tropical sour lime (C. aurantifolia Swingle) produced morphological anomalies in fungal compartments and, as a consequence, suppressed aflatoxin production of A. parasiticus (Razzaghi-Abyaneh et al. 2009); Citrus reticulata and Cymbopogon citratus EOs completely inhibited aflatoxin B1 production at 750 and 500 ppm, respectively (Singh et al. 2010); and Rammanee and Hongpattarakere (2011) demonstrated by immunoenzymatic methods for A. flavus and A. parasiticus the significant reduction of aflatoxins production by the application of the kaffir and acid lime EOs.

Citrus peel residues have recently attracted the attention of scientists in light of their industrial potential uses. Indeed, several studies have highlighted the importance of citrus waste valorization for enhancing economic competitiveness of Mediterranean agriculture (Raimondo et al. 2018; Salas et al. 2016). Previous studies pointed out that the extract of some Citrus spp. had inhibitory activity against A. flavus and aflatoxin accumulation. Bejarano Rodriguez and Centeno Briceño (2009) demonstrated that 20 mL/g of Citrus limon extract reduced (74%) in poultry feedstuffs after 1 h of treatment. Dos Santos Oliveira and Badiale (2008) report that the EOs from orange peel inhibited the production of AFB1 and AFB2 at a concentration of 250 mg/mL in in vitro test. Velázquez-Nuñez et al. (2013) found that the minimum inhibitory concentration for A. flavus growth was 16,000 mg/L of orange’s EO (Citrus sinensis var. Valencia), when added in the culture media.

Then, the aim of this study was to investigate the efficacy of five citrus EOs, namely bergamot (Citrus bergamia Risso), bitter orange (Citrus aurantium L.), sweet orange (Citrus sinensis (L.) Osbeck.), lemon (Citrus limon (L.) Burm. f.), and mandarin (Citrus deliciosa Ten.), whose cultivation is very widespread in Sicily (Italy) and, in general, in all Mediterranean countries, to inhibit the growth and AFB1 synthesis of an aflatoxigenic strain of A. flavus through in vitro tests for a possible future application in food industry that will represent also an interesting option for improving the profitability of Sicilian agriculture.

Materials and methods

Essential oils

Commercial pure EOs from bergamot (Citrus bergamia Risso), bitter orange (Citrus aurantium L.), sweet orange (Citrus sinensis (L.) Osbeck.), mandarin (Citrus deliciosa Ten.), and lemon (Citrus limon (L.) Burm. f.) were purchased from a local certified herbalist’s shop. Technical data sheets supplied by the producer report the concentration of the components characterized in used EOs (Table 1). The EOs were extracted by peel cold-pressing method: the peel and cuticle oils are removed mechanically, and the yield is a watery emulsion, which is then centrifuged to recover the EOs (Ferhat et al. 2007). The oils are commercialized in sealed glass bottles at room temperature.

Table 1 Main physicochemical and compositional data for tested citrus EOs (source: technical data sheets supplied by the producer)
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Fungal strains

The culture of A. flavus var. flavus CBS 573.65 producer AFB1 was obtained from the Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversity Centre (Utrecht, The Netherlands). The fungus was maintained on potato dextrose agar (PDA; Oxoid, Basingstoke, UK) at 4 °C until its use.

Effects of citrus EOs on fungal growth and aflatoxin production

The liquid medium yeast extract–sucrose (YES) containing 2 g yeast extract and 15 g sucrose in 100 mL of distilled water was used for aflatoxin production (Nogueira et al. 2010; Vergopoulou et al. 2001). Conidial suspension (in sterile physiologic solution) was prepared from A. flavus cultivated in YES agar at 30 °C for 7 days. Conidia concentration was determined using a Thoma counting chamber. The conidial suspensions (500 μL), containing 106 conidia/mL, were transferred to 20 mL of YES broth, with the addition of 0.25, 0.75, 1, and 2% (v/v) of EOs. A final concentration of 0.5% (v/v) Tween-20 (Sigma-Aldrich S.r.l., Milan, Italy) was added to YES broth after autoclaving to enhance oil solubility. Inoculated YES broth without EOs (0% v/v) was used as positive growth and aflatoxin production control. Each vial was incubated at 30 °C for 72 h under rotator shaken speed of 200 rpm. Three replicates were performed for each concentration, and the experiment was repeated three times.

