The Effects of Encapsulation on the In Vitro Anti-Clostridial Activity of Olive Mill Wastewater Polyphenolic Extracts: A Promising Strategy to Limit Microbial Growth in Food Systems.

Despite the technologies applied to food production, microbial contamination and chemical deterioration are still matters of great concern. In order to limit these phenomena, new natural approaches should be applied. In this context, the present study aimed to assess the antioxidant and anti-Clostridial effects of two different polyphenolic extracts derived from olive mill vegetation water, one liquid (LE) and one encapsulated (EE). The extracts have been preliminary characterized using Liquid Chromatography Quadrupole Time-Of Flight spectrometry. The Oxygen Radical Absorbance Capacity method was used to determine the antioxidant capacity, registering a higher value for EE compared to that for LE (3256 ± 85 and 2446 ± 13 µgTE/g, respectively). The antibacterial activity against C. perfringens, C. botulinum and C. difficile was studied by the agar well diffusion method, MIC and MBC determination and a time-kill test. The results confirm that EE and LE are able to limit microbial growth, albeit with minor effects when the phenolic compounds are encapsulated. Further studies are needed to evaluate the possible application of these extracts in food systems.

Occurrence of Hydroxytyrosol, Tyrosol and Their Metabolites in Italian Cheese.

Tyrosol (T) and hydroxytyrosol (HT) are phenyl alcohol polyphenols with well-recognized health-promoting properties. They are widely diffused in several vegetables, especially in olive products (leaves, fruits and oil). Therefore, they could be present in food produced from herbivorous animals such as in milk and cheese. In this study, an analytical method to determine T, HT and some of their phase II metabolites (sulphates and glucuronides) in cheese was developed and validated. Samples were extracted with an acidic mixture of MeOH/water 80/20 (v/v) and, after a low temperature clean-up, the extracts were evaporated and injected in a liquid-chromatography coupled with high resolution mass spectrometry (LC-Q-Orbitrap). A validation study demonstrated satisfactory method performance characteristics (selectivity, linearity, precision, recovery factors, detection and quantification limits). The developed protocol was then applied to analyze 36 Italian cheeses made from ewe, goat and cow milk. The sum of detected compounds (T, tyrosol sulfate, hydroxytyrosol-3-O-sulfate and hydroxytyrosol-4-O-sulfate) reached as high as 2300 µg kg-1 on a dry weight basis, although in about 45% of cow cheeses it did not exceed 50 µg kg-1. Ewe cheeses were significantly richer of polyphenols (sum) as well as HT sulfate metabolites than cow cheeses. In conclusion, results shows that cheese cannot be considered an important dietary source of these valuable compounds.

Dietary supplementation with olive mill wastewaters induces modifications on chicken jejunum epithelial cell transcriptome and modulates jejunum morphology.

BACKGROUND: The Mediterranean diet is considered one of the healthier food habits and olive oil is one of its key components. Olive oil polyphenols are known to induce beneficial effects in several pathological conditions, such as inflammatory bowel disease, and to contrast the proliferation of cancer cells or hypercholesterolemia. Polyphenols are also present in waste products derived from the olive industry: olive mill wastewaters (OMWW) are rich in polyphenols and there is an increasing interest in using OMWW in animal nutrition. OMWW are attributed with positive effects in promoting chicken performance and the quality of food-derived products. However, a tissue-specific transcriptome target analysis of chickens fed with OMWW has never been attempted. RESULTS: We explored the effect of dietary OMWW on the intestinal function in broilers. A morphological analysis of the jejunum revealed that OMWW reduced crypt depth, whereas no significant modifications were observed for villus height and the villus height/crypt depth ratio. An RNA Sequencing analysis was performed on isolated, intestinal, epithelial cells and 280 differentially expressed genes were found using a count-based approach. An enrichment analysis revealed that the majority of up regulated genes in the OMWW group were over-represented by the regulation of viral genome replication-related GO-Terms, whereas down regulated genes were mainly involved in cholesterol and lipid metabolism. CONCLUSIONS: Our study showed how an industrial waste product can be recycled as a feed additive with a positive relapse. OMWW dietary supplementation can be a nutritional strategy to improve chicken performance and health, prevent intestinal damage, enhance innate immunity and regulate cholesterol metabolism and fat deposition.

