Reduction of Alternaria Toxins via the Extrusion Processing of Whole-Grain Red Sorghum Flour.

This study delved into the impact of two extrusion processing parameters-screw speed (SS at 400, 600, 800 RPM) and material moisture content in the extruder barrel (M at 12, 15, 18%) at constant feed rate (50 kg/h)-on reducing the content of alternariol (AOH), alternariol monomethyl ether (AME), tenuazonic acid (TeA), and tentoxin (TEN) in whole-grain red sorghum flour. Ultra-performance liquid chromatography combined with a triple-quadrupole mass spectrometer (UPLC-MS/MS) was employed for the determination of Alternaria toxin levels. The extruder die temperature fluctuated between 136 and 177 °C, with die pressures ranging from 0.16 to 6.23 MPa. The specific mechanical energy spanned from 83.5 to 152.3 kWh/t, the torque varied between 88 and 162.8 Nm, and the average material retention time in the barrel ranged from 5.6 to 13 s. The optimal parameters for reducing the concentration of all Alternaria toxins with a satisfactory quality of the sorghum snacks were: SS = 400 RPM, M = 12%, with a reduction of 61.4, 76.4, 12.1, and 50.8% for AOH, AME, TeA, and TEN, respectively.

Moniliformin Occurrence in Serbian Maize over Four Years: Understanding Weather-Dependent Variability.

Moniliformin (MON) represents one of the most widespread emerging mycotoxins, whose presence in food and feed can potentially cause harmful effects on the health of both the public and animals. In order to investigate MON occurrence, a total of four hundred (n = 400) samples of unprocessed maize were sampled from different regions (Backa, Banat, and Srem) of Serbia during a period of four years (2018-2021) and were analyzed using a validated liquid chromatography with tandem mass spectrometry (LC-MS/MS) method. The influence of regional differences and variations from year to year in terms of weather conditions on the occurrence of MON was also assessed. The obtained validation parameters indicated that the LC-MS/MS method is applicable to the determination of MON in maize samples. It can be observed from the acquired findings that all samples were contaminated with MON, while concentration levels varied between the samples, especially from different sampling years. The maize samples showed the greatest levels of MON concentration during the dry and hot climatic conditions experienced in 2021. In maize samples harvested in the period 2018-2020, the MON concentration levels detected were about two to three times lower compared to the year 2021. Additionally, a comprehensive investigation into the correlation between weather conditions and the occurrence of MON in maize grown in Serbia was undertaken by reviewing the outcomes of research undertaken in the past decade.

Climate Change-A Global Threat Resulting in Increasing Mycotoxin Occurrence.

During the last decade, scientists have given increasingly frequent warnings about global warming, linking it to mycotoxin-producing moulds in various geographical regions across the world. In the future, more pronounced climate change could alter host resilience and host-pathogen interaction and have a significant impact on the development of toxicogenic moulds and the production of their secondary metabolites, known as mycotoxins. The current climate attracts attention and calls for novel diagnostic tools and notions about the biological features of agricultural cultivars and toxicogenic moulds. Since European climate environments offer steadily rising opportunities for Aspergillus flavus growth, an increased risk of cereal contamination with highly toxic aflatoxins shall be witnessed in the future. On top of that, the profile (representation) of certain mycotoxigenic Fusarium species is changing ever more substantially, while the rise in frequency of Fusarium graminearum contamination, as a species which is able to produce several toxic mycotoxins, seen in northern and central Europe, is becoming a major concern. In the following paper, a high-quality approach to a preventative strategy is tailored to put a stop to the toxicogenic mould- and mycotoxin-induced contamination of foods and feeds in the foreseeable future.

Impact of Climate Changes on the Natural Prevalence of Fusarium Mycotoxins in Maize Harvested in Serbia and Croatia.

