Salmonella Enteritidis survival in different temperatures and nutrient solution pH levels in hydroponically grown lettuce.

Due to climate change, with contaminated and less fertile soils, and intense weather phenomena, a turn towards hydroponic vegetable production has been made. Hydroponic cultivation of vegetables is considered to be a clean, safe and environmentally friendly growing technique; however, incidence of microbial contamination i.e. foodborne pathogens, might occur, endangering human health. The aim of this study was to investigate the effects of different plant growth stages, pH (values 5, 6, 7, 8) and bacterial inoculum levels (3 and 6 log cfu/mL) on hydroponically cultivated lettuce spiked with Salmonella Enteritidis. The results revealed that the pH and inoculum levels affected the internalization and survival of the pathogen in the hydroponic environment and plant tissue. Younger plants were found to be more susceptible to pathogen internalization compared to older ones. Under the current growing conditions (hydroponics, pH and inoculum levels), no leaf internalization was observed at all lettuce growth stages, despite the bacterium presence in the hydroponic solution. Noticeably, bacteria load at the nutrient solution was lower in low pH levels. These results showed that bacterium presence initiates plant response as indicated by the increased phenols, antioxidants and damage index markers (H2O2, MDA) in order for the plant to resist contamination by the invader. Nutrient solution management can result in Taylor-made recipes for plant growth and possible controlling the survival and growth of S. Enteritidis by pH levels.

Variation of microbial load and biochemical activity of ready-to-eat salads in Cyprus as affected by vegetable type, season, and producer.

Fresh vegetables are important components of an everyday balanced diet making ready to-eat-salads (RTE) a commodity widely consumed. However, in the past few years these products have been linked with outbreaks of salmonellosis and listeriosis; thus the continuous investigation of their safety is an essential requirement. A total of 216 samples of ready-to-eat salads from the Cypriot market were analysed to determine the microbiological quality and safety, along with physicochemical attributes of the salads and identify possible correlations between them. The samples were randomly collected from four retail outlets and correspond to five different salad producing companies. Furthermore, the effects of season, salad producer and type of salad and/or their interactions with the tested parameters were investigated. The results revealed that the higher microbial load among seasons was observed in samples collected during spring. Escherichia coli was found in 11.57% of samples and 2.62% of isolates were found to be able to produce extended spectrum ß-lactamase (ESBL). All samples were found negative for Salmonella enterica, whereas Listeria monocytogenes was present in 3.70% of samples. Higher levels of spoilage bacteria (lactic acid bacteria and Pseudomonas spp.) were detected during winter and spring. Additionally, the %CO2 production was affected by the type of salad, while the interaction between producer and type of salad, affected total phenolic content and antioxidant activity of samples. A positive correlation of phenols and antioxidants with the presence of Staphylococcus spp., Pseudomonas spp., E. coli and Bacillus cereus was observed, suggesting that excessive handling increases microbial load and plant stress.

Valorization of Carob Fruit Residues for the Preparation of Novel Bi-Functional Polyphenolic Coating for Food Packaging Applications.

The food industry has become interested in the development of innovative biomaterials with antioxidant and antimicrobial properties. Although several biopolymers have been evaluated for food packaging, the use of polyphenolic coatings has been unexplored. The purpose of this work was to develop an antioxidant and antimicrobial coating for food packaging through the polymerization of carob phenolics. At first, the polyphenolic coatings were deposited in glass surfaces polymerizing different concentrations of carob extracts (2 and 4 mg mL-1) at three pH values (7, 8 and 9). Results demonstrated that the coating produced at pH 8 and at a concentration of 4 mg mL-1 had the most potent antioxidant and antimicrobial potential. Then, the coating was applied directly on the salmon fillet (coating) and on the plastic container (active packaging). Peroxide and thiobarbituric acid-reactive substances (TBARS) methods were used to measure the potency to inhibit lipid oxidation in salmon fillets. Furthermore, the anti-Listeria activity of coatings was also assessed. Results showed a significant decrease of lipid oxidation during cold storage of salmon fillets for both treatments; the superiority of applied coating directly on the salmon fillets was also highlighted. Regarding the antimicrobial potency, the polyphenolic coating depleted the growth of Listeria monocytogenes after 10 days storage; while the active packaging had no effect on Listeria monocytogenes. Overall, we describe the use of low-cost carob polyphenols as precursors for the formation of bifunctional coatings with promising applications in food packaging.