YERSINIA PSEUDOTUBERCULOSIS, SEROGROUP O:1A, INFECTION IN TWO AMAZON PARROTS (AMAZONA AESTIVA AND AMAZONA ORATRIX) WITH HEPATIC HEMOSIDEROSIS.

Necropsies were conducted on a female blue-fronted Amazon (Amazona aestiva) and a female yellow-headed Amazon (Amazona oratrix) that died after depression, ruffled feathers, diarrhea, and biliverdin in the urine. Gross and microscopic examinations revealed multifocal necrosis in the liver, spleen, lungs, kidneys, intestines, and heart caused by acute bacteremia. Yersinia pseudotuberculosis, serogroup O:1a, was isolated by culturing from the visceral lesions in the liver, intestines, and spleen. Virulence gene analysis showed the presence of the inv gene and the complete pathogenicity island: IS100, psn, yptE, irp1, irp2 ybtP-ybtQ, ybtX-ybtS, and int asnT-Int. Histopathologic findings and chemical analysis also demonstrated hepatic hemosiderosis. As has been demonstrated in other species, hemosiderosis may predispose Amazona spp. to systemic infection with Y. pseudotuberculosis after enteric disease.

Influence of manufacturing procedure on the compositional and sensory properties of n-3 fatty acid-enriched pecorino cheese.

Pecorino cheeses were produced using two different manufacturing procedures (either from raw milk or from milk previously subject to a thermisation at 65 °C for 10 s with the addition of starter culture) using the milk obtained from three groups of ewes fed three different concentrates: (1) control, (2) control enriched with 100 g/kg of extruded linseed, and (3) control enriched with 200 g/kg of extruded linseed. All concentrates were administered with alfalfa hay ad libitum. The extruded linseed-enriched diets increased the concentration of n-3 fatty acids, conjugated linoleic acid, and monounsaturated and polyunsaturated acids in raw milk and thermised milk cheese and decreased the concentration of saturated fatty acids. Changes in the manufacturing procedure did not influence the fatty acid composition of the cheeses but markedly influenced their sensory properties. In particular, heat-treating the milk and adding a starter reduced the differences in cheese odour, flavour and toughness induced by the diet when raw milk was used. Cheeses made with thermised milk and the addition of starter culture were more uniform and obtained a higher score in the preference test compared with the corresponding raw milk cheeses. An appropriate manufacturing process, therefore, can be a possible strategy to obtain cheeses with improved health-promoting properties and an unaltered acceptance level by consumers.

Surface carcass treatment with olive mill wastewater polyphenolic extract against Salmonella Enteritidis and Listeria monocytogenes: in vitro and in situ assessment.

In recent years, there has been an increased interest in substances that could inhibit or reduce microbial growth in food products. Olive oil industry by-products, due to bioactive compounds with potential antimicrobial properties such as polyphenols, could be used in carcass treatment to enhance hygienic and quality traits. The assessment of the antimicrobial efficacy of bioactive molecules against pathogens should be determined with in vitro and in situ models since it is not possible to evaluate it directly on carcasses at the slaughterhouse. This study aimed to evaluate the effect of an olive mill wastewater polyphenolic extract against Salmonella Enteritidis and Listeria monocytogenes, simulating carcass surfaces using bovine dermis samples that were experimentally contaminated with the selected pathogens. The minimum inhibitory concentration and minimum bactericidal concentration were first determined for S. Enteritidis and L. monocytogenes. In situ, bactericidal activity assessment was performed using 20 cm2 derma samples contaminated with 5 Log CFU/20 cm2 of S. Enteritidis and L. monocytogenes in separate trials. Treatment with the polyphenolic extract was not effective for either microorganism. In order to establish the bacteriostatic activity of the polyphenolic extract, suspensions of about 2 Log CFU/20 cm2 of S. Enteritidis and L. monocytogenes were used. Polyphenolic extract treatment was not effective against Salmonella, while for Listeria it allowed microbial growth to delay (around 1 Log CFU/cm2 difference at 3, 7, and 14 days between treated and control groups). Further investigations are needed to evaluate the application of polyphenolic compounds on carcass surfaces and their effects on sensory traits.

