DISRUPTION OF ASPERGILLUS FLAVUS CELLS: A BEAD MILL HOMOGENIZATION METHOD

The most important mycotoxigenic fungus involved in pre- and post-contamination of crops is Aspergillus flavus which causes great health and economic loses worldwide due to production of the most potent natural hepatocarcinogen – aflatoxin B1. Contamination with this secondary metabolite is getting even worst by global climate changes and other abiotic stressors present in environment. Accordingly, researches with the aim of synthesis or identifying the antiaflatoxigenic and antifungal compounds are of interest. For such efforts realization, use and manipulation with intracellular content of A. flavus cells is necessary. The aim of this study was to apply Omni® Bead Ruptor 12 Homogenizer on disintegration of A. flavus cells, to find optimal parameters of homogenization and prepare biologically active extracts which can be used for determination of possible strategies for control of contamination with aflatoxins. Results of study showed that bead mill homogenizer Omni® Bead Ruptor 12 Homogenizer can be applied for disintegration of A. flavus mycelia and preparation of enzymatically active cell-free extracts. The homogenization mixture in 2 mL homogenization tubes should contain 100 mg of fresh wet mycelia, 1 g of precooled acid washed glass beads of 0.5 mm in diameter and 1 mL of ice-cold buffer. Such mixture should be homogenized at speed of 6 m/s during 120 s, in six cycles of 20 s with cooling of samples in ice-bath between cycles

Monitoring of sodium adsorption ratio (SAR) in groundwater of Eastern Croatia

Natrij je kation koji ima jedinstven utjecaj na tlo. Ovisno o svojoj koncentraciji, može uzrokovati štetne fizikalno-kemijske promjene u strukturi tla, što rezultira raspršenjem čestica i smanjenjem brzine infiltracije vode i zraka u tlo. Zajedno s kalcijem definira alkalitet i salinitet tla što znatno utječe na rast biljke i njenu mogućnost upijanja vlage iz tla. Višak natrija u odnosu na kalcij i magnezij u tlu dovodi do oštećenja u strukturi glinastih tala, te dolazi do bubrenja čestica čime se mijenja hidraulički kapacitet tla. Ovakva tla nalaze se u disperznom stanju, ljepljiva su, stvaraju koru i zadržavaju vodu, a sušenjem postaju vrlo teška. U ovom radu prikazani su rezultati ispitivanja koeficijenta SAR (engl. Sodium adsorption ratio), tj. omjera koncentracije natrija (štetni element) i koncentracije kalcija i magnezija (korisni elementi). Iako se prilikom izračuna ovog koeficijenta ne uzimaju u obzir promjene koncentracija kalcija u vodi uslijed promjena topljivosti zbog taloženja ili otapanja tijekom navodnjavanja, SAR se smatra prihatljiv za ocjenu većine voda koje se koriste za navodnjavanje. Praćenje stanja je obavljeno tijekom pet godina na 40 lokacija istočne Hrvatske, na kojima je prikupljana neprerađena podzemna voda.Sodium is cation with the unique effect on the soil. Depending on its concentration it can cause detrimental physicochemical effects on soil structure, which results in particle dispersing and the reduction of water and air infiltration in soil.When combined with calcium, it effects alcalinity and salinity of thesoil, effecting plant growth and its water apsorption. The excess of sodium in regard to calcium and magnesium in the soil, leads to breakdown of clay soils structure and particle swelling which changes hydraulic capacity of the soil. These soils are disperged, sticky, create a crust and retain water, and upon drying become very hard. In this paper the results of monitoring of SAR (Sodium adsorption ratio), i.e. ratio of sodium (detrimental element) to the combination of calcium and magnesium (beneficial elements), are presented. Although the calculation of this ratio does not take into account changes in the concentration of calcium in the water due to changes in solubility due to precipitation or dissolution during irrigation, SAR is considered acceptable indicator for the assessment of most of the water used for irrigation. Monitoring was carried out over the period of five years at 40 different locations in Eastern Croatia, and collected groundwater was not processed

Antifungalno i antiaflatoksikogeno djelovanje kumarinil tiosemikarbazida na plijesan Aspergillus flavus NRRL 3251

