Antifungal, antitoxigenic, and antioxidant activities of the essential oil from laurel (Laurus nobilis L.): Potential use as wheat preservative

Abstract Essential oils (EOs) are widely used in the food industry as natural food preservatives to extend product shelf life and as flavoring agents. The aim of this work was to study the chemical profile of the EO from laurel (Laurus nobilis) and its antifungal, antitoxigenic, and antioxidant activities. The extractive yield of the EO from Algerian laurel was 1.13% being 1,8‐cineole the most dominant compound (35.5%) by gas chromatography–mass spectrometry analysis. The values of minimum inhibitory concentration and minimum fungicidal concentration (MFC) against Aspergillus flavus were 1.75 and 2 mg/ml, respectively. The production of aflatoxin B1 was inhibited by EO concentrations between 0.25 mg/ml (15% decrease) and 1.50 mg/ml (86% decrease), and it was totally inhibited at the MFC value. The EO showed a wide antifungal spectrum against other species in a dose‐dependent manner. In a food‐model study, the L. nobilis EO showed remarkable efficacy in fumigated wheat grains, providing from 51.5% to 76.7% protection against A. flavus during 6‐month storage. The L. nobilis EO showed good free radical scavenging activity by DPPH assay (IC50 value of 602 μg/ml) and moderate antioxidant activity in the β‐carotene bleaching assay (46% inhibition of linoleic acid oxidation). The conclusions of this study justify future research for the application of EO from laurel as a natural preservative to improve food safety and extend shelf life by controlling spoilage and toxigenic molds as well as oxidative damage.


| INTRODUC TI ON
The aflatoxins are mycotoxins produced primarily by toxigenic strains of the fungi Aspergillus flavus and Aspergillus parasiticus. The most frequently found aflatoxin in contaminated food samples is aflatoxin B1 (AFB 1 ), which is a genotoxic and carcinogenic substance (IARC, 2012). In Africa, about 250,000 hepatocarcinoma-related deaths occur annually due to aflatoxin ingestion (Wild & Gong, 2010).
Climate change is anticipated to impact on the presence of aflatoxins in food in Europe.
The Joint FAO/WHO Expert Committee on Food Additives has recently calculated international estimates of chronic dietary exposure to aflatoxins (FAO/WHO, 2018). The mean upper bound (UB) dietary exposure to aflatoxins ranged from 1.3 ng/kg body weight per day (many European countries) to 34.8 ng/kg body weight per day (many African countries). For many European countries, wheat was the main contributor to the UB dietary exposure to aflatoxins, which reached between 37% and 76.5%.
The frequency of contamination of world crops by aflatoxins shows that the strategies currently used are insufficient to guarantee the security of the foods and that it is necessary to develop others, as a complement or substitution of those already existing (Byakika et al., 2019;Lasram et al., 2019;Yassein, El-Said, & El-Dawy, 2020). In this context, strategies based on the use of compounds naturally recognized as not harmful to the environment and to health, seem interesting. Indeed, plants produce different secondary metabolites for their protection against pathogenic attacks and environmental stresses (mechanical, biological, or climatic). These compounds could possibly be used as a means of combating fungal contamination and/or mycotoxins . Essential oils (EOs) are molecules of natural origin, biodegradable and are therefore considered as a possible alternative to synthetic pesticides (Ben Miri, Ariño, & Djenane, 2018). In view of their different biological properties, EOs have shown important antimicrobial activities that can be used for the control of the contamination by molds and mycotoxins in agricultural commodities (Abd El-Aziz, Mahmoud, Al-Othman, & Al-Gahtani, 2015;Ben Miri et al., 2018;Bluma, Amaiden, Daghero, & Etcheverry, 2008;Tian et al., 2012). EOs can also be used as flavoring or food additives (Taoudiat, Djenane, Ferhat, & Spigno, 2018), as well as antioxidants and antibacterial agents in foods (Djenane, Gómez, Yangüela, Roncalés, & Ariño, 2019), whose utilization has been authorized in the USA (FDA, 2019).
Lauraceae comprises numerous aromatic and medicinal plants, including Laurus nobilis, commonly known as laurel or bay laurel. It is a plant native to the southern Mediterranean region that grows to a height from 6 to 10 m, which is grown commercially for its aromatic leaves. L. nobilis is one of the cultivated and endemic species to Algeria, which is used mostly in cuisine and traditional medicine. The EO from Algerian wild-growing laurel has shown effective antimicrobial and antioxidant properties (Boughendjioua, 2017). A direct relationship between the content of bioactive phenols and the biological activity of the L. nobilis EO has been previously reported, in which 1,8-cineole was identified as a major phenolic compound (Caputo et al., 2017). However, it is difficult to correlate the antifungal activity of EOs with single compounds. There is evidence that even minor components play a critical role in antimicrobial activities, and it appears that the inhibitory effects are the result of their synergistic action (Cabral, Pinto, & Patriarca, 2013;Djenane et al., 2019).
The aims of the present study were to investigate the antifungal, antitoxigenic, and antioxidant activities of the EO from laurel and to assess the potential use as wheat grain preservative during long storage.