Survival of Mycobacterium avium subspecies paratuberculosis in retail pasteurised milk.

A survey of retail purchased semi-skimmed pasteurised milk (n = 368) for Mycobacterium avium subspecies paratuberculosis (MAP) was conducted between May 2014 and June 2015 across the midlands of England using the Phage-PCR assay. Overall, 10.3% of the total samples collected contained viable MAP cells, confirming that pasteurisation is not capable of fully eliminating human exposure to viable MAP through milk. Comparison of the results gained using the Phage-PCR assay with the results of surveys using either culture or direct PCR suggest that the phage-PCR assay is able to detect lower numbers of cells, resulting in an increase in the number of MAP-positive samples detected. Comparison of viable count and levels of MAP detected in bulk milk samples suggest that MAP is not primarily introduced into the milk by faecal contamination but rather are shed directly into the milk within the udder. In addition results detected an asymmetric distribution of MAP exists in the milk matrix prior to somatic cell lysis, indicating that the bacterial cells in naturally contaminated milk are clustered together and may primarily be located within somatic cells. These latter two results lead to the hypothesis that intracellular MAP within the somatic cells may be protected against heat inactivation during pasteurisation, accounting for the presence of low levels of MAP detected in retail milk.

Antioxidant and Antimicrobial Effects of Pistacia lentiscus L. Extracts in Pork Sausages.

Pistacia lentiscus fruits are ingredients of traditional Cypriot sausages. The objective of this study is to evaluate P. lentiscus extracts as natural additives to the sausages. First, the phenolic content and antioxidant activity of fruit and leaf extracts were determined. Results revealed that leaves are richer source of polyphenolic antioxidants than fruits, with methanol being the better extraction solvent. In the next step, the antioxidant and antimicrobial effects of methanolic extracts (300 mg/kg) in the pork sausage formulation were investigated. Peroxide, acid and thiobarbituric acid-reactive substance values demonstrated that both fruit and leaf extracts reduced the rate of lipid oxidation of sausages at 4 °C. Total viable count revealed significant differences on the fifth day of storage, with better microbial inhibition by leaf extract. No significant differences between the extracts were observed after the tenth day of storage. Overall, the extracts can be used to prevent lipid oxidation and reduce microbial spoilage during the first days of storage of fresh traditional pork sausages.

Microbial quality and molecular identification of cultivable microorganisms isolated from an urban drinking water distribution system (Limassol, Cyprus).

Microorganisms can survive and multiply in aged urban drinking water distribution systems, leading to potential health risks. The objective of this work was to investigate the microbial quality of tap water and molecularly identify its predominant cultivable microorganisms. Tap water samples collected from 24 different households scattered in the urban area of Limassol, Cyprus, were microbiologically tested following standard protocols for coliforms, E. coli, Pseudomonas spp., Enterococcus spp., and total viable count at 22 and 37 °C. Molecular identification was performed on isolated predominant single colonies using 16SrRNA sequencing. Approximately 85% of the household water samples were contaminated with one or more microorganisms belonging to the genera of Pseudomonas, Corynebacterium, Agrobacterium, Staphylococcus, Bacillus, Delftia, Acinetobacter, Enterococcus, Enterobacter, and Aeromonas. However, all samples tested were free from E. coli. This is the first report in Cyprus molecularly confirming specific genera of relevant microbial communities in tap water.

Knowledge and Practices of Cypriot Bovine Farmers towards Effective and Safe Manure Management.

