**3.3 Probiotics biosurfactants activity**

Probiotics are: "Live microorganisms which when administered in adequate amounts confer a health benefit on the host". They have been reported to have positive effects on the maintenance of human health (Gupta & Garg 2009). Interest in probiotics has gained great significance due to the increasing antimicrobial resistance of bacteria worldwide. Evidence suggests that probiotic organisms may have a role in lowering the incidence or the duration of antibiotic-related diarrhea, contributing to the prevention or treatment of vaginal candidiasis, bacterial vaginosis and recurrent lower urinary tract infections. Furthermore, they encourage improved immunological defense responses and can decrease the activity of numerous toxic antimetabolites (Falagas et al., 2006a, 2006b, 2007).

Probiotics mechanisms of action vary, however, some are known to produce various antimicrobial agents such as organic acids, hydrogen peroxide, carbon peroxide, diacetyl, low molecular weight antimicrobial substances and bacteriocins (Merk et al., 2005). In addition, probiotics have long been known also for the capacity to interfere with the adhesion and formation of biofilms of pathogens to epithelial cells of urogenital and intestinal tracts (Reid et al., 1998, 2001). The mechanisms of this interference include the release of surface active molecules (Gudiña et al., 2010; Rodrigues et al., 2006d). Hong et al., (2005) reported the production of antimicrobial lipopeptides by *Bacillus* probiotics products as the main mechanisms by which they inhibit the growth of pathogenic microorganisms in the gastrointestinal tract. Similarly, competition with other microorganisms for adherence to epithelial cells as well as biosurfactants production are well known mechanisms used by *Lactobacillus* probiotics to interfere with vaginal pathogens (Barrons & Tassone, 2008; Cribby et al., 2008; Falagas et al., 2007).

Similarly, biosurfactants obtained from *Lactobacillus helveticus* and *P. fluorescens* were able to inhibit the adhesion of four *Listeria* strains to stainless steel (Meylheuc et al., 2006a). Whichever strain of *L. monocytogenes* used in combination with biosurfactants, the antiadhesive biological coating developed both reduced the total adhering flora and the viable and culturable adherent bacteria on stainless steel surfaces. More recently, another group investigated the effect of rhamnolipid and surfactin biosurfactants on the adhesion of the food pathogens *E. sakazakii*, *L. monocytogenes* and *S. enteritidis* to polypropylene and stainless steel surfaces (Nitschke et al., 2009). Preconditioning with surfactin, rather than rhamnolipid, caused a reduction in the number of adhering cells particularly of *L. monocytogenes* and to some extent *E. sakazakii* on stainless steel. Surfactin showed a significant decrease in the adhesion on polypropylene of all strains. The adsorption of surfactin on polystyrene also reduced the adhesion of S*. enteritidis*- and *L. monocytogenes*growing cells. In addition, surfactin was able to delay bacterial adhesion within short contact periods using non-growing cells or longer contact periods using growing cells. Other antimicrobial and antiadhesive properties of a biosurfactant produced by *Lactobacillus paracasei* ssp. *paracasei* A20 isolated from a Portuguese dairy plant were also described (Gudiña et al., 2010). The biosurfactant had antimicrobial activity against a broad range of microorganisms including the pathogenic *C. albicans*, *E. coli*, *S. aureus*, *S. epidermidis* and *Streptococcus agalactiae* while exhibiting a considerable antiadhesive activity against a wide

The activity demonstrated by biosurfactants suggests that they could be considered as new tools in developing strategies to prevent or delay microbiological colonization of industrial

Probiotics are: "Live microorganisms which when administered in adequate amounts confer a health benefit on the host". They have been reported to have positive effects on the maintenance of human health (Gupta & Garg 2009). Interest in probiotics has gained great significance due to the increasing antimicrobial resistance of bacteria worldwide. Evidence suggests that probiotic organisms may have a role in lowering the incidence or the duration of antibiotic-related diarrhea, contributing to the prevention or treatment of vaginal candidiasis, bacterial vaginosis and recurrent lower urinary tract infections. Furthermore, they encourage improved immunological defense responses and can decrease the activity of

Probiotics mechanisms of action vary, however, some are known to produce various antimicrobial agents such as organic acids, hydrogen peroxide, carbon peroxide, diacetyl, low molecular weight antimicrobial substances and bacteriocins (Merk et al., 2005). In addition, probiotics have long been known also for the capacity to interfere with the adhesion and formation of biofilms of pathogens to epithelial cells of urogenital and intestinal tracts (Reid et al., 1998, 2001). The mechanisms of this interference include the release of surface active molecules (Gudiña et al., 2010; Rodrigues et al., 2006d). Hong et al., (2005) reported the production of antimicrobial lipopeptides by *Bacillus* probiotics products as the main mechanisms by which they inhibit the growth of pathogenic microorganisms in the gastrointestinal tract. Similarly, competition with other microorganisms for adherence to epithelial cells as well as biosurfactants production are well known mechanisms used by *Lactobacillus* probiotics to interfere with vaginal pathogens (Barrons & Tassone, 2008; Cribby

range of microorganisms.

plant surfaces used in foodstuffs preparation.

numerous toxic antimetabolites (Falagas et al., 2006a, 2006b, 2007).

**3.3 Probiotics biosurfactants activity** 

et al., 2008; Falagas et al., 2007).