The antifungal and antiaflatoxigenic effects of two series of coumarinyl thiosemicarbazides on Aspergillus flavus NRRL 3251 were studied. Fungi were grown in YES medium for 72 h at 29 °C in the presence of 0, 0.1, 1, and 10 μg mL-1 of coumarinyl thiosemicarbazides: one series with substitution in position 7 and another with substitution in position 4 of the coumarin core. Dry mycelia weight determination was used for antifungal activity estimation, while the aflatoxin B1 content in YES media, determined by the dilute and shoot LC-MS/MS technique, was used for the antiaflatoxigenic effect estimation. Standard biochemical assays were used for oxidative status marker (TBARS, SOD, CAT, and GPX) determination in A. flavus NRRL 3251 mycelia. Results show that 7-substituted-coumarinyl thiosemicarbazides possess a better antifungal and antiaflatoxigenic activity than 4-substituted ones. The most prominent substituted compound was the compound 3, N-(4-chlorophenyl)-2-(2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy)acetyl)hydrazine-1-carbothioamide, which completely inhibited aflatoxin production at the concentration of 10 μg mL-1. Oxidative stress response of A. flavus exposed to the selected compounds points to the modulation of oxidative stress as a possible reason of aflatoxin production inhibition.Ispitano je antifungalno i antiaflatoksikogeno djelovanje dviju serija kumarinskih tiosemikarbazida na plijesan Aspergillus flavus NRRL 3251. Plijesan je uzgojena u YES mediju tijekom 72 sata na 29 °C u prisutnosti kumarinskih tiosemikarbazida, koncentracija 0, 0,1, 1 i 10 μg mL-1, jedne serije gdje je kumarin supstituiran u položaju 4 i druge sa supstitucijom u položaju 7 kumarinske jezgre. Antifungalna aktivnost određena je mjerenjem mase suhog micelija, a sadržaj aflatoksina B1 određen je u YES mediju dilute and shoot LC/MS-MS metodom. Za određivanje markera oksidativnoga statusa u miceliju A. flavus NRRL 3251 koristili su se standardni biokemijski testovi (TBARS, SOD, CAT i GPX). Rezultati su pokazali da tiosemikarbazidi na kumarinu supstituiranom u položaju 7 imaju bolju antifungalnu i antimikotoksigenu aktivnost nego oni s kumarinom supstituiranom u položaju 4. Od ispitivanih spojeva valja istaknuti spoj 3, N-(4-klorofenil)-2-(2-((4-metil-2-okso-2H-kromen-7-il)oksi)acetil)hidrazin-1-karbotioamid, koji je pri koncentraciji 10 μg mL-1 potpuno inhibirao produkciju aflatoksina. Iz dobivenih rezultata može se pretpostaviti da je vjerojatni uzrok inhibicije produkcije aflatoksina oksidacijski stres uzrokovan ispitanim spojevima

Water for all : Proceedings of the 7th international scientific and professional conference Water for all

The 7th International Scientific and Professional Conference Water for all is organized to honour the World Water Day by the Josip Juraj Strossmayer University of Osijek, European Hygienic Engineering & Design Group (EHEDG), Danube Parks, Croatian Food Agency, Croatian Water, Faculty of Food Technology Osijek, Faculty of Agriculture in Osijek, Faculty of Civil Engineering Osijek, Josip Juraj Strossmayer University of Osijek Department of Biology, Josip Juraj Strossmayer University of Osijek Department of Chemistry, Nature Park “Kopački rit”, Osijek- Baranja County, Public Health Institute of the Osijek- Baranja County and „Vodovod-Osijek“ -water supply company in Osijek. The topic of World Water Day 2017 was "Wastewater" emphasizing the importance and influence of wastewater treatments on global environment. The international scientific and professional conference Water for all is a gathering of scientists and experts in the field of water management, including chemists, biologists, civil and agriculture engineers, with a goal to remind people about the significance of fresh water and to promote an interdisciplinary approach and sustainability for fresh water resource management. The Conference has been held since 2011. About 300 scientists and engineers submitted 95 abstracts to the 7th International Scientific and Professional Conference Water for all, out of which 33 was presented orally and 62 as posters. 47 full papers were accepted by the Scientific Committee. 38 full papers became the part of the this Proceedings while 9 papers were accepted for publication in Croatian Journal of Food Science and Technology and Electronic Journal of the Faculty of Civil Engineering Osijek - e-GFOS

