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.     

Application of Matrix Solid-Phase Dispersion and Liquid Chromatography–Ion Trap Mass Spectrometry for the Analysis of Pesticide Residues in Fruits

This study presents an application of rapid and sensitive multiresidue method for the analysis of acephate, acetamipride, atrazine, carbendazim, carbaryl, carbofuran, dimethoate, imidacloprid, linuron, malathion, monocrotophos, monuron, propazine, simazine, and tebufenozide in fruits. The method involves an extraction procedure based on matrix solid-phase dispersion using diatomaceous earth as a dispersant and dichloromethane as the eluent. The target pesticides were determined using liquid chromatography–ion trap mass spectrometry. Quantification of the analytes was carried out using the most sensitive ion transition. Ion trap parameters, like activation q and time, were found to have a prominent influence on method sensitivity for some pesticides and they were optimized accordingly. The confirmation of residues detected in real samples was performed by repeated injection and acquiring additional ion transitions besides the ones used for quantification. The method was validated for accuracy, linearity, reproducibility, and sensitivity. Mean values for recoveries were in the range of 70–120 % for all tested matrices. Repeatability of the method, expressed as the relative standard deviation, was in general lower than 20 %. The applicability of the method to routine analysis was tested in real fruit samples with good performance.     

Anthocyanin composition of the red wine Babić affected by maceration treatment

Identification of anthocyanins in the wine made of the Croatian autochthonous grape variety of Babić (Vitis vinifera L.) was carried out and their profile was determined by means of high-performance liquid chromatography with diode array and mass spectrometric detection. Dependence of anthocyanins content and profile on maceration treatment conditions was investigated. Statistically significant differences of anthocyanins concentration in wines Babić produced by various maceration treatments were confirmed by the use of multivariate analysis of variances. The investigation results indicated that the maceration temperature exerts higher influence on anthocyanins concentration than the duration of maceration. In addition, on the basis of anthocyanin composition and using different multivariate statistical analyses, differentiation of wines Babić according to maceration treatments was procured.     

Ability of Lactobacillus gasseri K 7 to inhibit Escherichia coli adhesion in vitro on Caco-2 cells and ex vivo on pigs' jejunal tissue

The ability of Lactobacillus (Lb.) gasseri K 7 to inhibit adhesion of Escherichia coli O8:K88 to intestinal mucosa was studied on cultured Caco-2 cells and ex vivo on pigs' small intestinal tissue. Lactobacilli were added simultaneously with E. coli, before E. coli and after E. coli for competition, exclusion and displacement assays. The concentration of lactobacilli on fully differentiated Caco-2 cells was 4.5+/-0.3 x 10(8) cfu/well, while the concentration of E. coli varied from 1.5 x 10(6) to 4.3 x 10(8) cfu/well. The number of E. coli adhered to Caco-2 monolayer (cfu/well) was lineary correlated (R(2)=0.97) to the concentration of added cells. In the assay simulating exclusion, E. coli adhesion was reduced by Lb. gasseri K 7 strain by 0.1 to 0.6 log cfu/well. The binding of E. coli was inhibited even more when incubated simultaneously with lactobacilli, particularly at the lowest concentration of E. coli (ratio E. coli/lactobacilli 1:248), where five-times reduction (or 0.7 log) was observed. When adhesion to tissue derived from pigs' jejunum was tested, concentration of E. coli was constant (6.9+/-0.14 x 10(7) cfu/ml), while the concentration of Lb. gasseri K 7 was 5.9 x 10(7) and 1.3 x 10(7) cfu/ml in two independent experiments, respectively. The adhesion of E. coli and Lb. gasseri K 7 cells to jejunal mucosa was similar (1.0+/-0.17 x 10(6) and 1.54+/-0.10 x 10(6) cfu/cm(2)) when the concentrations of single strains in suspensions were approximately the same. No significant competition, exclusion or displacement of E. coli by lactobacilli was observed on jejunal tissue. In conclusion, Lb. gasseri K 7 was found to be effective in reducing E. coli adhesion to Caco-2 enterocytes, but it was not able to do so in ex vivo conditions tested for pig jejunal tissue.     

