Multi-mycotoxins and Fungal Contaminants of Wheat from Anambra State, Nigeria

This study was carried out to determine the fungal and mycotoxins contamination of 36 Wheat ( Triticum aestivium ) samples purchased randomly from the seller of the agricultural produce in local markets of Anambra State, Nigeria. Results from the studies showed that two hundred and three fungal isolates consisting of 18 species of moulds and 5 species of yeasts contaminated the wheat samples at varying degrees. For moulds, Aspergillus species contaminated the samples mostly with (28) isolates followed by Penicillum species (19) isolates while Verticillium species and Cladosporium species had equal least contaminations with (3) isolates each. Among the yeast species, Candida rugosa had the highest number of contamination with (37) isolates followed by Cryptococcus laurentii (31) isolates while Candida stellatoides (9) isolates had the least contamination. Twenty-four fungal metabolites were also recovered. The concentration of trichothecene mycotoxin Deoxynivalenol (2067µg/kg), a protein synthesis and cell proliferation inhibitor in animals exceeded the maximum acceptable limits for human consumption. It can be deduced therefore that wheat circulating in Anambra State, Nigeria are variously contaminated with different xerophilic moulds and mycotoxins which can exert adverse health problems to consumers.


Introduction
Wheat (Triticum spp)(order poales), is a cereal crop belonging to the family poaceae and is ranked as the third main staple food and industrial cash crop after maize and rice in Nigeria (Tanno andWillcox, 2006, Kolawole, et al., 2013). Wheat is widely cultivated in North Africa and in Sudan and Ethiopia and it is grown on around 10 million ha in Africa and it is also an imported commodity in all of Africa. African countries are the world's biggest wheat importer with more than 45 m t in 2013 at around 15 billion US$ Wheat imports account for 60% of African's wheat consumption and 80% of Sub-Saharan (SSA) countries (Macauley, 2015). Wheat consumption is steadily increased in all African countries from the past 20 years as a result of growing population, changing food preferences and socioeconomic change associated with urbanization. In rapidly urbanizing in sub-Saharan Africa, wheat consumption is expected to grow to 38% by 2023 with imports already at 23 m tons of wheat in 2013 at a cost of $7.5 billion. With the growing importance of wheat in food security in Africa, the African Union Heads of State in January 2013, endorsed to add wheat to the list of strategic crops for Africa (Macauley, 2015). Wheat grains are milled and are used to produce thick porridges and "swallows", which are known by various names in different parts of African continents (Ukwuru et al, 2018). Wheat is not widely grown in Nigeria and it serves as additional food or feed supplement that provides vegetable protein, minerals and vitamins to humans and livestocks (Kolawole et al, 2013). It is a major component of many other foods including breads, porridge, cracker and biscuits (Tanno andWillcox, 2006, Kolawole, et al., 2013).
However, the practices associated with production, processing and post processing handling of wheat, may exacerbate fungal contamination. Thus, the fungi contaminate the wheat samples on field, indeed accompany them to their storage points and may express the capacity to produce associated mycotoxins thus leading to a progressive deterioration in food quality, leading to alarming economic loss reflected as reduced productivity, discolouration, production of off-odours, direct commodity loss, livestock losses due to death and lower growth rates and human illness (Ogiehor and Ikenebomeh, 2006, Kolawole, et al., 2013). Mycotoxins are natural products (poison) produced by filamentous fungi which evoke toxic responses when introduced in low concentrations to higher vertebrates by natural routes (Oyeka, 2003). Mycotoxins are produced by fungi for a variety of reasons (1) to attack or gain access to hosts by helping to dissolve cell membranes. (2) As a protective measures against encroaching organisms (Zain, 2011). The occurrence of mycotoxins within the fungus and commodities depend on the particular crops, the environmental conditions under which a particular crop is grown, harvested, stored and or from the raw materials used in processing them which can be controlled. Other extrinsic factors like climate or intrinsic factors such as fungal strain, specifically strain variation, the particular enzymes of the fungus and instability of toxigenic properties which are more difficult to control (Zain, 2011). The fungi that produce mycotoxins do not have to be present to cause harm because they may be killed by heat (Oyeka, 2003) but the mycotoxins they produced on foods are stable under most food processing conditions and therefore, persist to the final products. It is therefore, difficult to eliminate them once the food stuffs are contaminated and usually there is no treatment for mycotoxin poisoning (Zain, 2011) All mycotoxins are of fungal origin but not all toxic compounds produced by fungi are called mycotoxins. The target and the concentration of the metabolites are both important (Zain, 2011). Local food markets in developing countries like Nigeria are usually characterized by the absence of regulatory agencies and limited information on food quality; hence the constant selling of these fungal contaminated grains to consumers. Poverty, coupled with ignorance about mycotoxin contamination is also very important predisposing factors to consumers of contaminated food (Oyeka et al, 2019). Lack or absence of routine laboratory checks and diagnosis of mycotoxicosis has been complicated by a disregard on awareness of mycotoxin effects, acceptance of chronic low productivity and disease they cause. Also, lack of feed samples, sampling difficulties, analytical complications, species specific responses, nonspecific effects of mycotoxins on various body systems, universal symptoms that may be similar to other disorders and a cascading of animal responses (as seen with immune suppression) resulting in disorders not thought to be associated with mycotoxins hinder diagnosis of mycotoxins (CAST, 2003, whitlow andHagler, 2005).
The survey for mycotoxigenic fungal contaminants of wheat consume in Anambra State, Nigeria would no doubt give an insight into the possible mycotoxins which can be produced in nature and their toxicological roles in human and animal health.

