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      New developments in the study of the microbiota of raw-milk, long-ripened cheeses by molecular methods: the case of Grana Padano and Parmigiano Reggiano

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          Abstract

          Microorganisms are an essential component of cheeses and play important roles during both cheese manufacture and ripening. Both starter and secondary flora modify the physical and chemical properties of cheese, contributing and reacting to changes that occur during the manufacture and ripening of cheese. As the composition of microbial population changes under the influence of continuous shifts in environmental conditions and microorganisms interactions during manufacturing and ripening, the characteristics of a given cheese depend also on microflora dynamics. The microbiota present in cheese is complex and its growth and activity represent the most important, but the least controllable steps. In the past, research in this area was dependent on classical microbiological techniques. However, culture-dependent methods are time-consuming and approaches that include a culturing step can lead to inaccuracies due to species present in low numbers or simply uncultivable. Therefore, they cannot be used as a unique tool to monitor community dynamics. For these reasons approaches to cheese microbiology had to change dramatically. To address this, in recent years the focus on the use of culture-independent methods based on the direct analysis of DNA (or RNA) has rapidly increased. Application of such techniques to the study of cheese microbiology represents a rapid, sound, reliable, and effective way for the detection and identification of the microorganisms present in dairy products, leading to major advances in understanding this complex microbial ecosystem and its impact on cheese ripening and quality. In this article, an overview on the recent advances in the use of molecular methods for thorough analysis of microbial communities in cheeses is given. Furthermore, applications of culture-independent approaches to study the microbiology of two important raw-milk, long-ripened cheeses such as Grana Padano and Parmigiano Reggiano, are presented.

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          Most cited references88

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          Use of PCR in the field of molecular diagnostics has increased to the point where it is now accepted as the standard method for detecting nucleic acids from a number of sample and microbial types. However, conventional PCR was already an essential tool in the research laboratory. Real-time PCR has catalysed wider acceptance of PCR because it is more rapid, sensitive and reproducible, while the risk of carryover contamination is minimised. There is an increasing number of chemistries which are used to detect PCR products as they accumulate within a closed reaction vessel during real-time PCR. These include the non-specific DNA-binding fluorophores and the specific, fluorophore-labelled oligonucleotide probes, some of which will be discussed in detail. It is not only the technology that has changed with the introduction of real-time PCR. Accompanying changes have occurred in the traditional terminology of PCR, and these changes will be highlighted as they occur. Factors that have restricted the development of multiplex real-time PCR, as well as the role of real-time PCR in the quantitation and genotyping of the microbial causes of infectious disease, will also be discussed. Because the amplification hardware and the fluorogenic detection chemistries have evolved rapidly, this review aims to update the scientist on the current state of the art. Additionally, the advantages, limitations and general background of real-time PCR technology will be reviewed in the context of the microbiology laboratory.
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            Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms.

            We developed mobility shift analysis of single-stranded DNAs on neutral polyacrylamide gel electrophoresis to detect DNA polymorphisms. This method follows digestion of genomic DNA with restriction endonucleases, denaturation in alkaline solution, and electrophoresis on a neutral polyacrylamide gel. After transfer to a nylon membrane, the mobility shift due to a nucleotide substitution of a single-stranded DNA fragment could be detected by hybridization with a nick-translated DNA fragment or more clearly with RNA copies synthesized on each strand of the DNA fragment as probes. As the mobility shift caused by nucleotide substitutions might be due to a conformational change of single-stranded DNAs, we designate the features of single-stranded DNAs as single-strand conformation polymorphisms (SSCPs). Like restriction fragment length polymorphisms (RFLPs), SSCPs were found to be allelic variants of true Mendelian traits, and therefore they should be useful genetic markers. Moreover, SSCP analysis has the advantage over RFLP analysis that it can detect DNA polymorphisms and point mutations at a variety of positions in DNA fragments. Since DNA polymorphisms have been estimated to occur every few hundred nucleotides in the human genome, SSCPs may provide many genetic markers.
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              Bacterial strain typing, or identifying bacteria at the strain level, is particularly important for diagnosis, treatment, and epidemiological surveillance of bacterial infections. This is especially the case for bacteria exhibiting high levels of antibiotic resistance or virulence, and those involved in nosocomial or pandemic infections. Strain typing also has applications in studying bacterial population dynamics. Over the last two decades, molecular methods have progressively replaced phenotypic assays to type bacterial strains. In this article, we review the current bacterial genotyping methods and classify them into three main categories: (1) DNA banding pattern-based methods, which classify bacteria according to the size of fragments generated by amplification and/or enzymatic digestion of genomic DNA, (2) DNA sequencing-based methods, which study the polymorphism of DNA sequences, and (3) DNA hybridization-based methods using nucleotidic probes. We described and compared the applications of genotyping methods to the study of bacterial strain diversity. We also discussed the selection of appropriate genotyping methods and the challenges of bacterial strain typing, described the current trends of genotyping methods, and investigated the progresses allowed by the availability of genomic sequences.
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                Author and article information

                Journal
                Front Microbiol
                Front Microbiol
                Front. Microbiol.
                Frontiers in Microbiology
                Frontiers Media S.A.
                1664-302X
                28 February 2013
                2013
                : 4
                : 36
                Affiliations
                Department of Food Science, University of Parma Parma, Italy
                Author notes

                Edited by: Danilo Ercolini, Università degli Studi di Napoli Federico II, Italy

                Reviewed by: Aspasia Nisiotou, Technological Educational Institute of Athens, Greece; Panagiotis Skandamis, Agricultural University of Athens, Greece

                *Correspondence: Erasmo Neviani, Department of Food Science, University of Parma, Parco Area delle Scienze 11/A, 43124 Parma, Italy. e-mail: erasmo.neviani@ 123456unipr.it

                This article was submitted to Frontiers in Food Microbiology, a specialty of Frontiers in Microbiology.

                Article
                10.3389/fmicb.2013.00036
                3584316
                23450500
                fcd37b3f-a8ea-4c4b-8d9e-2ce059cd5eea
                Copyright © Neviani, Bottari, Lazzi and Gatti.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc.

                History
                : 18 October 2012
                : 09 February 2013
                Page count
                Figures: 2, Tables: 0, Equations: 0, References: 119, Pages: 14, Words: 0
                Categories
                Microbiology
                Review Article

                Microbiology & Virology
                culture-dependent methods,culture-independent methods,long-ripened cheeses microbiota,non-starter lactic acid bacteria,raw-milk,starter lactic acid bacteria,whey starter

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