Validation of a 1-Day Analytical Diagnostic Real-Time PCR for the Detection of Salmonella in Different Food Meat Categories

Foodborne disease caused by Salmonella represents a worldwide public health problem. In Europe, salmonellosis is still the second most commonly recorded zoonosis. Since the standard culture method for detecting Salmonella (ISO 6579:2002) requires up to 5 days to produce results, the need to develop rapid methods represents an important issue for the authorities and the producers. The aim of the present study was the in-house validation, according to ISO 16140, of an open-formula diagnostic real-time PCR for the detection of Salmonella in all the different meat categories reported in the EU Regulations relative to microbiological criteria for food safety. The assay employed specific primers and a probe target within the ttrRSBCA locus, which allows the tetrathionate respiration in Salmonella. Selectivity, relative accuracy, relative sensitivity and relative specificity were established by testing 110 bacterial strains and 175 various edible meat samples. Results showed 100 % selectivity, 100 % relative accuracy, 100 % relative sensitivity and 100 % relative specificity of the real-time PCR when compared to the standard culture method used as reference. In addition, in order to minimize the effect of the competitive micro-flora naturally present on meat samples, a highly nutritious and selective commercial medium (ONE Broth Salmonella, Oxoid) was evaluated in comparison with the classical non-selective pre-enrichment broth (buffered peptone water). Results demonstrated that the ONE Broth Salmonella medium increases the growth of Salmonella in the presence of competitive micro-flora.     

Evaluation of Pseudomonas spp. through O2 and CO2 headspace analysis

This article proposes an approach to determine the level of Pseudomonas spp. in milk, based on the evaluation of the content of oxygen and carbon dioxide produced in the headspace of sealed vials; the research was divided into two phases: model building and preliminary validation. Three different strains of Pseudomonas spp., Ps. putida (wild strain) and Ps. fluorescens (wild and collection isolates), were used as targets. Data of CO₂ and O₂ were modelled through a modified positive (CO₂) or a negative Gompertz equation (O₂) to estimate the Minimum Detection Time (MDT), defined as the time to attain 3% of CO₂ (MDT₁) or a decrease in the content of O₂ by 3% (MDT₂). Then, MDT₁ and MDT₂ were submitted to a linear regression procedure, using cell concentration as independent variable; the correlations ‘MDT₁/cell concentration’ and ‘MDT₂/cell concentration’ showed high determination coefficients (>0.983). Moreover, the regression procedure pointed out that both MDT₁ and MDT₂ decreased by ca. 3 h for an increase in cell count of 1 log cfu mL^?1. Preliminary validation in milk pointed out that the error associated with the regression line ‘MDT₂/cell concentration’ was below 5%.     

Zinc supplementation of Friesian cows: Effect on chemical-nutritional composition and aromatic profile of dairy products

Zinc represents an essential microelement for several biochemical mechanisms. The body's inability to store zinc necessarily requires a constant dietary supply to avoid alteration of physiological functions. The aim of the present study was to investigate the effect of dietary enrichment with zinc on chemical-nutritional and aromatic properties of milk and cheese. Thirty commercial dairy cows, balanced for parity, milk production, and days in milk, were randomly assigned to 2 groups. The control group was fed with a conventional complete diet (22 kg of dry matter/animal per day), whereas the experimental group received a daily zinc supplementation of 60 mg per kg of dry complete feed. During the experimental period, the milk yield was monitored and samples of milk and caciotta cheese were collected to obtain information about the chemical-nutritional composition and aromatic profile. Dietary zinc integration did not influence milk yield and composition, but induced a marked reduction of somatic cell count and improved the oxidative stability of ripened caciotta cheese. In both milk and cheese, the experimental group samples were characterized by a lower concentration of saturated fatty acids and an increase in oleic acid, vaccenic acid, and rumenic acid. The aromatic profile of dairy products was also positively affected by dietary zinc intake, with an increase in concentration of carboxylic acids, aldehydes, and esters. The present results suggest a positive role of zinc in improving animal health and nutraceutical properties of milk and corresponding cheese. Taking into account the analysis of volatile compounds, zinc dietary supplementation of dairy cows should contribute to the production of cheeses with interesting organoleptic properties, although more studies are necessary to confirm the consumer acceptability of these changes.     

