Jovaišienė, Jurgita

Mycotoxins are low molecular weight, toxic secondary metabolites naturally produced by various species of filamentous fungi. The occurrence of multiple mycotoxins is particularly concerning due to their potential impact on animal health and productivity, as well as their direct effects on reproductive disorders. The overall toxicity of mycotoxins arises from both individual mycotoxins and their combinations, making individual evaluation insufficient and requiring comprehensive in vitro assessment of their collective impact. This study aims to assess the cytotoxic effects and interaction patterns (additive, antagonistic, synergistic) of individual and combined mycotoxins on the Chinese hamster ovary (CHO-K1) cell line. CHO-K1 cells were selected to evaluate the effects of the mycotoxin’s aflatoxin B1 (AFB1), zearalenone (ZEA), deoxynivalenol (DON), and T-2 toxin (T-2), and their combinations. CHO-K1 cells were maintained in Ham’s F-12K medium with L-glutamine, 10% Fetal Calf Serum and 50 mg/mL gentamicin. AFB1 (5 and 7 µ/L), ZEA (500 and 1000 µg/L), DON (600 and 5000 µg/L), T-2 (250 and 500 µg/L), and their concentration combinations were prepared in DMSO, applied to cell cultures, and incubated at 37 °C for 24- 72 hours. Cytotoxic effects of mycotoxins and their combinations were assesed based on inhibitory concentration (IC50) values, with ≥ 50% cell death considered a positive response (n = 138). Cell viability was assessed by the MTT method. The absorbance was measured using an ELISA microplate reader at wavelengths of 540 nm and 580 nm. The interaction type was assessed in cell cultures using the Chou-Talalay method (n = 105). Fa-CI plots, combination index (CI) values and isobolograms were generated using CompuSyn software. In evaluating the effects of mycotoxin combinations on CHO-K1 cells, the highest IC50 after 72 h was observed with the ZEA/T-2 combination at 1000/500 µg/L (p < 0.05). Conversely, the AFB1/ ZEA/DON combination at 7/1000/5000 µg/L produced the lowest IC50 after the same exposure period (p < 0.05). Statistically significant interactions, ranging from pronounced antagonism to strong synergism, were detected across all tested mycotoxin combinations and concentrations in CHO-K1 cells throughout the experimental period. The strongest synergistic effect on CHO-K1 cells after 72 h was observed with the AFB1/ZEN/T-2 combination at 7/1000/500 µg/L (p < 0.05). In conclusion, our findings demonstrate that specific mycotoxin combinations exert varying cytotoxic effects on CHO-K1 cells. Also, this study exhibits an evident synergistic effect of specific mycotoxin combinations on CHO-K1 cells, suggesting potential risks on female reproduction associated with co-exposure to multiple mycotoxins.

