Biocontrol of Occurrence Ochratoxin A in Wine: A Review.

Viticulture has been an important economic sector for centuries. In recent decades, global wine production has fluctuated between 250 and almost 300 million hectoliters, and in 2022, the value of wine exports reached EUR 37.6 billion. Climate change and the associated higher temperatures could favor the occurrence of ochratoxin A (OTA) in wine. OTA is a mycotoxin produced by some species of the genera Aspergillus and Penicillium and has nephrotoxic, immunotoxic, teratogenic, hepatotoxic, and carcinogenic effects on animals and humans. The presence of this toxin in wine is related to the type of wine-red wines are more frequently contaminated with OTA-and the geographical location of the vineyard. In Europe, the lower the latitude, the greater the risk of OTA contamination in wine. However, climate change could increase the risk of OTA contamination in wine in other regions. Due to their toxic effects, the development of effective and environmentally friendly methods to prevent, decontaminate, and degrade OTA is essential. This review summarises the available research on biological aspects of OTA prevention, removal, and degradation.

The Application of Probiotic Bacteria from Strawberry (Fragaria ananassa × Duch.) in the Fermentation of Strawberry Tree Fruit (Arbutus unedo L.) Extract.

The search for unexplored plant resources that would provide a good basis for the development of novel probiotic functional foods is rapidly increasing. In this context, the strawberry tree fruit (Arbutus unedo L.) is particularly interesting, as it is rich in numerous antioxidant bioactive compounds that have been shown to be beneficial to health, but have not yet found industrial applications. In this work, the probiotic characterization of lactic acid bacteria strain Lactiplantibacillus plantarum DB2, isolated from strawberries (Fragaria ananassa × Duch.), was performed. The tested strain proved to be safe to use, displaying no antibiotic resistance or hemolytic activity. Due to its proven probiotic potential during simulated gastrointestinal transit, its antimicrobial activity, and its coaggregation with pathogens, it was selected for fermentation of an aqueous Arbutus unedo L. extract, which was subsequently microencapsulated and freeze-dried to extend its shelf life and preserve its functional properties. The antioxidant activity of the ferment obtained was maintained (80%), while after microencapsulation and freeze-drying, about 50% and 20% of the antioxidant activity was retained, respectively. In conclusion, this study demonstrates for the first time the application of probiotics isolated from strawberries in the fermentation of strawberry tree extract and monitors the antioxidant activity during post-fermentation formulation, paving the way for a potential industrial application of this underutilized plant.

Lactic Acid Bacteria of Marine Origin as a Tool for Successful Shellfish Farming and Adaptation to Climate Change Conditions.

Climate change, especially in the form of temperature increase and sea acidification, poses a serious challenge to the sustainability of aquaculture and shellfish farming. In this context, lactic acid bacteria (LAB) of marine origin have attracted attention due to their ability to improve water quality, stimulate the growth and immunity of organisms, and reduce the impact of stress caused by environmental changes. Through a review of relevant research, this paper summarizes previous knowledge on this group of bacteria, their application as protective probiotic cultures in mollusks, and also highlights their potential in reducing the negative impacts of climate change during shellfish farming. Furthermore, opportunities for further research and implementation of LAB as a sustainable and effective solution for adapting mariculture to changing climate conditions were identified.

A Predictive Assessment of Ochratoxin A's Effects on Oxidative Stress Parameters and the Fermentation Ability of Yeasts Using Neural Networks.

The aim of this paper was to examine the effect of different OTA concentrations on the parameters of oxidative stress (glutathione (GSH) and malondialdehyde (MDA) concentrations) and glucose utilization in ethanol production by wine yeasts. In addition to the above, artificial neural networks (ANN) were used to predict the effects of different OTA concentrations on the fermentation ability of yeasts and oxidative stress parameters. The obtained results indicate a negative influence of OTA (4 µg mL-1) on ethanol production after 12 h. For example, K. marxianus produced 1.320 mg mL-1 of ethanol, while in the control sample 1.603 µg mL-1 of ethanol was detected. However, after 24 h, OTA had no negative effect on ethanol production, since it was higher (7.490 and 3.845 mg mL-1) in comparison to control samples. Even low concentrations of OTA affect GSH concentrations, with the highest being detected after 12 and 24 h (up to 16.54 µM), while MDA concentrations are affected by higher OTA concentrations, with the highest being detected at 24 h (1.19 µM). The obtained results with the use of ANNs showed their potential for quantification purposes based on experimental data, while the results of ANN prediction models have shown to be useful for predictions of what outcomes different concentrations of OTA that were not part of experiment will have on the fermentation capacity and oxidative stress parameters of yeasts.

