Microbial and Physico-Chemical Characterization of Cold Smoked Sea Bass (Dicentrarchus labrax), a New Product of Fishery.

The aim of this study was to investigate the microbial and physico-chemical characteristics of cold smoked sea bass (CSSB), a novel italian fish product. The microbiological analyses showed the presence of bacterial contamination from the raw material, the environment, and the production process. The microbial spoilage population was dominated by lactic acid bacteria (LAB) associated with Gram-negative fermenting bacteria, including Photobacterium phosphoreum and psychrotrophic Enterobacteriaceae. Brochotrix thermospacta and Aeromonas spp. were also present; in contrast, mould and yeast were not detected (<2 CFU/g). High levels (6-7 log CFU/g) of LAB and total bacteria count (TBC) were observed from day 45 of storage; however, their presence does not seem to have influenced the total volatile basic nitrogen (TVB-N), which always remained below 35 mg N/100 g. Consequently, the product is acceptable until day 60 of storage, considering that the malonaldehyde index (TBARS) was lower than 6.5 nmol/g. Pathogenic bacteria such as Salmonella spp. and Listeria monocytogenes were not detected. Currently, there is a growing demand for seafood due to its high quality and nutritional value. Cold smoked sea bass offers a source of macro- and micronutrients essential for the proper functioning of the human body. It is also rich in protein and omega-3 fatty acids. The WHO and FAO evaluated the benefits and risks and concluded that there is convincing evidence of health benefits from fish consumption, such as a reduction in the risk of heart failure and improved neurodevelopment in infants and young children when fish is consumed by the mother before and during pregnancy. The CSSB analysed in this study demonstrated to have health benefits due to long-chain omega-3 PUFAs and other nutrients, such as proteins, minerals, and vitamin D, which are sometimes difficult to obtain from other sources. The results show that CSSB has a high nutritional value and excellent microbial quality.

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.

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.

The Occurrence of Five Unregulated Mycotoxins Most Important for Traditional Dry-Cured Meat Products.

This study investigated the occurrence of 5 unregulated mycotoxins in a total of 250 traditional dry-cured meat products sampled in 2020 and 2021 in five Croatian regions (eastern, northern, central, western, and southern). Aflatoxin B1 (AFB1), ochratoxin A (OTA), sterigmatocystin (STC), citrinin (CIT), and cyclopiazonic acid (CPA) concentrations were related to the geographical region of the product's origin and to local weather. The results revealed the contamination of 27% of samples, namely, STC in 4% of samples in concentrations of up to 3.93 µg/kg, OTA in 10% of samples in concentrations of up to 4.81 µg/kg, and CPA in 13% of samples in concentrations of up to 335.5 µg/kg. No AFB1 or CIT contamination was seen. Although no statistically significant differences in concentrations of individual mycotoxins across the production regions were found, differences in mycotoxin occurrence were revealed. The eastern and western regions, with moderate climate, delivered the largest number of contaminated samples, while the southern region, often compared with subtropics, delivered the smallest, so that the determined mycotoxins were probably mainly produced by the Penicillium rather than the Aspergillus species. Due to the interaction of various factors that may affect mycotoxin biosynthesis during production, the detected concentrations cannot be related solely to the weather.

Mycobiota and Mycotoxin Contamination of Traditional and Industrial Dry-Fermented Sausage Kulen.

The aim of this study was to identify and compare surface mycobiota of traditional and industrial Croatian dry-fermented sausage Kulen, especially toxicogenic species, and to detect contamination with mycotoxins recognized as the most important for meat products. Identification of mould species was performed by sequence analysis of beta- tubulin and calmodulin gene, while the determination of mycotoxins aflatoxin B1 (AFB1), ochratoxin A (OTA), and cyclopiazonic acid (CPA) was carried out using the LC-MS/MS (liquid chromatography-tandem mass spectrometry) method. The results showed a significantly higher number of mould isolates and greater species (including of those mycotoxigenic) diversity in traditional Kulen samples in comparison with the industrial ones. P. commune, as a potential CPA-producer, was the most represented in traditional Kulen (19.0%), followed by P. solitum (16.6%), which was the most represented in industrial Kulen samples (23.8%). The results also showed that 69% of the traditional sausage samples were contaminated with either CPA or OTA in concentrations of up to 13.35 µg/kg and 6.95 µg/kg, respectively, while in the industrial samples only OTA was detected (in a single sample in the concentration of 0.42 µg/kg). Mycotoxin AFB1 and its producers were not detected in any of the analysed samples (

A Two-Year Occurrence of Fusarium T-2 and HT-2 Toxin in Croatian Cereals Relative of the Regional Weather.

