**1. Introduction**

Despite that their use in foods is dated, in the last decades, Lactic Acid Bacteria (LAB) have attracted much attention for their documented beneficial properties and for potential useful applications. Among LAB, several strains are currently claimed as probiotics [1], i.e., live microorganisms that, when administered in adequate amounts, confer a health benefit on the host [2]. According to the consensus statement, there are some bacterial species with a long history of safe use and a well-recognized health effect, such as *Bifidobacterium adolescentis*, *B. animalis, B. bifidum*, *B. breve*, *B. longum*, *Lactobacillus acidophilus*, *L. reuteri, L. casei*, *L. fermentum*, *L. gasseri,* etc. [2]; some strains, such as *B. longum* subsp. *infantis* and *L. reuteri*, are widespread due to the strong evidence of their effect on health [3]. Probiotics are able to colonize, stably or transiently, host mucosal surfaces, including the gut, where they may contribute to host health; the capacity of probiotics to colonize biotic and abiotic surfaces by forming structured communities (i.e., biofilms), could have great potentials for human

health and food safety biotechnologies, although this aspect has is in fact barely been explored. It has recently been shown that microbial biofilms may play several "useful" roles such as biodegradation of toxic compounds and pollutants, bioremediation, toxic effluents treatment [4], despite being initially considered only a negative phenomenon. These applications suggest that microbial biofilms could be successfully used for new applications in the biomedical, industrial, food, and environmental field [4].

In the biomedical field, for example, a biofilm formed by probiotic microorganisms could be potentially useful to hinder the development of microorganisms responsible for infections, especially those caused by microorganisms of hospitals, typically resistant to common antibiotic treatments. Indeed, it is widely accepted that in the development of direct and airborne transmission of nosocomial infections, the hospital environment (infection reservoir) plays a key role [5]. In fact, it can be anticipated that a probiotic biofilm left to form ad hoc on several surfaces (e.g., toilets, air conditioning systems) could reduce the spread of pathogenic species that may harbor thereon. Other potential applications in the biomedical field could be: preparations used in skin lesions for the healing processes to add antibacterial capacity, the coating of implants and catheters, medical devices applied to the oral cavity which might hinder the growth of bacterial species associated with caries and periodontal disease [6–9].

On the other hand, regarding potential applications in the food industry, biofilms can be used to ensure the hygienic-sanitary safety of food products, as well as an extension of their shelf-life. The formation of biofilms by "useful/probiotic" microorganisms may be stimulated on materials commonly used to package food (plastic films, pellicles, combinations for packaging, paper, etc.) in order to develop an innovative active packaging system. Although the scientific community is very active in the production of research related to the ability of microorganisms to form biofilms, most studies have focused on biofilm formation by pathogens and/or spoilage microorganisms (*Enterobacter*, *Listeria*, *Micrococcus*, *Streptococcus*, *Bacillus* and *Pseudomonas*) [10–13]. It has been also shown that certain species of LAB are able to form biofilms and some of them are capable of exhibiting antimicrobial activity against pathogenic microorganisms [14–16]; some research was conducted on the possibility of using new methods of sanitation, exploiting the principle of biological competition using probiotic products [17], but this aspect needs to be explored further. In a previous study, we have described the optimization of the production of a probiotic biofilm through intermediate steps by fixing some valuable key points about the probiotics' ability to adhere to surfaces and to form biofilms [18]. These results were used to file a patent covering the use of probiotic biofilms as a means to control pathogen growth [19]. Even if some studies in literature present the use of LAB (mainly lactobacilli) biofilms to control pathogen growth in food and superficies [20–27], most of them propose the use of bio-surfactants and compounds with antimicrobial activity produced in greater quantities by lactobacilli when growing in sessile form. Indeed, our study proposes a probiotic biofilm that exploits the in vivo metabolism of two selected probiotic strains able to adhere rapidly on abiotic surfaces, and not the substances secreted by them and subsequently recovered and used, as in the prior art. To the best of our knowledge, only one study has previously proposed a similar approach evaluating the use of potential probiotic LAB (isolated from Brazilian s foods) biofilms to control *Listeria monocytogenes*, *Salmonella* Typhimurium, and *Escherichia coli* O157:H7 biofilms formation and suggesting that LAB strains can be excellent candidates to form protective biofilms to be used as biocontroller of contamination into the food chain [28].

Besides the use as an innovative active packaging to ensure the safety of food products, as well as an extension of their shelf-life, the proposed probiotic biofilm formed ad hoc on medical devices (catheters, implants, braces, bite blocks or condoms) and on bathrooms' surfaces (sink, bidet, toilet bowl, water closet or piece of furniture) could be considered a tool against colonizing strains, since these surfaces are often implicated in nosocomial infections. Our proposal could lead to the development of a useful means to control the growth of pathogenic and spoilage bacteria for industrial and medical applications. In the following, some specific applications of the developed probiotic biofilm are described, focusing on two different aspects: 1) effect of probiotic biofilms on pathogen sessile growth; 2) application as potential active packaging.
