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.

Estimation of acrylamide exposure in Italian schoolchildren consuming a canteen menu: health concern in three age groups.

Acrylamide (AA) is a food process contaminant with carcinogenic and genotoxic properties that is formed in thermally treated foods, especially carbohydrate-rich. Dietary exposure to AA, due to school canteen foods, was estimated in schoolchildren aged 3-13 years by combining the AA concentration in foods with the amount and frequency of food consumption. Potato products and bakery products presented the highest mean levels of AA (841 and 244 µg/kg, respectively) followed by meat (222 µg/kg) and egg products (151 µg/kg). The mean total AA intake was estimated at 2.16 µg/kg bw/d, with the highest percentages provided by potato products (34.5%), meat products (26.1%) and bread (24.5%). The calculated margins of exposure (MOEs) for the average AA exposure (84 and 212 for the benchmark dose lower confidence limits (BMDL) of 0.17 and 0.43 mg/kg bw/d) suggest that there is a health concern with respect to students eating in school canteens.

Comparison of PCR assays targeting the multi-copy targets B1 gene and 529 bp repetitive element for detection of Toxoplasma gondii in swine muscle.

The comparison of the sensitivities of two molecular assays designed to target the multi-copy sequences of the Toxoplasma gondii genomic B1 region and 529 bp-RE respectively, in detecting T. gondii in swine muscle was assessed. Diaphragm pillars were obtained from 498 slaughtered pigs managed in intensive farms in Central Italy. Genomic DNA was extracted from the tissues and T. gondii-B1 and 529 bp-RE sequences were amplified by specific PCR protocols. Toxoplasma gondii DNA was detected in 165 samples (33.13%). There was a good correlation (κ = 0.77) between the results obtained targeting the two different genetic markers, however the 529 bp RE-PCR assay overall detected a significantly higher (P < 0.05) number of T. gondii-positive samples (150 samples) than the B1-PCR protocol (134). Our results show that: i) standardized B1 and 529 bp-RE PCRs applied to muscle tissues can detect a high rate of T. gondii-infection; ii) a multi-target PCR approach is recommended for the accurate diagnosis of infection in swine and can also be used in food testing.

Seasonal variations of Italian Mediterranean Buffalo (Bubalus bubalis) Mozzarella cheese quality.

Water buffalo Mozzarella cheese is more appreciated in the summer, but milk production is higher during wintertime, as water buffalo are seasonally polyoestrous animals. The aim of the study reported in this Research Communication was to evaluate the effect of the reversing of the calving period on Mozzarella cheese composition and quality traits. Ten batches of Mozzarella cheeses produced during the summer and winter periods were analysed for pH, colour, chemical composition. Seasonal differences were observed for cheese yield (26·66% in winter and 25·61% in summer), moisture content (66·54% in winter and 61·18% in summer) colour and consumer evaluation.

Influence of Maternal and Postweaning Linseed Dietary Supplementation on Growth Rate, Lipid Profile, and Meat Quality Traits of Light Sarda Lambs.

The effects of dietary extruded linseed (EL) on growth performance, meat quality, and lipid profile of Semimembranosus and Longissimus lumborum muscles of 81 Sarda lambs were studied in a 3 × 3 design: EL content (0%, 10%, and 20%) of maternal dietary concentrate fed from 20 d to parturition to 60 d of lactation and EL content (0%, 10%, 20%) of lamb concentrate fed after weaning for 30 d. The basal diet was composed of alfalfa and meadow hay during pregnancy and alfalfa hay during lactation. At slaughter, carcass and meat quality were evaluated. Sensory quality of Semimembranosus from 0% and 20% EL lambs was assessed. Both maternal and postweaning diets affected growth performance, with higher body weights recorded with the 10% EL concentrate. Carcass and meat quality were not affected by diet. Saturated and monounsaturated FA decreased and n-3 polyunsaturated FA increased with increasing EL content in lamb diet. An increase in vaccenic and rumenic acid was associated with the EL content of the maternal diet. Both diets increased the n-6/n-3 FA ratio. No differences in acceptability were detected by consumers among groups. It is concluded that EL supplementation and early life nutrition can influence performance and FA metabolism in growing lambs.