To investigate into the T-2 and HT-2 toxin occurrence, 240 samples of unprocessed cereals (maize, wheat, barley, and oats) were sampled from different fields located in three Croatian regions during 2017-2018. In all samples, sum concentrations of T-2/HT-2 toxin were determined using the ELISA method, while the LC-MS/MS was used as a confirmatory method for both mycotoxins in positive samples (>LOD) and the establishment of T-2 over HT-2 toxin ratios. The results showed oats to be the most contaminated cereal, with T-2/HT-2 toxins detected in 70.0% of samples, followed by barley (40.9%), maize (26.8%) and wheat (19.2%), with the mean T-2/HT-2 ratio ranging from 1:2.7 in maize to 1:4.4 in oats. Sum T-2/HT-2 concentrations in two maize samples were higher than the indicative level recommended by the European Commission, necessitating subsequent investigations into the conditions under which these poorly investigated mycotoxins are produced. Statistically significantly (p < 0.05) higher concentrations of T-2/HT-2 toxin were determined in oats throughout study regions as compared to those found in wheat, but not maize and barley, while the concentrations of these mycotoxins were related to the regional weather in Croatia.

Aflatoxins and ochratoxin A in dry-fermented sausages in Croatia, by LC-MS/MS.

The aim of this study was to develop a highly-sensitive liquid chromatographic - tandem mass spectrometric (LC-MS/MS) method to investigate the presence of aflatoxins (AFB1, AFB2, AFG1 and AFG2) and ochratoxin A (OTA) in traditional homemade sausages (n = 88) collected from small family farms situated in different regions in Croatia. Aflatoxins were not detected (

Efficacy of low pressure DBD plasma in the reduction of T-2 and HT-2 toxin in oat flour.

T-2 and its major metabolite HT-2 toxin are naturally occurring contaminants in cereals, with the highest concentrations determined in oats. Because of their toxicity and resistance to conventional methods used in mycotoxin degradation, development of new effective procedures for reduction of T-2/HT-2 toxin levels is needed. The aim of this study was to investigate the effect of different gas types within various timeframes on the efficiency of T-2 and HT-2 toxin degradation by low pressure dielectric barrier discharge (DBD) plasma in oat flour. Although humidity of the sample influences the intensity of the plasma, oxygen atoms efficiently oxidize the sample and produce CO and N2. Before and after treatment, T-2 and HT-2 toxin concentrations were analysed by the confirmatory liquid chromatography tandem-mass spectrometry (LC-MS/MS) method. The highest reduction of T-2 and HT-2 toxin of 43.25% and 38.54%, respectively, was achieved using nitrogen plasma and this proved to be time-dependent.

Cytotoxicity of gamma irradiated aflatoxin B1 and ochratoxin A.

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

Analytical methods used for the authentication of food of animal origin.

Since adulteration can have serious consequences on human health, it affects market growth by destroying consumer confidence. Therefore, authentication of food is important for food processors, retailers and consumers, but also for regulatory authorities. However, a complex nature of food and an increase in types of adulterants make their detection difficult, so that food authentication often poses a challenge. This review focuses on analytical approaches to authentication of food of animal origin, with an emphasis put on determination of specific ingredients, geographical origin and adulteration by virtue of substitution. This review highlights a current overview of the application of target approaches in cases when the compound of interest is known and non-target approaches for screening issues. Papers cited herein mainly concern milk, cheese, meat and honey. Moreover, advantages, disadvantages as well as challenges regarding the use of both approaches in official food control but also in food industry are investigated.

Assessment of possible human exposure to ochratoxin A in Croatia due to the consumption of dry-cured and fermented meat products.

Ochratoxin A (OTA) is a toxic secondary metabolite produced by the fungi of Aspergillus and Penicillium species. Data indicate a frequent OTA contamination of cereals and cereal products, and consequently also the contamination of meat and meat products. The aim of this study was to determine a possible level of meat product consumers' exposure to OTA through the consumption of dry-cured and fermented meat products available on the Croatian market. Data showed the weekly OTA intake of 90% of male dry-cured ham consumers to be a maximum of 51.9 ng kg(-1) b.w., i.e., far below the tolerable weekly intake (TWI) of 120 ng kg(-1) b.w. weekly set out by the European Food Safety Authority (EFSA). OTA intake coming from the consumption of other meat products under study is lower and ranges from 0.1 to 42.1 ng kg(-1) b.w. weekly, dependent on the study. The study demonstrated that meat products in Croatia do not constitute a notable source of OTA in the human diet, so that the human health risk coming from the consumption of dry-cured and fermented meat products is negligible.

