**1. Introduction**

Live probiotic microorganisms obtained often with food are thought to improve human health. Thus, probiotics are usually defined as live microbial cells that provide a health benefit to the host when administered in sufficient quantities [1]. Among most prominent probiotic microorganisms are Gram-positive lactic acid bacteria (LAB), which mainly belong to the *Lactobacillus* and *Bifidobacterium* genera [2]. The essential probiotic requirement in terms of the health benefits is a positive influence on the digestion and immune systems [3]. Moreover, probiotics also have a protective role, directly competing with pathogens through signaling interference [4], releasing antimicrobial substances [5] or

metabolites such as acids [6–8]. Nevertheless, to exert its beneficial effects, any probiotic organism must survive, establish, and multiply in the host.

Probiotic bacteria are usually delivered as dried cultures, but the process used to prepare them may damage the cell's structure, vitality, and functionality [9,10]. Drying processes involve the removal of a large amount of fluid from the cell, which affects the cellular structure and may, therefore, cause cell death [10,11]. Moreover, probiotic cells must survive shelf life and transit in the gastrointestinal tract, including acid stress in the stomach [11], as well as degradation by enzymes and bile salt in the intestine [12].

One of the main strategies of bacteria to deal with environmental stresses is the formation of a complex structure called a biofilm [13]. In most natural settings, bacteria do not grow as free-living cells but, instead, they form complex polymicrobial structures [14]. The biofilm structures contain less than 10% microorganisms, while the other 90% is the extracellular matrix (ECM) produced by the bacteria themselves. This ECM mainly consists of polysaccharides and other macromolecules such as proteins, enzymes, surfactants, DNA, and lipids [15]. Thus, the biofilm structure is capable of resisting extreme environmental conditions such as transit through the gastrointestinal tract or desiccation [16]. The ECM creates a microenvironment, which might lead to enhanced survival during desiccation [17]. Apparently, hygroscopic polysaccharides are thought to promote biofilm fluidity and resistance to desiccation [18].

*Bacillus subtilis*, a spore-forming nonpathogenic Gram-positive bacterium, is commonly found in the soil and the gastrointestinal tract (GIT) of some mammals [19]. This bacterium can effectively maintain a favorable balance of microflora in the GIT of the mammalian host [20]. As one of the physiological hallmarks, *B. subtilis* can form a robust biofilm through activation of a dedicated signaling pathway to coordinate expression of genes encoding the ECM [21,22]. Its ECM relies mainly on exopolysaccharides (EPS) synthesized by the *epsA-O* operon and amyloid fibers encoded by the *tasA* located in the *tapA–sipW–tasA* operon [23].

According to recent studies, the use of *Bacillus* species and especially *B. subtilis* as probiotics gained vast interest. Thus, *Bacillus* species were reported to be effective in preventing respiratory infections and gastrointestinal disorders, and overcoming symptoms associated with irritable bowel syndrome [24,25]. However, the mechanism(s) via which *Bacillus* species act as probiotics remains unclear. It appears that the presence of *B. subtilis* helps to maintain a favorable balanced microbiota in the gut and enhances probiotic LAB cell growth and viability [26]. It was also suggested that these probiotic properties are related to its ability to stimulate the immune system [24] and the production of antimicrobial substances [27,28], or even inducing signaling interference against pathogenic microorganisms [4].

Cells of *B. subtilis* produce a vast diversity of antimicrobial substances, amongst them, relatively well-characterized groups of lipopeptides, for instance, surfactins, iturins, and fengycins. These compounds have a wide variety of biological activities such as anti-bacterial, anti-fungal, anti-viral, and anti-tumor activities [29]. They can work in different mechanisms such as disrupting the structure of bacteria members, decreasing the surface and interfacial tension of biofilms [30], and inhibiting quorum sensing, which inhibits biofilm formation [4]. Furthermore, lipopeptides have an essential role in signaling for biofilm formation in *B. subtilis* [31,32].

A model system was recently developed, which enhances biofilm formation by *B. subtilis* through mutual growth with LAB [33]. This system seems to be beneficial for the protection of LAB during heat treatment and through passage in the gastrointestinal tract [33]. The current study presents a further development of the biofilm-based protective coating for probiotic cells via a process defined as a bio-coating. Furthermore, this study provides evidence for two different probiotic functionalities of *B. subtilis*: (i) protecting the LAB during their exposure to desiccation conditions and acid stress; (ii) showing potent anti-microbial activity against pathogenic bacteria such as *Staphylococcus aureus*.