Soil chemicals properties and wheat genotype impact on micronutrient and toxic elements content in wheat integral flour

Aim To determine impact of soil chemical properties and different wheat genotypes in Croatia on micronutrient and toxic elements content in wheat integral flour. Methods Research was conducted and soil samples were collected from two different production areas in the Republic of Croatia: Ovčara and Dalj. Besides soil samples, grain samples of four different Croatian wheat genotypes were also collected and analyzed. In total, 40 samples of soil and 40 samples of wheat grain were analysed for total (aqua regia) and plant available (EDTA extraction) heavy metal content of Fe, Mn, Zn, Cu, Pb, Cd. Results Determined soil pHKCl ranged from 5.63 to 6.25 at Ovčara and from 6.95 to 7.37 at Dalj sampling sites. The highest total concentration of heavy metals in soil were determined for Fe, followed by Mn, Zn, Cu, Pb and the lowest total concentration wasrecorded for Cd. The highest EDTA concentrations in soil were determined for Mn, than followed by Fe, Cu, Pb, and the lowest EDTA concentration was recorded for Cd. The highest concentration in integral wheat flour was found for Fe, than lower for Mn, Zn, Cu, Pb and the lowest concentration was found for Cd. If consumers in Croatia used daily 203 g of bread made of integral flour, they would take 2.31 to 8.44 µg Cd daily, depending on soil and wheat genotype.Conclusion The analysed soil and winter wheat genotypes have significant impact on potential daily intake of toxic and essentialheavy metals by integral flour or bread

Regulated Mycotoxin Occurrence and Co-Occurrence in Croatian Cereals

A total of 209 samples of various cereal crops (maize, wheat, barley, rye and oats) grown in Croatian fields during 2016 and 2017 were collected to analyze and determine the occurrence and co-occurrence of EU regulated mycotoxins in cereals (AFB1, AFB2, AFG1, AFG2, DON, FB1, FB2, ZEA, T-2, HT-2 and OTA). The analysis, performed by a validated confirmatory LC-MS/MS method based on a dilute and shoot principle, highlighted Fusarium mycotoxins as the main contaminants, often co-occurring in samples from both years (50.0% in 2016 and 33.7% in 2017). DON was found to be the most frequent mycotoxin, present in 72.5% of the 2016 samples and 32.6% of the 2017 samples, while maize proved to be the most contaminated cereal type of both years with FUM as the most abundant mycotoxins, with an average concentration of 1180 µg/kg. Moderate temperatures with periods of high humidity favored the accumulation of DON in wheat samples instead of other Fusarium mycotoxins, while similar conditions favored maize contamination with FUM. A total of 8.3% of all the 2016 harvest samples and 7.9% of the 2017 harvest samples were assessed as non-compliant, containing mycotoxins in concentrations higher than the levels set by the EU legislation for food

Development and Validation of an UHPLC-MS/MS Method for the Simultaneous Determination of 11 EU-Regulated Mycotoxins in Selected Cereals

The availability of reliable sensitive multi-analyte methods for unambiguous determination of mycotoxins is crucial for ensuring food and feed safety, considering their adverse health effects and (co-)occurrence in various foods. Accordingly, a multi-mycotoxin confirmatory method for simultaneous determination of 11 mycotoxins regulated in cereals within the European Union (EU) using ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) was developed and in-house validated to fit the EU legislation requirements for analytical methods. A simple sample preparation was based on a solid–liquid extraction using a solvent mixture acetonitrile/water/formic acid (79/20/1, v/v/v) and a dilution of raw extract using water/acetonitrile/formic acid (79/20/1, v/v/v) before instrumental analysis. Average recoveries in all three validated cereal crop types (maize, wheat, and barley), spiked at multiple levels, were found acceptable for all analytes when matrix-matched calibration was used, ranging from 63.2% to 111.2% and also showing very good repeatability, with relative standard deviations below 20%. Matrix effect (SSE) evaluation revealed maize as the most complex of the three analyzed cereal matrices, with strong SSE (<50% and >150%) recorded for all 11 analyzed mycotoxins. An additional method verification was performed through successful participation in proficiency testing schemes, with the achieved z-scores generally in the acceptable range of −2 ≤ z ≤ 2. The obtained validation results demonstrated the suitability of the developed confirmatory multi-mycotoxin UHPLC-MS/MS method based on a dilute-and-shoot principle for the simultaneous determination of low concentrations of 11 EU-regulated mycotoxins in cereals, including aflatoxins B1, B2, G1 and G2, deoxynivalenol, fumonisins B1 and B2, zearalenone, T-2 and HT-2 toxins, and ochratoxin A

