Monitoring Food Safety in Armenia. Assessment of Mycotoxins in Cereals.

Widespread use of cereal grains and cereal-based products as main sources of food and feeds a prerequisite for settle the strategic importance of their safety [16, 17, 18, 20, 39, 48, 49].

As suggested by literature data the most important of various microflora of cereals and cereal products /molds, yeasts, bacteria/ are the filamentous fungi or molds [4, 5]. Produced by a range of mycotoxicogenic fungies the toxic substances – mycotoxins mostly described as secondary or storage pathogens. Notably, that cereal grains are more susceptible to soiled with fungies Aspergillus or Penicillium families, mainly Penicillium verrucosum, Aspergillus ochraceus, A. westerdijkiae and A. carbonarius produced Ochratoxin A (OTA), and various Fusarium species with the secondary fungal metabolite T-2 toxin. These fungi appear toward or at harvest and grow on the product if the conditions are suitable [14, 31].

According to the Food and Agricultural Organization (FAO) of the United Nations, up to 25% of the world’s food crops have been estimated to be significantly contaminated with mycotoxins [17, 22, 50]. Significant losses due to mycotoxins and their impact on human and animal health have been linked with national economic implications and all these factors have combined to make mycotoxins important worldwide. Consumption of commodities contaminated with mycotoxins leads to chronic mycotoxicoses which results in acute poisoning resulting in death. Therefore, as the global occurrence and importance of mycotoxins cannot be overemphasized, methods for preventing and reducing them before entering the food chain must be given continuous attention as 40% of the productivity lost associated with diseases [1, 27, 29, 37].

International organizations such as Joint WHO/FAO Expert Committee on Food Additives (JECFA) and European Food Safety Authority (EFSA) evaluated risk related with cereals that was based on contamination with different fungies and their metabolities and several risk assessments of these have been made [10, 11, 15, 35, 40].

In regards to food safety is one of the most important fungal toxic substance the mycotoxin Ochratoxin A (OTA) was detected in a great number of various commodities including cereals and cereal-based foods [9, 28, 32, 42].

Biological effects of OTA are presented by carcinogenic, nephrotoxic, immunotoxic, teratogenic and possibly neurotoxic and genotoxic properties, and also associated with Balcan Endemic Nephropathy [3, 21, 41, 46]. Research data in regard to T-2 toxin biological effects have shown induced haematotoxicity and myelotoxicity associated with impairment of haematopoiesis in bone marrow [11, 12, 44]. Recent data also indicate that T-2 toxin inhibits protein, RNA and DNA synthesis, induces apoptosis, and in some cell types necrosis, as well as lipid peroxidation affecting cell membrane integrity [6, 25, 30, 38, 48].

The European Food Safety Authority (EFSA) recently assessed OTA, established a Tolerable Weekly Intake (TWI) of 120 ng/kg bodyweight, and estimated maximum limits for OTA 5,0 µg/kg in raw cereal grains and 3.0 µg/kg for finished products intended for human consumption [10, 34]. Worldwide, 13 countries have reported legal maximum levels (MLs) or recommendations for T-2 and HT-2 toxins in food and/or feed products, including Republic of Armenia, where established for all foods T-2 toxin ML of 100 μg/kg, but for unprocessed cereals level up to 1,0 mg/kg  [8, 17].

In accordance with investigations of many authors contamination of cereal commodities by moulds and mycotoxins results in dry matter, quality, and nutritional losses and represents a significant hazard to the food chain. Most grain is harvested, dried and then stored on farm or in silos for medium/long term storage. A number of interacting abiotic and biotic factors influence on cereal quality. The most important abiotic factors which influence growth and OTA production by such spoilage fungi include water availability, temperature, and when grain is moist, gas composition. The interaction between these variables primarily determines relative development of the fungal community and can be used to provide guidelines of the level of risk of contamination of the grain through the food chain. Prevention strategies post-harvest can only be effective for mycotoxins that are formed during this component of the food chain. Pre-harvest natural contamination can only be minimized postharvest by application of processing techniques which will minimize subsequent entry into the food and feed chain where possible. These include accurate and regular moisture measurements to ensure safe thresholds, efficient drying of wet cereals for medium and long term storage in hygienic silos free of insect pests and moldy material [7, 13, 26, 31, 43, 45].