Antilisterial activity of olive-derived polyphenols: an experimental study on meat preparations.

Pork meat and processed pork products have been linked to multiple listeriosis outbreaks worldwide during the past few years. Specifically, it has been highlighted that minced pork meat is easily perishable and may increase the growth of Listeria monocytogenes, which could be harmful to the general public's health. This study aimed to investigate the potential application of olive oil mill wastewater polyphenolic and red beet extracts as natural antimicrobial agents for L. monocytogenes growth control in burgers. The minced pork meat was mixed with the extracts and experimentally inoculated with L. monocytogenes, then molded into vacuum-packaged and cold-stored (4±1°C) burgers kept under alternating exposure to fluorescent light. The L. monocytogenes enumeration was performed on burgers at 0, 2, 5, and 10 days of shelf life. In uninoculated burgers, physicochemical (pH, water activity, color) and sensory determination (descriptive sensory analysis) were also conducted. At the end of storage, the samples treated with olive-derived extract showed the lowest value of L. monocytogenes (approximately 1.3 Log CFU/g). The physicochemical and sensory traits of burgers have benefited from the addition of both olive-derived and red beet extracts. Results suggest that olive mill wastewater polyphenolic extracts could be added to minced pork meat products to act as a natural antimicrobial agent.

Development and characterization of an emulgel based on a snail slime useful for dermatological applications.

Snail slime is an interesting material for effective dermatological use (e.g. wounds). Its properties are stricly connected to the origin. In this paper a snail slime, deriving from the species Helix aspersa Muller and obtained from a company, was deeply characterized and then properly formulated. The slime, obtained by Donatella Veroni method, was firstly submitted to NMR analysis in order to evaluate the chemical composition. The main molecules found are glycolate and allantoin, well known for their activities in wound healing promotion. In vitro experiments performed on keratinocytes, revealed the snail slime ability to promote cellular well-being. Moreover, the microbiological analysis showed high activity against many strains involved in wounds infections such as gram+ (e.g. S. aureus, S. pyogenes), gram- (e.g. P. aeruginosa, E. coli) and the yeast C. albicans. The effect on skin elasticity was evaluated as well by the instrument Cutometer® dualMPA580. The snail slime was then formulated as hydrophilic gel, using a combination of corn starch and sodium hyaluronate as polymers, then used as external water phase of an O/W emulgel. The formulation is physically stable and easily spreadable and demonstrated antimicrobial activity as observed for slime alone, suggesting its suitability to be used for wound treatment.

Detection of Hepatitis E Virus in Game Meat (Wild Boar) Supply Chain in Umbria Region, Central Italy.

Consumption of raw or undercooked wild boar (WB) meat is considered an important risk factor for hepatitis E virus (HEV) infection in humans. The possibility of HEV contamination during the slaughtering practices may pose an additional risk. Based on these assumptions, we evaluated HEV contamination of WB meat hunted in Umbria (central Italy) during the 2022-2023 hunting season by real-time RT-PCR. Herein, we show that 10.8% of livers from slaughtered WB were positive for HEV RNA, thus providing an estimate of HEV infection in WB in the Umbria region. Then, by evaluating paired liver-muscle samples from both HEV-positive and HEV-negative animals, we found evidence of muscle HEV contamination in 33% and 14% of cases, respectively. This is the first report on the detection of HEV in WB meat in Umbria, an Italian region with diffuse WB hunting and consumption. The evidence of contamination provided by our study underscores the importance of adopting good hygienic practices in the processing stages of hunted WB carcasses to significantly reduce meat contamination and the risk posed for the final consumer.