| Plant material, extraction, and chemical characterization of L. nobilis EO
The fresh aerial parts of L. nobilis (only leaves) were collected during sunny days at the end of June 2015, in the province of Tizi-Ouzou (Mesghana), 100 km north-east from Algiers (Algeria). The geographic coordinates of collection sites were latitude 36°42′42″N, longitude 4°02′45″E, elevation above sea level 206 m, and 40 km from Mediterranean Sea. The identification was firstly given based on their morphological appearances and according to the flora of Algeria (Quézel & Santa, 1963). A voucher specimen was deposited at the Laboratory of Microbial Systems Biology (voucher number LBSM-BY15). The collected leaves were dried at ambient temperature in shady and dry conditions for 3 months, then packed, and stored in darkness until the extraction of EO.
Essential oil of two hundred grams (200 g) of dried leaves was extracted by hydrodistillation in Clevenger's apparatus during 3 hr until no more EO was obtained ( Figure 1). The water traces in the EO were removed with anhydrous sodium sulfate (Na 2 SO 4; Sigma-Aldrich). EOs were weighted and conserved at 4°C until use. Total phenolic compounds in the L. nobilis EO were estimated spectrophotometrically according to the Folin-Ciocalteu method with some modifications . Gallic acid (GA) was used as phenolic compound standard for the calibration curve. Results were expressed as micrograms of GA equivalents per milligram of sample dry weight (μg GAE/mg), using the equation of the linear regression line of the calibration curve. The same EO was characterized by GC-MS (Agilent model 6,850 and 7,890). The EO (10 μl) was dissolved in hexane (100 μl), and 2 μl of the solution was injected on apolar capillary column DB-5 (length 30 m × 0.25 mm i.d., film thickness 0.25 μm). Helium was used as the carrier gas at a flow rate of 1.0 ml/min. The column inlet pressure was 8.07 psi. The GC column oven temperature was increased from 60 to 245°C at 3°C/min, with

| Fungal material
Aspergillus flavus E73 strain was obtained from the culture collection of the Laboratory of Microbial Systems Biology at Kouba.
Additionally, fungal strains of Aspergillus carbonarius, A. fumigatus, A. niger, A. ochraceus, A. tamarii, A. terreus, Fusarium sp., Penicillium sp., and Rhizopus sp., isolated in our laboratory during mycological analysis of cereals and spices, were used to study the antifungal spectrum of the EO. Spore inoculum of each fungi was prepared from a culture on PDA at 25ºC for 7 days where spores were obtained by washing the Petri dish with 20 ml of 0.1% Tween 80 solution. The number of spores, determined using a hemocytometer slide (depth 0.2 mm, 1/400 mm 2 ) under a light microscope, was adjusted to 1 × 10 6 /ml throughout the study.
For the confirmation of the aflatoxinogenicity, 10 μl of spore suspension of A. flavus E73 strain was cultivated in Erlenmeyer flask containing 25 ml of the SMKY liquid medium at 25ºC for 10 days.
The content was filtered (Whatman N° 1) and extracted with 20 ml chloroform. After stirring and then decanting, the chloroform phase was evaporated to 1 ml. A volume of 50 µl was spotted on a thin layer chromatography plate (TLC). The development was carried out in a standard tank (20 × 20 cm) previously saturated with the solvent system toluene-isoamyl alcohol-methanol (90/32/2; v/v/v).
After migration, the plate was removed and dried at 60°C for 24 hr.

