The effect of pH, bile and calcium on the adhesion ability of probiotic enterococci of animal origin to the porcine jejunal epithelial cell line IPEC-J2.

Examination of adhesion ability using a quantitative assay based on radiolabelled bacteria showed that 10 Enterococcus strains exhibited adhesion ability from 2 to 4%. Enterococcus faecium EF2019 (isolate from rabbit faeces, deponed to Czech Culture of Microorganisms in Brno, CCM 7420) showed the highest adhesion ability (4.0+/-0.4%). With regard to survival, all strains displayed good resistance towards 0.3% oxgall and HCl (pH 3.0). Pretreatment of strains with HCl (pH 3.0) significantly reduced their adhesion. Pretreatment of strains by oxgall significantly reduced the adhesion capacity of E. faecium EF2019, EF1839 and EF319 strains, while the adhesion ability of E. faecium EE3 (isolate from canine feed) slightly increased. Furthermore, addition of calcium (200 mmol/l) significantly increased (P<0.001) the adhesion ability for all strains tested. The adhesion ability of the isolates from rabbits, EF1839 and EF529, as well as the isolate EE3 (strain from canine feed) increased from 2-3% up to 50-55% upon calcium addition. Despite, in general low adhesive properties, strains can survive passage through the gastrointestinal tract.

A novel eukaryotic cell culture model to study antiviral activity of potential probiotic bacteria.

As shown in many intervention studies, probiotic bacteria can have a beneficial effect on rotavirus and HIV-induced diarrhoea. In spite of that fact, antiviral effects of probiotic bacteria have not been systematically studied yet. Non-tumorigenic porcine intestinal epithelial cells (IPEC-J2) and alveolar macrophages (3D4/2) were treated in different experimental designs with probiotic and other lactic bacteria and their metabolic products. Vesicular stomatitis virus (VSV) was used in the study as a model virus. Cell survival and viral inhibition were determined by antiviral assay and confirmed by immunofluorescence. Pre-incubation of cell monolayers with probiotic bacteria reduced viral infectivity up to 60%. All bacteria used prevented VSV binding to the cell monolayers by direct binding of VSV to their surface. Probiotic and other lactic bacteria prevented viral infection also by establishment of the antiviral state in pre-treated cell monolayers. Probiotic and other lactic bacteria secreted antiviral substances during their growth, as the infectivity of the virus was diminished by 68% when bacterial supernatants were tested. It was shown for the first time that probiotic and other lactic bacteria exhibit an antiviral activity in a cell culture model. Possible mechanisms of antiviral activity include: 1) hindering the adsorption and cell internalisation of the VSV due to the direct trapping of the virus by the bacteria, 2) "cross-talk" with the cells in establishing the antiviral protection and 3) production of metabolites with a direct antiviral effect.

The survival and persistence in the human gastrointestinal tract of five potential probiotic lactobacilli consumed as freeze-dried cultures or as probiotic sausage.

Among five lactobacilli (L. plantarum MF1291, MF1298, DC13, L. pentosus MF1300 and L. salivarius DC5) which were administrated as freeze-dried cultures for 17 volunteers, MF1298 and DC13 were the most frequently reisolated strains in faeces demonstrating the human gastric survival of these strains. Furthermore, MF1298 and DC13 persisted in the same volunteer after ended intake, suggesting host-specific persistence behaviour. When MF1298 was administrated as sausage fermented with this strain, the number of volunteers harbouring MF1298 increased from 4 to 10 indicating that the sausage matrix protects the survival through the gastrointestinal tract (GIT).

Identification of potential probiotic starter cultures for Scandinavian-type fermented sausages.

Potential probiotic cultures suitable as starter cultures for the Scandinavian-type fermented sausages were identified among strains well-adapted to fermented meats as well as strains originating from a culture collection. From 15 different fermented meat products, 22 strains were isolated as dominant non-starter lactic acid bacteria (NSLAB). The isolates were identified by RAPD, API and sequence analysis of 16S rRNA and showed to be five strains of Lactobacillus sakei, five strains of Lactobacillus farciminis, five strains belonging to the group of Lactobacillus plantarum/pentosus, four strains of Lactobacillus alimentarius, two strains of Lactobacillus brevis and one strain of Lactobacillus versmoldensis. Heterofermentative strains as well as strains not growing at 37 degrees C and not lowering pH below 5.1 in a meat model were excluded leaving 9 strains for further studies. These strains together with 19 strains from a culture collection were evaluated by in vitro methods including survival upon exposure to pH 2.5 or 0.3% oxgall and adhesion to the human colon adenocarcinoma cell line Caco-2 as well as antimicrobial activity against potential pathogens. Strains that fulfilled all the probiotic criteria and showed to be fast acid producers in a meat model included three strains belonging to the group of Lb. plantarum/pentosus (MF1291, MF1298, MF1300) which originated from the dominant NSLAB of fermented meat products. MF1291 and MF 1298 were further identified as Lb. plantarum and MF1300 as Lb. pentosus. The three strains were all successfully applied as starter cultures for the production of fermented sausage. The viable count at the end of the processing period reached high cell numbers (4.7x10(7)-2.9x10(8) cfu/g) and pH of the sausages decreased to pH 4.8-4.9 without any flavour deviation compared to sausage fermented by a commercial meat starter culture.