Introduction. Fresh fruits are susceptible to fungal contamination at multiple stages, including cultivation, harvesting, transport, marketing, and storage by consumers. Identification of fungal contaminants is important because certain moulds can grow and produce mycotoxins, while certain yeasts and moulds may lead to fruit spoilage, resulting in reduced product quality and economic losses. Their naturally low pH further increases their susceptibility to fungal spoilage (1). Aim of the study. To evaluate the prevalence of moulds and mycotoxins in cherries, blackcurrants, and grape berries in relation to berry pH. Methods and materials. Fresh berries (cherries, blackcurrants, and grapes) were obtained from local farmers' markets. Yeast and mould counts were enumerated according to the standard Microbiology of food and animal feeding stuffs - Horizontal method forthe enumeration ofyeasts and moulds (ISO21527-2:2008). Mould genera were identified and characterised based on colony morphology and light microscopy. pH was measured using a WTW InoLab pH 720 pH meter. Mycotoxins were determined by thin-layer chromatography according to the Romer Labs (USA) method. Results. The total yeast and mould counts ranged from 4.68 to 6.27 ± 0.28 log10 CFU/g in blackcurrant samples, from 3.22 to 5.72 ± 0.48 log10 CFU/g in grape samples, and from 2.30 to 2.68 ± 0.08 log10 CFU/g in cherry samples (p <0.05). ThepH values ranged from 3.75 to 3.78 in grape samples, from 3.44 to 3.47 in cherry samples, and from 3.53 to 3.54 in blackcurrant samples. A weak positive correlation between yeast and mould counts and pH values was observed (r = 0.232), although the relationship was not statistically significant (p > 0.05). The following mould genera were identified: Penicillium, Cladosporium, Mucor, Rhizopus, Botrytis, Fusarium, and Monilia. The samples were predominantly contaminated with Penicillium (62%) and Cladosporium (22%) (p > 0.05). the saiiples Zearalenone (ZEN) and deoxynivalenol (DON) concentrations ranged from 165 to 250 µg/kg and LOD to 125 µg/kg in cherry samples, from 165 to 200 µg/kg and 125 µg/kg in grape samples, and from LOD to 130 µg/kg and LOD to 125 µg/kg in blackcurrant samples. Conclusions. The study demonstrated that yeast and mould contamination levels differed significantlyamong the analysed berry types (p < 0.05), with the highest counts detected in blackcurrant samples and the lowest in cherry samples. A weak positive correlation between pH values and yeast and mould counts was observed; however, this relationship was not statistically significant (p > 0.05), indicating that pH was not a determining factor for fungal contamination in the analysed samples. The predominant mould genera identified werePenicillium and Cladosporium. Afiatoxin B1 (AFL B1) was not detected in any of the samples, whereas zearalenone (ZEN) and deoxynivalenol (DON) were present in varying concentrations across allberry types. No statistically significant relationship was observed between yeast and mould counts and mycotoxin concentrations (p> 0.05).

ntroduction: Zearalenone (ZEN) is a nonsteroidal estrogenic mycotoxin characterized by a high binding affinity for estrogen receptors. In living organisms, ZEN is rapidly absorbed and metabolized into azearalenol (a-ZEL) and ẞzearalenol (B-ZEL), both of which may coexist in biological systems and pose potential health risks to animals and humans. The aim of the present study was to assess the overall metabolic activity and the nature of interactionsadditive, antagonistic, or synergistic among the five primary ZEN metabolites (a-ZEL, azearalanol (a-ZAL), B-zearalanol (B-ZAL), B-ZEL, and zearalanone (ZAN)) in the CHO-K1 cell line under in vitro conditions. Materials and methods: The Chinese hamster ovary cell line (CHO-K1) was cultured in Ham's F-12K (Kaighn's Medium) medium supplemented with L-glutamine, 10% fetal calf serum (FCS), Gentamicin (50 mg/mL). Cells were grown in 75 cm³ culture flaks at 37 °C in a humidified incubator with 5% CO2 and 98% relative humidity. The initial cell density used was 2.5x105 to 3.0x105 cells/mL, determined using a Neubauer haemocytometer. Cells were exposed individually to a-ZEL, ẞ-ZEL, a-ZAL, ẞ-ZAL, and ZAN for 24, 48, and 72 h at the following concentrations: 1 and 4 µg/L for a-ZEL; 2 and 3 µg/L for B-ZEL; 0.1 and 0.8 µg/L for B-ZAL; 0.5 and 1 µg/L for a-ZAL; and 3.5 and 8 µg/L for ZAN. Cytotoxicity was assessed using the MTT assay. Optical density was measured at wavelengths of 540 and 580 nm, and the ratio of these values was used to calculate the percentage of metabolically active cells,with control absorbance set to 100%. Each mycotoxin treatment and the control were tested in three independent experiments. Mean inhibitory concentration (IC) values were calculated from full dose-response curves. Isobologram analysis was used to determine the combination index (CI), a quantitative measure of the degree of interaction between two, three, four, or five mycotoxins. Interaction types were evaluated using CalcuSyn software version 2.1. Cl values < 1, = 1, and > 1 indicate synergistic, additive, and antagonistic effects, respectively. Results: The highest IC value among single compounds was observed for aZAL at 2 µg/L in CHO-K1 cells (p < 0.05). For twocompound combinations, the highest ICg after 72 h was observed for a-ZEL/ZAN at 1/3.5 µg/L (p < 0.05), while the lowest ICg, after 72 h occurred for ẞ-ZAL/a-ZAL at 0.1/0.5 µg/L (p < 0.05). For threecompound combinations, the highest ICg after 72 h was observed for a-ZEL/ẞ-ZEL/a-ZAL at 1/2/0.5 µg/L (p<0.05).The strongest synergistic effects in CHO-K1 cells were detected for a-ZEL/B-ZEL (4/2 and 4/3 µg/L) (p < 0.05), a-ZEL/ZAN (1/3.5 and 1/81 µg/L) (p<0.05), and B-ZEL/ZAN (3/3.5 µg/L). Conclusions: The strongest synergistic interactions between ZEN metabolites were observed in CHO-K1 cells after 72 hours of exposure, particularly for combinations containing a-ZEL, ẞ-ZEL, or a-ZAL (p < 0.05).