| In vitro antifungal and antiaflatoxigenic assays of L. nobilis EO
For the antifungal activity of L. nobilis EO against A. flavus, different concentrations of EO were added to 15 ml PDA at 45-50°C to obtain final concentrations in the range 0.25-2.00 mg/ml and poured into Petri dishes. Thereafter, 10 μl of spore suspension was spotted in the center of each Petri dish and incubated at 25°C for 7 days. The controls were prepared in parallel without EO. Measurements were F I G U R E 1 Scheme of Laurus nobilis essential oils extraction (hydrodistillation) made daily by taking the average of two perpendicular diameters of each colony. The comparison of the dimensions obtained with those of the controls made it possible to calculate the percentage inhibition (I%) at day 7, according to the following formula: C: average colony diameter (mm) in the control; T: average colony diameter (mm) in the treatment.
Additionally, the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) were assessed for a series of foodborne fungi by the broth dilution method (Shukla, Kumar, Singh, & Dubey, 2009). Different concentrations (0.25-2.00 mg/ml) of L. nobilis EO were added to 10 ml SMKY broth medium in test tubes. Tubes with only SMKY medium (10 ml) were used as controls.
The tubes were inoculated with 10 µl of spore suspensions and incubated at 25°C for 7 days. The MIC was determined as the lowest concentration of EO that inhibited fungal growth. After determining the MIC, 100 μl from the corresponding tubes was subcultured on PDA plates for the determination of the MFC. After 72 hr incubation at 25ºC, MFC was determined as the lowest EO concentration that prevented visible growth.
For the antiaflatoxigenic assay, 10 μl of spore suspension of A.
flavus was inoculated in Erlenmeyer flask containing 25 ml of SMKY medium with different concentrations (0.25-2.00 mg/ml) of L. nobilis EO and incubated at 25°C for 10 days (Mishra et al., 2013) together with the corresponding control tubes without EO. After incubation, the culture media was filtered (Whatman N° 1), and then, the mycelium dry weight was determined after desiccation at 80°C for 12 hr.
For the extraction of AFB 1 , the same procedure was followed as described in section of test confirming the aflatoxinogenicity of the strain. The intensity of the fluorescence of the spots confirmed the presence of AFB 1 . For the quantification of AFB 1 , the blue spots on TLC plates were scraped out, dissolved in 5 ml cold methanol, and centrifuged at 3,000 g for 5 min. The absorbance of the supernatant was made using a UV-visible spectrophotometer at 360 nm. The

| Activity of L. nobilis EO against A. flavus growth in stored wheat grains
Two varieties of wheat (Triticum aestivum), namely AS 81,189 A (Ain Abid) and HD 1,220 (Hiddab), harvested during 2016 in Dar El Beida, Algiers (Algeria), were used. The geographic coordinates of collection sites were latitude 36°42′51″ (N), longitude 3°12′45″ (E), elevation above sea level 17 m, and 10 km from Mediterranean Sea. Wheat grains were collected (around 10 kg of each variety), placed in sterile bags, and brought to the laboratory. The moisture content of the grains was 12% as determined by oven drying. The effect of L. nobilis EO on wheat was evaluated according to the method recommended by Prakash, Singh, Kedia, and Dubey (2012). The wheat grains of each variety were surface-sterilized with a 1% solution of sodium hypochlorite and rinsed 3-5 times with sterilized distilled water. One kilogram of each variety was kept separately in plastic boxes with an aerial volume of 2 L. One milliliter (1 ml) of spore suspension of A.
flavus was inoculated into grain samples through uniform spraying.
Then, the grain samples in the plastic boxes were fumigated with the EO at the MIC value with respect to aerial volume of container.
Control samples were prepared in parallel without EO addition. All the containers were kept airtight and stored for 6 months at 15°C and 62% relative humidity.
For mycological analysis, the spread-plate method was used. Ten grams of treated and control grain samples was placed in 250-ml flasks containing 90 ml of sterile Tween water (0.1%) and homogenized in Stomacher for 2 min. Serial decimal dilutions up to 10 -3 were made, and 0.1 ml of each dilution was inoculated on prepoured, solidified DRBC agar plates, and the inoculum spread with a sterile Drigalski spatula. Plates were incubated at 25ºC for 5 days, and the count was expressed as cfu/g of A. flavus.