Materials and Methods:
Study Location:

Sample collection
A total of thirty six (36) wheat samples ((Triticum aestivium) sample lots were purchased at random from six local market zones of Anambra State, Nigeria. The wheat samples were securely packaged in polythene bags, labeled properly and transported to the laboratory, stored under 4 o C for later use for research analysis.

Processing of samples
The fungi were isolated using modified standard methods of Dubey and Maheshwari (2005). Duplicate samples of surface sterilized, blended, and serially diluted wheat samples were cultured in potato dextrose agar fortified with 0.5mg/ml chloramphenicol. These were incubated for 5-7 days and regularly examined daily for fungal growth. The remaining portions of the wheat powder were stored for mycotoxins quantification Twenty (20) grams of twelve selected stored samples of ground wheat sample lots were chosen at random, packaged in zip lock paper envelopes, properly labeled and sent to Center for Analytical Chemistry, Department of Agro Biotechnology, University of Natural Resources and Life Sciences, Tulin, Austria for quantification of mycotoxins.

Identification of isolates
Identification of the isolates was based on extensive study of macroscopic and microscopic morphologies which was compared with standard texts of (Buhmer 1978; Frey et al 1979; De Hoog and Guarro 1995; Summerbell 1996; David et al,2007). Moulds isolates were identified with Slide culture technique of (Errol et al 2012) while yeasts isolates were identified using Sugar fermentation tests of (Chessbrough 2004), urease test of (Mcginnis 1980), germ tube tests of (Chessbrough 2004), Corn meal ager test of (Acumedia 2011) and Chromogenic agar tests of (Massoud 2010).

Quantification of mycotoxin from wheat samples
Duplicate portions of the 12 randomly selected ground representative wheat samples were used for quantification of mycotoxins using Liquid chromatography tandem Mass Spectrometry (LC-MS/MS) method of (Malachova et al 2014). Five (5) grams of each representative samples were weighed into a 50-ml polypropylene tube (Sarstedt, Nümbrecht, Germany) and 20ml of the extraction solvent (acetonitrile/water/acetic acid 79:20:1, v/v/v) added. Samples were extracted for 90 minutes on a GFL 3017 rotary shaker (GFL, Burgwedel,) and later diluted with the same volume of dilution solvent (acetonitrile/water/acetic acid 79:20:1, v/v/v) and subsequently centrifuged for 2 minutes at 3000 rpm (radius 15 cm) on a GS-6 centrifuge (Beckman Coulter Inc., Fullerton, CA). After appropriate mixing, 50µl of the diluted extracts were injected into the LC-MS/MS system without further pre-treatment.

Spiking of model matrices
For spiking the model matrices, 0.25g of a representative of the samples that were not contaminated with mycotoxins were weighed into 50ml of polypropylene tube. Multi -analyte standard in one concentration level was added in each of the tubes. The samples were placed in darkness to avoid analyte degradation and stored overnight at room temperature to allow evaporation of the solvent and to establish equilibrium between analytes and samples. The concentration range of the spiked samples were chosen to cover the respective limits of detection of each toxins, estimation linear calibration range, legislation limits of regulated toxins as well as the levels of commonly found toxins in naturally contaminated samples. External calibration was prepared by dilution of appropriate amounts of the final working solution with acetonitrile/water/acetic acid (49.5/49.5/1, v/v/v) at level corresponding to those of the spiked samples. The corresponding peak areas of the spiked samples were used for the estimation of apparent recoveries by comparison to a multi-analytes standard on one concentration level prepared and diluted in neat solvent. All concentrations of the naturally contaminated samples were corrected by a factor equivalent to the reciprocal of apparent recovery (i.e 1/RA, where R is the apparent recovery of each analyte) Malachova et al 2014.

LC-MS/MS parameters
Determination and quantification of target mycotoxins was performed with a QTrap 5500MS/MS system (Applied Biosystems, Foster City, CA) equipped with a TurboV electrospray ionization (ESI) source and a 1290 series HPLC system (Agilent Technologies, Waldbronn, Germany). Chromatographic separation was performed at 25 • C on a Gemini®C18-column, 150 × 4.6 mm i.d., 5µm particle size, equipped with a C18 security guard cartridge, 4 × 3 mm i.

STATISTICAL ANALYSIS
The data collected were analysed with SPSS (Statistical Package for Social Sciences) version 21 (SPPS Inc., Chicago, IL, USA).

