Regional distribution and diversity of Aspergillus and Penicillium species on Croatian traditional meat products.

Various factors, such as weather and production practices (e.g., environmental hygiene, process duration, raw material quality, ripening temperature, and relative humidity), in combination with the intrinsic product properties (e.g., pH, aw, salt content), significantly affect the growth of surface moulds. The aim of this study was to isolate and identify surface moulds retrieved from traditional meat products (TMPs) and correlate these data to the production region and production technology. The surface of 250 TMPs (dry-fermented sausages, n = 108; dry-cured meat products, n = 142) from five Croatian regions were sampled during a two-year period. Dry-fermented sausages had a significantly higher pH and a lower salt concentration when compared to dry-cured meat products. In total, 528 isolates were obtained, comprising 20 Penicillium and 17 Aspergillus species. The species most frequently isolated from the dry-fermented sausages were P. commune (32.4 %), A. proliferans (33 %), and P. solitum (14.8 %), while A. proliferans (52.1 %), P. commune (28.9 %) and P. citrinum (19.7 %) predominated in dry-cured meat products. Aspergillus predominated on the TMPs from southern Croatia, while Penicillium was prevalent on products from the other four regions, possibly due to differences in weather conditions. Seven potentially mycotoxigenic species (A. creber, A. flavus, A. niger, A. westerdijkiae, P. citrinum, P. commune, and P. nordicum) were isolated and identified. Regular monitoring of mould species and their toxigenic metabolites present on traditional meat products is of the utmost importance from the public health perspective, while the results of such a monitoring can prove beneficial for the tailoring of the production technology development.

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

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

Introduction to new guidelines for validation of methods to examine visually recognisable substances.

Visual examination of visually recognisable substances, including microscopy, focus on targets or contaminants such as particles of animal origin, plant seeds, spore bodies of moulds, sclerotia, packaging material, microplastic and 'Besatz' (everything that differs from the norm). The two principal results are counts (numbers) and weights for macroscopic methods, or presence/absence for microscopic methods. The level of detection equals at least the size of one unit, usually with a weight exceeding 1 mg, which is in the range of parts per million (ppm). These parameters do not follow a normal distribution but Poisson (counts), lognormal (weights) or binomial (Booleans) distributions, with effect on the interpretation of validation parameters. As for other domains, examination methods for visual monitoring need to be properly validated and quality control during actual application is needed. In most cases procedures for validation of visual methods are based on principles adopted from other domains, such as chemical analysis. A series of examples from publications show inconsistent or not correct implementations of these validation procedures, which stress the need for dedicated validation procedures. Identification of legal ingredients and composition analysis in the domain of visual examination relies on the expertise of the laboratory staff, therefore validation of a method usually includes the validation of the expert. In the view of these specific circumstances, a Guidance for quality assurance and control of visual methods has been developed, which are being presented and discussed in this paper. The general framework of the Guidance is adopted from ISO standards (17023, 17043, 13528). Part 1 of the Guidance includes the general background, theory and principles. Part 2 presents the actual validation procedures with experimental designs and equations for calculating the relevant parameters, and can be used as blueprint for a SOP in a quality management system. An EURL and NRL network for physical hazards is strongly recommended.

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 (

Characterization of Traditional Croatian Household-Produced Dry-Fermented Sausages.

Characterization of five types of traditional Croatian dry-fermented sausages produced by family farms was performed via identification of superficial mycobiota, physicochemical, sensory, instrumental color, fatty acids & fat quality indices. Detailed characterization of these sausages aimed to achieve standardization of their production and composition and to establish and/or improve their specification protocols. Traditional sausages varied significantly (p < 0.05) in all analyzed parameters except for the number of mold isolates. Sausages coming from eastern Croatia had a greater mold species diversity, with the highest number of isolated mycotoxigenic species in Slavonian domestic sausage. Sensory evaluation showed good acceptability of all sausages. According to health recommendations, Kulenova Seka showed the most representable values for most of fat quality indices. The results suggest the need for certain modifications in fat & fatty acid composition and, to a lesser extent, in salt content, however not at the expense of product safety, quality and acceptability.

A study of surface moulds and mycotoxins in Croatian traditional dry-cured meat products.

Xerophilic species of Aspergillus, Penicillium and Eurotium genera from surfaces of dry-cured traditional meat products (TMPs) can cause mycotoxin contamination during uncontrolled household processing. The aim of this study was to investigate into surface moulds growing on Croatian prosciuttos and fermented sausages produced in different climate regions using different technologies (n = 160), and to relate the occurrence of aflatoxin B1 (AFB1) and ochratoxin A (OTA) to their presence. The results revealed the Penicillium (79%) to be the dominating contaminating mould, while Aspergillus (11%), Eurotium (7%) and Mucor (4%) species were present in a significantly lower number of isolates, with higher prevalence and greater diversity in prosciuttos than in sausages, relative of the production technology and regional climate. OTA contamination (14% of samples) was significantly more frequent than that with AFB1 (8% of samples), with OTA concentration rising to the maximal 6.86 µg/kg, whereas AFB1 concentrations were slightly higher than, or around, the limit of quantification of the method in use, with the maximal value of 1.92 µg/kg. The presence of AFB1 in absence of toxicogenic moulds, observed in some samples, can be attributed to contaminated spices used in TMP production or an indirect contamination via a carry-over effect.