Salmonella in Wild Boar Meat: Prevalence and Risk Assessment in Central Italy (Umbria and Marche Region).

A survey was conducted from 2018 to 2023 to assess the presence of Salmonella in 280 hunted wild boar (carcasses after evisceration and skinning, N = 226; liver, N = 258; and fecal samples, N = 174). The overall prevalence was 2.86% (confidence interval 95%, 1.45-5.45%) with five positive samples detected in carcasses, three in the liver, and one in a fecal sample. This prevalence was in line with those found in nearby areas denoting a low number of positive samples. Positive animals were over 24 months of age and weighed, before skinning, 59.00 ± 9.11 Kg and no difference was detected in microbial loads between samples positive and negative for Salmonella (aerobic colony count of 4.59 and 4.66 log CFU/400 cm2, and Enterobacteriaceae count of 2.89 and 2.73 log CFU/400 cm2 (mean values) in positive and negative subjects, respectively). Salmonella Stanleyville was the most frequently isolated serotype. A semiquantitative risk assessment was conducted for the first time in game meat considering two products, meat cuts intended for cooking and fermented dry sausages. Only proper cooking can reduce the risk of ingestion of Salmonella to the minimum for consumers, whereas ready-to-eat dry sausages constitute risk products in terms of foodborne Salmonellosis (risk score of 64 out of 100).

Use of milk amyloid A in the diagnosis of subclinical mastitis in dairy ewes.

Subclinical mastitis (SM) is one of the most important diseases affecting dairy ewes worldwide, with negative impact on the animal health, farm income and public health. Animals with SM often remain untreated because the disease may not be revealed. Increase in somatic cell count (SCC) and positive bacteriology for mastitis pathogens in milk samples are indicative of SM but the evidence of only one of these alterations must suggest an uncertain SM (UM). UM is defined when positive bacteriological examination (Latent-SM) or SCC>500 000 cells/ml (non-specific-SM) are detected in milk. Nevertheless, SCC and bacteriological examination are expensive, time consuming and are not yet in use at the farm level in dairy ewes. Recently, a sensitive acute phase protein, amyloid A, displaying multiple isoforms in plasma and different body fluids including mammary secretion (milk amyloid A-MAA), has been investigated as a marker of mastitis in cows and, in a few studies, in sheep. The aim of this trial was to compare the concentration of MAA of single udder-halves in ewes with healthy udder-halves (HU-control group) and naturally occurring subclinical mastitis, both confirmed (SM group) and uncertain (UM groups: Latent-SM and non-specific-SM), for monitoring udder health. The reliability of a specific ELISA kit for the measurement of MAA was also tested. During a 3-month trial period, 153 udder halves were assigned to the experimental groups based on their health status: 25 with SM, 40 with UM (11 with latent-SM and 29 with non-specific-SM) and 88 HU. SCC and bacteriological analysis were performed to establish the control and subclinical mastitis groups. MAA concentrations in milk samples were measured using a specific commercially milk ELISA kit. The data were submitted to statistical analysis. Significant (P<0·05) differences among the groups SM, non-specific-SM and HU were detected with the SM having the highest level and HU the lowest. MAA concentration is affected by the udder health status and is a useful indicator of subclinical mastitis and increased SCC in sheep.

Retrospective study on the prevalence of Yersinia enterocolitica in food collected in Umbria region (central Italy).