Manure from bovine farms is commonly used as an organic fertiliser. However, if not properly managed, it can spread significant biological and chemical hazards, threatening both human and animal health. The effectiveness of risk control hugely relies on farmers' knowledge regarding safe manure management and on the application of suitable management practices. This study aims to evaluate the knowledge and practices of Cypriot bovine farmers towards safer manure management, from its generation to its final use, in line with the One Health approach. Factors affecting farmers' knowledge and applied practices are also investigated through a questionnaire survey. The questionnaire was developed and sent to all eligible bovine farmers in Cyprus (n = 353), and 30% (n = 105) of them returned the completed questionnaire. Results revealed there are some gaps in farmers' knowledge. The use of manure for fertilising crops dominated. Only half of the farmers stored manure in appropriate facilities, with 28.5% of them using a dedicated area with cement floors and 21.5% utilising leakproof tanks. The majority (65.7%) stored manure for more than three months before its use as a fertiliser in a dried form. In multiple regression analysis, education level and farming purpose were significant determinants of farmer knowledge. In conclusion, Cypriot farmers' knowledge must be reinforced to ensure proper manure management. The results highlight the importance of providing relevant training to farmers. Although the current practices partially decrease manure pathogens, interventions to promote the use of more effective treatment methods, such as biogas transformation and composting, would be beneficial.

Beneficial Effects of Yoghurts and Probiotic Fermented Milks and Their Functional Food Potential.

Probiotic fermented milks and yoghurts are acidified and fermented by viable bacteria, usually L. bulgaricus and S. thermophilus, resulting in a thicker product with a longer shelf life. They are a nutrition-dense food, providing a good source of calcium, phosphorus, potassium, vitamin A, vitamin B2, and vitamin B12. Additionally, they deliver high biological value proteins and essential fatty acids. There is accumulating evidence suggesting that yoghurt and fermented milk consumption is related to a number of health advantages, including the prevention of osteoporosis, diabetes, and cardiovascular diseases, as well as the promotion of gut health and immune system modulation. This review aims at presenting and critically reviewing the beneficial effects from the consumption of probiotic fermented milks in human health, whilst revealing potential applications in the food industry.

Expiration Date of Ready-to-Eat Salads: Effects on Microbial Load and Biochemical Attributes.

When minimally processed vegetables reach their expiration date, expose an increased microbial load. This includes mainly spoilage microorganisms but also foodborne pathogens, thus affecting the quality and safety of highly consumed ready-to-eat salads. A total of 144 ready-to-eat salads from the Cypriot market were analyzed in an attempt to determine the effects of the expiration date on the microbial load and plant metabolic variables of the salads. Possible correlations between them were also investigated for the first time. Furthermore, the impacts of the season (winter, summer), salad producing companies and type of salad and/or their interactions with the tested parameters were investigated. Results revealed that the microbial load (mainly spoilage microorganisms, such as Pseudomonas spp., yeasts and molds) increased towards the end of the shelf life. The microbial load was differentiated among the five salad producers and/or the salad types, highlighting the importance of a common and safe sanitation-processing chain in the preparation of ready-to-eat salads. Summer was the season in which Escherichia coli counts were found to be higher for plain lettuce, while Staphylococcus spp. was increased numbers for the lettuce+endive/radicchio, lettuce+rocket and lettuce+chives type of salads. Additionally, an increased Staphylococcus spp. was observed for plain rocket salads in winter. All samples examined were found negative for Salmonella enterica and Listeria monocytogenes. Moreover, carbon dioxide production and damage indexes (hydrogen peroxide and lipid peroxidation) increased on expiration date on both winter and summer seasons, indicating plant tissue stress at the end of shelf life. These findings indicate that the expiration date and relevant shelf life of processed vegetables are important parameters to be considered when postharvest management is applied to these products, ensuring safety and quality.

The Potential of Sun-Dried Grape Pomace as a Multi-Functional Ingredient for Herbal Infusion: Effects of Brewing Parameters on Composition and Bioactivity.

Wine and by-products are essential elements of a Mediterranean diet and considered as a reservoir of bioactive compounds with various health effects. Grape pomace, an easily available natural material of low cost, shares a similar wealth of health benefiting bioactive phytochemicals. The objective of this study was to explore the utilization of grape pomace from Commandaria dessert wine as main ingredient for functional infusions. Therefore, the ratio of water to grape pomace powder (40-200 mL g-1), infusion time (3-15 min) and temperature (55-95 °C) were optimized in terms of composition and bioactivity. Multiple response optimization indicated that brewing 200 mL water per g of material for 12.2 min at 95 °C, was the optimum method for preparing the infusion. Results also revealed a significant impact of three parameters as well as quadratic and interactive effects on composition and bioactivity of infusions. Furthermore, the infusion presents antimicrobial effects against Listeria monocytogenes serotypes and other common food pathogenic bacteria. Finally, a sensory evaluation was performed to assess the organoleptic attributes of the infusion and its improvement, with the addition of Mediterranean aromatic plants. Overall, the present work describes a promising strategy for the re-use of sun-dried grape pomace as a functional ingredient of infusions.