Unlocking the Bioactive Potential of Pomegranate Peels: A Green Extraction Approach.

Pomegranate (Punica granatum L.) is well known for its high content of bioactives, including polyphenols, flavonoids, and tannins, which have been shown to exhibit a wide range of biological activities, such as antioxidant, antimicrobial, and anticancer effects. It is worth noting that the majority of these molecules are found in the peels, which are usually disposed of after processing, causing a significant amount of waste, amounting to more than 3.6 million t/y. This work investigates microwave-assisted extraction (MAE) in water for the recovery of antioxidants from pomegranate peels (PP), including the optimisation of temperature and extraction times. The total phenolic, anthocyanin, flavonoid, and tannin contents of the recovered extracts were determined, as well as their antioxidant activities, which were found to be 356.35 mgGAE/gExtr, 303.97 µgCy3G/gExtr, 37.28 mgQE/gExtr, 56.48 mgGAE/gExtr, and 5.72 mmolTE/gExtr, respectively (according to the adopted reference). All results were compared with those obtained using a conventional protocol. In addition, the potential for water recycling by means of downstream nanofiltration in optimised MAE was investigated, leading to overall water reuse of approx. 75%. Power consumption (20.92 W/mgGAE) and common green metrics, Reaction Mass Efficiency (RME), E-Factor, and the Process Mass Intensiti/efficiency (PMI, PME), were considered in evaluating the proposed PP valorisation strategy. Finally, the biological activities of the main products were assessed. The antimicrobial properties of the PP extracts against three Gram-positive and three Gram-negative bacteria and their antiproliferative activity towards human cancer cells were tested. S. aureus bacteria was the most susceptible to the PP extracts. All tested products displayed antiproliferative activity against HeLa cells when higher concentrations were tested, with D-PP/NF (obtained from dried PP and sequential nanofiltration) being the most effective. This result was also confirmed via clonogenic analysis, which generally indicated the possible anti-cancer activity of pomegranate peel extracts obtained using this green approach.

3D Printing of Functional Strawberry Snacks: Food Design, Texture, Antioxidant Bioactive Compounds, and Microbial Stability.

3D printing technology (3DP) as additive manufacturing is an innovative design technology that can meet the individual nutritional and sensory needs of consumers. Therefore, the aim of this work was to apply 3DP in the production of a strawberry-based functional product with the addition of two hydrocolloids (corn and wheat starch) in three proportions (10, 15 and 20%) and to investigate the influence of 3DP process parameters on physico-chemical and textural properties, as well as the bioactive and antioxidant potential and microbiological stability, with(out) the addition of natural antimicrobial agents. Starch type had a significant effect on all tested bioactive compounds, as well as on starch content, except for total phenolic and hydroxycinnamic acid contents. Considering the content of bioactive compounds and antioxidant capacity, program 2 proved to be more suitable than program 1. All samples exhibited good textural properties, a high degree of stability and minimal geometric deviations. Regarding microbiological safety, no pathogenic bacteria were found in the 3DP samples during storage. The 3DP sample with added citral at a concentration of 75 mg L-1 showed the best microbiological quality. Ultimately, 3DP can be successfully used for the production of new strawberry-based functional products.

Poly(ε-caprolactone) Titanium Dioxide and Cefuroxime Antimicrobial Scaffolds for Cultivation of Human Limbal Stem Cells.