Honey is one of the oldest known natural products in the world. The beneficial properties of honey affect not only human health, but also food products. However, only high-quality, natural, and unheated honey has beneficial properties. In order to control honey quality, it is necessary to constantly monitor chemical composition indicators such as acidity, diastase activity, and moisture content. In recent years, there have been increasing reports of opportunistic bacteria and fungi in honey, so chemical analysis alone is not sufficient to ensure honey quality; it is also important to pay close attention to the microbiological composition of honey. The aim of the study. To evaluate the chemical and microbiological composition of honey collected in different regions of Lithuania. Methods and materials. The study was conducted using honey samples from different geographical and botanical areas. The honey was collected in 2023–2024 from apiaries located in different regions of Lithuania (Aukštaitija, Žemaitija, Suvalkija). A total of 34 honey samples were tested (2 samples of each type). During laboratory tests, the chemical analysis of honey composition was performed using standard methods: diastase activity, soluble dry matter content, moisture, acidity. The physical parameter pH was also determined. In order to assess the microbiological contamination of honey samples, the total number of microorganisms and mold colonies was determined. Results. Chemical analysis of honey samples showed that the honey was of good quality and met the requirements of Technical Regulation No. 3D-262 on Honey. The diastase activity of 82.4% ofall honey samples was higher than 8 Gothe units. The moisture content did not exceed 20%, except for heather honey, which can have a moisture content of up to 23%. The total acidity ofall samples was less than 50 meq kg1. Microbiological analysis showed that the highest total number of colony-forming units was found in meadow honey collected in 2023 (Kaunas district) - 5.03±0.36 log, CFU/g,and in meadow and forest honey collected in 2024 (Marijampolė district) - 4.05±1.93 log,n CFU/g. Meadow honey had the highest mold contamination: 2.09±0.55 log, CFU/g (2023, Kaunas district) and 1.75±0.74 log, CFU/g (2024, Kėdainiai district). Conclusions. The evaluation of honey samples collected in various regions of Lithuania showed that their chemical and microbiological properties met the essential quality and safety criteria. The diversity of honey samples was mainly determined by their botanical origin and region of collection.

Airborne dust and mold spores are considered the primary cause of spoilage in bread products. Airborne contamination is a major source of food contamination because it can carry mold spores. Mold contamination of baked goods also results in economic losses for companies. Dust and the molds and mycotoxins it contains not only affect product quality, but the effects of dust have also been linked to several respiratory diseases in bakery workers. The aim of this study was to determine the levels of molds and mycotoxins in dust sampled in the bakery environment. Dust samples were collected from a bakery during all seasons of the year. Samples were collected using two different methods: gravimetric and passive. The mold species and mycotoxin concentrations were determined in the dust samples. All data analyses were performed using Statistica software (Microsoft Excel 2016 and SPSS). The amount of dust varied from 0.093 g to 0.433 g and 0.010 g/m3 to 0.051 g/m3 collected by passive and gravimetric methods, respectively. The dominant mold species were Rhizopus spp., Trichoderma spp., Penicillium spp., and Cladosporium spp. The mycotoxins OTA, AFL B1 were detected in the dust samples. No positive values were found for the mycotoxins ZEA and DON. Our research data show how crucial it is to identify mold hazards in bakery to ensure the safety of production and health of workers.