Occurrence of Patulin and Polyphenol Profile of Croatian Traditional and Conventional Apple Cultivars during Storage

Apples and apple-based products are among the most consumed fruits around the world. However, they are susceptible to infection with the fungi Penicilium expansum. In addition to the reduction of apple quality, secondary metabolism of this fungus produces a mycotoxin patulin that has a negative effect on human health. Currently, there is no available research in the literature on the resistance of Croatian traditional apple cultivars to contamination with P. expansum, and consequently, on the patulin content in apples and apple juice produced from those apples. Although the mechanism of apple resistance to fungal diseases has not yet been sufficiently investigated, some studies have shown that polyphenolic compounds have some impact on fungi growth. In order to contribute with new knowledge, this research deals with monitoring the growth of P. expansum on apples, patulin detection by LC/MS-MS, determination of polyphenol profile by validated HPLC method, and determining the effect of polyphenolic compounds on fungi growth and patulin production during apple storage. The results of this study have shown that Croatian traditional apple cultivars harvested from family farm Horvatić contain higher concentration of polyphenolic compounds and higher antioxidant activity. At the same time, they showed more resistance to infection by P. expansum than conventional ones. The higher content of dihydrochalcones and flavanols encouraged the biosynthesis of patulin in examined cultivars. However, the higher content of non-flavonoids such as 2-6 dimethoxybenzoic acid, 4-hydroxycinnamic acid and chlorogenic acid leads to decrease in content of patulin. In conclusion, it seems that content of polyphenols and patulin production are correlated

Penicillium expansum Impact and Patulin Accumulation on Conventional and Traditional Apple Cultivars

Penicillium expansum is a necrotrophic plant pathogen among the most ubiquitous fungi disseminated worldwide. It causes blue mould rot in apples during storage, transport and sale, threatening human health by secreting patulin, a toxic secondary metabolite that contaminates apples and apple-derived products. Nevertheless, there is still a lack of sufficient data regarding the resistance of different apple cultivars to P. expansum, especially ancient ones, which showed to possess certain resistance to plant diseases. In this work, we investigated the polyphenol profile of 12 traditional and 8 conventional apple cultivar and their resistance to P. expansum CBS 325.48. Eight polyphenolic compounds were detected; the most prominent were catechin, epicatechin and gallic acid. The highest content of catechin was detected in ‘Apistar’—91.26 mg/100 g of fresh weight (FW), epicatechin in ‘Bobovac’—67.00 mg/100 g of FW, and gallic acid in ‘Bobovac’ and ‘Kraljevčica’—8.35 and 7.40 mg/100 g of FW, respectively. The highest content of patulin was detected in ‘Kraljevčica’ followed by ‘Apistar’—1687 and 1435 µg/kg, respectively. In apple cultivars ‘Brčko’, ‘Adamčica’ and ‘Idared’, patulin was not detected. Furthermore, the patulin content was positively correlated with gallic acid (r = 0.4226; p = 0.002), catechin (r = 0.3717; p = 0.008) and epicatechin (r = 0.3305; p = 0.019). This fact indicates that higher contents of gallic acid, catechin and epicatechin negatively affected and boost patulin concentration in examined apple cultivars. This can be related to the prooxidant activity of polyphenolic compounds and sensitivity of P. expansum to the disturbance of oxidative status