Fungal growth was determined by measurement of dried fungal mycelium according to Razzaghi-Abyaneh et al. (2009). The culture broth was filtered through filter paper (Whatman no. 1); the mycelia were washed with sterile water and then brought to dryness at 80 °C until a constant weight was obtained. The inhibitory effect of EOs was estimated in terms of mycelial growth inhibition percentage (MGI %) compared with the control, calculated as follows: MGI (%) = (control weight − sample weight)/control weight × 100.

Aflatoxin analysis

Sample preparation

Aflatoxin content in 5 mL of culture filtrate was determined by adding 1 mL of acetonitrile to 1 mL of supernatant. A microdispersion liquid-liquid extraction (mLLE) in Eppendorf tubes (2 mL) was carried out using acetonitrile and ethyl acetate. The sample mixture was then vortexed for 5 min and then was filtered using a 0.2-μm PTFE syringe filter. An aliquot (100 μL) of filtrate was analyzed in LC-ESI-TQD for estimation of AFB1 concentration. Recovery test showed a good recovery of AFB1 (see Table 2).

Table 2 Data acquisition parameters of MRM transitions for AFB1 aflatoxin
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Chemicals and standards

Aflatoxin standard (AFB1, > 98% purity) was purchased from Sigma-Aldrich (Saint Louis, MO). All reagents were LC-MS grade. De-ionized water is generated by Milli-Q system (Millipore, Tokyo, Japan). Methanol and acetonitrile were purchased from Fisher Scientific (Fair Lawn, NJ). Acetic acid was purchased from Sigma-Aldrich (St. Louis, MO).

LC-ESI-TQD analysis

The AFB1 detection was carried out by LC-ESI-TQD analysis; this approach allowed both to elude the immunoenzymatic method limits (no mass determination, and possible presence of both false positives and negatives) (Li et al. 2015) and to detect also the AFB1 at ppt (ng/kg) concentration (see Figs. 1 and 2).

Fig. 1
figure 1

SIM chromatogram of daughter ions of AFB1 standard (50 ppt)

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Fig. 2
figure 2

AFB1 analysis in lemon EO (0.75%)–treated sample

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An LC-ESI-TQD Varian 320 was used for analysis of AFB1. In particular, an ESI source with a Varian 320 triple quadrupole mass spectrometer coupled to two Varian ProStar pumps, a ProStar 410 autosampler (all of Agilent Technologies, Santa Clara, CA) with a Metachem degassit apparatus were used. Chromatographic analysis was carried out in a Waters XBridge C18 Column (150 × 2.1 mm i.d. with a particle size of 5 μm) and a Waters XBridge C18 as precolumn (10 × 2.1 mm i.d. with a particle size of 5 μm). Settings of TQD detector were as follows: 0.700 dwell time, capillary − 50.000 V, needle voltage − 4500 V; shield − 600 V, drying gas temperature 250 °C, nebulizing gas pressure 19 psi; drying gas pressure 45 psi; scan time 0.200 s. A solvent gradient program was used to maximize the signal-to-noise ratio (sensitivity).

The initial isocratic (0.3 mL/min) pump’s program was 90:10 (1 min), programmed at 60:40 in 4 min, 40:60 held for 14 min, 90:10 in 20 min with A: MilliQ water and B: methanol, both with 0.1% in HPLC-MS grade acetic acid: scan range, 100–500 m/z; averages 3. SIM scan was used.

Injection was performed in μpickup mode (10 μL). Quantitative analysis was carried out using positive-negative polarity SIM mode; the parent (or precursor ion) and daughter ions (product ions) for SIM transitions, recovery, and linearity are shown in Table 2.

Data were acquired with Varian Workstation MS Software. Processing of reagent blank disclosed no trace of AFB1. Mean recovery of spiked matrices was 98%.

Statistical analysis

All experiments were repeated at least three times. A descriptive statistical analysis was carried out, using a statistical package IBM SPSS Statistics 20.0; data of three replicates were not enough for an ANOVA analysis.