Yersinia enterocolitica represents one of the main foodborne pathogens in Europe and the evaluation of possible sources of contamination and its prevalence in food is of considerable interest for risk analysis approach. The results of the search for Yersinia enterocolitica in food samples taken in Umbria region (central Italy) were evaluated during the years 2015-2018. Different types of foods were considered, both ready-to-eat (meat products, dairy products, and raw vegetables) and meat preparations to be eaten after cooking. Samples were assayed by molecular screening for the species indicator gene ompF. Screening positives were subjected to isolation and characterization by searching for specific virulence marker genes, including the ail gene responsible for invasiveness and the ystB gene for the production of enterotoxin. The total prevalence of positive samples for Yersinia enterocolitica was 16.86% with a higher percentage of positive samples in meat preparations (19.35%), followed by ready-to-eat vegetables (11.76%). Poultry meat samples had a higher prevalence than pork and beef samples. Neither positive samples were found in meat products and dairy, nor seasonality in positivity was observed. All isolated strains of Yersinia enterocolitica were biotype 1A, with absence of the ail virulence gene but presence of ystB gene. Since the strains isolated from human patients appear to be primarily biotypes that possess the ail marker, future investigations would be needed regarding the real role of biotype 1A in human disease. In this context, attention should certainly be paid to ready-to-eat vegetables and to careful cooking of meat preparations.

Eustrongylides spp. parasite risk management in Atherina boyeri from Lake Trasimeno.

Atherina boyeri is the primary source of fishing profit in Lake Trasimeno and a common host for Eustrongylides spp. larvae. The presence of Eustrongylides in fish is a public health concern, and effective risk management procedures are necessary to guarantee that infected products do not reach the market. Currently, in the European Union, there is no official sampling plan for fresh fish that defines sample size, inspection methods, and criteria for accepting or rejecting the product. An approach to Eustrongylides risk management is proposed in this study. A total of 270 batches of A. boyeri, each consisting of 29 specimens, were collected and examined visually in 3 years (2020-2023). The prevalence of the parasite was 20% in 2020, and in the first months of 2021, it grew up to ⁓40%, then dropped to 12.50% in December 2021 and settled at 16% in February 2022. In January and February 2023, the prevalence fell below 1%. The mean abundance was calculated and used to establish a threshold value to determine fish marketability. In 2020 and 2021, several batches were judged not marketable, and in some batches, a freezing treatment was implemented to ensure the inactivation of the parasite. In the last months of 2022 and in January and February of 2023, the presence of parasites in captured fish was negligible, and this allowed the marketability of fish as fresh. The proposed sampling plan was effective in preventing the commercialization of potentially hazardous products.

Assessment of the Microbiological Safety and Hygiene of Raw and Thermally Treated Milk Cheeses Marketed in Central Italy between 2013 and 2020.

A profile of the microbial safety and hygiene of cheese in central Italy was defined based on an analysis of 1373 cheeses sampled under the Italian National Control Plan for Food Safety spanning the years 2013 to 2020 and tested according to Commission Regulation (EC) No. 2073/2005 (as amended). A total of 97.4% of cheese samples were assessed as being satisfactory for food safety criteria and 80.5% for process hygiene criteria. Staphylococcal enterotoxin was found in 2/414 samples, while Salmonella spp. and Listeria monocytogenes were detected in 15 samples out of 373 and 437, respectively. Escherichia coli and coagulase-positive staphylococci counts were found unsatisfactory in 12/61 and 17/88 cheese samples, respectively. The impact of milking species, milk thermal treatment, and cheese hardness category was considered. A statistically significant association (p < 0.05) was found between milk thermal treatment and the prevalence of coagulase-positive staphylococci and Listeria monocytogenes and between hardness and unsatisfactory levels of Escherichia coli. The data depict a contained public health risk associated with these products and confirm, at the same time, the importance of strict compliance with good hygiene practices during milk and cheese production. These results can assist in bolstering risk analysis and providing insights for food safety decision making.

Carassius auratus as a bioindicator of the health status of Lake Trasimeno and risk assessment for consumers.