As it has been shown by different investigators climatic features of geographic zones could interfere on the level of cereals contamination by mycotoxins. In particular, climate changes related with humidity, temperature issues together with postharvest events like sorting, drying and storage conditions are most important contributors to fungies growth on cereal grains and subsequent pollution with mycotoxins [19, 36, 51].

The mountainous nature of Armenia results in a series of highly diverse landscapes, with variations in geological substrate, terrain, climate, soils, and water resources. Being located at the central dry sector of sub-tropical climate zone, Armenia should have enjoyed the desert and semi-desert climate within latitudinal zonality. However, due to the mountainous nature of the country, the altitude zonality has been formed on the prior zonality background and as a consequence a great variety of climatic zones is characteristic to Armenia [2, 24].

As it has been presented in our research proposal we offered the screening of winter soft wheat varieties and collected wheat samples (unprocessed, intended for human consumption and animal feed) within provinces of Armenia with different climatic features, namely Shirak, Lori, Tavush, Syunik and Ararat, for presence and level of OTA and T-2 toxin contamination, since various weather and storage conditions could affect to mold growth and pollution by mycotoxins.

This report provides an analysis of Ochratoxin A (OTA) and T-2 toxin contamination of cereals-based products and cereal grains /wheat and barley/ collected from the provinces of Armenia with distinct climatic features. 

This survey was performed in the Laboratory of Testing Genetically Modified Organisms and Mycotoxins at the Institute of Molecular Biology NAS RA (License certificate #AST-001.Q-0015-2011).

For experiments were selected 49/6 samples of known wheat/barley varieties (control samples) and 80/32 samples of wheat/barley varieties collected from the provinces of Armenia, correspondingly. Control samples of wheat varieties were kindly provided by "Sermeri Gortsakalutyun" SNCO (Ministry of Agriculture RA), Merdzavan. The following varieties of wheat and barley grains samples were included for analyses: "Krasnodarskaya-99", "Yubileynaya-100", "Lebed", "Yuka", "Sila", "Vassa", "Tanya" winter soft wheat varieties and "Vacula", "Rodnik" spring barleys provided to the governmental agency from corresponding provinces. All control and collected cereals samples had been disinfected before the providing to provinces before dissemination and had been stored under the same appropriate conditions during 3-6 months.

Cereal crops from the individual farmers fields of corresponding provinces have been collected for 2 periods in 2013: March- June /stored/ and in July-October /post-harvest period/. Collected samples of wheat/barley grains by provinces Shirak, Lori, Tavush, Ararat, Vayk and Syunik made up 26, 22, 20, 20, 6  and 18 samples, respectively. Provided barley samples were intended exclusively for home livestock feed.

As a group of experimental materials in Armenian markets were purchased 50 samples of cereals-based products /bread, flour and etc/.

The sampling and methods of analysis were conducted in compliance with ISO requirements [8, 23, 34]. Sampling procedures included the following steps: collected sample – divided into the subsamples – choosing and mixing subsample. Each subsample was weight of 250g ±20%. Representative sample is triturated and thoroughly grounded and mixed in a mixer-grinder prior to proceeding with the extraction procedure. Sample preparation also included extraction, filtration/or centrifugation and dilution [23, 33, 52].

For screening of representative samples on mycotoxins testing was used immunochemical method of analyses – ELISA (enzyme-linked immunosorbent assay), including samples preparation and data calculation for OTA and T-2 toxin determination in cereals in accordance with manufacture’s protocols RIDASCREEN® T-2 Toxin  enzyme immunoassay for the quantitative analysis of T-2 toxin in cereals and feed and  RIDASCREEN® Ochratoxin A 30/15 enzyme immunoassay for the quantitative analysis of ochratoxin A in cereals, feed and other (R-Biopharm AG, Darmstadt, Germany). The absorbance was measured at 450 nm by the Microplate Reader STAT FAX 3200 (Awareness Technology Inc., USA). Data obtained by RIDASCREEN® enzyme immunoassays were evaluated by special software, the RIDA®SOFT WIN (Art.N0Z9999). Data calculation and analysis was performed by using Microsoft Excel program. 

The experimental results in regard to screening of contamination by specified mycotoxins for control samples of wheat and barley varieties under storage conditions and provided in post-harvest period are  presented in tab.1.