Cow colostrum and early milk enriched with eicosapentaenoic and docosahexaenoic fatty acid

The objective of the study was to explore whether it is possible to alter cow colostrum and early milk fatty acid composition with a low level of fat supplement, high in docosahexaenoic (DHA) and eicosapentaenoic (EPA) fatty acid. Diets included a control diet and a diet supplemented with DHA- and EPA-enriched fat supplement. Addition of fat supplement significantly decreased saturated fatty acids, C14:0 and C16:0 and increased the values of monounsaturated fatty acids, polyunsaturated fatty acids (PUFA), n3 fatty acids, EPA, DHA, C18:1n9cis and C18:1n11trans. The percentage of short-chain fatty acids significantly increased with the progress of lactation, while the percentage of PUFA, n3 and n6 significantly decreased. These results showed that fat supplement, high in DHA and EPA, modified the fatty acid profile of colostrum and milk fat and increased the proportion of beneficial fatty acids for human health.     

Rheological and Breadmaking Properties of Wheat Flours Supplemented with Octenyl Succinic Anhydride-Modified Waxy Maize Starches

The feasibility of emulsifying starches as bread improvers was investigated by incorporating starch sodium octenyl succinate (OSA-st), pre-gelatinized OSA-st and hydrolysed spray-dried OSA-st at 2.5, 5 and 10 % into wheat flour. Dough rheological properties (creep and recovery measurements; Mixolab, Alveograph) and bread quality parameters (specific loaf volume, crust and crumb colour, crumb moisture, crumb grain features, texture) were evaluated. The substituted flours, except hydrolysed OSA-st, significantly increased water absorption measured by Mixolab. The study on the rheological behaviour of doughs containing emulsifying starches, performed using a rheometer and an Alveograph, showed that OSA-st incorporation yielded strengthened dough, whereas pre-gelatinized and hydrolysed OSA-st addition led to more extensible dough. With regard to the thermal behaviour, investigated in water-limited systems by Mixolab, doughs prepared from pre-gelatinized OSA-st and hydrolysed OSA-st exhibited lower maximum peak torque, whilst all three examined starches increased cooking stability and decreased the setback value. Specific volumes of loaves baked from the substituted flours increased, and the highest effect was observed with pre-gelatinized OSA-st, which consequently produced bread crumbs with the largest mean gas cell area. The bread crumbs baked with octenyl succinate starches were whiter and softer. Although upon 1 day of storage no significant moisture retention capacity of emulsifying starches was noticed, the firmness values of OSA-st and pre-gelatinized OSA-st-supplemented bread crumbs, after 24 h of storage, were similar to or significantly lower than those of the control determined 2 h after baking. The obtained results indicate a requirement for further optimization of the octenyl succinate starch-supplemented doughs in terms of the combination of different types and levels of modified starches in order to obtain maximum bread quality.     

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.     

Suitability of some selected maize hybrids from Serbia for the production of bioethanol and dried distillers' grains with solubles

BACKGROUND: Bioethanol is mostly produced from starchy parts of the corn grain kernel leaving significant amounts of valuable by‐products such as dried distillers' grains with solubles (DDGS) which can be used as a substitute for traditional feedstuff. The suitability of six maize hybrids from Serbia was investigated for bioethanol and DDGS production. The correlation between physical and chemical characteristics of the grain, bioethanol yield and quality of the corresponding DDGS was assessed. RESULTS: All hybrids had very different chemical composition and physical characteristics which could allow various applications. The highest bioethanol yield (94.5% of theoretical) and volumetric productivity (2.01 g l^?1 h^?1) were obtained with hybrid ZP 434 and the lowest with ZP 611k. Regarding chemical composition, all DDGS samples manifested good properties as feed components. Their protein content was higher compared to the kernel. In addition, the samples showed high digestibility and high mineral content, especially of calcium and phosphorus. CONCLUSION: A hybrid ZP 434 was selected as the most promising bioethanol producer. This property is attributed to the highest level of soft endosperm which is more susceptible to starch‐hydrolysing enzymes. A high yield potential per hectare makes it the best candidate for commercial bioethanol production. © 2012 Society of Chemical Industry