Several investigators have pointed to evidence that probiotic type microorganisms and their biosurfactants may antagonize the growth of nosocomial pathogens on inanimate surfaces (Rodrigues et al., 2004a, 2004b, 2006b, 2006c; Walencka et al., 2008). Falagas & Makris, (2009) reviewed studies involving *in vitro* experiments on the potential role of probiotics microorganisms and their products in the inhibition of bacterial or fungal colonisation of artificial surfaces, such as vinyl urethral catheters and silicon rubber voice prostheses (Busscher et al., 1997, 1998; Velraeds et al., 1996, 1997, 2000; Rodrigues et al., 2004a, 2004b, 2006b, Van der Mei et al., 2000). The majority of the investigators examined the preconditioning of the materials surfaces with probiotic biosurfactants, while others added probiotic biosurfactant producing strains to examin adhesion or biofilm development. It was generally demonstrated that both probiotics microorganisms alone (mainly *Streptococcus thermophilus* and *Lactobacillus* spp. strains) or their biosurfactants were able to antagonize growth and development of potentially pathogenic microorganisms including *Staphylococcus aureus*, *Staphylococcus epidermidis*, *Streptococcus* spp., *Enterococcus faecalis, Candida albicans, Candida tropicalis* (Busscher et al., 1997; Van Hoogmoed et al., 2000).

Rodrigues et al., (2004a) demonstrated that the biosurfactant obtained from the probiotic bacterium *Lactococcus lactis* 53 was able to inhibit the adhesion of bacterial pathogens to silicone rubber with an adsorbed biosurfactant layer. Adhesion of yeasts was also decreased in the presence of biosurfactant, but to a lesser extent. In another work, using an artificial throat model, the same authors showed that biosurfactants obtained from probiotic strains greatly reduced microbial numbers on voice prostheses and induced a decrease in the airflow resistance of voice prostheses after biofilm formation, which may prolong the lifetime of indwelling silicone rubber voice prostheses (Rodrigues et al., 2004b).

In a more recent work, it was demonstrated that the preconditioning of silicon rubber with a biosurfactant produced by the strain *Streptococcus thermophilus* A reduced adhering bacterial pathogens by up to 97% and adhering *Candida* spp. by up to 70% (Rodrigues et al., 2006b). Velraeds et al., (1996) also reported on the inhibition of adhesion of pathogenic enteric bacteria by a biosurfactant produced by a *Lactobacillus* strain and later showed that the biosurfactant caused an important dose-related inhibition of the initial deposition rate of *E. coli* and other bacteria adherent on both hydrophobic and hydrophilic substrata (Velraeds et al., 1997).

Another interesting application area that is gaining increased interest relates to probiotics use in preventing oral infections (Çaglar et al., 2005; Hatakka et al., 2007; Kõll et al., 2008; Meurman, 2005; Meurman & Stamatova, 2007). Van Hoogmoed et al., (2004) demonstrated that *Streptococcus mitis* biosurfactant inhibited adhesion of *Streptococcus sobrinus* HG 1025 and *Streptococcus mutans* ATCC 25175 to bare enamel, while *S. mitis* biosurfactant was able to inhibit the adhesion of *S. sobrinus* HG 1025 to salivary pellicles. The authors later reported that these reductions may be attributed to increased electrostatic repulsion between the bacteria and the biosurfactant-coated pellicles (Van Hoogmoed et al., 2006).

New biosurfactant molecules produced by probiotic bacteria are reported from dairy products and environment. Recent work by Walencka et al., (2008) demonstrated that surfactants obtained from three *Lactobacillus acidophilus* strains inhibited *S. epidermidis* and *S. aureus* biofilm integrity and formation. Moreover, surfactant addition to preformed mature biofilms accelerated their dispersal and altered the characteristics of the biofilm morphology. A novel xylolipid biosurfactant from *Lactococcus lactis*, a probiotic strain isolated from a traditional Indian fermented dairy product, showed a good antibacterial activity against clinical pathogens of *E. coli* and MRSA strains (Saravanakumari & Mani, 2010). Xylolipid was non-pathogenic and safe for oral consumption and dermal applications, suggesting that it could be safely used as a therapeutic agent or as a preservative in food or cosmetic products.

In another recent work, a biosurfactant producing strain, *Lactobacillus* sp. CV8LAC, isolated from fresh cabbage, showed interesting antiadhesive activity against two *C. albicans* pathogenic biofilm-producing strains (CA-2894 and DSMZ 11225) (Fracchia et al., 2010). The CV8LAC biosurfactant significantly inhibited the adhesion of fungal pathogens to polystyrene microtiter plates in pre-coating and co-incubation experiments. In pre-coating assays, biofilm formation of strain CA-2894 was reduced by 82% at concentration of 312.5 µg/mL while that of strain DSMZ 11225 was reduced by 81% at 625 µg/mL. In co-incubation assays, biofilm formation of the two strains was inhibited by 70% at 160.5 µg/well and by 81% at 19.95 µg/well, respectively. It was interesting to note that no inhibition of both *C. albicans* planktonic cells was observed, thus indicating that the biosurfactant displayed specific anti-biofilm formation but not antimicrobial activity.

Considering their importance for human health and their recognized safety, environmental probiotic organisms may, thus, represent a safe and effective intervention for infection control purposes. Probiotics themselves or their products (biosurfactants), could be applied to patient care equipment, such as tubes or catheters, with the aim of decreasing the colonisation of these sites by nosocomial pathogens and potentially impede a central step in the pathogenesis of nosocomial infections (Falagas & Makris, 2009).
