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Assessment of the Exposure Risk to Mycotoxins from Stored Maize (Zea mays L.) in Triple Bags with Aromatic Plants Leaves (Lippia multiflora and Hyptis suaveolens) in Côte d’Ivoire

In Côte d’Ivoire, maize is second cereal most cultivated and consumed after rice. In bad storage conditions, corn may be contaminated by mycotoxins (aflatoxin B1, total aflatoxins, ochratoxin A, fuminosin B1, zearalenone). The aim of this study was to assess the exposure risk of these Original Research Article Gaël et al.; ARJA, 13(2): 13-25, 2020; Article no.ARJA.60981 14 mycotoxins for maize stored in triple bags in presence of aromatic plants leaves (Lippia multiflora and Hyptis suaveolens). The contents of water activity, aflatoxin B1 (AFB1), total aflatoxins (AFT), ochratoxin A (OTA), fuminosin B1 (FB1) and zearalenone (ZEA) were studied and monitored. The levels of AFB1, AFT, OTA, FB1 and ZEA resulted from maize grains treated with plants leaves were significantly lower than those recorded with untreated maize of control bags. The estimated daily intakes in AFB1, AFT, OTA, FB1 and ZEA, deriving with consumption of maize from experimental batches stored for 18 months are respectively 1.69 ± 0.00 2.09 ± 0.01 ng/kg bw/day, 8.66 – 10.91 ng/kg bw/day, 1.86 ± 0.01 2.47 ± 0.01 ng/kg bw/day, 2.01 ± 0.05 3.01± 0.05 ng/kg bw/day and 1.89 ± 0.12 3.56 ± 0.04 ng/kg bw/day. These levels are lower than the estimated intakes from maximal reference value (EDILM) for OTA, FB1 and ZEA. For aflatoxin B1 and total aflatoxins, the estimated daily intakes are lower than the estimated intakes from maximal reference value (EDILM) during, respectively, 15 storage months and 10 storage months. However, after 18 storage months, exposure risk of aflatoxin B1 is higher than the estimated intakes from maximal reference value. This inexpensive and easy-to-use treatment should be popularized among farmers


INTRODUCTION
Ranked third in the world after wheat and rice, maize (Zea mays L.) is one of the most widely grown cereals in the world. In Côte d'Ivoire, of all food crops, it is the second most cultivated cereal after rice (Oryza spp.), with an annual national production of 1.006.000 t in 2018 [1]. It plays an essential role as subsistence, commercial and socio-cultural culture [2]. Hence, maize is the staple food of a large segment of the Ivorian populations. In Côte d'Ivoire, the mean daily consumption of maize grains is estimated at 28.4 g [3]. It allows diverse dishes such as porridge, couscous or dense paste (tô) eaten with sauce [4]. Despite the growth in its production and its socio-economic importance, post-harvest losses during storage remain a real challenge for farmers [5]. Indeed, post-harvest losses can reach more than 60% in maize stored in traditional storage structures [6]. These losses are mainly due to insects and molds under inadequate storage conditions. The activity of insect pests creates an environment favorable to the growth of molds of the genus Aspergillus, Penicillium and Fusarium [7]; [8].These molds produce mycotoxins responsible for many diseases in humans and animals such as cancer [9]. Maize can be contaminated with two or more mycotoxins. In Côte d'Ivoire, studies revealed mycotoxin co-contamination in maize [10]; [11]. The consumption of maize contaminated by mycotoxins could represent an increased risk with carcinogenic and hepatotoxic effects for consumer [12]. There are reports that infants and children are even more sensitive to the effects of mycotoxins in general due to their immature immune systems coupled with high ingestion of maize-based foods [13]; [14]. It is very difficult or almost impossible to eliminate mycotoxins (aflatoxins, ochratoxin A…) from raw foods such as maize, however a good storage system would help maintain food safety. Studies showed the efficiency of aromatic plants Lippia multiflora and Hyptis suaveolens on sanitary quality of maize stored in granaries, polypropylene bags, and triple bags thanks to their insecticidal and fungicidal effects [15]; [16]; [17]. However, the exposure risk to mycotoxins from maize grains stored in triple bags containing aromatic plants has not yet been assessed. Thus, the current study was initiated to assess the exposures risk to aflatoxin B1, total aflatoxins, ochratoxin A, fuminosin B1 and zearalenone by the consumption of stored maize in triple bags in presence of leaves of Lippia multiflora and Hyptis suaveolens during 18 months by the ivorian adult.

Experimental Site
The experiment was carried out in the storeroom of Research Unit Laboratory of Biochemistry and Food Sciences (URBSA) UFR Biosciences at the University Félix HOUPHOUET-BOIGNY. The different bags were kept in a laboratory storeroom to 27.78 ± 0.19°C temperature and 75.0 ± 0.99% relative humidity. Wooden pallets were arranged floored as support for triple bagging system.