Ongoing climate change may affect the susceptibility of plants to attacks by pathogenic, mostly mycotoxigenic fungi with a consequent increase in the presence of mycotoxins. Fusarium fungi represent one of the most important producers of mycotoxins, and are also important pathogens of agricultural crops. Therefore, the main aim of the study was to estimate the impact of weather parameters on the natural occurrence of Fusarium mycotoxins, such as deoxynivalenol (DON), fumonisins B1 and B2 (FUMs), zearalenone (ZEN), T-2, and HT-2 toxins (T-2/HT-2) in maize samples harvested from two neighboring countries, Serbia and Croatia, during a four-year production period (2018-2021). The frequency and contamination level of examined Fusarium mycotoxins varied by maize year of production and could be linked to weather conditions per investigated country. Among them, FUMs were found to be the most common contaminants (84-100%) of maize in both Serbia and Croatia. Additionally, a critical assessment of Fusarium mycotoxins occurrence in the last 10 years (2012-2021), for both Serbia and Croatia, was done. Results pointed out the highest contamination of maize from 2014, especially with DON and ZEN, in connection to extreme levels of precipitation observed in both Serbia and Croatia, whereas FUMs occurred with high prevalence from each of the ten investigated years.

Influence of Biotreatment on Hordeum vulgare L. Cereal Wholemeal Contamination and Enzymatic Activities.

Crop contamination with mycotoxins is a global problem with a negative impact on human and animal health as well as causing economical losses in food and feed chains. This study was focused on the evaluation of the effect of lactic acid bacteria (LAB) strain (Levilactobacillus brevis-LUHS173, Liquorilactobacillus uvarum-LUHS245, Lactiplantibacillus plantarum-LUHS135, Lacticaseibacillus paracasei-LUHS244 and Lacticaseibacillus casei-LUHS210) fermentation on the changes in the level of deoxynivalenol (DON) and its conjugates in Fusarium spp.-contaminated barley wholemeal (BWP). Samples, with different contamination of DON and its conjugates, were treated separately (for 48 h). In addition to mycotoxin content, enzymatic activities (amylolytic, xylanolytic, and proteolytic) of BWP (before and after fermentation) were evaluated. It was established that the effect of decontamination depends on the LAB strain used, and a significant reduction in DON and the concentration of its conjugates in Lc. casei fermented samples was achieved: the amount of DON decreased on average by 47%, and the amount of D3G, 15-ADON and 3-ADON decreased by 82.4, 46.1, and 55.0%, respectively. Lc. casei also showed viability in the contaminated fermentation medium and an effective production of organic acids was obtained. Additionally, it was found that enzymes are involved to the detoxification mechanism of DON and its conjugates in BWP. These findings indicate that fermentation with selected LAB strains could be applied for contaminated barley treatment in order to significantly reduce Fusarium spp. mycotoxin levels in BWP and improve the sustainability of grain production.

Aflatoxins in Maize from Serbia and Croatia: Implications of Climate Change.

Aflatoxins (AFs) represent the most important mycotoxin group, whose presence in food and feed poses significant global health and economic issues. The occurrence of AFs in maize is a burning problem worldwide, mainly attributed to droughts. In recent years, Serbia and Croatia faced climate changes followed by a warming trend. Therefore, the main aim of this study was to estimate the influence of weather on AFs occurrence in maize from Serbia and Croatia in the 2018-2021 period. The results indicate that hot and dry weather witnessed in the year 2021 resulted in the highest prevalence of AFs in maize samples in both Serbia (84%) and Croatia (40%). In maize harvested in 2018-2020, AFs occurred in less than, or around, 10% of Serbian and 20% of Croatian samples. In order to conduct a comprehensive study on the implications of climate change for the occurrence of AFs in maize grown in these two countries, the results of available studies performed in the last thirteen years were searched for and discussed.

Changes in Fusarium and Aspergillus Mycotoxin Content and Fatty Acid Composition after the Application of Ozone in Different Maize Hybrids.

Mycotoxins in maize represent a great threat to human health. For this reason, novel technics such as ozone treatment are used to reduce the content of maize mycotoxins. However, there is little knowledge about the effect of ozone treatment on maize quality parameters. This study investigated the changes in Fusarium and Aspergillus mycotoxins and the changes in fatty acids during the ozone treatment of maize samples. Sixteen maize hybrids were visually tested for the naturally occurring ear rot severity and treated with three different concentrations of ozone (40, 70, and 85 mg/L). Mycotoxin content in maize samples was determined using a high-performance liquid chromatography (HPLC) system, whereas dominant fatty acids were determined using gas chromatography coupled with a flame ionization detector (GC-FID). Ozone treatments could be successfully applied to reduce the content of mycotoxins in maize below the detection limit. Ozone treatments increased the content of monounsaturated fatty acids (MUFAs) and decreased the content of polyunsaturated fatty acids (PUFAs), i.e., linoleic acid (36.7% in relation to the lowest applied ozone concentration), which negatively affected the nutritional value of maize.

