Training in tools to develop quantitative microbial risk assessment of ready-to-eat food with a comparison between the Romanian and Spanish food supply chains.

The prevention and control of bacterial contamination on ready-to-eat (RTE) fresh produce is an essential task to ensure food safety. Therefore, the development of novel and effective decontamination technologies to ensure microbiological safety of fruits and vegetables has gained considerable attention and new sanitisation methods are needed. The antimicrobial activity of essential oils (EOs) is well documented, but their application in fresh produce remains a challenge due to their hydrophobic nature. Thus, nanoemulsions efficiently contribute to support the use of EOs in foods by enhancing their dispersibility, their contact area and facilitating the introduction into bacterial cells. The combination of these factors ultimately increases their antimicrobial activity. Quantitative microbial risk assessment (QMRA) is gaining more attention as an effective tool to assess and prevent potential risks associated with food-borne pathogens. In this context, the current project aims to study the effectiveness of different washing methods based on nanoemulsified EOs, comparing them against traditional methods, using a QMRA model for Escherichia coli O157:H7 on cherry tomatoes. Different simulations within a stochastic risk assessment model were implemented using the biorisk package for R, aiming to describe microbial behaviour and biological risk along the Romanian and Spanish food supply chains of RTE fresh produce. Nanoemulsions were prepared using oregano and rosemary EOs, each from Romania and Spain. The four nanoemulsions were evaluated as decontamination treatments to control the growth of E. coli O157:H7 on artificially contaminated cherry tomatoes. The decontamination treatments showed encouraging results, comparable to commonly used chlorine solutions. Therefore, oregano and rosemary nanoemulsions are promising and could be a feasible alternative for chlorine solutions in the reduction of microbiological contaminants.

Hazard characterization of graphene nanomaterials in the frame of their food risk assessment: A review.

Different applications have been suggested for graphene nanomaterials (GFNs) in the food and feed chain. However, it is necessary to perform a risk assessment before they become market-ready, and when consumer exposure is demonstrated. For this purpose, the European Food Safety Authority (EFSA) has published a guidance that has been recently updated. In this sense, the aim of this study is to identify and characterise toxicological hazards related to GFNs after oral exposure. Thus, existing scientific literature in relation to in vitro degradation studies, in vitro and in vivo genotoxicity, toxicokinetics data, in vivo oral studies, and other in-depth studies such as effects on the microbiome has been revised. The obtained results showed that the investigations performed up to now did not follow internationally agreed-upon test guidelines. Moreover, GFNs seemed to resist gastrointestinal digestion and were able to be absorbed, distributed, and excreted, inducing toxic effects at different levels, including genotoxicity. Also, dose has an important role as it has been reported that low doses are more toxic than high doses because GFNs tend to aggregate in the digestive system, changing the internal exposure scenario. Thus, further studies including a thorough toxicological evaluation are required to protect consumer's safety.

Antimicrobial Properties of Bacterial Cellulose Films Enriched with Bioactive Herbal Extracts Obtained by Microwave-Assisted Extraction.

The use of bacterial cellulose (BC) as scaffold for active biofilms is one of the most interesting applications, especially for the biomedical and food industries. However, there are currently few studies evaluating the potential of incorporating herbal extracts into various biomaterials, including BC. Thus, the aim of this study is to report a screening of the total phenolic content and antioxidant and antimicrobial activity of ethanolic extracts of oregano, rosemary, parsley, and lovage. At the same time, the bioactive potential of BC enriched with the four ethanolic extracts is described. Microwave-assisted extraction was used to extract bioactive compounds from the four selected herbs. The physical, mechanical, structural, and chemical properties of BC were also assessed. Next, BC was enriched with the extracts, and their effect against Escherichia coli, Staphylococcus aureus, and Candida albicans was evaluated. The results showed that the bioactivity of the herbs varied significantly, with rosemary extract being the most bioactive. The BC films possessed good mechanical properties, and a three-dimensional network fibrillar structure appropriate for ethanolic-extract incorporation. The BC samples enriched with rosemary extracts had the highest antibacterial activity against S. aureus, while E. coli. and C. albicans seemed to be resistant to all extracts, regardless of herbs.

Correction: Bodea et al. Optimization of Moist and Oven-Dried Bacterial Cellulose Production for Functional Properties. Polymers 2021, 13, 2088.

The authors wish to make a change to the published paper [...].

Simultaneous determination of Allium compounds (Propyl propane thiosulfonate and thiosulfinate) in animal feed using UPLC-MS/MS.