| Antioxidant activity of L. nobilis EO
The antioxidant activity of EO was evaluated by two methods: the DPPH free radical scavenging assay and the β-carotene/linoleic acid bleaching assay. DPPH test evaluates the capacity of the EO to scavenge 2,2-diphenyl-1-picrylhydrazil radical (DPPH•). Briefly, in clean and dry tubes, volumes of 50 µl of different concentrations (100, 200, 400, 600, 800, and 1,000 µg/ml) of L. nobilis EO and standard BHT were added to 5 ml of 0.004% (w/v) methanolic solution of DPPH and incubated in darkness at room temperature for 30 min.
Thereafter, the absorbance was measured against a blank at 517 nm. DPPH radical scavenging activity was expressed in terms of inhibition percentage (I%) and was calculated using the following formula: A blank : absorbance of the control; A sample : absorbance of the sample.
The value of the inhibitory concentration (IC 50 ) represents the dose of the EO that causes the neutralization of 50% of the DPPH radicals. IC 50 was estimated by extrapolation by plotting the percent inhibition (I%) versus concentration curves.
The β-carotene/linoleic acid bleaching assay is a complementary method to assess the antioxidant activity of compounds (Miraliakbari & Shahidi, 2008). Briefly, 0.5 mg of β-carotene was dissolved in 1 ml of chloroform, 25 μl of linoleic acid, and 200 mg Tween 40. The chloroform was completely evaporated; then, 100 ml of aerated distilled water was added and the mixture was shaken. The sample (2 g/L) was dissolved in DMSO, and 350 µl of sample solution was added to 2.5 ml of the resulted mixture and then incubated in a water bath at 50°C for 2 hr with blanks. BHT was used as a positive control and DMSO as a negative control. The absorbance was measured at 470 nm, and the antioxidant activities (I%) were calculated using the following formula: A β-carotene after 2 hr assay : absorbance of β-carotene after 2 hr assay; A initial β-Carotene : absorbance of β-carotene at the beginning of the experiments.

| Statistical analysis
All experiments were repeated three times, and the results were analyzed by one-way ANOVA test (ρ < .05) using STATISTICA version 6.

| Yield, chemical, physical, and organoleptic properties of L. nobilis EO
The yield of L. nobilis EO was calculated according to the plant dry matter. The yield was 1.13 ± 0.03% (Table 1). This value was comparable to that of 1.2% obtained by Haddouchi, Lazouni, Meziane, and Benmansour (2009)  However, the yield of our EO was higher than that of 0.6% found in L.
nobilis from El Kala, Algeria (Ouibrahim et al., 2013). On the contrary, the EO yield was lower than that given by the same species in different regions of Morocco, whose performance can reach 2.5% (Yilmaz, Timur, & Aslim, 2013). Vekiari et al. (2002)  The presence of phenolic compounds in botanical products such as EOs has been gaining much attention due to their antioxidant, antimicrobial, and flavoring properties in foods. Yilmaz et al. (2013) found that phenol contents in L. nobilis EO were 112.3 μg/mg, which are higher than that found in our study (26 μg/mg).