Results and discussion

Fungal dry mass reduction by citrus EOs

The effects of the citrus EOs on dry biomass in liquid culture of YES medium after an incubation period of 72 h are shown in Fig. 3. The mycelial growth was significantly reduced by the tested EOs at concentrations ranging from 0.5 to 2% (v/v) when compared with the control. The inhibitory effect increased with increasing EO concentrations and was significantly related to the type of EO (p < 0.05). Results demonstrated that lemon and bergamot EOs, causing respectively a 97.88% and 97.04% reduction in mycelial growth at the concentration of 2% (v/v) compared with the control (p < 0.05), were the most effective EOs, followed by bitter orange EO which, at the same concentration, reduced the mycelial growth of 96.43% compared with the control. Sweet orange and, particularly, mandarin EOs showed the lowest percentage of mycelial growth reduction compared with other EOs at the tested concentrations. The antimicrobial or antifungal activity of EOs might be caused by the properties of terpenes/terpenoids that due to their highly lipophilic nature and low molecular weight are able to disrupt the cell membrane, causing cell death or inhibiting the sporulation and germination of fungi. In Table 1 were reported the concentrations of terpenes and terpenoids that characterize the EOs used in this study.

Fig. 3
figure 3

Mycelial growth inhibition (MGI %) of A. flavus by citrus EOs after 72-h incubation at 30 ± 0.5 °C. Mean values and LSD intervals

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Previous studies reported the effectiveness of citrus EOs on the inhibition of conidial germination of Aspergillus spp. In this sense, Sharma and Tripathi (2008) recorded a complete inhibition of conidial germination of A. niger when C. sinensis oil was used at a concentration of 1.5 mg/mL, while Rammanee and Hongpattarakere proved for the essential oils from kaffir and acid lime epicarps a strong antifungal activity against aflatoxin-producing A. flavus and A. parasiticus with minimum inhibitory concentration of 0.56 mg/mL. The EOs used in this study have been previously tested by Aloui et al. (2014), as a natural alternative to control the growth of A. flavus in dates, demonstrating a higher efficacy for bergamot and bitter orange EOs. Our results partially confirmed these last findings, although in our study lemon EO was also able to strongly reduce the A. flavus growth at 2% concentration. The observed antifungal activity of lemon EO can be explained on the basis of the different A. flavus targeted strain and of the differences in chemical composition of the volatile fractions, which can be dependent on the harvest time (Derakhshan et al. 2018; Caccioni et al. 1998), as previously reported by Caccioni et al. (1998) for orange, mandarin, citrange, and lemon EOs against Penicillium digitatum and P. italicum and by Morcia et al. (2017) for bergamot and lemon EOs and for five natural compounds recurrent in EOs (citronellal, citral, cinnamaldehyde, cuminaldehyde, and limonene) against Fusarium sporotrichioides, F. langsethiae, and F. graminearum.

AFB1 reduction by citrus EOs exposure of mycelium

The five citrus EOs showed variable levels of efficacy to the different doses of treatment (0 to 2%) (Fig. 4).

Fig. 4
figure 4

Effect of different concentrations of citrus EOs on A. flavus B1 aflatoxigenesis (ppb)

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The citrus EOs showed different capacity of AFB1 inhibition, and the following scale of efficacy can be indicated: lemon > bitter orange > bergamot > sweet orange > mandarin.

In particular a linear regression analysis showed a linear dose (%)–inhibition for lemon and bitter orange EOs with R2 = 0.7293 and R2 = 0.7184 respectively.

Data obtained indicated that not all tested citrus EOs are able to contrast the AFB1 production simultaneously with the reduction of mycelia growth. In fact, a good control of AFB1 was obtained by lemon and bitter orange EOs, confirming their ability to inhibit both mycelium growth and AFB1 production; instead, bergamot EO showed a minor capacity of inhibition to AFB1 production compared with its showed efficacy on mycelia growth inhibition; also, a minor linearity was detected for this EO (R2 = 0.5798). Sweet orange and mandarin EOs were not able to contrast AFB1 production as well as to inhibit the mycelium growth. In particular, mandarin EO applied to 0.75% enhanced the AFB1 production (Fig. 5). In general, inhibition of mycelium growth due to essential oil exposure significantly causes a decrease of mycotoxin production; however, these two effects do not always occur together and with the same efficiency and, in some cases, the addition of sub-lethal concentrations of some natural products can enhance mycotoxin production (Morcia et al. 2017). In fact, also Hope et al. (2005) found that cinnamon oil at specific concentration can significatively reduce the mycelium growth of F. culmorum and F. graminearum, but enhance their toxin production and Dambolena et al. (2008) found that limonene and EOs of aromatic plants could increase ergosterol production by F. verticillioides. With regard to Aspergillus spp., Prakash et al. (2015a) found that Piper betle essential oil induces overproduction of AFB1 in A. flavus. Finally, a stimulation of aflatoxigenesis in A. flavus has been reported even by Garcia et al. (2012) in presence of Equisetum arvense and Stevia rebaudiana extracts.