Fusarium metabolites in maize from regions of Northern Serbia in 2016-2017.

The main objective of this study was to determine the presence of Fusarium metabolites in maize samples collected from different regions of Northern Serbia (Backa, Banat and Srem) during a period of two years (2016-2017). A total of 458 maize samples were analysed by liquid chromatography-tandem mass spectrometry. A total of 40 metabolites were detected, where 94% of the samples contained at least 5 metabolites. Fumonisins (including B1, B2, B3 and B4), moniliformin and bikaverin were the most frequent (80-98%) Fusarium metabolites in both years. Furthermore, in samples from 2016, fumonisin A1 and A2, deoxynivalenol, deoxynivalenol-3-glucoside, zearalenone, culmorin, 15-hydroxyculmorin, fusapyron, fusaproliferin and aurofusarin were detected with frequencies of 58-80%. Levels of certain Fusarium metabolites in 2016 were higher on average due to increased humidity when compared to 2017, which was characterised by warm and dry conditions.

Preliminary Survey of Alternaria Toxins Reduction during Fermentation of Whole Wheat Dough.

The aim of this study was to investigate the fate of the most common Alternaria toxins found in wheat-tenuazonic acid (TeA), alternariol (AOH), and alternariol monomethyl ether (AME) during sourdough processing. For this purpose, spiked whole wheat flour, 3% sourdough starter, 0.5% of baker's yeast, and 105% of water calculated on flour weight as a base were used as raw materials. Spiked whole wheat dough was fermented for 4 h, 8 h, 12 h, 24 h, and 48 h at 25 °C, and at each point the fermented dough samples were taken, frozen, lyophilized, grounded, and stored until further analysis. To study the effect of sourdough processing on TeA, AOH and AME content, the validated method of high-performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) for these mycotoxins was used. Mathematical models of Alternaria toxins reduction were developed in the form of Four Parameter Logistic Regression function. The maximum reduction of TeA, AOH, and AME levels was archived at 48 h of dough fermentation at 25 °C compared with dough after kneading (0 h). Under these conditions, a reduction of the toxin levels of 60.3 %, 41.5%, and 24.1% was observed for TeA, AOH, and AME, respectively.

Mycotoxins in maize harvested in Serbia in the period 2012-2015. Part 2: Non-regulated mycotoxins and other fungal metabolites.

The main objective of this study was to screen, for the first time, the natural occurrence of non-regulated fungal metabolites in 204 maize samples harvested in Serbia in maize growing seasons with extreme drought (2012), extreme precipitation and flood (2014) and moderate drought conditions (2013 and 2015). In total, 109 non-regulated fungal metabolites were detected in examined samples, whereby each sample was contaminated between 13 and 55 non-regulated fungal metabolites. Moniliformin and beauvericin occurred in all samples collected from each year. In samples from year 2012, oxaline, questiomycin A, cyclo (l-Pro-l-Val), cyclo (l-Pro-l-Tyr), bikaverin, kojic acid and 3-nitropropionic acid were the most predominant (98.0-100%). All samples from 2014 were contaminated with 7-hydroxypestalotin, 15-hydroxyculmorin, culmorin, butenolid and aurofusarin. Bikaverin and oxaline were quantified in 100% samples from 2013 and 2015, while 3-nitropropionic acid additionally occurred in 100% samples from 2015.

Mycotoxins in maize harvested in Republic of Serbia in the period 2012-2015. Part 1: Regulated mycotoxins and its derivatives.

The main objective of this study was to apply a liquid chromatography-tandem mass spectrometric method to investigate the presence of 20 mycotoxins in 204 maize samples harvested in Northern Serbia in the period 2012-2015, including seasons with extreme drought (2012), hot and dry conditions (2013 and 2015) and extreme precipitation (2014). Between 2 and 20 mycotoxins contaminated examined samples. In samples collected from each year, all of six examined fumonisins were detected with very high prevalence (from 76% to 100%). Aflatoxin B1 was detected in 94% and 90% maize samples from 2012 and 2015, respectively. In samples from year 2014, deoxynivalenol, zearalenone and its derivatives were detected in 100% of samples. Furthermore, ochratoxin A (25%) was the most predominant in samples from 2012. The obtained results indicate that changes in weather conditions, recorded in the period of four years, had significant influence on the occurrence of examined mycotoxins in maize.