Results
A total of two hundred and three (203) fungal isolates were recovered from the study and Aspergillus species (28) had the highest number of moulds isolates followed by Penicillum species (19) isolates while the least Verticilium sp. and Cladosporium carrionii had 3 isolates each. In addition to moulds recovered, the study showed that yeasts were also very important fungal contaminants of the samples screened. Among the yeast species, Candida rugosa had the highest number of contamination with (37) isolates followed by Cryptococcus laurentii (31) isolates while Candida stellatoides had the least number of contamination with (9) isolates. Wheat from zone (1) had the highest fungal contamination with (76) isolates followed by those from zone (5) with (50) isolates while zone (2) had the least fungal contamination with (12) isolates. Statistical result showed that the frequency of moulds recovery was significant P < 0.05, whereas the recovery of yeasts was not significant P > 0.05 (Table 1) .  Twenty-four fungal metabolites were recovered from the studied wheat samples with 14 Fusaria metabolites being the highly recovered followed by 5 Alternaria metabolites and 1 Penicillium metabolite as the least fungal meyabolite recovered. Analytical results of wheat samples showed high percentage recoveries (77.6-202.4%) of fungal metabolites. The minimal quantification concentrations of fungal metabolites in the analyzed wheat samples ranged between 0.01 μg/kg (enniatin B3) and 409.8 μg/kg (Deoxynivalenol) ( Table 2).

Discussions
The genus Aspergillus dominated other mould species recovered from wheat samples in this study; this was followed by Penicillium, Fusarium, Malbranchae, Alternaria, Rhizopus, Trichoderma, Cladosporium and Verticillium. Many factors in field conditions and during storage act together in the contamination of crops and production of mycotoxins by these fungi, (Enyiukwu et al, 2014). Important mycotoxins producing fungi belonging to the genus Aspergillus, Penicillium, Fusarium and Alternaria are the major contributor of food contamination. Elaaraj et al (2015) reported Aspergillus as the most predominantly isolated fungal organisms in wheat samples followed by Penicillium, Fusarium, Alternaria and Cladosporium. Some of the mould species obtained in this study from wheat samples belong to genera they isolated. The fungi isolated from the six market zones of Anambra State showed that wheat bought from market zone 1 had the highest fungal isolates (76), followed by those form zone 5 (50) isolates while the least was from market zones 2 (12 isolates) ( Table 1). Yeasts also implicated as contaminants of the samples metabolize some food components and produce metabolites which cause the physical, chemical, and sensible properties of a food to change and thus food spoilage (Fleet and Praphailong 2001) though they do not produce mycotoxins. Saccharomyces cerevisiae is common food and environmental saprophyte and was isolated in this study, Candida tropicalis is found as part of the normal human mucocutaneous flora, its environmental isolations may be that the samples were contaminated with faeces, and contaminated soil (David et al, 2007). Candida tropicalis and Saccharomyces cerevisiae were recovered by Desiye and Abegas, 2013 from teff batter (Ethiopian pancake). Environmental isolations of Candida Stellatoides and Candida.rugosa may be that the sources of the samples are contaminated by human or animal excreta, polluted water, soil, air and plants. Candida rugosa has been associated with catheter related fungemia, (David et al, 2007). Wheat samples sold in Anambra State local markets were unique; rare mycotoxins were recovered from the samples. The conventional mycotoxins (Aflatoxins, Ochratoxins and Fumnosins) were not recovered in the study. This may be because wheat are not produced in Anambra State but are bought from the northern part of the country and they are usually stored for short period in bags in the market places by the traders thus limiting the conditions that favour the mycotoxigenic fungi that produce Aflatoxins, Fumnosin and Ochratoxins (Oyeka et al, 2019 (CAST, 2003). Deoxynivalenol glycoside; a masked form of deoxynivalenol contaminated the samples with concentration range of (19.7-33.9 µg/kg). Regular consumption of wheat containing low levels of deoxynivalenol and its masked form toxin may lead to acute toxicity to human and animals.
Nivalenol, another type B immunosuppressive, protein inhibitor trichothecene and a non-regulated fungal metabolite contaminated the samples with concentration range of (6.2 -10.9 µg/kg) and mean level of (8.22µg/kg) respectively. The metabolite nivalenol, is as toxicity as deoxynivalenol, therefore, any synergistic reaction of nivalenol and deoxynivalenol recovered in this study in animals will be a public health risk significance Oyeka (Taylor, 1970). Enniatins were thought to be similar but are chemically distinct antibiotics. They are mixtures of isomeric and homologous cyclodepsipeptides to which the general name "enniatin" is applied.
Enniatins are powerfully bioactive, apparently by interaction with membrane components, these properties they shared to some extent by the related cyclo-octadepsipeptide-valinomycin but there are differences, notably with regard to ion-selectivity (Audhya and Russel (1974 Penicillium metabolites Emodin has antibiotic property and had 75% contamination of the samples. Emodin has been reported to be toxic to cells (Ezekiel et al, 2012).

Conclusion
The co-presence of mycotoxigenic fungi, the studied mycotoxins and other fungal metabolites in the samples as demonstrated in this study implies that the samples could contain fungal toxins that can cause human and animal mycotoxicosis. The concentration of the immunosuppressive deoxynivalenol (409.8 -2067µg/kg) above the acceptable limit of human consumption in this study is a source of worry. Therefore, the consumption of the contaminated samples could have adverse consequences on national livestock production, human and economy.