Rapid Detection of Mycoplasma bovis, Staphylococcus aureus and Streptococcus agalactiae in Cattle Bulk Tank Milk in Cyprus and Relations with Somatic Cell Counts.

One hundred and seventy-seven (177) bulk tank milk samples were analyzed with a commercially available real-time polymerase chain reaction kit and 11 (6.21%), 41 (23.16%), and 58 (32.77%) tested positive for Mycoplasma bovis, Staphylococcus aureus, and Streptococcus agalactiae, respectively. Statistical analysis revealed a significant relationship between the presence of S. aureus and S. agalactiae. Enumeration of somatic cells was performed in the same samples by flow cytometry. The somatic cell counts were found higher in S. aureus and S. agalactiae positive samples. No association was found between M. bovis presence and somatic cells counts. Low internal assay control Ct values were found to be related with high somatic cell counts. Noticeably, this is the first report for the presence of M. bovis in Cyprus. Therefore, its presence was confirmed by bulk tank milk culture, conventional PCR, and next generation sequencing. Furthermore, M. bovis was typed with multilocus sequencing typing and was allocated to sequence type 29 (ST 29). Real-time PCR in bulk tank milk samples is a useful tool to detect mammary infections, especially for neglected pathogens such as M. bovis.

Processing Wastewaters from Spanish-Style cv. Chalkidiki Green Olives: A Potential Source of Enterococcus Casseliflavus and Hydroxytyrosol.

The purpose of this study was to examine the isolation of indigenous lactic acid bacteria (LAB) with functional properties from Spanish-style cv. Chalkidiki green olive processing wastewaters (GOW). Predominant indigenous LAB could serve as bioaugmentation agents/starter culture for table olives production and protected designation of origin specification. Spontaneous fermentation of fresh GOW over different temperatures (15 °C to 50 °C) and pH values (3.5 to 11.5) for 30 d enabled the isolation/molecular identification of the lactic acid bacterium Enterococcus casseliflavus and the plant-associated bacterium Bacillus amyloliquefaciens subsp. plantarum. E. casseliflavus was found to reduce chemical oxygen demand by 72%. Its resistance to extreme pH values, salinity, and temperature was successfully modeled and the minimum inhibitory concentration of oleuropein against the bacterial growth was determined (0.9 g/L). Furthermore, hydroxytyrosol content was doubled (up to 553 mg/L) after GOW spontaneous fermentation under acidic conditions at 15 °C to 30 °C for 120 d, creating an additional source of input. These results highlight the significance and potential of E. casseliflavus in Spanish-style cv. Chalkidiki green olive processing.

Application of the urban exposome framework using drinking water and quality of life indicators: a proof-of-concept study in Limassol, Cyprus.

BACKGROUND: Cities face rapid changes leading to increasing inequalities and emerging public health issues that require cost-effective interventions. The urban exposome concept refers to the continuous monitoring of urban environmental and health indicators using the city and smaller intra-city areas as measurement units in an interdisciplinary approach that combines qualitative and quantitative methods from social sciences, to epidemiology and exposure assessment. METHODS: In this proof of concept study, drinking water and quality of life indicators were described as part of the development of the urban exposome of Limassol (Cyprus) and were combined with agnostic environment-wide association analysis. This study was conducted as a two-part project with a qualitative part assessing the perceptions of city stakeholders, and quantitative part using a cross-sectional study design (an urban population study). We mapped the water quality parameters and participants' opinions on city life (i.e., neighborhood life, health care, and green space access) using quarters (small administrative areas) as the reference unit of the city. In an exploratory, agnostic, environment-wide association study analysis, we used all variables (questionnaire responses and water quality metrics) to describe correlations between them. RESULTS: Overall, urban drinking-water quality using conventional indicators of chemical (disinfection byproducts-trihalomethanes (THM)) and microbial (coliforms, E. coli, and Enterococci) quality did not raise particular concerns. The general health and chronic health status of the urban participants were significantly (false discovery rate corrected p-value < 0.1) associated with different health conditions such as hypertension and asthma, as well as having financial issues in access to dental care. Additionally, correlations between THM exposures and participant behavioral characteristics (e.g., household cleaning, drinking water habits) were documented. CONCLUSION: This proof-of-concept study showed the potential of using integrative approaches to develop urban exposomic profiles and identifying within-city differences in environmental and health indicators. The characterization of the urban exposome of Limassol will be expanded via the inclusion of biomonitoring tools and untargeted metabolomics.