The aim of this study was to evaluate the efficiency of ozonation in reducing aflatoxin B₁ and ochratoxin A in nuts. During the study, mold colony-forming units were determined using a dilution method based on ISO 21527-2:2008, and a mycotoxicological analysis was also performed. In total, 22 samples were analyzed: 6 shelled hazelnuts, 8 hazelnuts with shell and 8 peanuts with shell. The study was carried out in two stages. In the first stage, the number of mold CFU before and after ozonation (2.5 ppm, 30 or 60 min) was determined. In the second stage, the concentration of mycotoxins (aflatoxin B₁ and ochratoxin) was determined before and after ozonation treatment. The results of the study showed that hazelnuts with shell samples were the most infected with mold, from 0.6 ± 0.1 log₁₀ CFU/g to 1.6 ± 0.1 log₁₀ CFU/g. After 30 minutes of ozonation, molds were completely destroyed in all shelled hazelnuts samples (P > 0.05). After 60 minutes of ozonation, all molds in nuts were destroyed (P < 0.05). Before ozonation, the highest AFB₁ concentrations were detected in shelled hazelnuts samples (up to 5.0 μg/kg), while hazelnuts with shell (75% of all samples) showed the highest levels of contamination with OTA. After 30 minutes of ozonation at 2.5 ppm, AFB₁ concentrations significantly decreased in peanuts and hazelnuts with shell samples (P < 0.05). After 30 minutes of ozonation, OTA concentrations were significantly lower in peanuts and hazelnuts with shell samples (P > 0.05). Similar results were obtained after 60 minutes of ozonation, although in shelled hazelnut samples, OTA reduction was not statistically significant (P > 0.05).

Cereal grains are one of the most used raw materials in the food industry. According to statistical data, the highest global production volumes are recorded for maize (1116.34 million tons) and wheat (764.49 million tons). In addition to these major cereal crops, barley, oats, and rye are also in high demand. Therefore, efforts are focused on producing high-quality and safe raw materials for use in the food and feed industries. This study evaluates the impact of molds, their volatile organic compounds (VOCs), and mycotoxins on the qualitative indicators of wheat, barley, triticale, oat, and rye during storage. Grain samples were collected from a farm in Pasvalys district in September, February, and July. Total mold contamination (CFU/g) was determined, and mycotoxins (AFB1 , ZEA, DON, OTA, T-2) were determined using thin-layer chromatography. Qualitative grain indicators were analyzed using an NIRS 6500 spectrometer, while external and internal mold contamination was assessed by light microscopy. Mold fatty acids and VOCs were examined via gas chromatography-mass spectrometry. Results showed that mold contamination increased by 14.94% (P < 0.01) from the beginning to the end of storage. Mycotoxin levels also increased: AFB1 by 18.65, OTA by 9.5, and T-2 by 3.5 times (P < 0.01). T-2 toxin increased the least, i.e., by 3.5 times (P < 0.01). Chemical composition analysis indicated reductions in ash, fiber, and protein levels (P < 0.01), with a strong negative correlation between T-2 toxin and protein content. Fat (P ≥ 0.05), moisture, and starch (P < 0.01) levels increased, showing a positive correlation with T-2 toxin and Rhizopus spp. spread. Specific VOCs were associated to mold species, such as 3-ethoxy-1,1,1,5,5,5-hexamethyl-3-(trimethylsilyloxy) trisiloxane (Alternaria alternata) and 2,2,4,6,6-pentamethylheptane (Fusarium sporotrichioides). Penicillium viridicatum exhibited polyunsaturated fatty acids, while Aspergillus flavus, dominated by monounsaturated fatty acids, synthesized 2,2,5,5-tetramethylhexane. These biomarkers are valuable for early grain mold contamination detection and prevention.