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.

Preventing Microbial Growth in Game Meat by Applying Polyphenolic Extracts from Olive Mill Vegetation Water.

We studied the efficacy of different formulations of polyphenol extracts (mainly containing hydroxytyrosol and tyrosol) from olive mill vegetation water on the microflora on the surfaces of game meat cuts with high or low initial bacterial loads. Meat with a high microbial load (>5 Log cfu/g; mean value = 6.83 ± 0.45 standard deviation) was immersed for 10 or 60 sec into 25% and 10% solutions of microencapsulated freeze-dried and non-encapsulated polyphenolic extracts. Aerobic colony, Enterobacteriaceae, Pseudomonas spp., and lactic acid bacteria counts were determined on treated samples compared to controls after 7 days of storage (in vacuum-packed conditions at +3 °C). Significant differences were registered only for aerobic colony count for a 10% liquid extract treatment (0.64 log reduction). In contrast, the dipping or immersion of game meat with low initial microbial loads (<5 Log cfu/g; mean value = 3.58 ± 0.72 standard deviation) in 10% solutions of the polyphenol extracts effectuated significant reductions in all bacteria counts (p < 0.002) at 7 and 14 days of storage for different extracts, independently from the application methods. The use of the extracts to inhibit bacterial growth in game meat should only be considered if a good hygienic baseline is guaranteed.

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.

Olive Mill Wastewater Extract: In Vitro Genotoxicity/Antigenotoxicity Assessment on HepaRG Cells.

Olive mill wastewater (OMWW), with its high level of phenolic compounds, simultaneously represents a serious environmental challenge and a great resource with potential nutraceutical activities. To increase the knowledge of OMWW's biological effects, with an aim to developing a food supplement, we performed a chemical characterisation of the extract using the Liquid Chromatography-Quadrupole Time-of-flight spectrometry (LC-QTOF) and an in vitro genotoxicity/antigenotoxicity assessment on HepaRG ™ cells. Chemical analysis revealed that the most abundant phenolic compound was hydroxytyrosol. Biological tests showed that the extract was not cytotoxic at the lowest tested concentrations (from 0.25 to 2.5 mg/mL), unlike the highest concentrations (from 5 to 20 mg/mL). Regarding genotoxic activity, when tested at non-cytotoxic concentrations, the extract did not display any effect. Additionally, the lowest tested OMWW concentrations showed antigenotoxic activity (J-shaped dose-response effect) against a known mutagenic substance, reducing the extent of DNA damage in the co-exposure treatment. The antigenotoxic effect was also obtained in the post-exposure procedure, although only at the extract concentrations of 0.015625 and 0.03125 mg/mL. This behaviour was not confirmed in the pre-exposure protocol. In conclusion, the present study established a maximum non-toxic OMWW extract dose for the HepaRG cell model, smoothing the path for future research.

Does hunted wild boar meat meet modern consumer nutritional expectations?

The present study aimed to evaluate the nutritional characteristics of hunted wild boar (WB) meat and compare them with those of meat from analogous domestic animals (pigs) reared in two different rearing systems: indoor-intensive (PI) and outdoor-extensive (PO). WB meat showed a lower amount of lipid content compared to pork and a higher antioxidant activity compared to PI and PO. The comparison of the fatty acid composition of WB and domestic pig reveals significant differences in saturated fatty acids (SFA) and polyunsaturated fatty acids (PUFA), with WB having the highest PUFA level and the lowest SFA level. The omega 6/omega 3 (n-6/n-3) PUFA ratio, PUFA/SFA, atherogenic and thrombogenic indices (AI and TI), as well as the hypocholesterolemic/ hypercholesterolemic index (h/H), were calculated. The n-6/n-3 PUFA ratio was higher in pork independently of the rearing system. The PUFA/SFA ratio of WB meat was above the minimum ratio of 0.40 recommended to contribute to a reduction in the risk of coronary diseases in pork from both rearing systems. AI and TI were lower in WB meat compared to commercially reared pigs, while h/H was higher in WB in comparison with pork meat. WB meat shows good nutritional quality; therefore, the use of game meat as a food source could be appropriate and could benefit contemporary consumers looking for "green" and high-nutritional products.

