Climate Change and Mycotoxins Trends in Serbia and Croatia: A 15-Year Review.

This review examines the 15-year presence of mycotoxins in food from Serbia and Croatia to provide a comprehensive overview of trends. Encompassing the timeframe from 2009 to 2023, this study integrates data from both countries and investigates climate change patterns. The results from Serbia focus primarily on maize and milk and show a strong dependence of contamination on weather conditions. However, there is limited data on mycotoxins in cereals other than maize, as well as in other food categories. Conversely, Croatia has a broader spectrum of studies, with significant attention given to milk and maize, along with more research on other cereals, meat, and meat products compared to Serbia. Over the investigated 15-year period, both Serbia and Croatia have experienced notable shifts in climate, including fluctuations in temperature, precipitation, and humidity levels. These changes have significantly influenced agriculture, consequently affecting the occurrence of mycotoxins in various food products. The results summarized in this 15-year review indicate the urgent need for further research and action to address mycotoxins contamination in Serbian and Croatian food supply chains. This urgency is further emphasized by the changing climatic conditions and their potential to exacerbate public health and food safety risks associated with mycotoxins.

Aflatoxins in maize, milk and dairy products from Serbia.

This study presents data on the occurrence of aflatoxins B1, B2, G1 and G2 in maize and aflatoxin M1 (AFM1) in milk and dairy products from Serbia in 2022. A total of 100 maize samples were analysed using liquid chromatography tandem mass spectrometry, while 107 cow and goat milk, besides dairy products were tested with an ELISA method. Aflatoxins contaminated 78% of the maize samples, with aflatoxin B1 as the most prevalent aflatoxin, also at the highest determined level. All milk samples tested positive for AFM1, while contamination in dairy products ranged from 14% to 100%. The hot and dry weather during the 2022 maize growing season, characterised by moderate to extreme drought conditions, significantly increased maize contamination with aflatoxins, impacting subsequent milk and dairy products contamination. The findings of this study confirm the continuous trend and persistent challenge in Serbia concerning the prevalence of aflatoxins in maize and milk, closely linked to climate change.

Application of biodegradable cholinium ionic liquids for the extraction of 5-hydroxymethylfurfural (HMF) from honey.

5-Hydroxymethylfurfural (HMF), a Maillard reaction product, can be formed when honey is subjected to heat treatment or a long storage time, becoming volatile and toxic depending on its concentration. The fact that, until today, there is no literature data on the extraction of 5-hydroxymethylfurfural (HMF) from honey using ionic liquids directed the investigation of the influence of biodegradable cholinium ionic liquids on the formation of aqueous biphasic systems and the application of these systems for the extraction of HMF from honey. The influence of anions of synthesised ionic liquids on the construction of biphasic systems in which an inorganic salt was used as a salting agent was investigated. Then, the extraction of HMF in these systems was examined, and the mechanisms of HMF extraction using ionic liquids were explained using computer simulations. Examining the effect of cholinium ionic liquids (choline chloride ([Ch][Cl]), cholinium nicotinate ([Ch][Nic]), cholinium propionate ([Ch][Prop]), and cholinium butyrate ([Ch][But])) on the formation of aqueous biphasic systems by comparing the phase diagrams, it was concluded that the ability of ionic liquids to form an aqueous biphasic system with tripotassium phosphate (K3PO4) decreases in the following order: [Ch][But] ≈ [Ch][Prop] > [Ch][Nic] > [Ch][Cl]. By applying all tested aqueous biphasic systems for the extraction of HMF from honey, an extraction efficiency of more than 89% was achieved. Complete extraction was achieved using the extraction system with [Ch][But], while the weakest ability to extract HMF was exhibited by the system with [Ch][Cl]. The mechanisms of HMF extraction using ionic liquids are explained on the basis of the optimised structures of the ionic liquid systems with HMF, together with the visualisation of non-covalent interactions, and on the basis of the calculated binding energies ΔGbin, which can be used as a good predictor of the extraction potential of newly synthesised ionic liquids.

