Biocontrol of Occurrence Ochratoxin A in Wine: A Review.

Viticulture has been an important economic sector for centuries. In recent decades, global wine production has fluctuated between 250 and almost 300 million hectoliters, and in 2022, the value of wine exports reached EUR 37.6 billion. Climate change and the associated higher temperatures could favor the occurrence of ochratoxin A (OTA) in wine. OTA is a mycotoxin produced by some species of the genera Aspergillus and Penicillium and has nephrotoxic, immunotoxic, teratogenic, hepatotoxic, and carcinogenic effects on animals and humans. The presence of this toxin in wine is related to the type of wine-red wines are more frequently contaminated with OTA-and the geographical location of the vineyard. In Europe, the lower the latitude, the greater the risk of OTA contamination in wine. However, climate change could increase the risk of OTA contamination in wine in other regions. Due to their toxic effects, the development of effective and environmentally friendly methods to prevent, decontaminate, and degrade OTA is essential. This review summarises the available research on biological aspects of OTA prevention, removal, and degradation.

Lactic Acid Bacteria of Marine Origin as a Tool for Successful Shellfish Farming and Adaptation to Climate Change Conditions.

Climate change, especially in the form of temperature increase and sea acidification, poses a serious challenge to the sustainability of aquaculture and shellfish farming. In this context, lactic acid bacteria (LAB) of marine origin have attracted attention due to their ability to improve water quality, stimulate the growth and immunity of organisms, and reduce the impact of stress caused by environmental changes. Through a review of relevant research, this paper summarizes previous knowledge on this group of bacteria, their application as protective probiotic cultures in mollusks, and also highlights their potential in reducing the negative impacts of climate change during shellfish farming. Furthermore, opportunities for further research and implementation of LAB as a sustainable and effective solution for adapting mariculture to changing climate conditions were identified.

A Predictive Assessment of Ochratoxin A's Effects on Oxidative Stress Parameters and the Fermentation Ability of Yeasts Using Neural Networks.

The aim of this paper was to examine the effect of different OTA concentrations on the parameters of oxidative stress (glutathione (GSH) and malondialdehyde (MDA) concentrations) and glucose utilization in ethanol production by wine yeasts. In addition to the above, artificial neural networks (ANN) were used to predict the effects of different OTA concentrations on the fermentation ability of yeasts and oxidative stress parameters. The obtained results indicate a negative influence of OTA (4 µg mL-1) on ethanol production after 12 h. For example, K. marxianus produced 1.320 mg mL-1 of ethanol, while in the control sample 1.603 µg mL-1 of ethanol was detected. However, after 24 h, OTA had no negative effect on ethanol production, since it was higher (7.490 and 3.845 mg mL-1) in comparison to control samples. Even low concentrations of OTA affect GSH concentrations, with the highest being detected after 12 and 24 h (up to 16.54 µM), while MDA concentrations are affected by higher OTA concentrations, with the highest being detected at 24 h (1.19 µM). The obtained results with the use of ANNs showed their potential for quantification purposes based on experimental data, while the results of ANN prediction models have shown to be useful for predictions of what outcomes different concentrations of OTA that were not part of experiment will have on the fermentation capacity and oxidative stress parameters of yeasts.

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.

Unlocking the Bioactive Potential of Pomegranate Peels: A Green Extraction Approach.

Pomegranate (Punica granatum L.) is well known for its high content of bioactives, including polyphenols, flavonoids, and tannins, which have been shown to exhibit a wide range of biological activities, such as antioxidant, antimicrobial, and anticancer effects. It is worth noting that the majority of these molecules are found in the peels, which are usually disposed of after processing, causing a significant amount of waste, amounting to more than 3.6 million t/y. This work investigates microwave-assisted extraction (MAE) in water for the recovery of antioxidants from pomegranate peels (PP), including the optimisation of temperature and extraction times. The total phenolic, anthocyanin, flavonoid, and tannin contents of the recovered extracts were determined, as well as their antioxidant activities, which were found to be 356.35 mgGAE/gExtr, 303.97 µgCy3G/gExtr, 37.28 mgQE/gExtr, 56.48 mgGAE/gExtr, and 5.72 mmolTE/gExtr, respectively (according to the adopted reference). All results were compared with those obtained using a conventional protocol. In addition, the potential for water recycling by means of downstream nanofiltration in optimised MAE was investigated, leading to overall water reuse of approx. 75%. Power consumption (20.92 W/mgGAE) and common green metrics, Reaction Mass Efficiency (RME), E-Factor, and the Process Mass Intensiti/efficiency (PMI, PME), were considered in evaluating the proposed PP valorisation strategy. Finally, the biological activities of the main products were assessed. The antimicrobial properties of the PP extracts against three Gram-positive and three Gram-negative bacteria and their antiproliferative activity towards human cancer cells were tested. S. aureus bacteria was the most susceptible to the PP extracts. All tested products displayed antiproliferative activity against HeLa cells when higher concentrations were tested, with D-PP/NF (obtained from dried PP and sequential nanofiltration) being the most effective. This result was also confirmed via clonogenic analysis, which generally indicated the possible anti-cancer activity of pomegranate peel extracts obtained using this green approach.

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 (

SPME-GC-MS and Multivariate Analysis of Sensory Properties of Cheese in a Sack Matured with Probiotic Starter Cultures.

