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Rivolta d'Adda, Italy

Feligini M.,Laboratorio Qualita` dei Prodotti | Panelli S.,Laboratorio Qualita` dei Prodotti | Sacchi R.,Universita` Degli Studi Of Pavia | Ghitti M.,Universita` Degli Studi Of Pavia | Capelli E.,Universita` Degli Studi Of Pavia
Food Control

The aim of the study was to distinguish the raw milk from different farms in relation to their geographical sites within a narrowed territorial district. The goal was achieved by applying a molecular-based system for traceability that uses microbial DNA barcodes present in milk. Microbiota of milk were fingerprinted by PCR of the 16S-23S intergenic transcribed spacer using the Automated Ribosomal Intergenic Spacer Analysis (ARISA). A total of 64 markers within the range 279-756 bp were detected on the thirty-eight bulk milk samples, none of which was common to all the patterns. Overall samples did not show relevant differences across the two years of sampling. In fact, every farm maintained a specific core profile over time, thus demonstrating that the interaction between site and year of sampling is not significant and that the variability between years does not affect the distinction between grouping of farms. The system was able to trace the geographical origin of raw milk with a resolution of less than 5 km. According to the European regulations for the protection of the geographical names of foodstuffs which have a tangible link to the territory, the ARISA system described here may represent a suitable analytical tool for tracing the origin of milk integrating and reinforcing traceability processes of the dairy chain. © 2014 2014 Published by Elsevier Ltd. Source

Feligini M.,Laboratorio Qualita` dei Prodotti | Brambati E.,Laboratorio Qualita` dei Prodotti | Panelli S.,Laboratorio Qualita` dei Prodotti | Ghitti M.,University of Pavia | And 3 more authors.
Food Control

Late blowing is a defect of semi-hard and hard cheeses induced by butyric clostridia that, through the formation of endospores, contaminate raw milk via silage, fresh forage and animal bedding. Generally it becomes evident during ripening, when the physical and chemical conditions of the cheese core support spores outgrowth. This causes the production of undesirable off-odors and an anomalous organization of the loaf, that seriously prejudices cheeses, with significant economic losses for the dairy industry.This study describes the development of an original application of the automated ribosomal intergenic spacer analysis (ARISA) that, relying on a multi-species-specific primer set, allows to simultaneously displaying distinctive fingerprints, diagnostic for Clostridium tyrobutyricum, generally considered the main responsible of late blowing, and for the other species so far associated with the defect: Clostridium butyricum, Clostridium sporogenes, Clostridium beijerinckii, Clostridium tertium and Clostridium perfringens, also providing relative abundance estimations. This approach has been applied to the seasonal analysis of tank raw milk and curd used for Grana Padano PDO cheese production in dairies located in different areas of the Northern Padan plain. C.beijerinckii and C. tertium, occurring both in 100% milks and curds, together with C.perfringens, retrieved in 98.7% milks and 100% curds, resulted the most abundant species in all the seasons. On the other hand, C.sporogenes always resulted the less abundant species, occurring in 77.2% of milks and 86.1% of curds. Highly significant seasonal effects were detected for C.tyrobutyricum and C.beijerinckii that displayed the most marked and opposite trends. The occurrence of a spatial pattern of variation for curd was also observed, thus envisaging relevant effects presumably linked to differential breeder practices (feeding and hygiene). This ARISA approach presents important perspectives in terms of applicability along the entire dairy production chain for diagnostic purposes and for providing an estimate of the microbiological quality of milk. © 2014 Elsevier Ltd. Source

Feligini M.,Laboratorio Qualita` dei Prodotti | Panelli S.,Laboratorio Qualita` dei Prodotti | Buffoni J.N.,Laboratorio Qualita` dei Prodotti | Bonacina C.,Laboratorio Qualita` dei Prodotti | And 2 more authors.
Journal of Food Science