By data of analyses (tab.1) for wheat samples of variety “Krasnodarskaya -99” collected in postharvest period within provinces and stored /before dissemination/ didn’t observe certain contamination of OTA or  the level of this mycotoxins was lower of quantification limit of determination. The levels of OTA contamination in wheat samples of varieties “Lebed”, “Yuka”, “Sila”, “Vassa” and “Tanya” also have been below the limit of 1.25 μg/kg in accordance with data of experiments.

Notably, that wheat variety of “Yubileynaya-100” collected from the provinces Lori and Aragatsotn was contaminated with OTA; at that in case of collection in provice of Aragatsotn (under storage) level of this mycotoxin four times exceeded the permissible limit of 5 μg/kg for unprocessed cereals up to 4 times, while in case of Lori it was lower than the level of 1.25 μg/kg. It also has been detected quantitatively low threshold of OTA contamination for tested barleys samples of varieties “Vacula” and “Rodnik”.

Contamination by T-2 toxin above mentioned varieties of cereals demonstrated a wide range of variation (3.5-850 μg/kg)  between groups of under storage and collected in post-harvest period, as well as in the same "stored" group, with the highest content of this mycotoxin in wheat of variety “Tanya”.

Thus, wheat of variety “Krasnodarskaya-99” in group of post-harvest collected from provinces Shirak, Ararat and Aragatsotn revealed the same T-2 toxin low level of contamination about 4.0 μg/kg, while for stored samples of this variety noted as “before dissemination” and from province of Shirak were detected two levels of T-2 toxin content: about 20 μg/kg and level, exceeding up to 25-35 times (500-700 μg/kg). The same consistent pattern we observed in wheat samples of variety “Yuka” and “Yubileynaya-100” collected from corresponding provinces. It should be noted extremely (up to 40 times) different levels of T-2 toxin in wheat samples of variety “Tanya” between groups of “stored” and “post-harvest”.

Collected  with Ararat and Aragatsotn provinces wheat samples of varieties “Lebed”, “Vassa” and “Sila”, correspondingly, have indicated low levels of T-2 toxin contamination in post-harvest period.  By data obtained in regard to “stored” barley samples varieties “Vacula” and “Rodnik” were detected high levels of T-2 toxin. 

Obtained data for distribution of investigated mycotoxins in wheat and barley samples collected with  provinces of Armenia for two periods are presented in table 2. 

Analysis of data obtained (tab.2) for the first period revealed no certain contamination of OTA in wheat samples with considered provinces, but in case of 2 samples from Syunik  indicated very high contamination by this mycotoxin (up to 4.5 time exceeded maximum limit).  Contamination of these wheat samples by T-2 toxin was in range of 80-300 μg/kg, with low level in samples of Shirak and high – in Lori, Tavush and Syunik provinces.

Data obtained for second period have shown changes in character of contamination:  increasing OTA level, especially in samples of provinces Tavush, Ararat, Vayk and Syunik, and reduction of T-2 toxin level to range of 7.0-19.0 μg/kg in all considered provinces. Notably, those samples of Shirak province indicated the lower levels of contamination OTA and T-2 toxin in two periods.

For two periods of investigation most of samples of barleys intended for food and feed collected with considered provinces was observed no more than 1.25 μg/kg contamination by OTA with exception one sample from Lori at contamination level of 22.0 μg/kg. Contamination of all investigated barley samples by T-2 toxins was in range of 8.0 - 40.0 μg/kg with low level in samples of Shirak and high – in Lori provinces, be noted that for feed intended samples 2 times more contaminated in compare with food samples.

On the average of data obtained for two periods have been shown that OTA contamination of  wheat or barleys by investigated provinces was lower of limit of quantification (1,25 μg/kg), and for T-2 toxin were detected two distinct (up to 10 times) ranges of contamination in investigated provinces, but they didn’t exceed limit of regulation.

By summarizing of experimental data should be noted, that mycotoxins-free samples of cereals made up 46,1% (for OTA) and 38,5% (for T-2 toxin) in Shirak;  30,0% (for OTA) and 10,0% (for T-2 toxin) in Tavush; 18,2% (for OTA) and 27,3% (for T-2 toxin) in Ararat; 4,5% (for OTA) in Lori; 5,6% (for OTA) and 27,8% (for T-2 toxin) in Syunik, whereas in provinces of Lori and Vayk weren’t revealed samples without contamination of T-2 toxin. Thus, most of tested samples (83%) were contaminated with T-2 toxin at the different levels ranging from 8.0 to 300 µg/kg for  samples of wheat and barley intended for food and feed.

