SUPPRESSION OF AFLATOXINS PRODUCTION IN ARTIFICIALLY INFESTED MAIZE GRAINS WITH ASPERGILLUS FLAVUS DURING STORAGE CONDITIONS

Maize is the one of important crops in Egypt. Aflatoxins (AFs) are the foremost cancer present compounds by Aspergillus flavus (A. flavus) and cause health risks to human and animals. This study aimed to suppression of aflatoxins production by A. flavus by using different concentrations of natural substances (carnation oil, lemongrass oil, propolis and beewax) and chemical substances (salicylic acid and potassium sorbate) on maize grains. The strains of A. flavus were isolated from local maize grains on Potato Dextrose Agar (PDA) and detect its ability of aflatoxins production on coconut agar media. Samples (100g) of sterilized maize grains were treated individually with different concentration of carnation oil, lemongrass oil, salicylic acid and potassium sorbate (0.25, 0.5, 1.0, 2.0, 4.0 and 6.0%) and at concentration 1% and 4% for propolis and beeswax each, then inoculated with A. flavus and stored for 30 days at 28±2 ̊C. AFs reduction was determined by using High Performance liquid Chromatography (HPLC). All the tested substances had active effect in inhibition of AFs production by A. flavus in stored maize grains. The production of Aflatoxin B1 and B2 (AFB1 and AFB2) was decreased to about 93% and 99% at concentration of 0.25% carnation oil. Lemongrass oil almost completely inhibited AFB1and AFB2 production (99.12% 99.98% and 99.98% -99.99%, respectively) at concentration of 2% 6%. Potassium sorbate and salicylic acid (0.25%6%) that were significantly effective controlling aflatoxins production on maize grains compared with control. While, the propolis and beewax found to be the most active to protect maize grains against fungi. Natural substances, carnation oil, lemongrass oil, beewax and propolis had higher active effect at low concentration on aflatoxin production more than salicylic acid and more safe for human used.


INTRODUCTION
Maize (Zea mays) is that the third field crop within the world, also considered as one of important crops in Egypt. It's planted on 378,000 Fadden of land (FAO, 2013). Within the storage, amount several pests and parasites attack maize. However, fungi also are vital and thought of because the second explanation for grain losses (Ominski et al., 1994 and Kumari et al., 2019). Aspergillus species could be a quite common fungus within the setting, and may be a problem in hold on grains. Fungus genus, A. parasiticus and A. flavus synthesize aflatoxins once they grow on a variety of vulnerable food and feed crops. These fungi are reported together of the extreme contaminants of varied plants and plant material like maize, peanuts, rice, cotton seeds and spices, in addition to exploit product (El-Nagerabi et al., 2012). Aflatoxins are among the foremost cancer present compounds acknowledged (or identified) and that they cause vital health risks to humans and animals (Elshafie et al., 2011 andQureshi et al., 2015).
Over the past few years, there are varieties of approaches that may be taken to attenuate aflatoxins contamination in grains and these involve prevention of fungal growth and therefore aflatoxins formation to reduce or eliminate aflatoxins from contaminated grains. It's a well-established undeniable fact that, some plant based mostly essential oils contain compounds that are able to inhibit plant growth, there's appreciable interest in these essential oils from aromatic plants with antimicrobial properties to manage pathogens and toxin-producing molds (Soliman andBadeaa, 2002 andTepe et al., 2005). Though the bulk of the essential oils are classified as usually Generally Recognized As Safe (GRAS), their use in foods as preservatives is usually restricted because of flavor issues (Lambert et al., 2001). At the present many product are used as antifungal agents together with common preservatives as essential oils like carnation and lemongrass (Smith-palmer et al., 2001), organic acid (salicylic acid) (Rajesh and Mubasshirin, 2018), organic salt (potassium salt) (Merck, 2015) and natural products as beeswax and propolis (Buchta et al., 2011). Propolis is that the resinous substance collected honey bees from numerous plant sources. The antifungal activity of propolis has been evaluated by Quiroga et al., 2006;Aly and Elewa, 2007;Ghasem et al., 2007 andYang et al., 2010. The chemical composition of propolis is extremely complex, containing over 150 elements like flavonoids, phenolic resin acids and their esters, alcohols, ketones, amino acids, and inorganic compounds (Hegazi et al., 2000;Banskota et al., 2001;Marcucci et al., 2001 andBankova, 2005a). The honeybee's wax has a very wide spectrum of useful applications, cosmetics, food process (food packaging, process and preservationnatural additive) and medication (coating pills, antibiotic properties) (Krell, 1996). Therefore, in the present investigation, suppression of aflatoxin production of A. flavus by several substances either natural as carnation oil and lemongrass oil, propolis and beewax or chemical as salicylic acid and potassium sorbate were evaluated for their efficacy as preservative against aflatoxigenic fungi in maize grains.

