A Lactobacillus-derived biosurfactant inhibits biofilm formation of human pathogenic Candida albicans biofilm producers L. Fracchia 1,2 , M. Cavallo 1,2 , G. Allegrone 1,2 , and M.G. Martinotti 1,2 1 Department of Chemical, Food, Pharmaceutical and Pharmacological Sciences, University of Eastern Piedmont, Via Bovio 6, 28100, Novara, Italy. 2 Drug and Food Biotechnology Center, Via Bovio 6, 28100, Novara, Italy. Fifteen lactic-acid bacteria, isolated from fresh fruits and vegetables produced biosurfactants in the mid-exponential phase (5 hours). Twelve isolates were genotipically identified to belong to the genus Lactobacillus. Among these, the Lactobacillus sp. CV8LAC, isolated from cabbage, showed the largest oil spreading halo. Extracted CV8LAC biosurfactant reduced the water surface tension from 70.92 mN/m to 47.68 mN/m and its CMC was 106 µg/mL. The CV8LAC biosurfactant significantly (p<0.05) inhibited the adhesion of two Candida albicans pathogenic biofilm- producer strains (CA-2894 and DSMZ 11225) in pre-coating and co-incubation experiments. In pre-coating assays, biofilm formation of the strain CA-2894 was reduced by 82% at concentration of 312.5 µg/ml and that of DSMZ 11225 by 81% at 625 µg/ml. In co-incubation assays, biofilm formation of CA-2894 and DSMZ 11225 was inhibited by 70% at 160.5 µg/well and by 81% at 19.95 µg/well, respectively. No inhibition of both C. albicans planktonic cells was observed, thus indicating that the biosurfactant displayed anti-biofilm formation but not antimicrobial activity. Keywords Lactobacillus sp. CV8LAC; biosurfactant; Candida albicans; biofilm 1. Introduction Probiotic bacteria, such as lactobacilli, are well known to have a positive effect on the maintenance of human health [1- 3]. These bacteria, which constitute an important part of natural microbiota, are recognized as potential interfering bacteria by producing various antimicrobial agents such as organic acids, hydrogen peroxide, carbon peroxide, diacetyl, low molecular weight antimicrobial substances, bacteriocins, and adhesion inhibitors, such as biosurfactants [3]. In particular, lactobacilli have long been known for their antimicrobial activity and capability to interfere with the pathogens adhesion on epithelial cells of urogenital and intestinal tracts [4-6], and for their anti-biofilm production on catheter devices [7] and voice prostheses [8, 9]. The mechanisms of this interference have been demonstrated to include, among others, the release of biosurfactants [10-12]. Biosurfactants have recently become an important product of biotechnology for industrial and medical applications [13-15]. Adsorption of biosurfactants to a substratum surface modifies its hydrophobicity, interfering in the microbial adhesion and desorption processes [16]; in that sense, the release of biosurfactants by probiotic bacteria in vivo can be considered as a defence weapon against other colonizing strains in the urogenital and gastrointestinal tracts [17] and on medical devices. Biosurfactants produced by lactobacilli, in fact, have been shown to reduce adhesion of pathogenic micro-organisms to glass [18], silicone rubber [19], surgical implants [20] and voice prostheses [8, 9]. Consequently, previous adsorption of biosurfactants can be used as a preventive strategy to delay the onset of pathogenic biofilm growth on catheters and other medical insertional materials, reducing the use of synthetic drugs and chemicals [16, 21, 22]. Candida species are of increasing concern as causative agents of fungal biofilm related infections on prosthesis in odontoiatry and otorinolaringoiatry [23-25]. Development of new technologies based on the control of the Candida spp. biofilm growth is, thus, foreseen as a major breakthrough in medicine and will have a strong impact in the clinical practice and preventive medicine. Many lactobacilli are known to inhibit the growth of Candida spp. in different ways, such as competition for adhesion sites or production of different antagonistic metabolites which inhibit its growth [26, 27] however, the specific role of lactobacilli-produced biosurfactant on Candida albicans biofilm has been rarely investigated [28, 12]. The aim of this study was to determine the anti-biofilm capability of a biosurfactant produced by a Lactobacillus sp., isolated from cabbage, against two pathogenic strains of C. albicans biofilm producers. 2. Materials and Methods 2.1 Collection of samples and lactic acid bacteria isolation Three cucumbers and one head of lettuce were collected from different local markets of Novara in Italy, seven apples, one cabbage and five pears were directly obtained from a producer of biological fruit and vegetable in a rural area of _______________________________________________________________________________________