Propyl-propane-thiosulfonate (PTSO) and Propyl-propane-thiosulfinate (PTS) are organosulfur compounds used to supplement the diet of livestock because of their beneficial effects on feed palatability, their antibacterial, anti-inflammatory, and antimethanogenic activities. Besides, antibiotic residues in the environment can be reduced by using these natural bioactive compounds. The objective of this study was to optimize the extraction parameters for the analysis of PTSO and PTS in feed matrices by performing a solid-liquid extraction and quantification by Ultra performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS). Optimization was performed using the Response Surface Methodology on a Box-Behnken experimental design, optimizing the following parameters: solvent:sample ratios and evaporation temperature set for the rotary evaporator. The method was validated for 3 concentration levels for both PTSO (100, 500, 1000 ng g-1) and PTS (500, 1150, 2300 ng g-1). The highest recoveries of PTSO and PTS were obtained using 12.5 mL of 100% acetonitrile, stirring for 15 min, and an evaporation temperature of 20 °C. The validated method was further applied to detect and quantify these compounds in different feed matrices. In conclusion, this is the first study to simultaneously analyze PTSO and PTS at low concentrations, employing a sensitive technique such as UPLC-MS/MS.

Optimization of Moist and Oven-Dried Bacterial Cellulose Production for Functional Properties.

Bacterial cellulose (BC) is a natural polymer with properties suitable for tissue engineering and possible applications in scaffold production. However, current procedures have limitations in obtaining BC pellicles with the desired structural, physical, and mechanical properties. Thus, this study analyzed the optimal culture conditions of BC membranes and two types of processing: draining and oven-drying. The aim was to obtain BC membranes with properties suitable for a wound dressing material. Two studies were carried out. In the preliminary study, the medium (100 mL) was inoculated with varying volumes (1, 2, 3, 4, and 5 mL) and incubated statically for different periods (3, 6, 9, 12, and 18 days), using a full factorial experimental design. Thickness, uniformity, weight, and yield were evaluated. In the optimization study, a Box-Behnken design was used. Two independent variables were used: inoculum volume (X1: 1, 3, and 5 mL) and fermentation period (X2: 6, 12, and 18 d) to determine the target response variables: thickness, swelling ratio, drug release, fiber diameter, tensile strength, and Young's modulus for both dry and moist BC membranes. The mathematical modelling of the effect of the two independent variables was performed by response surface methodology (RSM). The obtained models were validated with new experimental values and confirmed for all tested properties, except Young's modulus of oven-dried BC. Thus, the optimal properties in terms of a scaffold material of the moist BC were obtained with an inoculum volume of 5% (v/v) and 16 d of fermentation. While, for the oven-dried membranes, optimal properties were obtained with a 4% (v/v) and 14 d of fermentation.

Anthocyanins in Blueberries Grown in Hot Climate Exert Strong Antioxidant Activity and May Be Effective against Urinary Tract Bacteria.

Anthocyanins are extensively studied for their health-related properties, including antibacterial activity against urinary tract infections (UTI). Among common fruits, blueberries, with their remarkable antioxidant capacity, are one of the richest sources. Anthocyanin-rich extracts were obtained from four varieties: Snowchaser, Star, Stella Blue and Cristina Blue, grown in the hot climate of Southern Spain. Their total anthocyanins contents (TAC) were determined spectrophotometrically, and the anthocyanin profile by ultra high performance liquid chromatography - tandem mass spectrometer (UHPLC-MS/MS). Their antioxidant activity was assessed by oxygen radical absorbance capacity (ORAC) assay, while antibacterial activity against strains isolated from UTI patients was assessed in vitro, helping to select the varieties with the highest bioactive potential. Star showed the highest TAC and antioxidant activity (1663 ± 159 mg of cyanidin-3-O-glucoside (cy-3-O-glu) equivalents/100 g fresh weight (FW), 6345 ± 601 µmol Trolox equivalents (TE)/100 g FW, respectively), followed by Cristina Blue, Stella Blue and Snowchaser. As far as we know, this is the first time that cyanidin-3-rutinoside has been identified in blueberries. The extracts inhibited all the tested strains, MICs ranging from 0.4 mg/mL (for Stella Blue extract against UTI P. aeruginosa) to 9.5 mg/mL (for all extracts against UTI K. pneumoniae ssp. pneumoniae). This is the first study that assessed in vitro the antibacterial activity of blueberries against Klebsiella pneumoniae, Providencia stuartii and Micrococcus spp. strains isolated from UTI.

Cylindrospermopsin-Microcystin-LR Combinations May Induce Genotoxic and Histopathological Damage in Rats.