Microbial DNA from 9 batches of Taleggio PDO cheese sampled at various times during ripening, brines, swabs of wooden shelves used for cheese dry-salting, and 13 commercial cheeses were analyzed by denaturing gradient gel electrophoresis (PCR-DGGE) and/or random amplification of polymorphic DNA (RAPD-PCR). Sequencing allowed the detection of 12 genera, 27 species, and 2 unclassified bacteria. Molecular analysis allowed for the detection of microorganisms not previously associated with Taleggio such as Lactobacillus paracasei, Carnobacterium maltaromaticum, Bacillus licheniformis, Corynebacterium variabile, Psychrobacter cibarius, and Staphylococcus carnosus. For the first time Massilia spp. was detected in a dairy ecosystem. © 2012 Institute of Food Technologists®. Source

Panelli S.,Laboratorio Qualita` dei Prodotti | Brambati E.,Laboratorio Qualita` dei Prodotti | Bonacina C.,Laboratorio Qualita` dei Prodotti | Feligini M.,Laboratorio Qualita` dei Prodotti
Journal of Dairy Research

This work applies culture-independent methods for the characterization of fungal populations (yeasts and moulds) naturally occurring in Sardinian ewe's milk sampled in the Italian areas with the largest dairy production (Sardinia and Lazio regions). Sequences of the D1/D2 variable domains at the 5′ end of the 26S rRNA gene were obtained by amplification of DNA directly isolated from milk, and this allowed identification of a total of 6 genera and 15 species of fungi. Among the 6 identified genera Geotrichum spp., Candida spp., Phaeosphaeriopsis spp., Pestalotiopsis spp. and Cladosporium spp. belong to the phylum of Ascomycota, while Cryptococcus spp. is part of the phylum of Basidiomycota. In particular, two genera (Pestalotiopsis and Phaeosphaeriopsis) and two species (Plectosphaerella cucumerina and Pryceomyces carsonii) have never been reported in dairy ecosystems before. Results provide evidence that several moulds and yeasts, previously described only in ovine cheeses, are transferred directly from raw milk. The knowledge of fungal consortia inhabiting sheep raw milk is a particularly relevant issue because several species are directly involved in cheese making and ripening, determining the typical aroma. On the other hand, spoilage yeasts and moulds are involved in anomalous fermentation of cheese and may be responsible for considerable economic losses and serious risks for consumers' health. Copyright © Proprietors of Journal of Dairy Research 2014. Source

Panelli S.,Laboratorio Qualita` dei Prodotti | Brambati E.,Laboratorio Qualita` dei Prodotti | Bonacina C.,Laboratorio Qualita` dei Prodotti | Feligini M.,Laboratorio Qualita` dei Prodotti
Journal of Food Science

Clostridium tyrobutyricum has been identified as the main causal agent of the late blowing defect in cheese, with major effects on quality and commercial value. In this work, for the first time, we applied automated ribosomal intergenic spacer analysis (ARISA) approach to diagnose the presence of C. tyrobutyricum in raw milk before cheesemaking. A species-specific primer set was designed and used for this original application of the ARISA. Sensitivity of detection, reproducibility of the fluorescent PCR assay, and repeatability of the capillary electrophoretic analysis of amplicons were evaluated using DNA extracted from milk added with known amounts of C. tyrobutyricum genome copies, ranging from 3 × 106 to 3. Results indicated that the sensitivity of the technique permits to detect the bacterium in all the samples. The reproducibility, evaluated by analyzing 3 sets of serial dilutions, resulted satisfactory, with little deviation within PCR reactions amplifying the same starting amount of template (standard deviations ≤ 0.1, coefficients of variation ≤ 3%). The peaks' fluorescence displayed an evident correspondence with the number of genome copies contained in each dilution. The capillary electrophoretic analysis, tested by running a single PCR product per dilution point in 10 repeats, resulted efficient and highly repeatable, with excellent coefficients of variation ≤ 2% and standard deviations ≤ 0.1 in all the sample sets. This application of ARISA gives good estimates of the total C. tyrobutyricum DNA content allowing a specific, fine-scale resolution of this pollutant species in a complex system as milk. A further advantage linked to the automatization of the process. © 2013 Institute of Food Technologists®. Source

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