| Antifungal activity of the EO from L. nobilis
The antifungal activity of L. nobilis EO against A. flavus during the seven days of incubation is shown in Figure 2. As compared to the control, the colony diameter was significantly reduced (p < .05) with increasing concentrations of EO from 0.25 to 1.5 mg/ml in a dosedependent manner. Also, the onset of fungal growth was delayed 3 days at 1.25 mg/ml and 4 days at 1.5 mg/ml, while the fungus growth was totally inhibited with the higher treatments of 1.75 and 2 mg/ml. The percentage (%) inhibition of mycelia growth after day 7 of incubation is presented in Figure 3. The analysis of data revealed that L. nobilis EO at 1.5 mg/ml caused 76.1% inhibition in mycelial growth of A. flavus as compared with the control (p < .05), while the inhibition reached 100% at concentrations of 1.75 mg/ml and above.
The MIC and MFC techniques were employed to assess fungistatic and fungicidal properties of the EO against a series of foodborne fungi. L. nobilis EO was tested up to a maximum concentration of 2 mg/ml. The data of fungitoxic spectrum are shown in Table 3.
The MIC against the toxigenic strain of A. flavus was recorded at  (Table 2). Hmiri et al. (2011) reported that the antifungal activity of E. camaldulensis rich in 1,8-cineole was due at least partially to the action of this monoterpene, and the works of Shukla, Singh, Prakash, and Dubey (2012) and Caputo et al. (2017) showed that pure 1,8-cineole inhibited mycelial growth but at higher concentrations than complete EOs. According to Gusarov, Shatalin, Starodubtseva, and Nudler (2009) monoterpene alcohols such as linalool and terpineol. Several authors reported that these compounds increase the permeability of the plasma membrane and inhibit the respiration process on the mitochondrial membrane of fungi (Deba, Xuan, Yasuda, & Tawata, 2008;Imelouane et al., 2009).
The study of MIC and MFC is important to determine the minimum dose to control fungal populations and gives opportunity for EOs to come in close contact with fungal spores in the medium (Kumar, Shukla, Singh, Prasad, & Dubey, 2008).

| Antiaflatoxin activity of the EO from L. nobilis
The mycelium dry weight and the production of AFB 1 were measured after an incubation period of 10 days, as shown in Figure 4.
The recorded data explain an inversely proportional relationship between the amount of EO and the mycelium dry weight the AFB 1 production. As the dose of EO increased in the SMKY broth, the inhibition of mycelium dry weight was accompanied by a decrease in the synthesis of AFB 1 . The data revealed that both mycelium dry weight and AFB 1 production were significantly inhibited (p < .05) by EO concentrations between 0.25 and 1.50 mg/ml as compared to the control. The percentage of inhibition ranged from 11.1% to 80.9% for mycelium dry weight and from 14.9% to 85.7% for AFB 1 production.
A complete growth inhibition was observed at EO concentrations above 1.75 mg/ml, so no aflatoxin was detected.
A positive correlation was found between the decrease in mycelium dry weight and the inhibition of AFB 1 with increasing con- as reported by Chaudhari et al. (2020). The oil exhibited complete protection of stored maize from fungal infestation (without affecting seed germination) and subsequent AFB 1 production at 2.5 and 1.5 µl/ml, respectively.

| Application of L. nobilis EO in stored wheat grains: Protection against A. flavus by fumigation
The experimental design is shown in Figure 5. As described in Section 2.5, the EO was obtained from Algerian L. nobilis, and lots F I G U R E 4 Effect of Laurus nobilis essential oil on dry mycelium weight and aflatoxin B1 (AFB 1 ) production by Aspergillus flavus during 10 days of incubation of wheat grains samples were fumigated with this EO as potential natural preservative. Wheat grains were inoculated with pathogenic fungi (A. flavus) and stored during 6 months at 15°C and 62% relative humidity. Wheat grains samples were analyzed for A. flavus growth.
The potential use of L. nobilis EO as wheat preservative was based on the percentage of protection against A. flavus in L. nobilis EOfumigated wheat grains as compared to untreated samples (control).
The EO showed remarkable efficacy in fumigated wheat samples during storage for up to 6 months providing from 51.5% to 76.7% protection of wheat grains from A. flavus contamination (Table 4).
In general, to obtain similar protective effects in food products as those observed in vitro, higher concentrations of EOs should be used Tian et al., 2011). This can be explained by the fact that when the EOs are in contact with the food matrix, some active volatile substances are bound by food components. In addition, food lipids can form a coating around the microorganisms, protecting them from antimicrobial agents. Also, the lower water content of grains as compared to laboratory media could hinder the transfer of antimicrobial molecules to the active site in the microbial cell . Thus, it has been hypothesized that penetration of oils into the internal parts of the grain could improve in the presence of water, and thus, toxigenic microorganisms could be more easily controlled in the inner parts of moist seeds. Therefore, the nature of the food system, the density of the fungal inoculum, the storage conditions, and their moisture content must be taken into consideration when determining the in vivo concentration of EOs against food contamination by molds. thus be used in agricultural crops according to the specifications of organic farming. EOs are volatile, so vapors can easily be removed from fumigated foods after drying in the sun. In this regard, active packaging and microencapsulation technologies can be used with EOs for the storage of foodstuffs (Bouzidi, Lakhlef, Hellal, & Djenane, 2019;Djenane et al., 2019;García-Díaz, Patiño, Vázquez, & Gil-Serna, 2019;Li et al., 2019).
In conclusion, the EO of laurel leaves could play an important role in stored wheat grains protection, reducing risks associated with the use of synthetic insecticides. Given the presumed negative impact of synthetic fungicides on human health, the use of plant-based antifungal agents has generated considerable interest in the agri-food industries. In the β-carotene bleaching assay, the oxidation of linoleic acid was moderately inhibited by the EO (46 ± 1.4%) as compared to positive control BHT (95 ± 1.6%). Data of β-carotene bleaching assay were around 15% lower than that provided by the radical scavenging activity.

