Dynamics of microbial populations in food: a molecular contribution to the studies of “shelf-life”

Degradative bacteria are undesirable non pathogenic microorganisms which alter the colour, smell and taste of food, drastically limiting their “shelf life”. The “shelf life is the time-limit in which the progressing of the degradative events determines either only imperceptible sensory modifications or still acceptable modifications for the use safety. The durability of a product is a measurable characteristic and, in order to simply define it, various criteria can be adopted such as the threshold criteria, the mathematic criteria, the acceptability criteria, and the sensory criteria. These strategies try to estimate, in a quite simple and quick way, the time-limit of an alimentary product, however the interest towards the real composition and dynamics of the microflora, that develops during the conservation of food, is yet too low. In this work, used a molecular approach to the studies of “shelf life”. Our primary aim was to follow, from a qualitative and quantitative point of view, the dynamic of the microbial population in an alimentary model system (preparation of cooked vegetables in thermal sealed containers) during the normal period of conservation in a 4°C refrigerator (from production/date to expiration/date). This research will enable to check which kind of bacterial groups are over represented and therefore be considered the main responsible of the product degradation. For this purpose the following strategy has been adopted: 1) Viable count of mesophilic, psycrotrophic bacteria, and totals coliform bacilli, performed daily from portions of the same lot of production. 2) Amplification of the 16S rDNA by means of PCR. 3) Restriction analysis of amplified 16S rDNA (ARDRA) with the enzymes AluI of a daily isolated mesophilic bacteria representative number and grouping of isolates on the basis of their specific ARDRA profiles. 4) 16S rDNA sequencing and analysis of one strain for each ARDRA group, for species attribution. 5) Analysis of intraspecific genetic variability by using the RAPD technic. 6) Formulation of a descriptive model of microbial interactions established during the conservation of the product and planning of technological intervents that modify the microbial dynamics in order to preserve the quality of the alimentary product. Results of viable count indicated that the main populations are the total mesophilic bacteria and the psycrotrophic bacteria. In particular the latest ones show a greater growth rate than total mesophilic bacteria. However both populations seem to reach the plateau around day 4-5 from the production that correspond to the expiry date defined on sensory bases. Moreover, both populations show a fair phenotypic variability on PCA. The ARDRA data revealed the present of an over represented aplotype, very likely constituted by bacterial belonging to the same species.