Effect of goat milk composition on cheesemaking traits and daily cheese production

Cheese yield is strongly influenced by the composition of milk, especially fat and protein contents, and by the efficiency of the recovery of each milk component in the curd. The real effect of milk composition on cheesemaking ability of goat milk is still unknown. The aims of this study were to quantify the effects of milk composition; namely, fat, protein, and casein contents, on milk nutrient recovery in the curd, cheese yield, and average daily yield. Individual milk samples were collected from 560 goats of 6 different breeds. Each sample was analyzed in duplicate using the 9-laboratory milk cheesemaking assessment, a laboratory method that mimicked cheesemaking procedures, with milk heating, rennet addition, coagulation, curd cutting, and draining. Data were submitted to statistical analysis; results showed that the increase of milk fat content was associated with a large improvement of cheese yield because of the higher recovery of all milk nutrients in the curd, and thus a higher individual daily cheese yield. The increase of milk protein content affected the recovery of fat, total solids, and energy in the curd. Casein number, calculated as casein-to-protein ratio, did not affect protein recovery but strongly influenced the recovery of fat, showing a curvilinear pattern and the most favorable data for the intermediate values of casein number. In conclusion, increased fat and protein contents in the milk had an effect on cheese yield not only for the greater quantity of nutrients available but also for the improved efficiency of the recovery in the curd of all nutrients. These results are useful to improve knowledge on cheesemaking processes in the caprine dairy industry.     

Radical Scavenging,Total Antioxidant Capacity,and Antiproliferative Activity of Phenolic Extracts from Extra Virgin Olive Oil by Cultivar ‘Frantoio’

The beneficial health effects of extra virgin olive oil are due to both its high content of monounsaturated fatty acids and its high content of phenolic compounds, which have recently attracted research interest. In this context, the aim of this work was to examine the in vitro antioxidant and antiproliferative activities of the phenolic extract obtained from extra virgin olive oil from O. europea cultivar ‘Frantoio’ (samples 1–4), one of the main varieties cultivated in Italy. The total phenolic content was determined by Folin–Ciocalteu method and instead the phenolic profile was obtained by HPLC coupled to a diode array detector and mass spectrometry. Extra virgin olive oil extracts exhibited high antioxidant activity through different mechanisms of action and these activities are related to the phenolic content. Sample 3 demonstrated the strongest free radical scavenging activity with IC₅₀ value of 56.5 μg/mL. The reducing ability measured with FRAP assay revealed that samples ranged from 91.3 to 156 μM Fe(II)/g. The same interesting trend was observed with Trolox equivalent antioxidant capacity value. Moreover, the virgin olive oils showed a good oxidative stability ranging between 19 to 32 h. Antiproliferative activity evaluated by SRB assay revealed that phenolic extracts from the cultivar ‘Frantoio’ showed a strong antiproliferative activity against CORL-23 cell line with an IC₅₀ value of 14.5 and 55.9 μg/mL for samples 3 and 1, respectively, and these results are comparable to the positive control vinblastine. Overall, these results showed that extra virgin olive oils from the cultivar ‘Frantoio’, may represent an interesting source of phenolic compounds as functional components that could be consumed in diets and/or used for the elaboration of functional food and pharmaceutical industries.     

Microdroplet-based multiplex PCR on chip to detect foodborne bacteria producing biogenic amines

The development of fast, reliable and culture-independent molecular tools to detect bacteria producing biogenic amines deserves the attention of research and ultimately of the food industry in order to protect consumers' health. Here we present the application of a simple, low-cost, fast and sensitive method to perform microdroplet-based multiplex PCR, directly on a food matrix, for the simultaneous detection of bacterial genes involved in biogenic amine biosynthesis. After inoculating wine with Lactobacillus brevis IOEB 9809, cell lysis and DNA amplification are performed in one single step, without preliminary nucleic acid extraction or purification treatments. The assay is performed in about 30 min, requiring 150 nL of starting sample and it enables the detection of down to 15 bacterial cells. With respect to traditional culture techniques, the speed, the simplicity and the cheapness of this procedure allow an effective monitoring of microbial cells during food-making and processing.     