Straw Wine Melanoidins as Potential Multifunctional Agents: Insight into Antioxidant, Antibacterial, and Angiotensin-I-Converting Enzyme Inhibition Effects.

Numerous studies provide robust evidence for a protective effect of red wine against many diseases. This bioactivity has been mainly associated with phenolic fractions of wines. However, the health effects of melanoidins in red sweet wines has been ignored. The goal of the present work was to unravel the antioxidant, antimicrobial, and angiotensin-I-converting enzyme (ACE) inhibitory properties of straw sweet wine melanoidins. Results demonstrated that melanoidins have a potential antioxidant activity, determined by 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and Ferric reducing antioxidant power (FRAP) assays. The antimicrobial activity of melanoidins was also tested against Listeria monocytogenes, Salmonella Enteritidis, and Escherichia coli. Minimum inhibitory concentration (MIC) of isolated melanoidins against three bacterial strains ranged from 5 mg mL-1 to 10 mg mL-1. Finally, the ACE inhibitory effect of isolated melanoidins was evaluated, as it is linked with antihypertensive activity. Results showed that they have ACE-inhibitory activity ranging from 58.2 ± 5.4% to 75.3 ± 6.4% at a concentration level of 2 mg mL-1. Furthermore, the chemical properties of isolated melanoidins were determined. Results demonstrated that the skeleton of straw wine melanoidins is mainly composed of carbohydrates, and bear significant numbers of phenolic compounds that may play critical roles in their functional properties. Overall, this study describing the chemical composition and functional properties of melanoidin fractions isolated from a straw wine highlights that they can be exploited as functional agents for multiple purposes. Finally, melanoidins are an unexplored source of bioactive molecules in straw wines except from polyphenols that contribute to the health effects.

Adherence to the Mediterranean Diet and Lifestyle Characteristics of University Students in Cyprus: A Cross-Sectional Survey.

Objective. To assess dietary-related habits among young adults. Design and Setting. Dietary habits were assessed cross-sectionally, using a self-completed questionnaire in 193 students enrolled in public and private universities in Cyprus. Adherence to the Mediterranean diet was evaluated using the validated KIDMED index. BMI was estimated based on weight and height measurements. Results. The mean BMI was 23.31 (±3.98). The mean adherence score to the Mediterranean diet was 6.0 (IQR 4 to 8), with 26.9% of students being classified as high adherers and 21.8% as low adherers to the Mediterranean diet. About 32% of students consumed a second serving of fruit and vegetables more than once a day, whereas 26% reported going more than once a week to a fast-food restaurant and 31% consumed sweets and candy several times a day. On the other hand, 76% of participants reported consumption of at least two dairy products daily and 88% use olive oil at home. The majority consume coffee 2-3 times per day. Conclusions. Results support a shift from traditional healthy diets to more unhealthy eating patterns. However, we also report a high dairy intake and use of olive oil. Tailored-made strategies targeting the young adult population could be warranted.

Detection of viable Mycobacterium avium subspecies paratuberculosis in powdered infant formula by phage-PCR and confirmed by culture.

Surveys from different parts of the world have reported that viable Mycobacterium avium subsp. paratuberculosis (MAP) can be cultured from approximately 2% of samples of retail pasteurised milk samples. Pasteurised milk is used for the production of powdered infant formula (PIF) and therefore there is a concern that MAP may also be present in these products. Several studies have previously reported the detection of MAP in PIF using PCR-based assays. However, culture-based surveys of PIF have not detected viable MAP. Here we describe a phage amplification assay coupled with PCR (page-PCR) that can rapidly detect viable MAP in PIF. The results of a small survey showed that the phage-PCR assay detected viable MAP in 13% (4/32) of PIF samples. Culture detected viable MAP in 9% (3/32) PIF samples, all of which were also phage-PCR positive. Direct IS900 PCR detected MAP DNA in 22% (7/32) of PIF samples. The presence of viable MAP in PIF indicates that MAP either survived PIF manufacturing or that post-production contamination occurred. Irrespective of the route of MAP contamination, the presence of viable MAP in PIF is a potential public health concern.