Cylindrospermopsin (CYN) and microcystins (MC) are cyanotoxins that can occur simultaneously in contaminated water and food. CYN/MC-LR mixtures previously investigated in vitro showed an induction of micronucleus (MN) formation only in the presence of the metabolic fraction S9. When this is the case, the European Food Safety Authority recommends a follow up to in vivo testing. Thus, rats were orally exposed to 7.5 + 75, 23.7 + 237, and 75 + 750 µg CYN/MC-LR/kg body weight (b.w.). The MN test in bone marrow was performed, and the standard and modified comet assays were carried out to measure DNA strand breaks or oxidative DNA damage in stomach, liver, and blood cells. The results revealed an increase in MN formation in bone marrow, at all the assayed doses. However, no DNA strand breaks nor oxidative DNA damage were induced, as shown in the comet assays. The histopathological study indicated alterations only in the highest dose group. Liver was the target organ showing fatty degeneration and necrotic hepatocytes in centrilobular areas, as well as a light mononuclear inflammatory periportal infiltrate. Additionally, the stomach had flaking epithelium and mild necrosis of epithelial cells. Therefore, the combined exposure to cyanotoxins may induce genotoxic and histopathological damage in vivo.

A new method for the simultaneous determination of cyanotoxins (Microcystins and Cylindrospermopsin) in mussels using SPE-UPLC-MS/MS.

The aim of this study was to optimize the extraction conditions of Microcystin-LR (MC-LR), Microcystin-RR (MC-RR), Microcystin-YR (MC-YR) and Cylindrospermopsin (CYN) simultaneously from mussels by using response surface methodology (RSM) and to validate the method by a dual solid phase extraction (SPE) system combined with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The optimal parameters were: 90% MeOH (% v/v) for the extraction, a solvent/sample ratio of 75 and 15% MeOH in the extract before loading onto SPE. Mussels were spiked at 10; 37.5 and 75 ng g-1 fresh weight (f.w) of the 4 toxins, showing linear ranges of 0.5-75 ng g-1 f.w; low values for the limits of detection (0.01-0.39 ng g-1 f.w.) and quantification (0.23-0.40 ng g-1 f.w.); acceptable recoveries (70.37-114.03%) and relative standard deviation (%RSDIP) values (2.61-13.73%). The method was successfully applied to edible mussels exposed to cyanobacterial extracts under laboratory conditions, and it could allow the monitoring of these cyanotoxins in environmental mussel samples.

In vivo genotoxicity evaluation of cylindrospermopsin in rats using a combined micronucleus and comet assay.

Cylindrospermopsin (CYN) is a potent cyanotoxin recognized as an emerging human threat due to its cytotoxicity and potential carcinogenicity. Although the genotoxicity of CYN has been extensively studied in vitro, limited data are available on its in vivo genotoxicity. The aim of this study was to evaluate the in vivo genotoxicity of pure CYN (7.5-75 µg/kg body weight) after oral exposure of rats through a combined assay of the micronucleus test (MN) in bone marrow, and the standard and modified comet assay in stomach, liver and blood. Also, histopathological changes in stomach and liver were evaluated. Positive results in the MN test were observed in bone marrow in the exposed rats at all the tested concentrations. However, the comet assay revealed that CYN did not induce DNA strand breaks nor oxidative DNA damage in any of the tissues investigated. Finally, histopathological changes were observed in stomach and liver (7.5-75 µg/kg) in intoxicated rats. These results could indicate that CYN is able to induce irritation in stomach before its biotransformation in rats orally exposed, and genotoxicity in bone marrow.

Risk assessment methodologies in the field of contaminants, food contact materials, technological ingredients and nutritional risks.

The programme aimed at training the fellow in the risk assessment guidelines proposed by the EFSA in the field of contaminants, food contact materials, technological ingredients and nutritional risks. It had a modular 'learning by doing' approach and a balanced learning/case studies and theory. Module 1 offered an insight into chemical risk assessment and conferred transferable skills for a proper application of the framework. The hands-on activities consisted of three case studies that went from a simple exercise on an official opinion, to working in a team with experts to produce a new opinion, to an individual work to obtain a publishable review manuscript. Module 2 was a training in experimental toxicology designed to create a toxicological basis and to enable the fellow to perform toxicological studies for risk assessment purposes. She joined the team working on cyanotoxins, gained experience with both EFSA and Organization of Economic Cooperation and Development (OECD) guidelines on genotoxicity and an insight into the developing of analytical methods suitable for risk assessment purposes. During module 3, the fellow was trained in nutritional risk assessment and involved in experimental work in chemical characterisation, biomarkers and mechanisms of action of bioactive compounds. This developed the critical perspective when assessing nutritional and health claims related the design of experiments, methods used, interpretation of results and human relevance. Module 4 provided a 'hand-on experience' in scientific risk communication as the fellow was encouraged and supported in the participation at local, national and international workshops and congresses presenting the outcomes of the three modules. Thus, the fellow was successfully integrated in the day-by-day workflow of the department, gaining first-hand practical experience in risk assessment in a multicultural and interdisciplinary context. This enabled a productive exchange of good practices and contributed to building a European risk assessment community.