Techno-Functional Performance of Emmer, Spelt and Khorasan in Spontaneously Fermented Sourdough Bread.

The aim of this study was to test the suitability of three different ancient wheat varieties (emmer, spelt and khorasan) to produce spontaneously fermented sourdough bread and to evaluate the impact on the dough rheological properties, ultrastructure and baking quality. Modern wheat sourdough bread and bakery yeast fermented bread were used as controls. Sourdoughs produced from modern and ancient wheats exerted different effects on dough viscoelastic properties, bread specific volume, texture, firming rate, colour and sensory properties, while there was no influence on bread water activity. Both khorasan sourdough, being characterised with the highest dough strength and dense gluten protein matrix, and emmer sourdough, with loose and thin gluten strands of low strength, yielded breads characterised by low specific volume and hard crumb texture. Spelt and modern wheat sourdough were characterised by foam-like dough structures with entrapped gas cells leading to breads of similar specific volume and texture. Although the yeast-fermented wheat flour exerted a higher specific volume and the lowest firmness, the sourdough wheat flour bread had a lower firming rate. A comparison of sourdough bread prepared with modern and ancient wheats revealed that breads based on ancient varieties possess a less noticeable sour taste, odour and flavour, thus contributing to more sensory-appealing sourdough bread.

Potential health benefits of blueberry and raspberry pomace as functional food ingredients: Dietetic intervention study on healthy women volunteers.

The fruit juice industry generates pomace as a valuable by-product especially rich in polyphenols, dietary fibers, vitamins, minerals, and unsaturated fatty acids. In the cookies used in this study, 30% of the gluten-free flour was replaced with dried and ground blueberry and raspberry pomace, rich source of polyphenols, dietary fibers, linoleic and alpha-linolenic acid. In order to examine whether the addition of blueberry and raspberry pomace in cookie formulation can have beneficial effects on certain blood parameters and anthropometric measurements, the designed cookies were tested in 20 healthy, normally fed female subjects, aged 30-50 years (41.35 ± 8.58 years) over four-week dietetic intervention study. Significant changes in the composition of fatty acids serum phospholipids, decrease in LDL-cholesterol level (20.16%), increase in adiponectin level (25.52%) and decrease in ALT and AST values were observed, thus indicating that inclusion of cookies containing blueberry and raspberry dried and ground pomace to usual diet might have positive effects on certain cardiovascular risk factors and liver function indicators.

Antioxidative, Antibacterial and Antiproliferative Properties of Honey Types from the Western Balkans.