| Antioxidant activity of the EO from L. nobilis
When exposed to environmental oxidative stress, molds activate several lines of defense to protect against the cellular damage that reactive oxygen species (ROS) can cause. It has been suggested that the first line of defense is represented by the activation of antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD), and peroxidase. Modulation of CAT and SOD activity was directly associated with a change in ROS levels but also with a modulation of aflatoxin production in A. flavus (Narasaiah, Sashidhar, & Subramanyam, 2006). In addition, it has been shown that aflatoxin production is induced by an increase in oxidative stress (ROS) in A. flavus and A. parasiticus and that aflatoxinogenic strains require higher levels of oxygen compared to nonaflatoxinogenic strains (Jayashree & Subramanyam, 2000).
Lipid peroxidation is a consequence of ROS formation, and it has been shown to be involved in the synthesis of aflatoxin. Kim et al. (2008) showed that the biosynthesis of aflatoxins is related to the formation of ROS and the peroxidation of fungal cells. On the other hand, Ferreira et al. (2013) reported that the mechanism of inhibition of Curcuma longa EO and curcumin on the production of aflatoxins might be related to the inhibition of the ternary steps of aflatoxin biosynthesis involving the peroxidation and oxygenation of lipids. This could therefore have contributed to the observed antiaflatoxinogenic effect of the EO from L. nobilis. Our results showed significant antioxidant activity, so the high AFB 1 inhibitory efficacy may be partly due to its antioxidant properties on lipid peroxidation in the biosynthesis process of AFB 1 .

| CON CLUS ION
Essential oils are increasingly used in the food industry as an alternative of synthetic products, as natural food preservatives to extend product shelf life since they have antibacterial, antifungal, and antioxidant properties. The safety risk in stored grains, such as the presence of toxigenic molds and mycotoxins, is a major concern for food safety. The frequency of contamination of stored wheat by aflatoxins shows that current control strategies are insufficient to guarantee food safety and that it is necessary to develop other complementary agri-food technologies that are more sustainable than currently implemented ones. In this context, a natural alternative strategy was developed to prevent the growth of toxigenic molds during the storage of wheat grains. L. nobilis EO led to noticeably inhibition of A. flavus and AFB 1 production in vitro, as well as suppression of common foodborne fungal species. In stored wheat grains, the fumigation with L. nobilis EO reduced A. flavus counts in the treated grains. In addition, the EO showed high antioxidant activity.
In this respect, the use of biopreservation to extend shelf life during wheat storage could be a sustainable solution for the development of Algerian Agri-food industries to reduce losses of products due to fungal growth and mycotoxin contamination.
The conclusions of this study justify future research for the application of EOs as fumigants in food systems to improve their safety and shelf life by controlling spoilage and toxigenic molds as well as lipid peroxidation. These findings should be taken into account in the development of safe and eco-friendly specific technologies such as new biopolymers and nanoparticles coatings.

ACK N OWLED G M ENTS
The present research was carried out with the financial support of the Ministry of Higher Education and Scientific Research of Algeria (Grant D00L01UN150120180002) and the Government of Aragón and FEDER 2014-2020 (Grant Grupo A06_20R).

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

AUTH O R S' CO NTR I B UTI O N S
AB, YBM, MA, and LAO were involved in experimental and analytical work. LM, DD, and AA were involved in the designing of the experiment, data analysis, and interpretation of the results. All authors reviewed and approved the final manuscript.

E TH I C A L A PPROVA L
The experiment does not include any animal or human testing.