Limbal Stem Cell Deficiency (LSCD) is a very serious and painful disease that often results in impaired vision. Cultivation of limbal stem cells for clinical application is usually performed on carriers such as amniotic membrane or surgical fibrin gel. Transplantation of these grafts is associated with the risk of local postoperative infection that can destroy the graft and devoid therapeutic benefit. For this reason, electrospun scaffolds are good alternatives, as proven to mimic the natural cells surroundings, while their fabrication technique is versatile with regard to polymer functionalization and scaffolds architecture. This study considers the development of poly(ε-caprolactone) (PCL) immune-compatible and biodegradable electrospun scaffolds, comprising cefuroxime (CF) or titanium dioxide (TiO2) active components, that provide both bactericidal activity against eye infections and support of limbal stem cells growth in vitro. The PCL/CF scaffolds were prepared by blend electrospinning, while functionalization with the TiO2 particles was performed by ultrasonic post-processing treatment. The fabricated scaffolds were evaluated in regard to their physical structure, wetting ability, static and dynamic mechanical behaviour, antimicrobial efficiency and drug release, through scanning electron microscopy, water contact angle measurement, tensile testing and dynamic mechanical analysis, antimicrobial tests and UV-Vis spectroscopy, respectively. Human limbal stem cells, isolated from surgical remains of human cadaveric cornea, were cultured on the PCL/CF and PCL/TiO2 scaffolds and further identified through immunocytochemistry in terms of cell type thus were stained against p63 marker for limbal stem cells, a nuclear transcription factor and cytokeratin 3 (CK3), a corneal epithelial differentiation marker. The electrospun PCL/CF and PCL/TiO2 successfully supported the adhesion, proliferation and differentiation of the cultivated limbal cells and provided the antimicrobial effect against Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans.

SPME-GC-MS and Multivariate Analysis of Sensory Properties of Cheese in a Sack Matured with Probiotic Starter Cultures.

RESEARCH BACKGROUND: Cheese in a sack is a traditional cheese produced in Croatia. Types of cheese with similar production technology are made in other countries but chemical and microbiological composition varies between regions. Traditionally, cheese in a sack is produced without the addition of starter cultures. Addition of beneficial probiotic cultures to numerous dairy products has documented advantages. Effects that the addition of probiotic bacteria to traditional cheese have on aroma compounds and sensory properties have not been fully investigated. The aim of this study is to determine the sensory properties and differences in the aromatic profiles between cheese samples ripened in a lambskin sack, produced traditionally without the addition of any starter culture, or with the addition of probiotic bacteria. EXPERIMENTAL APPROACH: In this study, cheese in a sack was produced with the addition of probiotic cultures Lactobacillus plantarum B and L. lactis ssp. lactis S1. During ripening volatile aroma compounds were analysed with a solid-phase microextraction gas chromatography-mass spectrometry. Sensory properties were evaluated by trained tasters who are familiar with the traditional taste of the cheese from a sack. The results of aroma composition and taste scores were then compared using factorial and principal component analyses. RESULTS AND CONCLUSIONS: Chromatography showed differences in the composition of aroma compounds and the sensory properties between the cheese produced with Lactobacillus starter cultures and the control cheese, traditionally produced without a starter culture. The addition of probiotic cultures L. plantarum B and L. lactis ssp. lactis S1 resulted in products with better sensory properties and chemical profile of volatile aromatic compounds. NOVELTY AND SCIENTIFIC CONTRIBUTION: This study investigates the usage of naturally present probiotic cultures as starter cultures in cheese in a sack production. Their effects on aroma profiles and sensory characteristics have been compared for the first time using factorial and principal component analyses.

A study of surface moulds and mycotoxins in Croatian traditional dry-cured meat products.

Xerophilic species of Aspergillus, Penicillium and Eurotium genera from surfaces of dry-cured traditional meat products (TMPs) can cause mycotoxin contamination during uncontrolled household processing. The aim of this study was to investigate into surface moulds growing on Croatian prosciuttos and fermented sausages produced in different climate regions using different technologies (n = 160), and to relate the occurrence of aflatoxin B1 (AFB1) and ochratoxin A (OTA) to their presence. The results revealed the Penicillium (79%) to be the dominating contaminating mould, while Aspergillus (11%), Eurotium (7%) and Mucor (4%) species were present in a significantly lower number of isolates, with higher prevalence and greater diversity in prosciuttos than in sausages, relative of the production technology and regional climate. OTA contamination (14% of samples) was significantly more frequent than that with AFB1 (8% of samples), with OTA concentration rising to the maximal 6.86 µg/kg, whereas AFB1 concentrations were slightly higher than, or around, the limit of quantification of the method in use, with the maximal value of 1.92 µg/kg. The presence of AFB1 in absence of toxicogenic moulds, observed in some samples, can be attributed to contaminated spices used in TMP production or an indirect contamination via a carry-over effect.

Mycotoxins in food and feed.