This paper presents the physicochemical characteristics and antioxidative, antibacterial and antiproliferative effects of nineteen samples of different honey types (acacia, linden, heather, sunflower, phacelia, basil, anise, sage, chestnut, hawthorn, lavender and meadow) collected from different locations in the Western Balkans (Republic of Serbia, Kosovo, Bosnia and Herzegovina, and Northern Macedonia). Physicochemical parameters (moisture, pH, electrical conductivity, free acidity, and hydroxymethylfurfural [HMF]) were analysed. Based on the obtained results, all tested honey samples were in agreement with EU regulation. The antioxidant potential of honey samples was assessed by determination of total phenolic content (TPC) and evaluation of scavenging activity towards diphenilpicrylhydrazyl radicals (DPPH·). The highest phenolic content was found in basil honey (101 ± 2.72 mg GAE/100 g), while the lowest was registered in rapeseed honey (11.5 ± 0.70 mg GAE/100 g). Heather, anise, phacelia, sage, chestnut and lavender honey samples were also rich in TP, containing 80−100 mg GAE/100 g. DPPH scavenging activity varied among the samples being the highest for lavender honey (IC50 = 88.2 ± 2.11 mg/mL) and the lowest for rapeseed honey (IC50 = 646 ± 8.72 mg/mL). Antibacterial activity was estimated in vitro using agar diffusion tests and measuring minimal inhibitory concentration (MIC). Among investigated bacterial strains following resistant potencies were determined: Escherichia coli > Escherichia coli ATCC 8739 > Enterococcus faecalis > Proteus mirabilis > Staphylococcus aureus > Staphylococcus epidermidis. The linden honey from Fruska Gora (MIC values of 3.12% and 6.25% against Staphylococcus aureus and Staphylococcus epidermidis, respectively) and phacelia honey (MIC values of 6.25% and 3.12% against S.Staphylococcus aureus and Staphylococcus epidermidis, respectively) showed the strongest antibacterial activity. Antiproliferative activity was evaluated using the colorimetric sulforhodamine B (SRB) assay. The highest antiproliferative activity was obtained from linden honey sample 1 (IC50MCF7 = 7.46 ± 1.18 mg/mL and IC50HeLa =12.4 ± 2.00 mg/mL) and meadow sample 2 (IC50MCF7 = 12.0 ± 0.57 mg/mL, IC50HeLa = 16.9 ± 1.54 mg/mL and IC50HT−29 = 23.7 ± 1.33 mg/mL) towards breast (MCF7), cervix (HeLa) and colon (HT-29) cancer cells. Active components other than sugars contributed to cell growth activity.

High-Protein Bar as a Meal Replacement in Elite Sports Nutrition: A Pilot Study.

This study was focused on the creation of high-protein bars formulated using whey protein isolate (24%) and soy protein isolate (6%) as the sources of proteins; oat flakes and inulin, both abundant in dietary fibres, and creatine monohydrate and other minor ingredients (vitamin and mineral mixture, potassium sorbate) to achieve the requirements for a meal replacement formula for physically active people. The nutritional profile of the high-protein bar was examined (energy 1215 kJ/288 kcal; protein 34.1 ± 0.20 g, fat 6.01 ± 0.13 g of which was saturated 3.12 ± 0.08 g, fibre 3.10 ± 0.17 g carbohydrate 23.0 ± 0.16 g of which sugars 1.50 ± 0.19 g and starch 21.5 ± 0.11 g in 100 g), and sensory properties with instrumental parameters (texture and colour) were determined and compared with bars commercially available on the market. The created high-protein bar was sensorily acceptable in comparison to other commercially available bars. The dietary intervention study was conducted on elite athletes (professional handball players) to evaluate effects of created versus control bar consumption on their metabolic parameters. The baseline characteristics (mean age, body mass index (BMI), fat mass, muscle mass, lean mass and fat percentage) of the athletes (8) were determined at the start of the study. The cross-over intervention study was organized in two successive phases (5 days each) with a seven-day long washout period between phases. Bars were consumed after the afternoon training unit. Blood samples were collected at the start and the end of the intervention study to analyse the metabolic profiles of the athletes. Serum levels of high-density cholesterol (HDL), low-density cholesterol (LDL) and total cholesterol (HOL), glucose, triacylglycerides (TAG), total and direct bilirubin, creatine kinase (CK), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) were measured. The results showed that bar consumption significantly decreased serum aspartate transaminase (AST) and lactate dehydrogenase (LDH) and increased total and direct bilirubin levels, suggesting lower exercise-induced muscle damage and increased antioxidative response, respectively. Therefore, it can be concluded that the consumption of the created high-protein bar was able to improve physiological adaptation after training.

A comprehensive study of parameters correlated with honey health benefits.