RESEARCH BACKGROUND: Cheese in a sack is a traditional cheese produced in Croatia. Types of cheese with similar production technology are made in other countries but chemical and microbiological composition varies between regions. Traditionally, cheese in a sack is produced without the addition of starter cultures. Addition of beneficial probiotic cultures to numerous dairy products has documented advantages. Effects that the addition of probiotic bacteria to traditional cheese have on aroma compounds and sensory properties have not been fully investigated. The aim of this study is to determine the sensory properties and differences in the aromatic profiles between cheese samples ripened in a lambskin sack, produced traditionally without the addition of any starter culture, or with the addition of probiotic bacteria. EXPERIMENTAL APPROACH: In this study, cheese in a sack was produced with the addition of probiotic cultures Lactobacillus plantarum B and L. lactis ssp. lactis S1. During ripening volatile aroma compounds were analysed with a solid-phase microextraction gas chromatography-mass spectrometry. Sensory properties were evaluated by trained tasters who are familiar with the traditional taste of the cheese from a sack. The results of aroma composition and taste scores were then compared using factorial and principal component analyses. RESULTS AND CONCLUSIONS: Chromatography showed differences in the composition of aroma compounds and the sensory properties between the cheese produced with Lactobacillus starter cultures and the control cheese, traditionally produced without a starter culture. The addition of probiotic cultures L. plantarum B and L. lactis ssp. lactis S1 resulted in products with better sensory properties and chemical profile of volatile aromatic compounds. NOVELTY AND SCIENTIFIC CONTRIBUTION: This study investigates the usage of naturally present probiotic cultures as starter cultures in cheese in a sack production. Their effects on aroma profiles and sensory characteristics have been compared for the first time using factorial and principal component analyses.

Properties and Fermentation Activity of Industrial Yeasts Saccharomyces cerevisiae, S. uvarum, Candida utilis and Kluyveromyces marxianus Exposed to AFB1, OTA and ZEA.

In this paper the effect of aflatoxin B1, ochratoxin A and zearalenon on morphology, growth parameters and metabolic activity of yeasts Saccharomyces cerevisiae, Saccharomyces uvarum, Candida utilis and Kluyveromyces marxianus was determined. The results showed that the three mycotoxins affected the morphology of all these yeasts, primarily the cell diameter, but not their final cell count. Fourier transform infrared spectroscopy showed that the yeast membranes bound the mycotoxins, C. utilis in particular. The cell membranes of most yeasts underwent denaturation, except S. uvarum exposed to ochratoxin A and zearalenone. In the early stage of fermentation, all mycotoxin-exposed yeasts had lower metabolic activity and biomass growth than controls, but fermentation products and biomass concentrations reached the control levels by the end of the fermentation, except for C. utilis exposed to 20 µg/mL of zearalenone. The adaptive response to mycotoxins suggests that certain yeasts could be used to control mycotoxin concentrations in the production of fermented food and beverages.

Removing aflatoxin M1 from milk with native lactic acid bacteria, centrifugation, and filtration.

In order to minimise human exposure to aflatoxin M1 (AFM1) the levels of this highly carcinogenic mycotoxin in milk, heat-treated milk, and other dairy products have been limited to <0.05 µg kg-1. However, its removal from dairy products presents a challenge for dairy producers, as commercial additives change organoleptic properties, and filtration alone yields poor results. The aim of this study was to find a strain of lactic acid bacteria (LAB) from milk or dairy products that most effectively binds AFM1 and to see whether heat treatment of the selected LAB affects the binding efficiency. We also wanted to investigate whether centrifugation can improve filtering of the obtained AFM1-LAB complexes from milk. To do that, we isolated and identified 10 native LAB species/strains, incubated their viable or heat-treated cells (108 CFU mL-1) in milk spiked with 0.5 µg L-1of AFM1 at 4 °C for 0, 2, 4, and 24 h, and quantified the amount of unbound AFM1 with HPLC. AFM1 binding efficiency ranged from 21 to 92 % for viable cells and from 26 to 94 % for the heattreated ones. Since both viable and heat-treated Lactobacillus plantarum KM showed the best results, we used it for the next step in AFM1 removal from milk. Heat treatment in combination with filtration and centrifugation yielded removal as high as 96 %.

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.

Microbiological Quality and Variability of Natural Microbiota in Croatian Cheese Maturing in Lambskin Sacks.

As in the traditional production of cheese in lambskin sacks raw cow's or sheep's milk is mostly used, the purpose of this study is to see how the production affects the microbiological quality of the cheese. To do that, we tested 39 samples of raw cow's and sheep's milk, curd, ripened cheese (15, 30 and 45 days) and lambskin sacks for native microbial population. Two-thirds of the milk, curd and cheese samples had higher counts of staphylococci and enterobacteria than permitted by regulations. Not a single sample had Salmonella and Listeria monocytogenes, but we did find Escherichia coli in sheep's milk and cheese, and yeast and mould in both types of milk and cheese. Staphylococcus xylosus prevailed in lambskin sacks. Despite the high incidence of S. aureus, even in the final product, staphylococcal enterotoxin was detected in only two sheep's cheese samples. Among the lactic acid bacteria, Lactococcus lactis and Lactobacillus paracasei prevailed in cow's cheese, whereas Leuconostoc mesenteroides and Lactobacillus plantarum prevailed in sheep's cheese. In the lambskin sacks Leuconostoc mesenteroides and Lactobacillus plantarum were predominant. Our findings give an important insight into the fermentation and microbial ecology of the cheese in lambskin sacks.