Mycotoxins represent secondary fungal metabolites not essential to the normal growth and reproduction of a fungus, but capable of causing biochemical, physiological and pathological changes in many species. Harmful effects of mycotoxins observed in humans and animals include carcinogenicity, teratogenicity, immune toxicity, neurotoxicity, hepatotoxicity, nephrotoxicity, reproductive and developmental toxicity, indigestion and so forth. These substances can be found in a variety of very important agricultural and food products, primarily dependent of product moisture content, and its water activity, relative air humidity, temperature, pH value, composition of the food matrix, the degree of its physical damage, and the presence of mold spores. Given that industrial processing has no significant effect on their reduction and in order to be able to vouch for the absence of mycotoxins, it is necessary to process foodstuffs under standardized and well-controlled conditions and to control each and every loop of the food production and storage chain. Preventative measures capable of reducing the contamination to the minimum must be in place and should be exercised by all means. In case that contamination does happen, methods for mycotoxin reduction or elimination should be implemented in dependence on a number of parameters such as properties of food or feed. Further research is needed in order to identify conditions that facilitate the growth of mycotoxin-producing fungi and develop effective preventative measures that can reduce contamination of food and feed as also to recognize possible synergistic effects of different mycotoxins in organism.

Cytotoxicity of gamma irradiated aflatoxin B1 and ochratoxin A.

Toxicity of gamma irradiated mycotoxins aflatoxin B1 (AFB1) and ochratoxin A (OTA) was investigated in vitro. AFB1 and OTA stock solutions (50 mM, in methanol) were gamma irradiated (5 and 10 kGy) and non-irradiated and irradiated mycotoxins solutions were tested for cytotoxicity on Pk15, HepG2 and SH-SY5Y cell lines (MTT assay, 1-500 µM concentration range; 24 h exposure). Degradation of mycotoxin molecules was examined by liquid chromatography tandem mass spectrometry (HPLC-MS/MS). AFB1 and OTA radiolytic products were less toxic than the parent mycotoxins to all of the tested cell lines. Gamma irradiation even at 5 kGy had effect on AFB1 and OTA molecules however, this effect was dependent on chemical structure of mycotoxin. Since gamma irradiation at low dose reduced initial level of both mycotoxins, and gamma irradiated mycotoxins had lower toxicity in comparison to non-irradiated mycotoxins, it can be concluded that gamma irradiation could be used as decontamination method.

How sulphur dioxide and storage temperature contribute to patulin degradation in homemade apple juice.

Mycotoxin patulin is one of the quality indicators for apple juice. Like other mycotoxins, it raises consumer health concerns. The issue of low quality is particularly relevant for apples provided by small producers, whose quality control may not be standardised. As sulphur dioxide (SO2) is common in fruit preservation against fungi, the aim of this study was to determine how efficient it is in degrading patulin in apple juices stored in real-life conditions. This included refrigerated (4 °C) and non-refrigerated warehouses/environments (30 °C) over 8, 10, 12, and 20 weeks of storage. Apple juice was diluted to 0.010 µg g-1, 0.050 µg g-1, and 0.100 µg g-1 of patulin. SO2 was added to each sample in the amounts of 250 µg mL-1 and 50 µg mL-1. Untreated juice samples for each patulin concentration served as controls under the same experimental conditions. Patulin content was determined with high performance liquid chromatography. The best degradation was observed with 250 µg mL-1 of SO2 at 30 °C regardless of the patulin baseline concentration. Although treatment with SO2 and refrigeration did not fully remove patulin, it was highly efficient over twelve weeks of storage. Our results suggest that patulin levels can be reduced between 33 and 100 % at 30 °C and up to 100 % at 4 °C.

Properties and Fermentation Activity of Industrial Yeasts Saccharomyces cerevisiae, S. uvarum, Candida utilis and Kluyveromyces marxianus Exposed to AFB1, OTA and ZEA.

In this paper the effect of aflatoxin B1, ochratoxin A and zearalenon on morphology, growth parameters and metabolic activity of yeasts Saccharomyces cerevisiae, Saccharomyces uvarum, Candida utilis and Kluyveromyces marxianus was determined. The results showed that the three mycotoxins affected the morphology of all these yeasts, primarily the cell diameter, but not their final cell count. Fourier transform infrared spectroscopy showed that the yeast membranes bound the mycotoxins, C. utilis in particular. The cell membranes of most yeasts underwent denaturation, except S. uvarum exposed to ochratoxin A and zearalenone. In the early stage of fermentation, all mycotoxin-exposed yeasts had lower metabolic activity and biomass growth than controls, but fermentation products and biomass concentrations reached the control levels by the end of the fermentation, except for C. utilis exposed to 20 µg/mL of zearalenone. The adaptive response to mycotoxins suggests that certain yeasts could be used to control mycotoxin concentrations in the production of fermented food and beverages.