One hundred honey samples of different floral origin (acacia, sunflower, meadow, and forest) collected from nine European countries (Serbia, Albania, Croatia, Montenegro, Romania, Bulgaria, Bosnia and Herzegovina, North Macedonia and Hungary) were analysed for various physicochemical, sensory, antioxidant and antibacterial parameters. The relative antioxidant capacity index and relative antibacterial index were calculated, integrated and expressed as a new property - Power of Honey, intended to be used to predict the health potential of a honey based on its antioxidant and antibacterial activities. Free acidity and colour coordinates L* and a* were chosen for building an artificial neural network model for the prediction of honey health potential. These were chosen based on the obtained correlations between the investigated parameters and in light of the simplicity of the analysis. This model successfully predicted the Power of Honey with a gained coefficient of determination of 0.856. Forest honey samples exhibited the highest Power of Honey. This novel approach should make it possible for honey producers to predict the honey health potential of a particular honey based on a quick and simple analysis.

Prevalence, knowledge and attitudes towards using sports supplements among young athletes.

BACKGROUND: The aim of this international study was to investigate the prevalence of the use of sports supplements among young athletes, as well as their knowledge and attitudes towards sports supplementation. METHODS: Organized survey study testing the level of knowledge, attitudes, beliefs and practices concerning the use of sports supplements was administered to 348 athletes, 15-18 year olds from 4 countries competing in 18 sports at the international level. RESULTS: The prevalence rate of the intake of sports supplements was 82.2%, with the protein supplements being predominant (54.5%). Coaches were identified as the primary source of information regarding supplementation (41.4%). The enhancement of athletic performance (35.4%) was the major motivation for the supplements intake. The majority of athletes (72.1%) were aware of associated health risks. The young athletes possess varying levels of knowledge regarding their own supplementation. The obtained data about the level of knowledge were statistically analyzed using the correspondence analysis. Less than 40% of athletes had the knowledge about the proper and intended use of protein, creatine, amino acids, beta alanine and glutamine, while they had greater understanding about vitamins and minerals, sports drinks and caffeine. The athletes in developed countries had greater access and utilization of professional resources such as dieticians. Young athletes are still unfamiliar with WADA regulations (55.5%), and the misuse of sports supplements represents an ethical dilemma for some. CONCLUSION: These findings indicate the necessity of a comprehensive education of all team members about sports supplements and careful supervision of the athletic development of young athletes.

Physicochemical properties and mineral content of honey samples from Vojvodina (Republic of Serbia).

Forty-five honey samples of three honey types - two monofloral (acacia and sunflower) and one polyfloral (meadow) were collected from the Autonomous Province of Vojvodina (Republic of Serbia) and analysed for their physicochemical parameters (moisture, acidity, pH, ash, electrical conductivity, glucose, fructose, hydroxymethylfurfural (HMF), CIE L∗a∗b∗ colour and mineral content). All tested physicochemical parameters were in agreement with the EU regulation except for moisture (4 samples) and HMF content (1 sample). Principal component analysis (PCA) was used for assessing the effects of three honey types on all investigated physicochemical parameters. According to PCA, acacia honey samples can be clearly isolated and form a cluster, while, regarding other honey types, PCA can offer the possibility to distinguish sunflower and meadow honey samples regarding examined physicochemical parameters and mineral content.

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.

Differential influence of vitamin C on the peripheral and cerebral circulation after diving and exposure to hyperoxia.

We examined if the diving-induced vascular changes in the peripheral and cerebral circulation could be prevented by oral antioxidant supplementation. Fourteen divers performed a single scuba dive to eighteen meter sea water for 47 min. Twelve of the divers participated in a follow-up study involving breathing 60% of oxygen at ambient pressure for 47 min. Before both studies, participants ingested vitamin C (2 g/day) or a placebo capsule for 6 days. After a 2-wk washout, the study was repeated with the different condition. Endothelium-dependent vasodilator function of the brachial artery was assessed pre- and postintervention using the flow-mediated dilation (FMD) technique. Transcranial Doppler ultrasound was used to measure intracranial blood velocities pre- and 90 min postintervention. FMD was reduced by ∼32.8% and ∼21.2% postdive in the placebo and vitamin C trial and posthyperoxic condition in the placebo trial by ∼28.2% ( P < 0.05). This reduction in FMD was attenuated by ∼10% following vitamin C supplementation in the hyperoxic study ( P > 0.05). Elevations in intracranial blood velocities 30 min after surfacing from diving were reduced in the vitamin C study compared with the placebo trial ( P < 0.05). O2 breathing had no postintervention effects on intracranial velocities ( P > 0.05). Prophylactic ingestion of vitamin C effectively abrogated peripheral vascular dysfunction following exposure to 60% O2 but did not abolish the postdive decrease in FMD. Transient elevations of intracranial velocities postdive were reduced by vitamin C. These findings highlight the differential influence of vitamin C on peripheral and cerebral circulations following scuba diving, which are only partly mediated via hyperoxia.