Natural incidence of zearalenone in Croatian pig feed, urine and meat in 2014.

The aim of this study was to determine the levels of zearalenone (ZEN) in different feed materials and feedstuffs for pigs, as well as in pig urine and pig meat following contaminated feed consumption. In total, 253 feed material and feedstuff samples were collected from Croatian pig farms. The results revealed the presence of ZEN in significant concentrations, the maximal being found in maize (5522 µg/kg), wheat (3366 µg/kg) and pig fattening feed (1949 µg/kg). In farms in which high feed contamination and pig hyperestrogenism were observed, samples of pig urine (n=30) and meat (n=30) were retrieved as well. The mean ZEN concentrations in pig urine and pig meat were 206±20.6 µg/L and 0.62±0.14 µg/kg, respectively. Despite high contamination of feedstuffs responsible for farmed pigs' intoxication, ZEN levels determined in pig meat were shown to be of little significance for human safety.

Mutagenicity and DNA-damaging potential of clenbuterol and its metabolite 4-amino-3,5-dichlorobenzoic acid in vitro.

The aim of this study was to evaluate in vitro toxicity of clenbuterol and its metabolite 4-amino-3,5-dichlorobenzoic acid. Cytotoxicity and pro-oxidative effect of both compounds were studied on human colon adenocarcinoma cell line SW 480. No significant cytotoxic effect of either compound was observed. Results of an Ames test on Salmonella typhimurium did not indicate mutagenic activity of clenbuterol on TA 98 and TA 100 strains, regardless of metabolic activation. Potential mutagenic effects of the highest clenbuterol concentration (2500 ng/ml) were observed on the TA 1535 strain. The obtained results of alkaline comet assay on isolated human lymphocytes suggested that both compounds induced an increase of primary DNA damage in a concentration-dependent manner. 4-ADBA was a slightly more potent inducer of primary DNA damage as compared to clenbuterol. Chromosomal aberration analysis showed that clenbuterol caused a statistically significant increase in the total number of aberrant cells only at the highest concentration tested (3% vs. 0.7% in the negative control). The results of this study might represent a solid frame for designing and planning future studies with both compounds, which should further clarify their mechanisms of action and genotoxic/cytogenetic effects relevant for human risk assessment.

Ochratoxin A reduction in meat sausages using processing methods practiced in households.

The aim of this study was to investigate the possibilities of ochratoxin A (OTA) reduction in home-made meat products. Meat sausages (n = 50) produced from raw materials coming from pigs exposed to OTA-contaminated feed, were subject to common heat processes practiced in households (cooking, frying and baking). Concentrations of OTA in pre- and post-processed products were quantified using a validated immunoassay method, enzyme-linked immunosorbent assay, and confirmed using a high-performance liquid chromatography with fluorescence detection. In line with the differences in recipes used and the degree of OTA accumulation in raw materials, OTA concentrations established in Mediterranean and roast sausages were lower than those found in liver and blood sausages. Baking of contaminated sausages at the temperatures of 190-220°C (for 60 min) resulted in significant reduction of OTA levels (75.8%), while 30-min cooking (at 100°C) and frying (at 170°C) proved to be significantly less effective (e.g. yielding OTA reductions of 7.4% and 12.6%, respectively). The results pointed out that despite high OTA stability, heat processes are capable of reducing its concentration in home-made meat products, depending on the processing modality used.

Comparison of accumulation of clenbuterol and salbutamol residues in animal internal tissues, non-pigmented eyes and hair.

The aim of the study was to compare the accumulation of ß-adrenergic agonist residues clenbuterol (CLB) and salbutamol (SAL) in internal tissues, non-pigmented eyes and hair of laboratory animals repeatedly administered with CLB and SAL during 7 days. Experimental albino guinea pigs (n = 20) were treated with CLB (n = 10) and SAL (n = 10) in anabolic doses of 0.25 and 2.5 mg/kg, whereas the control animal group (n = 10) was left untreated. Methodology validation showed that the ELISA assay to be suitable for ß-agonists' semiquantitative determination. The results revealed a significantly higher (P < 0.05) accumulation potential of CLB in comparison with SAL in all investigated tissues. Despite of their lack of pigmentation and the applied dose, the highest residual CLB concentrations were determined in the eyes of the studied animals, followed by their hair, liver, lungs, kidney, heart and adipose and muscle tissue, whereas residual SAL concentrations found in the eyes and hair of the administered animals did not significantly differ (P > 0.05) from those obtained in their internal tissues.

Comparison of clenbuterol and salbutamol accumulation in the liver of two different mouse strains.

