Control of listeria monocytogenes biofilms in the food industryfighting a recurring problem by studying its formation and elimination

Abstract

The present work is included in the LISSA National Project (RTI2018-098267-R-C32), a project that focuses its objectives on two innovative strategies in the field of alternative antibiofilm applications. Different studies were carried out on this thesis, focused on understanding the influence of treatments for L. monocytogenes biofilms control. In the first place, starting from a standard in vitro model for L. monocytogenes biofilms, it was proceeded to evaluate the efficacy of an enzymatic product for the detachment of biofilms. The results showed that detachment effectiveness of mature biofilms was comprised between 85 and 99 % for the different L. monocytogenes strains. The results showed that the detachment of these structures may be directly related to the strain under study, and may be due to the fact that some strains are capable of generating biofilms with a more robust matrix. In a second study, eleven treatments were compared, including enzymatic agents applied at different temperatures and concentrations, alkaline and acid detergents, for the elimination of L. monocytogenes S2-bac. Results showed that L. monocytogenes cells conforming the biofilms diminished their counts after applying the acid, alkaline and chlorinated alkaline treatments on 6.03, 6.24 and 4.76 Log CFU/cm2, respectively. The observation of the biofilms remaining structure on the surface by direct epifluorescence microscopy (DEM) demonstrated that conventional treatments were not able to eliminate the formed structures. The enzymatic treatments applied at 50ºC obtained the highest detachment and biocidal activity, although without reaching what was shown by the combined treatment, which improved cell dispersion and increased the effectiveness. Thirdly, given that the main concern regarding biofilms consolidation in the food industry is cross-contamination to the product, it was considered necessary to evaluate the impact in terms of transference. A comparison was made on the effectiveness of chlorinated alkaline versus the combined treatment evaluated in the previous stage of this thesis, for the elimination of biofilms formed by different L. monocytogenes strains. Cross-contamination to chicken broth was evaluated too, targeting untreated and treated biofilms. The results showed that, when the biofilms were not subjected to treatment, the transference rates obtained led to cross-contamination (20.4 %), which represents a potential risk for food safety, since approximately 1.66x104 cells would be migrating to the product. The conventional treatment transferred rates similarly to those obtained for the untreated biofilms, contrary to what was obtained after the application of the combined treatment, which proved not to produce cross-contamination to the chicken broth due to its high detachment effectiveness. Afterwards, the impact of both the conventional and the combined treatment was evaluated. At the same time, the regeneration capacity of cells conforming the structure was studied after the application of the selected treatments to observe if the remaining residual load could consolidate the structure again after 24 hours. The alternative treatment was significantly (P < 0.05) more effective than the conventional one, but none of them completely eradicated the pathogen from the tested surfaces. Biofilm regeneration was found, achieving cell counts like controls. Finally, a study was carried out to determine the microbial contamination in different industrial surfaces of a meat industry. Subsequently, an antibiofilm treatment was applied for several weeks to determine the reduction impact of the different microbial groups, focusing on L. monocytogenes detection. The results obtained showed two main groups of zones with greater and lesser degree of microbiological contamination, being the total aerobic counts the microbial group with the highest contribution. L. monocytogenes presence was detected on five different surfaces throughout the sampling. The applied antibiofilm treatment showed a reduction in all the microbial groups.