Effect of Atmospheric Cold Plasma Treatments on Reduction of Alternaria Toxins Content in Wheat Flour.

Beside Fusarium toxins, Alternaria toxins are among the most commonly found mycotoxins in wheat and wheat products. Currently, investigations of possibilities of reduction of Alternaria toxins in the wheat-processing chain are limited. Therefore, the aim of this study was to explore the potency of cold atmospheric plasma treatments, as a new non-thermal approach, for reduction of alternariol (AOH), alternariol monomethyl ether (AME) and tentoxin (TEN) content in spiked white wheat flour samples. Samples were treated with plasma generated in the air during 30 s to 180 s, with an increment step of 30 s, and at four varying distances from the cold plasma source (6 mm, 21 mm, 36 mm and 51 mm). The reduction of the Alternaria toxins content in samples after treatment was monitored by high performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The maximum reduction of the examined Alternaria toxins was obtained by treatment performed at 6 mm distance from the plasma source, lasting 180 s, resulting in reductions of 60.6%, 73.8% and 54.5% for AOH, AME and TEN, respectively. According to the obtained experimental results, five empirical models in the form of the second-order polynomials were developed for the prediction of AOH, AME and TEN reduction, as well as the temperature and the moisture content of the wheat flour, that gave a good fit to experimental data and were able to predict the response variables successfully. The developed second-order polynomial models showed high coefficients of determination for prediction of experimental results (between 0.918 and 0.961).

Effect of Wheat Milling Process on the Distribution of Alternaria Toxins.

Alternaria toxins are mycotoxins produced by various Alternaria species which, besides the Fusarium species, represent the principal contaminants of wheat worldwide. As currently, only limited information on the behaviour of Alternaria toxins during processing of cereals is available, the objective of this study was to investigate the effect of the dry milling process of wheat on Alternaria toxins distribution. Alternariol (AOH), alternariol monomethyl ether (AME) and tenuazonic acid (TeA) content were analysed by high performance liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in all milling fractions of untreated (control), fungicide-treated, Alternaria tenuissima inoculated and commercial wheat sample. After dry milling process, in last break and milling flows and by-products, increased concentration of examined Alternaria toxins was detected. TeA was quantified in almost all milling fractions in all tested wheat samples, while AOH and AME were detectable mostly in last break and milling flows and by-products. In respect to the contamination with Alternaria toxins, white flour can be considered as relatively safe product. Since Alternaria toxins are concentrated mainly in the peripheral parts of the kernel, a special attention should be given to their content in low-grade flours and milling by-products.

The effect of storage temperature and water activity on aflatoxin B1 accumulation in hull-less and hulled spelt grains.

BACKGROUND: In concomitance with shifts in climate conditions in recent years, an increasingly frequent emergence of Aspergillus flavus and aflatoxins in cereals has been observed. In this study the effects of temperature (15, 23, 30 and 37 °C) and water activity (aw ) (0.85, 0.90, 0.95 and 0.99) on aflatoxin B1 (AFB1 ) production by A. flavus isolate inoculated on hull-less and hulled spelt grains were investigated. RESULTS: The optimal conditions for AFB1 biosynthesis were reached at 30 °C and aw value of 0.99 in the all tested samples (hull-less grains, dehulled spelt grains and hulls). The AFB1 accumulation was significantly higher in hull-less than in dehulled grains, that implicated a protective effect of spelt hulls. The levels of AFB1 were about 10-170 times higher in hulls than in grains. In order to determine the possibility of predicting the occurrence of AFB1 under different storage conditions mathematical models [second order polynomial (SOP) and artificial neural network (ANN)] were applied. CONCLUSION: The achievement of such estimation facilitates further decisions on continuous monitoring of the potential hazard related to AFB1 contamination of stored spelt-based food. The knowledge of the storage temperature and aw effects on the AFB1 content in spelt during the postharvest phase is of great practical importance. © 2019 Society of Chemical Industry.

Aflatoxins in maize harvested in the Republic of Serbia over the period 2012-2016.