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.

Isolation and Characterisation of L. plantarum O1 Producer of Plantaricin as Potential Starter Culture for the Biopreservation of Aquatic Food Products.

Lactobacillus plantarum O1 was isolated from the gut of sea bream (Sparus aurata) and identified with the API biochemical test and MALDI-TOF MS. This strain was further characterised according to the selection criteria for lactic acid bacteria as starter cultures for aquatic food production. L. plantarum O1 showed good antimicrobial activity against pathogenic test microorganisms. Further investigation confirmed it as the producer of the bacteriocin plantaricin. This strain also showed good growth at a wide range of temperatures (from 4 to 45 °C) and a wide range of pH (2-12), even in the presence of 3.5% NaCl. Its viability was also good after lyophilisation and in simulated gastric and small intestinal juice. The strain is a promising probiotic, and our further research will focus on its application in the biopreservation of fresh fish and shellfish.

Co-occurrence of ochratoxin A and citrinin in unprocessed cereals established during a three-year investigation period.

The aim of this study was to investigate ochratoxin A (OTA) and citrinin (CIT) co-occurrence in different unprocessed cereals (n = 189) originating from Croatia during a three-year investigation period (2014-2016) using validated enzyme immunoassay (ELISA) methods. CIT and OTA were determined in 49% and 7% of samples, respectively. Significantly higher (p < 0.05) overall mean concentrations were determined for CIT (66.8 ± 76.0 µg/kg) in comparison to OTA (5.2 ± 1.1 µg/kg). Based on the analysis of all investigated cereals, CIT was found about 15 times more frequently than OTA and in similarly (15-fold) higher concentrations, irrespective of the cultivation year. The results revealed a moderately positive correlation between OTA and CIT concentrations in maize (rs = 0.44) and wheat (rs = 0.59), whereas in barley and oat this correlation (p > 0.01) was not significant.

Deoxynivalenol and zearalenone in unprocessed cereals and soybean from different cultivation regions in Croatia.

The aim of this study was to investigate the occurrence of deoxynivalenol (DON) and zearalenone (ZEN) in unprocessed cereals and soybean sampled in 2014 and 2015 from different fields located in Croatian regions. A total of 306 samples were analysed for DON and 415 samples for ZEN concentrations using quantitative ELISA methods. In 2014, DON and ZEN were determined in all samples in the mean concentrations of 1,461 ± 2,265 µg/kg and 656 ± 853 µg/kg, respectively, while in 2015 these means were 2,687 ± 2,731 µg/kg and 1,140 ± 1,630 µg/kg, respectively. The cultivation year significantly (p < 0.05) influenced mycotoxin concentrations, whereas the influence of cultivation region was seen with ZEN for all cereals except soybean, and not seen with DON at all. A higher contamination determined during 2015 could be explained by high to extreme humidity evidenced in the period of cereals' growth and harvesting.

Mycotoxins in organic and conventional cereals and cereal products grown and marketed in Croatia.

In this study, the levels of aflatoxin B1 (AFB1), ochratoxin A (OTA), zearalenone (ZEN), deoxynivalenol (DON) and fumonisins (FUM) in unprocessed cereals (n = 189) and cereal-based products (n = 61) were determined using validated ELISA methods. All samples originated from either conventional or organic production corresponded to the 2015 harvest in Croatia. Based on the mean mycotoxin concentrations, the risk for the consumer to exceed the tolerable daily intake (TDI) for these toxins by the consumption of both types of cereals and cereal-based products was assessed. Mycotoxin contamination of organic cereals and organic cereal-based products was not significantly different (p > 0.05). Given that the exposure assessment resulted in a small fraction of the TDI (maximum: DON, 12% of TDI), the levels of the investigated mycotoxins in both types of cereals and cereal-based products from the 2015 harvest did not pose a human health hazard.

Moulds and mycotoxins detected in the regional speciality fermented sausage 'slavonski kulen' during a 1-year production period.