Cereal stalks are by-products commonly used as straw for animal bedding or as a dietary supplement. They can be a potential source of fungal and mycotoxin contamination, especially under humid climatic conditions [1]. The aim of this study was to screen cereal stalks for the presence of common mycotoxins produced by Fusarium, Penicillium, and Aspergillus species prior to harvesting and storage under field conditions. Stalk samples were collected on the same day the grains were harvested. A total of 5 wheat stalk samples, 5 barley stalk samples, and 5 triticale stalk samples were collected from completely different fields. The primary moisture content was determined by weighing samples of stalks. The total number of yeasts and molds in the collected samples was determined according to the LST ISO 21527-2:2008 standard [2]. Mycotoxin concentrations (AFL B1, ZEN, and DON) were assessed using thin-layer chromatography, following methods provided by Romer Labs [3, 4]. All tested stalks samples of wheat, barley and triticale were found to be contaminated with fungi, Fusarium spp. being the predominant genus. A positive correlation was observed between fungal contamination and the primary moisture content of the stalks. The highest average total number of yeasts and molds was found in triticale stalks (6.08 ± 0.15 log CFU/g). The highest average concentration of AFB1 was detected in barley and triticale stalks (0.85 ± 0.85 µg/kg). The highest concentration of ZEN was found in wheat stalks (175 ± 175 µg/kg), while the highest average concentration of DON was detected in triticale stalks (325 ± 175 µg/kg). It can be concluded that cereal stalks used for straw production can be a potential source of fungal and mycotoxin contamination. Exposure through the digestive or respiratory tract may lead to health disturbances and decreased productivity.

ABSTRACT Pre-clinical production animal practice (80 academic hours) is obligatory part of the Study Programme in Veterinary Medicine at the Faculty of Veterinary Medicine, Veterinary Academy, Lithuanian University of Health Sciences (LSMU VA FVM). During this practice the 2nd year students acquire the competencies in production animal Veterinary Hygiene and Welfare, Rearing and Breeding, Feeds and Animal Nutrition under supervision of a practice supervisor and following the Procedure (1). During the Practice each student should complete the Logbook, prepared the Report and Reflection.To obtain student feedback on the pre-clinical practice, a questionnaire was developed by the Coordinator of Practice and approved by the Study Committee of FVM. The questionnaires were voluntary completed by 17; 29 and 30 students in the 2021/2022, 2022/2023 and 2023/2024 academic year, respectively. The results showed that the purpose of the practice is clear and easy to understand. The data indicates that 20.66%, 41.41%, 45.5% of respondents strongly agreed, while 13.50%, 12.12%,18.75% agreed, respectively, in the years specified above. The duration of the practice was sufficient with strong agreement by 24.41%, 24.00% and 38.83% respondents, while 37.44%, 29.65%, 20.17% agreed, in the respective year indicated. 37.72%; 35.06%, 45.5% of respondents strongly agreed, and 16.28%, 20.31%,16.00% agreed that the practice gave them the opportunity to develop their personal qualities in working independently; 37.72%, 29.88%, 51.17% of respondent strongly agreed and 16.28%,17.69%, 16.19% agreed that the practice gave them the opportunity to develop their personal qualities in a team, in the years indicated, respectively. In previous academic years, 20.80%,18.49%,19.75% of students expressed strong agreement, and 16.75%, 20.80%, 18.49% expressed agreement that respondents applied the theoretical knowledge they obtained from the studies. The limitation of this study is the low response to the questionnaires, so the goal is to encourage students to be more active in providing feedback.

Due to global warming, heat-induced stress is becoming an increasingly important problem for dairy cows in Lithuania. A temperature-humidity index (THI) is a single value representing the combined effects of air temperature and humidity associated with the level of heat stress. The aim of this study was to evaluate the effects of THI on the level of stress hormone cortisol in cows and productivity of dairy cows in climatic conditions of two dairy farms in Lithuania. The study was conducted in 2019–2020 in two selected dairy farms in southern Lithuania. Necessary data on environmental conditions and productivity of cows on farms were collected, averages of temperaturehumidity index were calculated. 20 urine samples were tested to determine the level of stress hormone in cows. Statistics were processed using statistical data analysis. The largest differences between the average air temperatures (⁰C), relative humidity (%), air velocity (m/s) and temperature-humidity index values in the farm regions were observed in the winter season. A statistically significant dependence of cortisol concentration in the body on THI was also found (r = 0.902, P < 0.05). In both farms, the highest temperatures-humidity index averages were recorded in 2019 June (74.2 and 73.9). The statistical analysis demonstrated that the temperature-humidity index had a negative effect on the milk yield, fat content and protein content of cows (P < 0.05).