Hydrophilic interaction chromatography coupled to tandem mass spectrometry as a method for simultaneous determination of guanidinoacetate and creatine.

The biosynthesis of creatine (Cr) is closely related to the bioavailability of guanidinoacetate (GAA). The lack of one or the other may compromise their role in the energy transport and cell signaling. A reliable estimate of their levels in biological samples is imperative since they are important markers of many metabolic disorders. Therefore, a new LC-MS/MS method for simultaneous determination and quantification of GAA and Cr by multiple reaction monitoring (MRM) mode was developed based on the hydrophilic interaction chromatography (HILIC) and response surface methodology (RSM) for the optimization of chromatographic parameters. The optimized parameters ensured good separation of these similar, very polar molecules (chromatographic resolution > 1.5) without prior derivatization step in a short analysis run (6 min). The developed method was validated to ensure accurate (R, 75.1-101.6%), precise (RSD < 20%) and low quantification (LOQ of 0.025 µg mL-1 for GAA and 0.006 µg mL-1 for Cr) of the tested analytes and the use of matrix-matched calibration eliminated variable effects of complex matrices such as human plasma and urine. Therefore, this method can be implemented in medical laboratories as a tool for the diagnostics of creatine deficiencies and monitoring of guanidinoacetate and creatine supplementation regimes in biological samples.

Dietary guanidinoacetic acid increases brain creatine levels in healthy men.

OBJECTIVE: Guanidinoacetic acid (GAA) is an experimental dietary additive that might act as a creatine source in tissues with high-energy requirements. In this case study, we evaluated brain levels of creatine in white matter, gray matter, cerebellum, and thalamus during 8 wk oral GAA administration in five healthy men and monitored the prevalence and severity of side effects of the intervention. METHODS: Volunteers were supplemented daily with 36 mg/kg body weight (BW) of GAA for the first 4 wk of the intervention; afterward GAA dosage was titrated ≤60 mg/kg BW of GAA daily. At baseline, 4, and 8 wk, the participants underwent brain magnetic resonance spectroscopy, clinical chemistry studies, and open-ended questionnaire for side-effect prevalence and severity. RESULTS: Brain creatine levels increased in similar fashion in cerebellum, and white and gray matter after GAA supplementation, with an initial increase of 10.7% reported after 4 wk, and additional upsurge (7.7%) from the weeks 4 to 8 follow-up (P < 0.05). Thalamus creatine levels decreased after 4 wk for 6.5% (P = 0.02), and increased nonsignificantly after 8 wk for 8% (P = 0.09). GAA induced an increase in N-acetylaspartate levels at 8-wk follow-up in all brain areas evaluated (P < 0.05). No participants reported any neurologic adverse event (e.g., seizures, tingling, convulsions) during the intervention. CONCLUSIONS: Supplemental GAA led to a region-dependent increase of the creatine pool in the human brain. This might be relevant for restoring cellular bioenergetics in disorders characterized by low brain creatine and functional enzymatic machinery for creatine synthesis, including neurodegenerative diseases, brain tumors, or cerebrovascular disease.

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.

Comprehensive QSRR modeling as a starting point in characterization and further development of anticancer drugs based on 17α-picolyl and 17(E)-picolinylidene androstane structures.

