Microbial biofilms in food environments: study approaches and intervention strategies

The main objective of this study was to examine in-depth the problems related to the for-mation of microbial biofilms in food plants. Although it has long been known that the ability to form biofilms is a common trend in natural environments including food processing environments, most of the studies in the literature concern the medical context, where microbial biofilms are often the cause of serious hospital infections. In the food field, however, the interest in biofilms has only recently arisen, even though the formation of microbial biofilms appears to be the major cause of cross-contamination in food products. Currently, data on microbial communities in the different food processing areas are only limited, and the influence of environmental parameters on the char-acteristics of biofilms have been studied for only a few microbial species. This knowledge is, how-ever, necessary for the development of intervention strategies for the prevention and removal of biofilms, which allows at the same time to obtain a high degree of hygienic-sanitary safety of the surfaces, as well as to reduce the impact conventional strategies have on the environment and on the safety of operators. The study discussed in this thesis was carried out on specific groups of organisms known to be pathogenic or food spoilers, including Listeria monocytogenes, Staphylococcus aureus and Pseudomonas spp. The need for the availability of appropriate study models that allow to obtain, in as little time as possible, a high number of biofilm samples with homogeneous characteristics was addressed in Chapter 2, where the use of a microtiter plate system and a reactor able to grow mi-crobial biofilms was evaluated on materials widely used in the food industry. In addition, to facili-tate the microbiological laboratory activities, two techniques of plate counts were compared in or-der to highlight problems and benefits in the context of the study of biofilms for both. The micro-titer plate assay and the CDC biofilm reactor assay showed to be sufficiently reliable and repeatable tools to produce a number of samples large enough to provide sufficient information on the ability of food microorganisms to produce biofilms under different operating conditions. Moreover, the drop plate method has proved particularly suitable, sufficiently accurate and reliable, as well as ad-vantageous from the economic point of view, for the quantification of viable cells present in the biofilm. The influence of environmental parameters on biofilm formation was studied in Chapter 3 and Chapter 4. In particular, in Chapter 3 the effect of the synergy of multiple parameters on the formation of biofilms in a static model system was studied, both in terms of quantification of total the biomass (dead and live cells and EPS matrix) and of the only evaluation of the cell count. The use of a Central Composite Design allowed to mimic the real environmental conditions in the food industry and to obtain the greatest amount of information limiting the number of experiments to be carried out. Therefore, useful data were obtained to increase the information in the literature about the synergistic effects of the environmental parameters on biofilm formation regarding the food sector. In Chapter 4 the effect of temperature on the adhesion and on the biofilm structure, as well as on resistance to disinfectants commonly used in the food industry sanitation plans was studied. The study showed that temperature significantly affects the kinetics of adhesion, but also the cell density and the amount of EPS produced, and consequently the resistance to biocides. The use of CLSM technique for microscopic observation allowed the study of biofilms in undisturbed condi-tions, and thus it is well suited to a possible use during the biofilm growth. The evaluation of dif-ferent strategies for the removal of biofilms was the subject of Chapter 5 and Chapter 6, in which conventional and non-conventional approaches were considered. A comparison between chemical, physical and biological treatments shows that a hurdle-approach, in which different strategies are used in sequence, could help in limiting the health and hygiene problems related to microbial bio-films in the production of foodstuffs. Finally, in Chapter 7 the gained knowledge was used to study the problem of biofilms in specific food contexts.