Collection of Maize Grains and Aromatic Plants used in the Study
Maize grains and leaves of L. multiflora and H. suaveolens were collected in March 2016 from producers of Gbêkê region (7°50 North and 5°18 West in center of Côte d'Ivoire). Prior to the storage, maize were sun-dried for 2-3 days before being used for the experiment. While, the L. multiflora and H. suaveolens leaves were drying at an average temperature of 30•C for 6-7 days and kept away from direct sun exposure. The dried leaves were chopped into fine particles before being used for the experiment.

Using the triple bagging
Storage bags used in our study, were made of polypropylene bags and polyethylene bags (Purdue Improved Cowpea Storage: PICS) developed by Purdue University for storing cowpeas from Niger. These bags, obtained from suppliers, are composed of a triple bagging system.

Treatments
The storage method is based on the mixture of plants leaves. Method tested in this study, consisted in adding of aromatics plants leaves (0 -5% w/w) in the polypropylene bags and the triple bagging system containing 50 kg maize grains and storing on pallets in warehouses for 18 months. The filling of the bags was performed by alternately as maize grains strata and aromatics plants. The maize grains were stored as follows: -1 control batch of 50 kg of maize grain in polypropylene bag without plants leaves (TPPB0); -1 control batch of 50 kg of maize grain in triple bagging system without plants leaves (TPB0); -1 experimental batch of 50 kg of maize grain in

Sampling
The sampling was performed at the beginning of the storage (0 month), then 5, 10, 15 and 18 months later, in triplicate. Thus, 2 kg of maize samples from each bag was gathered through the top, the center and the bottom opening sides.

Determination of water activity
The water activity was ascertained using a hygrometer from HygroLab Rotronic according to the method of [18]. Thus, a sample of 5 g of maize grains was placed in 10 Aw containers void of any trace of water. After two minutes, value of water activity was directly carried out in the device.

Estimate of Intakes in Aflatoxin B1, Total Aflatoxins, Ochratoxin A, Fuminosin B1 and Zearalenone from Maize Stored
According to the definition of the Codex Alimentarius, the estimate of the exposure is the assessment of the quantitative exposure of the probable ingestion of chemical dangers through foods [24]. The mycotoxin intake was estimated from the data of maize consumption in Côte d'Ivoire [25]. The maize consumed daily by an Ivorian adult of 70 kg is estimated at 28.4 g per capita / day [3]. The mycotoxin intake was calculated using the following equation. The estimated values of the exposure were compared to estimated daily intakes calculated from limits maximal (EDILM). These limits were set at 5 µg/kg, 4 µg/kg, 5 µg/kg, 2000 µg/kg, and 500 µg/kg respectively for aflatoxin B1, for total aflatoxins, for ochratoxin A, for fuminosine B1 and for zearalenone [20]; [26]; [27]; [28].

EAI = (T x Q) / BW
With EAI: the estimated daily intake of AFB1, AFT, OTA, FB1 and ZEA in ng kg -1 of body weight (b.w.) day -1 T. The AFB1, AFT, OTA, FB1 and ZEA contents in maize stored (ng/kg); Q: the daily Intake of maize grains (g/day); BW: the Body weight of an adult person (70 kg).

Characterization of the Risk of Exposure Risk to Aflatoxin B1, Total Aflatoxins, Ochratoxin A, Fuminosin B1 and Zearalenone
The characterization of the Risk of Exposure (RE) to aflatoxin B1, total aflatoxins, ochratoxin A, fuminosin B1 and zearalenone is calculated with the following formula. This result allows only concluding on the potential occurrence of effects but not on their importance. RE = (EDI) / (EDILM) If: -RE <1 means that the exposed population is theoretically out of danger, that is, this exposed population is not likely to develop the health effects studied. -RE> 1 means that the toxic effect can occur without it being possible to predict the probability of the occurrence of this event.

Statistical Analysis
All analyses were performed in triplicate and the full data were statistically treated using SPSS software (version 20.0). It consisted in Analysis of Variance at repeated measures. Means derived from parameters were compared with the Tukey High Significant Difference test at 5% significance level. Table 2 presents the results of water activity, aflatoxin B1, total aflatoxins, ochratoxin A, fuminosin B1 and zearalenone contents of stored maize. For all mycotoxins, these parameters increase progressively during storage. Control batches (TPPB0 and TPB0) have high values than experimental batches (TB1, TB2, TB3, TB4, TB5, TB6, TB7 and TB8) after 18 months of storage for all parameters.