In the European Union, ß(2)-adrenergic agonists like clenbuterol and salbutamol are banned from use as growth promoters. Although clenbuterol and salbutamol both accumulate in the liver, differences in the accumulation rate can be seen among animal species due to different ß(2)-adrenoreceptor distributions. The aim of this study was to compare the accumulation of the two in the liver tissue of two different mouse strains. The study included 200 8-week-old BALB/c and C57/BL/6 mice. One group of BALB/c (40) and one group of C57/BL/6 (40) mice were treated with 2.5 mg/kg body mass clenbuterol per os for 28 days. The remaining two animal groups were treated with salbutamol in the same manner. The animals were then randomly sacrificed on day 1, 15 and 30 post treatments. Despite of the same treatment dose, the results revealed clenbuterol to persist in the liver tissue longer than salbutamol. On post treatment day 30, the concentration of clenbuterol residue in C57/BL/6 and BALB/c mice liver tissue were 0.23 ± 0.02 and 0.21 ± 0.03 ng/g, respectively, while residues of salbutamol were not detected. When comparing the accumulation of both compounds between the two mouse strains, it becomes apparent that no significant difference (P > 0.05) in the accumulation rate can be found.

Accumulation of ractopamine residues in hair and ocular tissues of animals during and after treatment.

The aim of the present study was to assess the accumulation of ractopamine residues in the hair and ocular tissues of guinea pigs during repeated ractopamine administration and after treatment. The experiment was conducted in 38 guinea pigs (30 treated and eight controls). Treated animals were orally administered ractopamine hydrochloride in a dose of 3.5 mg/kg body mass per day using probes for seven consecutive days. Ractopamine concentration was determined in hair during the treatment (Days 1, 3 and 7) with ractopamine hydrochloride and in ocular tissues and hair on defined days after exposure (Days 1, 10, 20 and 30). Residues were present in hair in high concentrations as early as Day 3 (86.15 ± 87.71 ng/g) and Day 7 (85.25 ± 56.97 ng/g). After exposure, residues were found to persist, having depleted from 68.06 ± 30.54 ng/g on Day 1 to 8.01 ± 2.22 ng/g on Day 30, with a significantly higher concentration in hair in contrast to low residue levels in ocular tissues (1.20-0.34 ng/g). The results of the study pointed to high ractopamine accumulation, even in non-pigmented hair, suggesting hair to be used as a matrix in the control of ractopamine abuse in farm animals because of its many advantages over ocular tissues.

Analysis of naturally occurring zearalenone in feeding stuffs and urine of farm animals in Croatia.

The aim of this study was to determine feed and urinary levels of zearalenone. A total of 114 samples, 64 feeding stuffs (commodities, pig and cattle feed), and 50 urine samples were analyzed by the use of enzyme-linked immunosorbent assay (ELISA). Zearalenone was detected in 68.7% of feeding stuffs, while all urine samples except for four yearling samples were positive for zearalenone. The maximum zearalenone concentration in feeding stuffs and urine was 577 ng/g and 241.1 ng/mL, respectively. Although zearalenone concentrations in some samples were high, the risk for humans was negligible since the calculated concentrations in meat were below the tolerable daily intake (TDI).

Correlation of deoxynivalenol and fumonisin concentration determined in maize by ELISA methods.

Maize (Zea mays) is one of the most common crops in Croatia that is often contaminated with mycotoxins. The enzyme linked immunosorbent assay (ELISA) method as a screening immunological method has become very popular in the determination of mycotoxins contamination of cereals, as it has many advantages such as a rapid and simple determination of their concentration. In this present study, 40 maize samples collected from animal feed factories were analyzed for the concentration of deoxynivalenol (DON) and fumonisin (FUM) using the validated ELISA method of two different kit manufacturers to investigate the correlation of the determined mycotoxins concentration. The correlation coefficients for the concentration of DON and FUM, obtained using different kit manufacturers, were r = 0.9698 and r = 0.9303, respectively, yielding a high correlation between the applied ELISA methods. Concerning the legislation, in one maize sample, the DON concentration higher than the maximum recommended value was determined (16.24 mg/kg), whereas the FUM concentration in all the analyzed samples, without exception, were in accordance with recommended values.

Biochemical changes in pig serum after ochratoxin A exposure.

The objective of this study was to investigate the effect of ochratoxin A (OTA) on serum biochemical parameters of pigs during subchronic treatment with 300 µg OTA/kg of feed for 30 days. OTA treatment resulted in significantly higher (p < 0.05) serum levels of creatinine, urea, potassium and alkaline phosphatase, and significantly lower levels of glucose and total protein. These changes in serum biochemical parameters in treated pigs were indicative of impaired liver and kidney function caused by OTA exposure.