The selection of the most promising anticancer compounds from the pool of the huge number of synthesized molecules is a quite complex task. There are many compounds characterization approaches which can suggest the best structural features of a molecule with the highest antiproliferative effect on the certain type of cancer cell lines. One of these approaches is the lipophilicity determination of compounds and the analysis of its correlation with the anticancer activity. Since the importance of the lipophilicity is underlined in many earlier studies, this study is focused on determination of lipophilicity of previously synthesized 17α-picolyl and 17(E)-picolinylidene androstane derivatives by using reversed-phase high performance liquid chromatography (RP-HPLC) as a very fast, effective and relatively cheap method. Determination of the chromatographic lipophilicity of the studied androstanes can be considered as the part of their physicochemical characterization, which is a very important step in their further selection as drug candidates. The present study does not neglect the in silico approach. The determined chromatographic lipophilicity was analyzed by quantitative structure-retention relationship (QSRR) approach in order to reveal which molecular characteristics contribute mostly to the typical behavior of the androstanes in the applied chromatographic system, and thus to their lipophilicity. Classical statistical approach and Sum of Ranking Differences method were used for selection of the best QSRR models which should be used in prediction of chromatographic lipophilicity of studied androstane derivatives.

Antioxidant Capacity, Mineral Content and Sensory Properties of Gluten-Free Rice and Buckwheat Cookies.

Light buckwheat flour was used to substitute rice flour at the level of 10, 20 and 30% to produce gluten-free cookies. The substitution of gluten-free cookie formulation with light buckwheat flour contributed to the significantly higher mineral content, especially magnesium, potassium, iron and copper, in comparison with the control rice cookies (p<0.05). Gluten-free cookies made with rice flour and buckwheat flour exhibited significantly higher total phenolic and rutin content, scavenging activity against 1,1-diphenyl-2-picrylhydrazyl radicals (DPPH•), antioxidant activity and reducing power than the control cookies (p<0.05). Comparing all evaluated sensory properties, cookies containing 20% of light buckwheat flour had the most acceptable sensory properties. The obtained results of principal component analysis showed that the cookies with 20 and 30% buckwheat flour had better antioxidant and sensory properties in comparison with other two cookie samples.

Multi-residue method for determination of selected neonicotinoid insecticides in honey using optimized dispersive liquid-liquid microextraction combined with liquid chromatography-tandem mass spectrometry.

The objective of this study was to develop analytical method based on optimized dispersive liquid-liquid microextraction (DLLME) as a pretreatment procedure combined with reversed phase liquid chromatographic separation on C18 column and isocratic elution for simultaneous MS/MS determination of selected neonicotinoid insecticides in honey. The LC-MS/MS parameters were optimized to unequivocally provide good chromatographic separation, low detection (LOD, 0.5-1.0 µg kg(-1)) and quantification (LOQ, 1.5-2.5 µg kg(-1)) limits for acetamiprid, clothianidin, thiamethoxam, imidacloprid, dinotefuran, thiacloprid and nitenpyram in honey samples. Using different types (chloroform, dichloromethane) and volumes of extraction (0.5-3.0 mL) and dispersive (acetonitrile; 0.0-1.0 mL) solvent and by mathematical modeling it was possible to establish the optimal sample preparation procedure. Matrix-matched calibration and blank honey sample spiked in the concentration range of LOQ-100.0 µg kg(-1) were used to compensate the matrix effect and to fulfill the requirements of SANCO/12495/2011 for the accuracy (R 74.3-113.9%) and precision (expressed in terms of repeatability (RSD 2.74-11.8%) and within-laboratory reproducibility (RSDs 6.64-16.2%)) of the proposed method. The rapid (retention times 1.5-9.9 min), sensitive and low solvent consumption procedure described in this work provides reliable, simultaneous, and quantitative method applicable for the routine laboratory analysis of seven neonicotinoid residues in real honey samples.