Estimated Intake of Aflatoxin B1, Ochratoxin A, Fuminosin B1 and Zearalenone of Stored Maize
The ratio of Estimated Daily Intake (EDI) to estimated daily intakes calculated from limits maximal (EDILM) was determined to assess the risk of exposure to consumers.

Estimated intake of total aflatoxins
In control batches, the estimated intakes of total aflatoxins (AFT) increase significantly during 18 storage months. Thus, exposure risk of total aflatoxins is high for control and treated lots after 18 storage months.

Estimated intake of ochratoxin A
The estimated intake of ochratoxin A (OTA) increase significantly during 18 storage months in control batches. These intakes vary from 0.63 to 7.51 ng/kg of bw/d for TPPB0 and from 0.03 to 3.10 ng/kg of bw/d for TPB0. In experimental batches, estimated intakes increase significantly at 10th storage's month but the means are lower than control batches. After 10 months of storage, values are between 0.22 and 0.34 ng/kg of bw/d. After 18 months of storage, estimated intakes for experimental batches varies respectively from 1.86 to 2.47 ng/kg of bw/d. The lot TB6 has the lowest intake in OTA. However, the contributions of experimental batches TB2, TB3, TB6 and TB8 are lower than the estimated daily intakes from limits maximal (2.02 ng/kg of bw/d). These lots have a low exposure risk for OTA (Table 3).  (Table 3).

Risk Exposure in Aflatoxin B1, Total Aflatoxins, Ochratoxin A, Fuminosin B1 and Zearalenone
For every mycotoxin, the ratio of Estimated Daily Intake (EDI) to Estimated Daily Intake from limits maximal (EDILM) was determined to assess the risk of exposure to consumers after 18 months of storage.

DISCUSSION
The results observed in this study showed that the system bags in the presence of an Aw lower than 0.65 being the accepted limit value [32]. In addition, authors have shown the correlation between the evolution of Aw and mycotoxins (OTA, FB1, ZEA) in studies on the storage of corn in granaries in the presence of L. multiflora and H. suaveolens [33] and storing cowpeas in bags with triple bagging in the presence of L. multiflora [31]. The mycotoxins contamination observed in this study was revealed in previous studies in Côte d'Ivoire [10]; [11]. Authors showed frequent co-exposure of major mycotoxins (AF, FB1 and OTA) [11]. For these authors, this co-exposure is related to the frequency of consumption of corn, peanuts, millet and attiéké. Co-exposure to these mycotoxins could cause negative effects on human health such as teratogenic, immunotoxic effects, etc [10]; [11]. For aflatoxin B1 (AFB1) and total aflatoxins, the estimated intakes, respectively, after 15 and 10 months of storage are lower than the estimated intakes from reference value (EDILM  [37]. These results indicated that foods currently contaminated by AFB1 had low health risks for residents and that dietary exposure to AFB1may not account for the occurrence of liver cancer in Guangzhou (China). In our study, the risk of exposure remains low for FB1 and ZEA in batches treated after 18 months of storage. While the estimated adult exposure for FB1 is extremely low and appears as little risk, the estimated exposure of children may be of concern. The standard child's body weight at 15 kg is one fifth that of the average adult and yet the amount of maize-based food products consumed has been assumed to be similar based on their consumption of corn-based breakfast cereal and snack food such as corn chips.
This results in the exposure of children being substantially higher than adults [38]. Some factors increasing risk of contamination such as weather variables are not entirely controllable, although there are a number of good agricultural practices (GAP) that will assist in reducing contamination [39]. Other factors such as insect pressure and storage conditions can be controlled [38]. The results of our work could contribute to control the storage conditions such as water activity.   TPPB0  TPB0  TB1  TB2  TB3  TB4  TB5  TB6  TB7 TPPB0  TPB0  TB1  TB2  TB3  TB4  TB5  TB6  TB7  TB8   5

CONCLUSION
This study contributes to improving corn storage and preserving the health of corn consumers. The results showed that the leaves of L. multiflora and H. suaveolens in the triple bagging system reduce exposure risk of mycotoxins (Aflatoxin B1, total Aflatoxin, Ochratoxin A, Zearalenone and Fuminosin B1). However, exposure risk to aflatoxin B1 (AFB1) and ochratoxin A (OTA) is low until 15 months of storage in experimental batches. The exposure risk to total aflatoxins (AFT) is low until 10 months of storage. The batches of maize stored in polypropylene bag (TPPB0) and triple bag without plants (TPB0), exposure risk is very high for AFB1, AFT and OTA. The storage technology used is inexpensive, easily applicable and environmentally friendly. However, for greater efficiency, it would be important to encourage good post-harvest practices.

CONSENT
It is not applicable.