Aflatoxins (AFs) are one of the most frequent contaminants in maize. The Republic of Serbia represents a leader in terms of maize production and exports in Europe. In recent years, Serbia faced climate changes. Therefore, the main objective of this study was to examine AF occurrence in 3000 maize samples collected during 2012-2016. In maize samples from 2012, 2013, 2015, and 2016, AFs were detected in 72.3%, 24.7%, 36.7%, and 5.0% of the samples, in concentration ranges of 1.0-111.2, 1.2-65.2, 1.1-76.2, and 1.3-6.9 µg kg-1, respectively. Contrary to this, in maize samples from 2014, no AFs were detected. Analysis of weather conditions indicates that 2012 was characterised by extreme drought conditions, 2013 and 2015 by lack of precipitation and high air temperatures, 2014 as the rainiest year in these years, while 2016 had moderate weather conditions.

Aflatoxins contamination of maize in Serbia: the impact of weather conditions in 2015.

In recent years climate changes recorded in temperate regions of Europe have led to aflatoxin (AF) contamination of maize. Thus, the aim of this study was to investigate the influence of weather conditions on levels of aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1) and aflatoxin G2 (AFG2) in 180 maize samples collected from the main maize-growing regions (Western Backa, North Banat, South Banat and Central Serbia) in Serbia after harvest in 2015. The concentrations of AFs were determined by a validated HPLC method with post-column derivatisation and fluorescence detection (HPLC-FLD). The presence of AFB1, AFB2, AFG1 and AFG2 was detected in 57.2%, 13.9%, 5.6% and 2.8% of maize samples in the concentration ranges of 1.3-88.8 µg kg-1, 0.60-2.8 µg kg-1, 1.8-28.5 µg kg-1 and 2.1-7.5 µg kg-1 respectively. The recorded smaller amount of precipitation and especially higher air temperatures during the summer of 2015 were favourable for AF production, which resulted in 32.2% and 21.1% of samples being unsuitable for human consumption, since AFB1 and the sum of AFs concentrations were above 5.0 and 10.0 µg kg-1 respectively. Furthermore, the findings in this study indicate that the microclimate conditions in the investigated regions had a great influence on the contamination frequency of maize with AFs. The highest percentage of samples unsuitable for human consumption, considering both AFB1 and total AFs content were 72.5% and 51.5% respectively from Central Serbia, whilst the lowest percentages of 15.6% and 6.2% respectively were found in Western Backa. These findings confirmed that maize should be continuously monitored in order to protect human and animal health from the harmful effects caused by AFs contamination.

Comparison of ELISA, HPLC-FLD and HPLC-MS/MS Methods for Determination of Aflatoxin M1 in Natural Contaminated Milk Samples.

Presence of aflatoxin M1 (AFM1) in milk should be continuously controlled in order to protect the population from risks associated with its proven toxicity and carcinogenicity. During recent years, there has been an increase in demand for development of sensitive, accurate, simple and fast method which is reliable for detection of AFM1 at low concentrations found in milk samples. For that purpose, enzyme linked immunosorbent asssay (ELISA), high performance liquid chromatography with fluorescence detector (HPLC-FLD) and high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) were optimized and validated in order to apply them for AFM1 analysis in naturally contaminated milk samples, and to assess the closeness of agreement between results of three different methods. The obtained validation parameters indicate that all three methods are suitable for determination of AFM1 in milk samples. The statistical analysis of variance between the methods and the obtained correlation coefficients indicate that there is a strong correlation between methods. All three methods are satisfactory in meeting the requirements for official control purposes. To the best of author's knowledge, this study represents the first report of an investigation and comparison of ELISA, HPLC-FLD and HPLC-MS/MS methods for determination of AFM1 in naturally contaminated milk samples.

Possibility of Alternaria toxins reduction by extrusion processing of whole wheat flour.

This study represents the first report about possibility of reduction of Alternaria toxins in wheat using the extrusion process. Effects of extrusion processing parameters - moisture content (w=16, 20, 24g/100g), feeding rate (q=15, 20, 25kg/h), and screw speed (v=300, 390, 480rpm), on reduction rate of tenuazonic acid (TeA), alternariol (AOH) and alternariol monomethyl ether (AME), in whole wheat flour were investigated. Temperature ranged between 111.1 and 160.8°C, while the absolute pressure was from 0.17 to 0.23MPa. The simultaneous influence of w and v was the most important for TeA reduction (p<0.05), while v and q were the most influential for AOH reduction (p<0.01). Level of AME reduction was mostly influenced by w and v (p<0.10). Optimal parameters for reduction of all three Alternaria toxins were as follows: w=24g/100g, q=25kg/h, v=390rpm, with a reduction of 65.6% for TeA, 87.9% for AOH and 94.5% for AME.