Fish are good bio-indicators of the health status of the aquatic environment and can be used as biomarkers to assess the aquatic behavior of environmental pollutants, the exposure of aquatic organisms, and the health risk for consumers. Goldfish are a significant bioindicator in the Lake Trasimeno aquatic system (Umbria, Italy). This study aimed to characterize the health status and the chemical and biotic contamination of Lake Trasimeno to define its anthropogenic and natural pressures and the risk associated with consuming its fishery products. 114 determinations were performed on Carassius auratus samples from 2018 to 2020, and the occurrence of brominated flame retardants, non-dioxin-like polychlorinated biphenyls, heavy metals, and microplastics was analytically investigated. Dietary exposure assessment, risk characterization, and benefit-risk evaluation were performed for schoolchildren from 3 to 10 years old. Flame-retardants registered high levels of non-detects (99% for polybrominated diphenyl ether and 76% for hexabromocyclododecanes), while polychlorinated biphenyls were found in all samples with a maximum level of 56.3 ng/g. Traces of at least one heavy metal were found in all samples, though always below the regulatory limit. Microplastics were found with a 75% frequency of fish ingesting at least one particle. Dietary exposure and risk characterization reveal negligible contributions to the reference values of all contaminants, except for mercury, which reached up to 25% of admissible daily intake. The benefit- risk assessment highlighted that the benefits of freshwater fish intake outweigh the associated risks. The examination of goldfish as indicator fish reveals the quality of Lake Trasimeno's aquatic environment and the safety of its products.

Polymeric Patches Based on Chitosan/Green Clay Composites and Hazelnut Shell Extract as Bio-Sustainable Medication for Wounds.

Hazelnut shells, the main waste deriving from hazelnut processing, represent an interesting source of active molecules useful in pharmaceutics, although they have not yet been examined in depth. A hydrosoluble extract (hazelnut shell extract, HSE) was prepared by the maceration method using a hydroalcoholic solution and used as the active ingredient of patches (prepared by casting method) consisting of composites of highly deacetylated chitosan and green clay. In vitro studies showed that the formulation containing HSE is able to stimulate keratinocyte growth, which is useful for healing purposes, and to inhibit the growth of S. aureus (Log CFU/mL 0.95 vs. 8.85 of the control after 48 h); this bacterium is often responsible for wound infections and is difficult to treat by conventional antibiotics due to its antibiotic resistance. The produced patches showed suitable tensile properties that are necessary to withstand mechanical stress during both the removal from the packaging and application. The obtained results suggest that the developed patch could be a suitable product to treat wounds.

Antibacterial Activity of Crocus sativus L. Petals Extracts against Foodborne Pathogenic and Spoilage Microorganisms, with a Special Focus on Clostridia.

In recent years, there has been a growing interest in the use of novel antimicrobial agents able to inhibit or kill food-borne bacteria or to interrupt the onset of food spoilage. Crocus sativus L. petals, typically considered as waste obtained from saffron spice production, could be a source of natural bioactive compounds to be used as food preservatives. The purpose of this work was to investigate the antibacterial properties of two hydroalcoholicsaffron petal extracts obtained by maceration (SPEA) and by ultrasonic bath (SPEB) methods. The main polyphenols identified in both extracts were gallic and chlorogenic acids, representing almost 70% of the phenolic fraction monitored. The antibacterial activity was studied by the agar well-diffusion method, against food-borne pathogenic and spoilage bacteria. Both extracts showed activity mainly against Gram-positive bacteria, in particular those belonging to the Clostridiaceae family (C. perfringens, C. botulinum and C. difficile), with inhibition zone diameters ranging from 13 to 18 mm. The antibacterial properties against Clostridia were further analyzed, determining MIC and MBC and performing a time-kill test. SPEA showed lower MIC/MBC values (250 mg/mL) compared to SPEB (500 mg/mL), suggesting that it could be more active against the assayed strains, probably because of its higher content of gallic acid. SPEA and SPEB, tested at a concentration of 1 × MIC, showed bactericidal activity against C. perfringens, C. botulinum and C. difficile and these results suggest that saffron petals could represent a valuable natural alternative source to conventional preservatives. Further investigations are needed to evaluate possible applications in the food industry.