Interestingly, that samples contamination by mycotoxins expressed distinct levels in cereals intended for food and feed. Thus, in figure 1 presented data for considered provinces in comparision with lowest value of contamination for T-2 toxin (by samples of Shirak province). 

As it has been shown in fig.1 the highest level of contamination by T-2 toxin was determinated in samples intended for feed from Tavush and Lori provinces. It should be noted that provided barley samples were intended exclusively for home livestock feed. It is worth noting, that by contamination of T-2 toxin collected samples of cereals intended for food and feed, taken correspondingly, from considered provinces are differ in 1.5-2 times. Should be taking into account, that in the so-called stored grain ecosystem factors include grain and contaminant mould respiration, insect pests, rodents and the key environmental factors of temperature, water availability and intergranular gas composition, and preservatives which are added to conserve moist grain for animal feed.

Substantial  part of Armenian farmers mainly produced cereals for self consumption and feed without appropriate implementation of post-harvest events, including the flour obtained after grinding without sorting and properly drying and storage in non-appropriate conditions. Hence, they have no incentive to concentrate on the quality of produce, so the failure to comply with storage condition, in this case, might be considered as a key risk factor for food and feed contamination with mycotoxins.

Next objective for evaluation OTA contamination in food chain was investigation of cereal-based products. Experimental results of tested cereals-based food products represented in table.3. It has been analyzed following groups of breadstuffs products: flour, bread, buns, lavash  /white bread into thin flat cakes from wheat flour/. 

Of all the 50 breadstuffs products samples, 70 % were positive for OTA. The incidence of  T-2 toxin in the white bread samples was 70.5 % in range of 0.25 –1.78 μg/kg, and in the black bread samples - 50%. Almost all types of buns (without stuffing, with raisins and chocolate)  were contaminated with T-2 toxin, concentrations of which varied between 0.8-4.6 μg/kg; for lavash and flour the incidence of this mycotoxin made up 77 and 80%, correspondingly, in the same range of 0.5-3.7 μg/kg.  Notably, that the highest level of contamination with T-2 toxin marked in bun with chocolate ( 2-3 times higher in compare with other kind of buns) and might be consequence of chocolate contamination by this mycotoxin.

Thus, as endpoint of cereals-based products in food chain from the field to consumers - breadstuffs products contamination by mycotoxin results of different factors, including not appropriated storage and manufacturing conditions, also weak control of quality.

Data of screening control samples varieties of wheat and barley (tab.1) provided by governmental agency indicated distinct levels of pollution by mycotoxins. Based on results of two groups  - "stored" and "post-harvest" – we have assumed that high level of contamination by mycotoxins in “under keeping” group could be consequence of influence different factors under storage conditions as moisture, temperature and etc. as described by Magan N., et al.  2007.

In regard to data of contamination by mycotoxins it is worth noting that control samples wheat of variety “Krasnodarskaya-99” provided with considered provinces (Shirak, Ararat, and Aragatsotn) showed the same lowest values for tested mycotoxins, specifying on good acclimatization possibility of this wheat variety. In opposite, wheat samples of variety “Yubileynaya-100” with Aragatsotn, revealed high level of OTA contamination, while samples of the same variety collected in province of Lori was observed trace amounts of this mycotoxin. Such differences may be resulted by influence of climatic features and the following important issues as good and timely harvest, drying, sorting, cooling and storage conditions in accordance with the Good Storage Practice to reduce Ochratoxin A in cereals [45].

Satisfactory results of wheat samples varieties of “Yuka” with Armavir, “Sila” with Aragatsotn, “Lebed”  and “Vassa” with Ararat with regard to contamination by mycotoxins in post-harvest period might be considered as promising for acclimatization of wheat varieties with corresponding provinces.

By data in regard with mycotoxins pollution of samples varieties of barleys we assumed about their susceptibility to contamination by fungies of Fusarium species and necessity for future monitoring.