Effect of modified atmosphere and temperature abuse on the growth from spores and cereulide production of Bacillus weihenstephanensis in a cooked chilled meat sausage.

The effect of modified atmosphere packaging (MAP) on the germination and growth of toxin producing psychrotolerant Bacillus spp is not well described. A model agar system mimicking a cooked meat product was used in initial experiments. Incubation at refrigeration temperature of 8 degrees C for 5 weeks of 26 Bacillus weihenstephanensis including two emetic toxin (cereulide) producing strains showed that B. weihenstephanensis is sensitive to MAP containing CO2. The sensitivity to 20% CO2 was dependent on strain and oxygen level, being increased when oxygen was excluded from the MAP. Growth from spores was observed at the earliest within 2 weeks when 20% CO2 was combined with 2% O2 and in 3 weeks when combined with "0"% O2 (the remaining atmosphere was made up from N2). Results were validated in a cooked meat sausage model for two non-emetic and one emetic B. weihenstephanensis strain. The packaging film oxygen transfer rates (OTR) were 1.3 and 40 ml/m(2)/24 h and the atmospheres were 2% O2/20% CO2 and "0"% O2/20% CO2. Oxygen availability had a large impact on the growth from spores in the MAP meat sausage, only the most oxygen restricted condition (OTR of 1.3 ml/m(2)/24 h and "0"% O2/20 % CO2 inhibited growth of the three strains during 4 weeks storage at 8 degrees C. Cereulide production was undetectable during storage at 8 degrees C irrespective of choice of the MAP (quantified by liquid chromatography mass spectrometry/mass spectrometry). MAP storage at 8 degrees C for 1 and 3 weeks followed by opening of packages and temperature abuse for 1.5 h daily at 20 degrees C during 1 week resulted in increased cell counts and variable cereulide production in the meat sausage. A pre-history at 8 degrees C for 1 week in MAP with OTR of 1.3 or 40 ml/m(2)/24 h and 2% O2 resulted in cereulide concentrations of 0.816-1.353 microg/g meat sausage, while a pre-history under the most oxygen restricted condition (OTR of 1.3 ml/m(2)/24 h, "0"% O2/20 % CO2 resulted in minimal cereulide production (0.004 microg/g meat sausage) at abuse condition. Extension of MAP storage at 8 degrees C for 3 weeks followed by abuse resulted in a substantially reduced cereulide production. Data demonstrates that MAP can be used to inhibit growth of a psychrotolerant toxin producing Bacillus spp. during chill storage at 8 degrees C, and substantially reduce the risk of emetic food poisoning at abuse condition. Results are of relevance for improving safety of ready to eat processed chilled foods of extended durability.

Comparison of Saccharomyces cerevisiae strains of clinical and nonclinical origin by molecular typing and determination of putative virulence traits.

Saccharomyces cerevisiae strains of clinical and nonclinical origin were compared by pulse field gel electrophoresis. Complete separation between strains of clinical origin and food strains by their chromosome length polymorphism was not obtained even though there was a tendency for the clinical and food strains to cluster separately. All the investigated strains, except for one food strain, were able to grow at temperatures > or =37 degrees C but not at 42 degrees C. Great strain variations were observed in pseudohyphal growth and invasiveness, but the characters were not linked to strains of clinical origin. The adhesion capacities of the yeast strains to a human intestinal epithelial cell line (Caco-2) in response to different nutritional availabilities were determined, as were the effects of the strains on the transepithelial electrical resistance (TER) across polarized monolayers of Caco-2 cells. The yeast strains displayed very low adhesion capacities to Caco-2 cells (0.6-6.2%), and no significant difference was observed between the strains of clinical and nonclinical origin. Both S. cerevisiae strains of clinical and non-clinical origin increased the TER of polarized monolayers of Caco-2 cells. Based on the results obtained in this study, no specific virulence factor was found that clearly separated the strains of clinical origin from the strains of nonclinical origin. On the contrary, all investigated strains of S. cerevisiae were found to strengthen the epithelial barrier function.

Application of measurements of transepithelial electrical resistance of intestinal epithelial cell monolayers to evaluate probiotic activity.

Among five potentially probiotic lactobacilli investigated, Lactobacillus plantarum MF1298 and Lactobacillus salivarius DC5 showed the highest increase in the transepithelial electrical resistance (TER) of polarized monolayers of Caco-2 cells, and this increase was shown to be dose dependent. Furthermore, preincubation with MF1298 attenuated a decrease in TER induced by Listeria monocytogenes.