Effect of the Application of a Green Preservative Strategy on Minced Meat Products: Antimicrobial Efficacy of Olive Mill Wastewater Polyphenolic Extract in Improving Beef Burger Shelf-Life.

The mincing process of raw meat favors microbial spoilage as well as chemical and enzymatic oxidation processes. In order to limit this degradative process, preservatives are routinely added to minced meat products. The role of olive mill wastewater polyphenolic extract as a replacement for synthetic preservatives in beef burger was assessed. The antioxidant capacity of the extract experimentally added to beef burger was evaluated using the oxygen radical absorbance capacity method (ORACFL) to assess the shelf-life, while the lipid oxidation was measured by thiobarbituric reactive substance (TBAR) determination. The antimicrobial activity was assayed by means of classical methods and predictive microbiology. The experimental addition of polyphenolic extract led to 62% lower lipid oxidation and 58% higher antioxidant capacity; it also successfully modulated spoilage microbial populations with an average growth reduction of 15% on day 7. Results indicate that olive mill wastewater polyphenolic extracts could be added to raw ground beef meat to act as natural antioxidants and to modulate microbial growth.

A Study of the Occurrence of Aflatoxin M1 in Milk Supply Chain over a Seven-Year Period (2014-2020): Human Exposure Assessment and Risk Characterization in the Population of Central Italy.

Aflatoxin food contamination represents a rising global issue that will continue to increase due to climate change. Aflatoxin M1 (AFM1) is of high concern for the whole dairy industry. In light of AFM1's harmful potential, a human health exposure assessment and risk characterization were performed for all age populations of central Italy with regard to milk and cheese consumption by means of the margin of exposure (MOE). In total, 16,934 cow and ewe's milk samples were collected from 2014 to 2020 and analyzed by an enzyme-linked immunosorbent assay (ELISA) screening method, confirmed by high-performance liquid chromatography with a fluorescence detector (HPLC-FLD). The average concentration of AFM1 in cow's milk ranged from 0.009 to 0.015 µg/kg, while in ewe's milk, the average concentration ranged from 0.009 to 0.013 µg/kg. The average amount of AFM1 exposure ranged from 0.00005 to 0.00195 g/kg bw/day, with the main contributor represented by drinking milk, followed by the consumption of soft cheeses. A high level of public health concern related to the youngest consumers has arisen from risk characterizations highlighting the need for constant monitoring of AFM1's occurrence in milk by inspection authorities, alongside regular updates with regard to exposure assessments.

Survival rate of Escherichia coli O157 in artificially contaminated raw and thermized ewe milk in different Pecorino cheese production processes.

Pecorino is a typical Italian cheese, mostly produced in central and southern Italy regions using ewe raw milk and following traditional procedures. The use of raw milk constitutes a risk linked to the potential survival or multiplication of pathogenic microorganisms, as Shiga toxin-producing Escherichia coli (STEC). The aim of this study was to compare different Italian traditional Pecorino production methods to determine if there were any phases that could influence the Escherichia coli O157 survival rate, but also if they could negatively influence lactic acid bacteria survival rate, during the phases of production and ripening. Therefore batches of Pecorino cheese were prepared using different production methods, representing the real and typical cheese production in southern and central Italy regions: 1) heating the milk at 37 °C for about 40 min before curding, 2) heating the milk at 60 °C (thermization) for 13 min, so that the alkaline phosphatase reaction is still positive before curding, 3) cooking curd at 41 °C and 4) at 45 °C, both for 5 min. Our results demonstrated that traditional milk treatments different from pasteurization can help but do not eliminate serious microbiological treats, as E. coli O157, especially if the raw milk is heavily contaminated. The heat treatment at 60 °C applied to raw milk was able to decrease the concentration of E. coli O157 of 1.7 log10CFU/ml and, according to the inactivation slope, it would be further reduced prolonging the heating treatment. The results obtained also showed that, during the Pecorino cheese ripening, E. coli O157 was always enumerable for 60 days, remaining detectable after 90 days of ripening.