The data of distribution for mycotoxins in wheat/barley grains by provinces (table 2) revealed the large scatter values of the occurrence of OTA and T-2 toxin, including contaminated and mycotoxin-free samples of cereals, which partially, could be considered as result of the impact of climatic features.

As data indicated samples of wheat collected from Shirak, characterized as slightly elevated land areas (heights of some points reach 1,800-2,200 m) with prevalence of cool or even cold climate, mostly detected no contamination or very low levels of tested mycotoxins. At the same time analysis of wheat samples collected in Lori province with prevalence of mild climate, which bordered in the east with Shirak, were observed high levels and widespread ranges of contamination in case of both mycotoxins.

Collected in postharvest period in Tavush (located in the north-east of Armenia, where the climate is moderately humid; the summers are warm and winters are mild) samples of cereal grains mostly detected OTA contamination below of 1.25 μg/kg, but content of T-2 toxin ranged up to 30 times (10.0 - 300,0 μg/kg).

Wheat samples contamination by OTA in post-harvest period were higher in provinces of Ararat, Vayk and Syunik in compare with Shirak, Lori and Tavush, while content of T-2 toxin was at the same level of pollution. Noticeably, that in Ararat province was shown wide range of climatic features: the territory of Ararat province is accurately divided into two parts – plains and mountains and there climate is continental with hot sunny summers and cold winters.

Since samples with both high and low level of contamination of tested mycotoxins were revealed in Syunik, we propose that it could be explained by range of microclimates characterized this region, from dry tropical to temperate warm, to the cold and snowy mountains, therefore the most part of the province is located in the zone of dry subtropics so the climate there is very warm.

It should be noted that contamination by T-2 toxin in groups of wheat collected with provinces of Armenia at the first (under storage) and second (post-harvest) periods (tab.2) was varied up to 10-20 times. Wide variations of T-2 toxin content were observed in groups of cereals intended for food and feed, and it is worth noting that the large portion of cereal grains farmers are mainly produced on their private fields and absence of implementation of strategical requirements for post-harvest events that, finally, affect on the quality cereals-based food or feed.

It worth pay attention on fact, that actually the lowest contamination of cereals by both mycotoxins occurred in Shirak province, which ecosystem may be unfavorable place for growth of mold and contamination by mycotoxins.

To clear understanding contamination processes in food chain of cereals we proposed analyses of some breadstuffs products purchased in the random markets. Interestingly, that most of them were contaminated by OTA at the level exceeding identified values of cereals samples even in storage. Considering wide spread use of cereals-based products, due to high level of their contamination tested breadstuffs products could be considered as a groups of risk, and should be taken under continued control, as well as their monitoring in food chain. 

Based on above results should be summarized that levels of contamination of cereals and cereals-based products by mycotoxins, OTA and T-2 toxin, and their distribution in wheat samples of different provinces are caused by various reasons. It has been shown that level of contamination depend on the variety of wheat, whereas distribution of mycotoxins in considered Armenian provinces was related mainly with climatic features of region, among which at first should be taken factors of temperature and humidity, which are the main determinants for level of emission mycotoxins by molds. It is also important to maintain post harvest events like sorting, drying and other, preventing subsequent pollution by mycotoxins.

The post-harvest control strategies should focused on: minimizing mycotoxins in the food chain by efficient and prompt drying of wet cereal grain, regular and accurate moisture measurement determinations and appropriate transportation  and storage conditions, ability to efficiently identify and reject material below specified standards in terms of  both fungal contamination and, at some stages, mycotoxin levels and etc. Besides, it should be conducted risk assessment studies for identification of critical control points during harvesting, drying and storage stages in the cereal production chain.

Risk assessment of contamination by mycotoxins and food safety should be associated with continuous monitoring of the mycotoxins level in cereals after harvesting, as well as under storage conditions by effective diagnostic tools.

These results of monitoring of cereals could be used to assess exposure to OTA and T-2 toxin in noted regions with high risk level of mycotoxins content in food and feed with points of view influence on human and animal health, as well as for identification of provinces with suitable and unfavorable climatic feature for cereals acclimatization. 

This work was made supported by an Armenian National Science and Education Fund (ANSEF) (New York, USA), research grant ID: molbio#3199.

Author expresses thanks to Mr. Khachatryan H., Deputy Head of "Seed Agency" (Sermeri Gortsakalutjun).