Evidence of arsenic release promoted by disinfection by-products within drinking-water distribution systems.

Changes in disinfectant type could trigger a cascade of reactions releasing pipe-anchored metals/metalloids into finished water. However, the effect of pre-formed disinfection by-products on the release of sorbed contaminants (arsenic-As in particular) from drinking water distribution system pipe scales remains unexplored. A bench-scale study using a factorial experimental design was performed to evaluate the independent and interaction effects of trihalomethanes (TTHM) and haloacetic acids (HAA) on arsenic (As) release from either scales-only or scale-biofilm conglomerates (SBC) both anchored on asbestos/cement pipe coupons. A model biofilm (Pseudomonas aeruginosa) was allowed to grow on select pipe coupons prior experimentation. Either TTHM or HAA individual dosing did not promote As release from either scales only or SBC, detecting <6 µg AsL(-1) in finished water. In the case of scales-only coupons, the combination of the highest spike level of TTHM and HAA significantly (p<0.001) increased dissolved and total As concentrations to levels up to 16 and 95 µg L(-1), respectively. Similar treatments in the presence of biofilm (SBC) resulted in significant (p<0.001) increase in dissolved and total recoverable As up to 20 and 47 µg L(-1), respectively, exceeding the regulatory As limit. Whether or not, our laboratory-based results truly represent mechanisms operating in disinfected finished water in pipe networks remains to be investigated in the field.

Detection of Mycobacterium avium subsp. paratuberculosis in bulk tank milk by combined phage-PCR assay: evidence that plaque number is a good predictor of MAP.

Conventional culture and a rapid phage-PCR method were used to detect Mycobacterium avium subsp. paratuberculosis (MAP) in bulk tank milk (BTM) samples. Only two of 225 samples (0.9%) were found to contain MAP by culture whereas 50 (22%) MAP-positive samples were identified using the phage-PCR assay, including both samples that were MAP-culture positive. Results using the phage-based method for independently tested duplicate samples indicated that the assay is very reproducible (r(2)=0.897), especially when low levels of mycobacteria are present. A relationship was established between plaque number and the presence of MAP in a sample. A cut-off value was determined allowing identification of MAP-positive samples based on plaque number alone (90% sensitivity, 99% specificity; area under the curve=0.976). These results indicate that the assay is a robust method for screening BTM, providing results within 24 h.

Rapid detection methods for viable Mycobacterium avium subspecies paratuberculosis in milk and cheese.

Mycobacterium avium subsp. paratuberculosis (MAP) may have a role in the development of Crohn's disease in humans via the consumption of contaminated milk and milk products. Detection of MAP from milk and dairy products has been reported from countries on the European continent, Argentina, the UK and Australia. In this study three different methods (quantitative real time PCR, combined phage IS900 PCR and conventional cultivation) were used to detect the presence of MAP in bulk tank milk (BTM) and cheese originating from sheep, goat and mixed milks from farms and products in Cyprus. During the first survey the presence of MAP was detected in 63 (28.6%) of cows' BTM samples by quantitative real time PCR. A second survey of BTM used a new combined phage IS900 PCR assay, and in this case MAP was detected in 50 (22.2%) samples showing a good level of agreement by both methods. None of the herds tested were known to be affected by Johne's disease and the presence of viable MAP was confirmed by conventional culture in only two cases of cows BTM. This suggests that either rapid method used is more sensitive than the conventional culture when testing raw milk samples for MAP. The two isolates recovered from BTM were identified by IS1311 PCR REA as cattle and sheep strains, respectively. In contrast when cheese samples were tested, MAP DNA was detected by quantitative real time PCR in seven (25.0%) samples (n=28). However no viable MAP was detected when either the combined phage IS900 PCR or conventional culture methods were used.