Fig. 5
figure 5

Efficacy of different concentrations of mandarin EO on A. flavus B1 aflatoxigenesis (ppb)

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About the efficacy of EOs against the AFB1 production, Velázquez-Nuñez et al. (2013) compared the antifungal activity efficacy of orange peel EOs at selected concentrations, applied both of by vapor exposure or through the direct addition on the growth of A. flavus. Velázquez-Nuñez et al. showed that A. flavus growth decreased as EO concentration increased. Although the effect of orange peel EO by direct addition was faster, orange peel EO vapors were more effective since lower concentrations of the latter were required to achieve the same antifungal effect. Dwivedy et al. (2017) reported Mentha cardiaca EO as preservative against fungal and aflatoxin contamination of stored dry fruits. Pandey et al. (2016) reported that the application of Lippia EO in storage system inhibited the fungal growth and aflatoxin production without affecting the seed germination rate. Prakash et al. (2015b) showed that a mixture containing Angelica archangelica EO, phenyl ethyl alcohol (PEA), and α-terpineol (1:1:1) have antiaflatoxigenic and antioxidant activity against A. flavus. Finally, Kazemi, through its study, showed the efficacy of Carum copticum EO both as antiaflatoxin B1 and growth inhibitor against Aspergillus strains (Kazemi 2015). Nazzaro et al. (2017) refer that EO from Citrus sinensis epicarp (composed by limonene at 84.2%) is capable of inhibiting the growth of A. niger; it also leads to irreversible deleterious morphological alterations (in particular the loss of cytoplasm in fungal hyphae, and budding of hyphal tip). The EOs affect AFB1 inhibition and/or mycelium growth by two different ways. Firstly, DNA binding formation of aflatoxins is reduced by EOs and also, aflatoxins cause an increase of reactive oxygen species and EOs react with ROS reducing them (Alpsoy 2010).

Referring to citrus EOs, limonene most likely plays an effective role in the AFB1 synthesis (Table 1). However, the total number of compounds present in citrus EOs together with their antagonistic, synergistic, or additive effect on fungal species and/or strains needs to be more deeply explored (Jing et al. 2014).

The findings of the present study may draw the attention of food industries to conduct further experiments regarding large-scale exploitation of essential oils as preservative of food commodities.

Conclusion

Data obtained in our study showed as a dose-dependent antifungal activity of lemon, bitter orange, and bergamot essential oils (EOs) can inhibit the fungal growth. These EOs, at proper concentrations, can inhibit both the in vitro A. flavus mycelium growth and AFB1 genesis. Currently, it is still difficult to control exposure of man and animals to mycotoxins, because these compounds naturally occur in the environment. EOs and natural products are generally categorized under “generally recognized as safe” as food additives (Kumar et al. 2017) focusing interest of both scientific community and stakeholders for integration of plant extracts or essential oils in both crop food protection and food safety management. In addition, the growing awareness towards the adverse effect of synthetic chemical preservatives on health and environment focused the interest of both scientific community and stakeholders for integration of plant extracts or essential oils in both crop food protection and food safety management.

This is the first study that evaluated effects of five citrus EOs both in mycelium growth and AFB1 synthesis, and this result can be applied in applied research in food protection and preservation by fungal and AFB1 contamination.

This specific effect deserves deeper study evaluating synergistic effects deriving by citrus EO mixtures, in view of their practical application as a future eco-friendly crop, storage system protection and aimed to reach a minor human health risks. Furthermore, the demonstrated efficacy of citrus EOs can be a pivotal key to recovery and reuse of citrus fruit waste permitting an integrated recycle of waste with economic advantages for the Mediterranean countries.