Probiotics are defined as mono or mixed cultures of living microoganisms which, when applied to animal or man, beneficially affect the host by improving the properties of the indigenous microflora. The aim of this study was to investigate the influence of S-layer proteins on probiotic properties of L. brevis D6, L. brevis SF15B, L. brevis K1, L. brevis ZG1_K7. Primarily, in this study was meant to examine the effect of S-layer proteins on the survival of L. brevis strains in simulated GIT conditions. The results indicated that the presence of S-layer proteins reduces cell death in simulated GIT conditions. All bacterial strains tested, with the S-layer proteins, show a high percentage of autoaggreagtion and coaggregation with bacteria Salmonella enteric serovar Typhimurium compared to strains which removed the S-layer proteins. To achieve a positive impact on the health of the host, there is need to ingest a well-defined number of living probiotic bacteria in a concentrated, powdered preparation that can be prepared by lyophilization. Therefore, lyophilization of L. brevis strains was carried out in this study. After lyophilisation the number of living cells, with S-layer proteins, was higher than number of living cells where the S-layer proteins were removed before the lyophilisation, suggesting a protective role of S-layer proteins under stress by lyophilisation drying of bacterial cells.