Isolation and identification of A. flavus
Maize grains were collected from local markets in Egypt. The surface sterilized maize grains were placed on Potato Dextrose Agar (PDA) medium and incubation at 28 ±2°C for 7 days. At the end of the incubation period, Aspegillia isolates of fungal were identified based on light macroscopic and microscopical characteristics. Aspegillia isolates of fungal colonies were transferred onto fresh PDA plates to study their morphological characteristics. The isolates were identified using the taxonomic key prepared by using fungal keys and manuals (Klich, 2002 and Samson et al. 2004).

Mycotoxicological analysis of A. flavus isolates
The ability of A. flavus isolates for production of AFs was examined using coconut agar media (CAM) (100 g of sliced coconut was homogenized for 5 min. with 300 ml of hot water. Then filtrate and adjust pH to 7.0 using 2N NaOH and adding 20g/L agar then autoclave, Davis et al., 1987).
Plug from A. flavus (39 isolates) was placed on the center of CAM plates and incubated for 7 days. After the incubation period, plates were examined under (UV) lamp in a dark room for fluorescence to detect the presence of aflatoxin production. If the mould fluoresced under UV light was considered to be aflatoxin positive and confirmed as an aflatoxigenic form of A. flavus.

Screening and detecting of aflatoxins produced by different isolates of A. flavus
To confirm the correlation between fluorescence and aflatoxin production, five of toxigenic strains of A. flavus (NOs.1, 2, 5, 7 and 8) were used in this test. The colonies were grown on Yeast Extract Sucrose (YES; 2 % yeast extract and 20% sucrose) (Abdollahi and Buchanan, 1981). Spore suspensions of the isolates were prepared and adjusted to approximately 10 6 spores /ml by using a hemocytometer. One ml spore suspension was inoculated into 50 mL of sterile YES and incubated at 25 °C for 14 days. The entire culture was filtrated by filter paper No.3, the filtrate was Natural products (propolis and beewax): purchased from local market. Propolis sample was kept at room temperature in dark. Crude propolis was grounded into powder and macerated in acetic acid (4g propolis + 96 ml acetic acid) and lactic acid (4g propolis + 96 ml lactic acid). The extract of propolis was filtered through Whatman No. 1 filter paper to obtain a stock solution (4%). Meanwhile beeswax was melted at 65°C in hot water, prior to use, at the rate of 4g beeswax: 96 ml water (Badawy, 2016 and Abdel-Kader, et al., 2019).
Prior maize grains treating, dilutions of working solutions were prepared at concentration 0.25, 0.5, 1.0, 2.0, 4.0 and 6.0% for carnation oil, lemongrass oil, salicylic acid and potassium sorbate and 1% and 4% for propolis and beeswax.

