Food microbiota diversity

Meat fermentation and fermented sausage manufacturing are ancient processes in Europe, and typical preparations based on fermentations driven by indigenous microflora are still produced by local meat factories or artisanal producers without the use of starter cultures. LAB (Lactic Acid Bacteria) and CNC (Coagulase Negative Cocci) are the two main groups of bacteria with technological value determining the several biochemical and physical reactions taking place during the fermentation and ripening of sausages. Interest in preserving the biodiversity of microorganisms involved in the fermentation of food products has been reported. As a consequence, and also taking into consideration the technological and economic importance of these microorganisms, the availability of methodologies able to unequivocally characterise single species, potentially right up to strain level, is called for. Identification of species, particularly within the genus Lactobacillus, using phenotypic methods such as sugar fermentation or other biochemical traits, may produce ambiguous results and be complicated because of the presence of several LAB species with similar characteristics. Molecular methods are nowadays increasingly employed to clarify the taxonomy of microbiota from sausages. The largest number of works are based on RAPD (Random Amplified Polymorphic DNA), 16S rDNA sequencing, and PCR-DGGE (Denaturing Gradient Gel Electrophoresis) analysis. The above DNA-based methodologies are easy, fast, and provide an accurate identification at species level, but are not as informative when it comes to characterisation of the bacterial population at strain level. Amplified Fragment Length Polymorphism (AFLPs) was used to analyse the naturally occurring flora of Lactic Acid Bacteria (LAB) and Coagulase Negative Cocci (CNC) in traditional North Italian naturally fermented sausages. More than five hundred presumed LAB and CNC were collected and cultured from nineteen different artisanal, naturally fermented sausages and, among the isolated colonies, a total of three hundred forty seven strains were analysed and separated into several clusters based on AFLP banding patterns. In order to precisely determine the species inside these clusters, partial sequences of fragments of the 16S ribosomal DNA gene of the isolated and of the standard strains were determined, and sequence homology searches were conducted through GenBank on few strains per cluster. The clusters were clearly identified with species specific strains. The most represented species of LAB was Lactobacillus sakei (131 isolates, 61 per cent of the total) and the most represented species of CNC was Staphylococcus equorum (66 isolates, 50 per cent of the total). This report gives a wide evaluation of the strain biodiversity present in naturally fermented sausages of North Italy. Moreover it demonstrates that AFLP is a robust and useful technique for characterising the strain levels of LAB and CNC microflora.