STRUCTURAL CHARACTERIZATION OF S-LAYER PROTEINS EXTRACTED FROM KEFIR LACTOBACILLI BY MEANS OF RAMAN SPECTROSCOPY

P. MOBILI; C. ARAUJO-ANDRADE; E. GERBINO; C. FRAUSTO-REYES; G. DE ANTONI; R. IVANOV-TZONCHEV; A. GÓMEZ-ZAVAGLIA

Manchester

Congreso; 6th International SPEC; 2010

SPEC

Kefir is a fermented milk that has been consumed for thousands of years in Eastern Europe. It was originated from the northern area of the Caucasus Mountains and it is prepared from kefir grains [1]. Kefir grains are clusters of microorganisms that include primarily lactic acid bacteria (lactobacilli, lactococci, leuconostoc), acetic acid bacteria, yeasts, and other microorganisms not yet described held together by a matrix of polysaccharides and proteins [2-4]. These microorganisms are usually associated with the beneficial effects of this fermented milk. This beneficial activity is at least partially related with bacterial surface properties, which in turn, may depend on the specific characteristics of their S-layers. S-layer structures are macromolecular paracrystalline arrays that completely cover the cell surface of some bacteria [1,2]. They are attached to the underlying cell wall by non-covalent bonds and usually may be dissociated and solubilized into protein monomers by dissociating agents such as 5 M LiCl. The presence of S-layer proteins in Lactobacillus kefir and Lactobacillus parakefir isolated from kefir grains has been reported for the first time in the last years [5] and some probiotic properties of these bacteria have been attributed to the S-layer [6]. In addition, structural differences in their S-layer proteins have been correlated with specific surface properties (i.e.: aggregation) of whole bacterial cells [7,8]. Taking into account that lactobacilli surface properties are not only species but also strain dependent, it turns out evident that the structural characterization of S-layer proteins from different lactobacilli strains could be very helpful in the interpretation of such strain-specific probiotic properties. In the present work, Raman spectroscopy has been used for the structural characterization of S-layers extracted from different strains of Lactobacillus kefir, Lactobacillus brevis and Lactobacillus parakefir. This vibrational spectroscopic methodology has already been used in Food Microbiology for the characterization of whole microorganisms and also for the analysis of proteins [9,10]. As in FTIR spectroscopy, the position of a given band in a Raman spectrum depends on inter- and intra-molecular interactions, including peptide-bond angles and hydrogen-bonding patterns. In this work the analysis of the Raman spectra of different lactobacilli strains allowed for the determination of their secondary structure. To fulfil this aim, amide I and III regions of the raw Raman spectra were fitted in order to determine the relative areas of the bands constituting each peak, thus determining the secondary structure components of the protein [5,7]. References [1]     M. Sára and U.B. Sleytr, J. Bacteriol. 182, 859-868 (2000). [2]     S. Avall-Jääskeläinen and A. Palva, FEMS Microbiol. Rev. 29, 511-529 (2005). [3]     G. Garrote, L. Delfederico, R. Bibiloni, A. Abraham, P. Pérez, L. Semorile and G. De Antoni, J. Dair. Res. 71, 222-230 (2004). [4]     M. Kahala, K. Savijoki and A. Palva, J. Bacteriol. 179, 284-286 (1997). [5]     J.T. Pelton and L.R.McLean, Anal. Biochem. 277, 167-176 (2000). [6]     M.A. Golowczyc, P. Mobili, G.L. Garrote, A.G. Abraham, G.L. De Antoni, Int J Food Microbiol. 118, 264-273 (2007). [7]     P. Mobili, A. Londero, T. Roseiro, E. Eusebio, G. De Antoni, R. Fausto and A. Gómez-Zavaglia, Vibrat. Spectrosc. 50, 68-77 (2009). [8]     P. Mobili, M. Serradell, S. Trejo, F. Avilés Puigvert, G. Abraham and G. De Antoni, Ant. van Leeuw. 95, 363-372 (2009). [9]     P. Rosch, M. Schmitt, W. Kiefer and J. Popp, J. Mol. Struc. 661-662, 363-369 (2003). [10]    A. Torreggiani and G. Fini, J. Raman Spectrosc. 29, 229-236 (1998).