Antimicrobial, cytotoxic and antioxidative evaluation of natural deep eutectic solvents.

Natural deep eutectic solvents (NADES) are a new generation of green solvents. They are mixtures of two or three compounds such as choline chloride as a cationic salt and alcohols, acids, amides, amines or sugars as hydrogen-bond donors. Although the majority of NADES' components are of natural origin and therefore NADES are often presumed to be non-toxic, the evaluation of their toxicity and biodegradability must accompany the research on their synthesis and application. Therefore, the aim of this work was to investigate the effect of ten synthesised NADES towards bacteria (i.e., Escherichia coli, Proteus mirabilis, Salmonella typhimurium, Pseudomonas aeruginosa, Staphylococcus aureus), yeast (i.e., Candida albicans) and human cell lines (i.e., HeLa, MCF-7 and HEK293T). In addition, oxygen radical absorbance capacity (ORAC) method was used to determine the antioxidative activity of the tested NADES. Differences in toxicity response between microorganisms and cell lines were observed, and only NADES that contained organic acid showed toxicity towards the test systems. Furthermore, the NADES containing compounds that possess antioxidative activity also showed antioxidative activity. However, research whose primary purpose is the synthesis and application of NADES must be followed by an evaluation of their biological properties (e.g., antimicrobial activity, toxicity towards animal cells and antioxidative or other biological activity) to find the solvent with the best profile for wider industrial applications.

Influence of high power ultrasound on selected moulds, yeasts and Alicyclobacillus acidoterrestris in apple, cranberry and blueberry juice and nectar.

The purpose of this study was to investigate the effect of non-thermal technology, high power ultrasound (HPU) on inactivation of Aspergillus ochraceus 318, Penicillium expansum 565, Rhodotorula sp. 74, Saccharomyces cerevisiae 5 and Alicyclobacillus acidoterrestris DSM 3922 in clear juices and nectars from apple, blueberry and cranberry juice concentrate. Inoculated juice and nectars were treated by high power ultrasound (20kHz) according to procedure set by central composite design (CCD). Three operational parameters, amplitude (60, 90 and 120µm), temperature (20, 40 and 60°C), and treatment time (3, 6 or 9min) were varied in order to observe the influence of ultrasound and combination of ultrasound and slight heating (thermosonication) on growth and inactivation of selected microorganisms. Number of vegetative cells of A. acidoterrestris DSM 3922 were not significantly reduced by high power ultrasound (p>0.05), except in apple juice, where statistical significant (p<0.05) influence of quadratic interaction of amplitude on bacteria reduction were observed. In all samples of fruit juices and nectars in terms of ultrasonic treatment at 60°C and times of 3, 6 and 9min, regardless of the value of the amplitude, complete inactivation of the growth of yeasts and moulds were achieved, while at 20 and 40°C it is not observed. The value of reduction of cells of selected yeasts and moulds for ultrasound treatments at 60°C and the duration of the 3, 6 and 9min ranged from 3.556 to 5.934 log units, depending on the initial number of selected yeasts and moulds before treatment.

Removing aflatoxin M1 from milk with native lactic acid bacteria, centrifugation, and filtration.

In order to minimise human exposure to aflatoxin M1 (AFM1) the levels of this highly carcinogenic mycotoxin in milk, heat-treated milk, and other dairy products have been limited to <0.05 µg kg-1. However, its removal from dairy products presents a challenge for dairy producers, as commercial additives change organoleptic properties, and filtration alone yields poor results. The aim of this study was to find a strain of lactic acid bacteria (LAB) from milk or dairy products that most effectively binds AFM1 and to see whether heat treatment of the selected LAB affects the binding efficiency. We also wanted to investigate whether centrifugation can improve filtering of the obtained AFM1-LAB complexes from milk. To do that, we isolated and identified 10 native LAB species/strains, incubated their viable or heat-treated cells (108 CFU mL-1) in milk spiked with 0.5 µg L-1of AFM1 at 4 °C for 0, 2, 4, and 24 h, and quantified the amount of unbound AFM1 with HPLC. AFM1 binding efficiency ranged from 21 to 92 % for viable cells and from 26 to 94 % for the heattreated ones. Since both viable and heat-treated Lactobacillus plantarum KM showed the best results, we used it for the next step in AFM1 removal from milk. Heat treatment in combination with filtration and centrifugation yielded removal as high as 96 %.