Evaluation of aflatoxins production in treated, stored maize grains
One hundred grams of maize grains was placed in flasks (500 ml) with 10 ml of dH2O and sterilized for 20 min in an autoclave. Each sterilized flask of maize grains were treated individually with the tested materials at the proposed concentrations and shaken for few min. The day after, all the treated maize grains were inoculated with 2 mL A. flavus (isolate No. 5 that recorded the highest aflatoxin production) spore suspension (10 6 spore /ml), shaken well. The treated and un-treated inoculated maize grains were stored for 30 days at 28±2ºC. After incubation period, the moldy maize grains were autoclaved at 100 °C for 30 min and used for the extraction of aflatoxins according to CB method (AOAC, 2016).

Determination of aflatoxins in maize grains
The determination of Aflatoxins (AFs) was performed using High Performance liquid Chromatography (HPLC), according to (AOAC, 2016). The HPLC system used for AFs determination was ultimate 3000 Thermo Fisher system (Germany) equipped with auto sampler, pump, fluorescence detector and a C18 column chromatography Phenomenex (250x4.6mm, 5μm). The mobile phase, water: methanol: acetonitrile (60:30:10, v/v/v), was isocratically flowed at 1.2 ml/min. AFs were measured at 360 nm excitation and 440 nm emission wave length.

Statistical analysis
Results were subjected to one-way analysis of variance (ANOVA) of the general liner model (GLM) using SAS (1999) statistical package. The results were the average of three replicates (p ≤ 0.05).

Screening and detecting of aflatoxins produced by different isolates of A. flavus
Based on the cultural and physiological characteristics, thirty-nine isolates were referred to as A. flavus from maize grain samples using a taxonomical key and species represented by (Klich, 2002 and Samson et al., 2004). The detection of aflatoxigenic and non aflatoxigenic A. flavus isolates by using ultraviolet light (UV) revealed that five (12.82%) of isolates (NOs. 1,2,5,7 and 8) were aflatoxigenic (positive) and 34 (87.18%) of isolates were non-aflatoxigenic (negative). The detection by UV at 365 nm recognized aflatoxigenic by turn out blue fluorescent colonies within the center of glass petri dish of CAM.
Thin layer chromatography analysis for aflatoxins production by Aflatoxigenic isolates on (YES) medium showed blue fluorescing spots (Fig 1) under long wave UV (365 nm) parallel to AFB1 and AFB2 standards. The results of TLC showed that, five tested of A. flavus isolates (NOs. 1, 2, 5, 7 and 8) were able to produce one or more types of aflatoxin(s). In this respect, isolates NOs.1, 2 and 8 produced AFB1 only, whereas, isolates NOs. 5 and 7 produced AFB1 and B2 (Fig 1). From the quantitative method of HPLC the concentration of AFB1 and AFB2 produced by isolates (NOs 5 and 7) were calculated. It was found that the concentration of AFB1 was calculated as 1060 and 608 µg/kg maize grains for strain 5 and 7, respectively. Meanwhile, the concentration of AFB2 was 460 and 700 µg/kg maize grains for strain 5 and 7, respectively. So, the total production of AFB1 and B2 calculated as 1520 and 1308 µg/kg maize grains for the two isolates 5 and 7, respectively, on the basis of capability to produce.  Table 1 represented the effect of essential oils (carnation, lemongrass) on AFB1 and AFB2 production by A. flavus in stored maize grains. The highest decrease in AFB1 production was observed in carnation oil at concentration 2-4% (0.21-0.01µg/kg) while; AFB2 was highly decreased at 1-4% of carnation oil. However, carnation oil suppressed the production of AFB2 to more than 99% from the concentration of 0.25%. The complete reduction of AFB1 and AFB2 production was recorded at 6% (Figs 2 and 3). Figs 2 and 3 illustrated the reduction percentage of AFB1 and B2 after treated with carnation oil and lemongrass oil. AFB1 production rate was reduced about 75.34%, 90.88% and 97.06% following treatment with lemongrass oil at a concentration of 0.25%, 0.5% and 1%, respectively.
However, AFB1 and AFB2 production by A. flavus was approximately complete inhibited by treatment with 2% of lemongrass oil (99.12%-99.98%), respectively and by 6% of lemongrass oil (99.98%-99.99%), respectively (Figs 2 and 3). There was a significant difference in aflatoxins production of control compared with the carnation oil and lemongrass oil treated samples (Table 1).In the current study, carnation oil had the highest reduction effect on AFB1 and B2 production were recorded form concentration (0.25%-1%) compared with lemongrass oil.   Table (2) showed that potassium sorbate and salicylic acid had a highly effect on the reduction of AFB1 and B2 at all concentration. Both potassium sorbate and salicylic acid at different concentrations from 0.25% to 6% that were significantly effective in controlling aflatoxin production on maize grains caused by A. flavus compared with control. The highest decrease of AFB1 and AFB2 were observed in 4% of potassium sorbate (99.97% and 99.98%) and salicylic acid 98.27% -99.95%). While, the completely reduction of AFs production observed at 6% of both.
Potassium sorbate was recorded a reduction percent (99.83% -100%) for AFB1 and (99.74% -100%) for AFB2 production. While, Salicylic acid, the minimum inhibition of AFB1(83.28%) and AFB2 (91.86%) caused by salicylic acid in 0.25%, whereas the maximum reduction of AFB1 and AFB2 were (100%) that exhibited by 6% salicylic acid, these results shown in Figs 4 and 5. Nevertheless, the propolis and beeswax found to be the most active substance as preservative material on maize grains.

