Andreja Leboš Pavunc

Functionality of the S-layer protein from the probiotic strain Lactobacillus helveticus M92

The objective of this study was the characterisation of the S-layer protein (SlpA) and its functional role in the probiotic activity of Lactobacillus helveticus M92. SlpA was isolated and identified by SDS-PAGE LC-MS/MS analysis. The slpA gene encoding the SlpA from L. helveticus M92 was sequenced and compared with other well characterised slpA genes. Sequence similarity searches revealed high homology with the SlpA of Lactobacillus strains. Purified SlpA showed significantly better immunomodulatory effects in orally immunised mice than L. helveticus M92 cells after SlpA removal. SlpA is involved in the autoaggregation of L. helveticus M92 cells and coaggregation of L. helveticus M92 with S. Typhimurium FP1 as these processes were negatively affected after SlpA removal from the cell surface. Therefore, the influence of oral treatment with L. helveticus M92 on an oral infection of mice by S. Typhimurium FP1 was investigated. Following the oral immunization of mice, with viable L. helveticus M92 and S. Typhimurium FP1 cells, the concentration in the luminal contents of total S-IgA and specific anti-Salmonella S-IgA antibodies, from all immunized mice was significantly higher compared to the control group or a group of mice infected only with S. Typhimurium FP1. These results demonstrate that the observed reduced infection by S. Typhimurium FP1 in mice with L. helveticus M92 is associated with competitive exclusion in the intestinal tract and enhanced immune protection conferred by the L. helveticus M92 and its SlpA.

Improved sauerkraut production with probiotic strain Lactobacillus plantarum L4 and Leuconostoc mesenteroides LMG 7954.

UNLABELLED Probiotic strain Lactobacillus plantarum L4 and strain Leuconostoc mesenteroides LMG 7954 were applied for the controlled fermentation of cabbage heads. The parameters of the controlled and spontaneous fermentations, including antimicrobial effect of cabbage brines obtained at the end of both fermentations, were monitored. To check out the influence of starter culture strains, 10 randomly chosen lactic acid bacteria, isolated at the end of controlled cabbage heads fermentation were identified by API 50 CHL test, and the presence of the probiotic culture was confirmed by Pulsed Field Gel Electrophoresis. The starter cultures applied for cabbage heads fermentation allowed lowering of NaCl concentrations from 4.0% to 2.5% (w/v), considerably accelerated fermentation process by 14 d, and improved the product quality. The produced sauerkraut heads are considered probiotic product as viable probiotic cells count in final product was higher than 10(6) colony-forming units (CFU) per gram of product. PRACTICAL APPLICATION The results of this research could be applied in the production of fermented cabbage heads with added functional (probiotic) value and with lower NaCl concentration with expected shortened fermentation time. This could not only be of economic but also of ecological importance.

Proteolytic activity of probiotic strain Lactobacillus helveticus M92

The aim of this research was to investigate the potential of previously defined probiotic strain Lactobacillus helveticus M92 as functional starter culture for fermented dairy products. Therefore, proteolytic activity of L. helveticus M92 was investigated and compared with those of different representatives of probiotic and starter culture strains. Cluster analysis of AFLP fingerprints showed a difference of L. helveticus M92 compared to five other L. helveticus strains, but the percentage of similarity confirmed the identification on species level. Casein hydrolysis by L. helveticus M92 was monitored by agar-well diffusion test, SDS-PAGE and Ansons method. L. helveticus M92 exhibited the highest proteolytic activity among tested probiotic and starter cultures strains with the fastest acidification rate and the highest pH decrease after overnight incubation in skim milk. The presence of prtH2 gene was confirmed by PCR amplification with specific primers, while PCR product was not obtained after amplification with primers specific to prtH. Furthermore, SDS-PAGE LC-MS/MS analysis of insoluble proteome of L. helveticus M92 enabled identification of several proteins involved in proteolytic system of L. helveticus such as protease PrtM as well as proteins involved in Opp peptide transport system and the intracellular peptidases PepE, PepN, and PepQ.