Isolation and Characterisation of L. plantarum O1 Producer of Plantaricin as Potential Starter Culture for the Biopreservation of Aquatic Food Products.

Lactobacillus plantarum O1 was isolated from the gut of sea bream (Sparus aurata) and identified with the API biochemical test and MALDI-TOF MS. This strain was further characterised according to the selection criteria for lactic acid bacteria as starter cultures for aquatic food production. L. plantarum O1 showed good antimicrobial activity against pathogenic test microorganisms. Further investigation confirmed it as the producer of the bacteriocin plantaricin. This strain also showed good growth at a wide range of temperatures (from 4 to 45 °C) and a wide range of pH (2-12), even in the presence of 3.5% NaCl. Its viability was also good after lyophilisation and in simulated gastric and small intestinal juice. The strain is a promising probiotic, and our further research will focus on its application in the biopreservation of fresh fish and shellfish.

Co-occurrence of ochratoxin A and citrinin in unprocessed cereals established during a three-year investigation period.

The aim of this study was to investigate ochratoxin A (OTA) and citrinin (CIT) co-occurrence in different unprocessed cereals (n = 189) originating from Croatia during a three-year investigation period (2014-2016) using validated enzyme immunoassay (ELISA) methods. CIT and OTA were determined in 49% and 7% of samples, respectively. Significantly higher (p < 0.05) overall mean concentrations were determined for CIT (66.8 ± 76.0 µg/kg) in comparison to OTA (5.2 ± 1.1 µg/kg). Based on the analysis of all investigated cereals, CIT was found about 15 times more frequently than OTA and in similarly (15-fold) higher concentrations, irrespective of the cultivation year. The results revealed a moderately positive correlation between OTA and CIT concentrations in maize (rs = 0.44) and wheat (rs = 0.59), whereas in barley and oat this correlation (p > 0.01) was not significant.

The Effect of Autochthonous Starter Culture, Sugars and Temperature on the Fermentation of Slavonian Kulen.

In this study, the effect of an isolated and well-characterised autochthonous starter culture, glucose and maltodextrin (w=0.8%) and temperatures of 12 and 20 °C on fermentation and quality of Slavonian kulen produced using the traditional technology and recipe were investigated. Physicochemical and microbiological analyses were carried out after 20 days of fermentation. Upon the completion of the production process (90 days), a sensory analysis was carried out. Furthermore, pH value was continuously measured throughout the twenty-day fermentation period. The addition of an autochthonous starter culture and sugars and different fermentation temperatures significantly (p<0.05) affected the instrumental colour and texture parameters of the Slavonian kulen. The fermentation was most intense in the samples with added autochthonous starter culture and 0.8% glucose, and fermented at 20 °C. Microbiological analysis showed that samples with added autochthonous starter culture and fermented at higher temperature contained a higher number of lactic acid bacteria and coagulase-negative staphylococci and were safe. Sensory evaluation confirmed the outcomes of physicochemical and microbiological analyses and showed differences among samples fermented at two different temperatures and with added glucose or maltodextrin and an autochthonous starter culture.

Deoxynivalenol and zearalenone in unprocessed cereals and soybean from different cultivation regions in Croatia.

The aim of this study was to investigate the occurrence of deoxynivalenol (DON) and zearalenone (ZEN) in unprocessed cereals and soybean sampled in 2014 and 2015 from different fields located in Croatian regions. A total of 306 samples were analysed for DON and 415 samples for ZEN concentrations using quantitative ELISA methods. In 2014, DON and ZEN were determined in all samples in the mean concentrations of 1,461 ± 2,265 µg/kg and 656 ± 853 µg/kg, respectively, while in 2015 these means were 2,687 ± 2,731 µg/kg and 1,140 ± 1,630 µg/kg, respectively. The cultivation year significantly (p < 0.05) influenced mycotoxin concentrations, whereas the influence of cultivation region was seen with ZEN for all cereals except soybean, and not seen with DON at all. A higher contamination determined during 2015 could be explained by high to extreme humidity evidenced in the period of cereals' growth and harvesting.