Chemical composition and functional characterisation of commercial pumpkin seed oil

BACKGROUND: Pumpkin (Cucurbita pepo L.) seed oil is a common product in Slovenia, Hungary and Austria and is considered a preventive agent for various pathologies, particularly prostate diseases. These properties are related to its high content of carotenoids and liposoluble vitamins. In this study the carotenoid (lutein and zeaxanthin), vitamin E (α‐ and γ‐tocopherol) and fatty acid contents of 12 samples of commercial pumpkin seed oil were investigated together with the composition of the volatile fraction resulting from the roasting process. RESULTS: The aromatic profile obtained from the commercial samples was directly related to the intensity of the roasting process of the crushed pumpkin seeds. The roasting temperature played a crucial role in the concentrations of volatile substances originating from Strecker degradation, lipid peroxidation and Maillard reaction. CONCLUSION: The findings suggest that high‐temperature roasting leads to the production of an oil with intense aromatic characteristics, while mild conditions, generally employed to obtain an oil with professed therapeutic characteristics, lead to a product with minor characteristic pumpkin seed oil aroma. The nutraceutical properties of the product are confirmed by the high content of α‐ and γ‐tocopherol and carotenoids. © 2012 Society of Chemical Industry     

Crude palm oil from interspecific hybrid Elaeis oleifera × Elaeis guineensis: Fatty acid regiodistribution and molecular species of glycerides

The composition and structure of triacylglycerols (TAGs) and partial glycerides of crude palm oil obtained from interspecific hybrid Elaeis oleifera × Elaeis guineensis, grown in Colombia, were fully characterised and compared to data obtained by analysing crude African palm oil. Hybridisation appears to substantially modify the biosynthesis of fatty acids (FAs) rather than their assembly in TAGs. In fact, total FAs analysis showed significant differences between these two types of oil, with hybrid palm oil having a higher percentage of oleic acid (54.6 ± 1.0 vs 41.4 ± 0.3), together with a lower saturated fatty acid content (33.5 ± 0.5 vs 47.3 ± 0.1), while the percentage of essential fatty acid, linoleic acid, does not undergo significant changes. Furthermore, 34 TAG types were identified, with no qualitative differences between African and E. guineensis × E. oleifera hybrid palm oil samples. Short and medium chain FAs (8:0, 10:0, 12:0, 14:0) were utilised, together, to build a restricted number of TAG molecular species. Oil samples from the E. guineensis × E. oleifera hybrid showed higher contents of monosaturated TAGs (47.5–51.0% vs 36.7–37.1%) and triunsaturated TAGs (15.5–15.6% vs 5.2–5.4%). The sn-2 position of TAGs in hybrid palm oil was shown to be predominantly esterified with oleic acid (64.7–66.0 mol% vs 55.1–58.2 mol% in African palm oil) with only 10–15% of total palmitic acid and 6–20% of stearic acid acylated in the secondary position. The total amount of diacylglycerols (DAGs) was in agreement with the values of free acidity; DAG types found were in agreement with the representativeness of different TAG species.     

Validation of a fast real-time PCR method to detect fraud and mislabeling in milk and dairy products

Fast real-time PCR TaqMan assays were developed and validated for species identification in dairy products. Based on the amplification of 12S rRNA and cytB partial genes of mitochondrial DNA, the methods were demonstrated to be sensitive, fast, and species-specific for Bos taurus, Ovis aries, Bubalus bubalis, and Capra hircus. The limit of detection calculated was lower than 1%, and the efficiency was reported to be higher than 96% in every assay. An internal amplification control was used to detect possible false negatives. The method was validated by means of laboratory-prepared samples mixing different species. Moreover, 18 commercial dairy samples were analyzed by both real-time PCR and isoelectric focusing, the official European Union reference method. The 4 TaqMan assays were confirmed to be a useful tool for milk and dairy product authentication.     

Maternal dietary habits and mycotoxin occurrence in human mature milk

During 2006, 82 samples of human mature milk were collected at Italian hospitals and checked for aflatoxin M1 (AFM1) and ochratoxin A (OTA) by immunoaffinity column extraction and HPLC. AFM1 was detected in four (5%) of milk samples (ranging from < 7 ng/L to 140 ng/L; mean level: 55.35 ng/L); OTA was detected in 61 (74%) of milk samples (ranging from < 5 ng/L to 405 ng/L; mean level: 30.43 ng/L. OTA levels were significantly higher (p less, not double equals 0.05) in milk of habitual consumers of bread, bakery products and cured pork meat. No other statistically significant differences were observed although habitual consumers of pasta (p = 0.059), cookies (p = 0.061) and juices (p = 0.063) had mean contamination values of OTA higher than the moderate consumer. The very few AFB1 positive samples did not allow statistical comparisons. The present study confirms that the occurrence of OTA in human milk is related to maternal dietary habits. The findings support the possibility of dietary recommendations to woman, during pregnancy and lactation, aimed to tentatively reduce the OTA contamination of human milk.