Potassium sorbate
Salicylic acid

DISCUSSION
In the present study, five strains of A. flavus isolated from maize grains were used to evaluate their ability to produce aflatoxin(s) in synthetic medium (YES).The most aflatoxigenic isolates of A. flavus was used to estimate the production of AFB1 and AFB2 in stored maize grains for 30 days treated with different concentrations of carnation oil, lemongrass oil, salicylic acid, potassium sorbate, beeswax and propolis. It is well known that aflatoxigenic fungi are frequently found in foodstuffs and animal feeds and are associated with a wide spectrum of stored agricultural commodities. However, not all Aspergillus species are able to produce aflatoxins .
In this concern, regional variations in biological weapon contamination of crops is also thanks to atmospheric condition and to agricultural irreversible injury in cell membrane, cytomembrane and cellular organelles once A. parasiticus and A. flavus were exposed to totally different essential oils.
It was found that organic acids such as salicylic acid have antimicrobial effects (Kupferwasser et al., 2003). It seems that salicylic acid prevented aflatoxin production due to reduced fungal growth and could delay AFB1 aggregation for a few days. In this regards, In this concern, there are some papers about the application of beeswax based edible films and coatings in the preservation of fruits and vegetables et al., 2019). However, according to our knowledge, there are no many papers about the incorporation of natural biologically active compounds in the beeswax coating. The beeswax could be an advanced mixture (more than three hundred components) of hydrocarbons, free fatty acids, esters of fatty acids and fatty alcohol, di-esters and exogenous substances (Tulloch, 1980). Crude beeswax showed antifungal (Al-Waili, 2004) and antibacterial activity against Gram-positive and Gram-negative bacteria (Nevine, 2011). Also, Fagundes et al. (2015) recorded that edible composite coating of sodium benzoate based on either hydroxypropyl methylcellulose or beeswax was the most effective than sodium methyl paraben and sodium ethyl paraben against Alternaria alternata black spot on artificially inoculated cherry tomatoes.

CONCLUSION
Our results indicate that food preservative from natural sources as carnation oil, lemongrass oil, beeswax and propolis were more effective against aflatoxigenic fungi and its toxins production during storage than chemical preservative as salicylic acid and potassium sorbate, at the concentrations (0.25 to 6%). It is suggested that natural substances could be used to prevent growth of aflatoxigenic fungi and its toxins production in stored maize grains.

ACKNOWLEGMENT
This research was financially supported in part by In-House project No. 11030132 of National Research Centre; Egypt entitled "Integrated Management of Diseases Affecting Maize Crop during Vegetative Growth and Storage Periods".