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).

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.

Wild boar captured in a large corral-style trap or hunted: preliminary comparison of meat quality traits.

The management and numerical control of wild boars mainly depend on hunting practices, even if other alternative strategies such as the use of traps and cages can be adopted. There is little information available on the quality of captured wild boar meat. The aim of this study was to evaluate the meat quality of wild boars captured with a large corral-style trap compared to still hunting and collective hunting methods. Longissimus dorsi samples were collected from 60 wild boars, 20 of which were obtained by trapping, 20 by still hunting, and 20 by collective hunting. The animals considered were 32 males and 28 females, weighing between 42 and 68 kg. Muscle pH has been recorded at 1, 24, and 48 hours post-mortem. Furthermore, after 24 hours, color, drip loss, cooking loss, and Warner-Bratzler shear force were also evaluated. Trapping with large enclosures such as corral-style traps, if properly managed, does not seem to adversely affect the quality traits of wild boar meat, which were found to be like those obtained by the still hunting method.

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.

Evaluation of ß-Lactamase Enzyme Activity in Outer Membrane Vesicles (OMVs) Isolated from Extended Spectrum ß-Lactamase (ESBL) Salmonella Infantis Strains.

Outer membrane vesicles (OMVs) are nanoparticles released by Gram-negative bacteria, which contain different cargo molecules and mediate several biological processes. Recent studies have shown that OMVs are involved in antibiotic-resistance (AR) mechanisms by including ß-lactamase enzymes in their lumen. Since no studies have as yet been conducted on Salmonella enterica subs. enterica serovar Infantis' OMVs, the aim of the work was to collect OMVs from five S. Infantis ß-lactam resistant strains isolated from a broiler meat production chain and to investigate whether ß-lactamase enzymes are included in OMVs during their biogenesis. OMVs were isolated by means of ultrafiltration and a Nitrocefin assay quantified the presence of ß-lactamase enzymes in the OMVs. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) were used to identify the OMVs. The results showed that all strains release spherical OMVs, ranging from 60 to 230 nm. The Nitrocefin assay highlighted the presence of ß-lactamase enzymes within the OMVs. This suggests that ß-lactamase enzymes also get packaged into OMVs from bacterial periplasm during OMV biogenesis. An investigation into the possible role played by OMVs in AR mechanisms would open the door for an opportunity to develop new, therapeutic strategies.

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.

Evaluation of an immunofluorescence antibody assay for the detection of antibodies against Toxoplasma gondii in meat juice samples from finishing pigs.

Serum and meat juice analyses for the detection of Toxoplasma gondii antibodies by an immunofluorescence antibody assay were compared in 100 seropositive and 100 seronegative slaughtered heavy swine. Meat juice was obtained from diaphragm and gracilis muscles of the serologically tested animals. Seventy-two diaphragmatic meat juice samples (36%, 95% interval confidence [IC] 29.4%-43.1%) and 63 gracilis meat juice samples (31.5%, 95% IC 25.1%-38.4%) tested positive for T. gondii antibodies. The average concordance between serum and meat juice derived from both muscles was "substantial" (K=0.6-0.8). The K-value was 1 when considering serum samples showing a titer >1/16, whereas it decreased to 0.62 and to 0.49 when considering serum samples with a 1/16 titer in meat juice from diaphragm and gracilis muscle, respectively.