This study investigated the surface-to-interior mycotoxin contamination of the regional speciality meat product 'slavonski kulen' that occurred during a 12-month period and identified moulds present on its surface during the final production stage. In total, 15 pieces of slavonski kulen were produced and sampled at production months 0, 3, 6, 9 and 12 (three samples per piece). Concentrations of aflatoxin B1 (AFB1) and ochratoxin A (OTA) were determined using the ELISA method, while the identification of moulds made use of both traditional and molecular methods. A 12-month-long production of dry-fermented sausages under controlled conditions that did not include product cleaning at any point caused significant mycotoxin contamination, with determined mean AFB1 and OTA concentrations of 11.79 ± 2.34 and 16.13 ± 3.32 µg kg-1, respectively. Significantly higher mycotoxin levels were observed in the external layers of the products in comparison with their interior and, even more so, central parts. In total, five Penicillium species (P. polonicum, P. terrigenum, P. solitum, P. jugoslavicum and P. corylophilum) and two Aspergillus species (A. versicolor and A. sydowii) were isolated. Moulds responsible for the production of AFB1 and OTA in the later production stages had probably been overgrown by non-toxicogenic moulds, so that the former did not last until the end of the sausages' production process and were therefore not identified.

Citrinin in cereals and feedstuffs coming from Croatia and Bosnia & Herzegovina.

The aim of this study was to investigate the occurrence of citrinin in different cereals (n = 341) and feedstuffs (n = 67) coming from farms and feed factories established in Croatia and Bosnia & Herzegovina. The highest mean citrinin concentration (103 ± 102 µg/kg) was observed in cereals sampled in Bosnia & Herzegovina during 2014, although significantly different levels between the two countries were not observed. Across the cereal samples, the maximal citrinin concentration was determined in wheat (429 µg/kg), while across the feedstuffs, the highest concentration was found in pig feed (63 µg/kg). Despite of the increased citrinin levels found in some samples, especially wheat, the obtained values cannot be compared against the maximum limits, since no such levels are stipulated under the applicable legislation. But, given that data on citrinin are very scarce, they can serve as an indicator of cereal and feed contamination in this part of Europe.

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.

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.

Toxic element concentrations in the bottlenose (Tursiops truncatus), striped (Stenella coeruleoalba) and Risso's (Grampus griseus) dolphins stranded in eastern Adriatic Sea.

Concentrations of cadmium (Cd), arsenic (As), mercury (Hg) and lead (Pb) were measured in muscle, liver and kidney of three cetacean species, the bottlenose (Tursiops truncatus), striped (Stenella coeruleoalba) and Risso's (Grampus griseus) dolphins from the Croatian waters of the Adriatic Sea. In all three dolphin species Cd levels decreased in tissues in the order: kidney > liver > muscle, while As and Pb decreased in the order: liver > kidney > muscle for striped and Risso's dolphins, but with the order reversed for liver and kidney in the bottlenose dolphin for Pb. Levels of Hg consistently followed the order: liver > muscle > kidney, with mean concentrations in the liver being 11-34 times higher than in the other tissues. The highest mean concentrations of trace elements were measured in Risso's dolphins at 14.9 µg/g wet weight, for Cd in the kidney, and concentrations in the liver of 2.41, 1,115 and 0.63 µg/g for As, Hg and Pb, respectively. Statistically significant differences between the three dolphin species were determined for Cd, Hg and Pb in liver tissues, for As in muscle and for Cd in kidney. Significant correlations of metals between tissues were determined in all three species. The results presented give an indication of the environmental condition with regard to the content of toxic metals along the eastern coast of the Adriatic Sea.

The natural occurrence of T-2 toxin and fumonisins in maize samples in Croatia.

Maize (Zea mays) considered to be one of the most frequent crops in Croatia, is often contaminated with Fusarium mycotoxins. The aim of this study was to investigate the possible contamination of maize with T-2 toxin and fumonisins on 46 samples from different regions of Croatia. The highest concentrations of T-2 toxin and fumonisins were 210 and 25,200 ng/g, with mean values of 110 and 4,509 ng/g, respectively, pointing to maize contamination with these mycotoxins after the period of the year with extremely high rainfall.

Mould and mycotoxin contamination of pig feed in northwest Croatia.

The aim of this study was to investigate the contamination of pig feed with moulds and the occurrence of mycotoxins. A total of 30 feed samples were collected at different animal feed factories in the north-western part of Croatia. Mycological analysis showed that the total number of moulds ranged from 1 × 10(3) to 1 × 10(5) cfu/g with samples contaminated with Aspergillus spp. (63 %), Penicillium spp. (80 %), and Fusarium spp. (77 %). A determination of aflatoxin B1 (AFB1), ochratoxin A (OTA), zearalenone (ZEA), deoxynivalenol (DON), T-2 toxin (T-2) and fumonisin (FUM) concentration was done using the validated ELISA method. The mean concentrations of AFB1 (0.5 ± 0.6 µg/kg), OTA (1.53 ± 0.42 µg/kg) and FUM (405 ± 298 µg/kg) were below the maximum levels or recommended values in the EU in all the investigated samples. The observed results indicated an increased contamination of pig feed with Fusarium mycotoxins DON and ZEA with mean concentrations of 817 ± 447 and 184 ± 214 µg/kg, higher than recommended in 40 and 17 % of the analysed samples, respectively.