Probiotic potential of Saccharomyces cerevisiae and Kluyveromyces marxianus isolated from West African spontaneously fermented cereal and milk products.

The yeast species Saccharomyces cerevisiae and Kluyveromyces marxianus are associated with fermentation of West African indigenous foods. The aim of this study was to characterize potential probiotic properties of S. cerevisiae and K. marxianus isolates from the West African milk products lait caillé and nunu and a cereal-based product mawè. The strains (14 in total) were identified by 26S rRNA gene sequencing and characterized for survival at gastrointestinal stress (bile salts and low pH) and adhesion to Caco-2 intestinal epithelial cells. Selected yeast isolates were tested for their effect on the transepithelial electrical resistance (TEER), using the intestinal epithelial cell line Caco-2 and for maintenance of intracellular pH (pHi ) during perfusion with gastrointestinal pH (3.5 and 6.5). All tested yeasts were able to grow in bile salts in a strain-dependent manner, exhibiting a maximum specific growth rate (µmax ) of 0.58-1.50 h-1 . At pH 2.5, slow growth was observed for the isolates from mawè (µmax of 0.06-0.80 h-1 ), whereas growth of yeasts from other sources was mostly inhibited. Yeast adhesion to Caco-2 cells was strain specific and varied between 8.0% and 36.2%. Selected strains of S. cerevisiae and K. marxianus were able to maintain the pHi homeostasis at gastrointestinal pH and to increase TEER across the Caco-2 monolayers, indicating their potential to improve intestinal barrier functions. Based on overall results, strains of K. marxianus and S. cerevisiae from mawè exhibited the highest probiotic potential and might be recommended for further development as starter cultures in West African fermented products.

Transcriptome analysis of Lactococcus lactis subsp. lactis during milk acidification as affected by dissolved oxygen and the redox potential.

Performance of Lactococcus lactis as a starter culture in dairy fermentations depends on the levels of dissolved oxygen and the redox state of milk. In this study the microarray analysis was used to investigate the global gene expression of L. lactis subsp. lactis DSM20481(T) during milk acidification as affected by oxygen depletion and the decrease of redox potential. Fermentations were carried out at different initial levels of dissolved oxygen (dO2) obtained by milk sparging with oxygen (high dO2, 63%) or nitrogen (low dO2, 6%). Bacterial exposure to high initial oxygen resulted in overexpression of genes involved in detoxification of reactive oxygen species (ROS), oxidation-reduction processes, biosynthesis of trehalose and down-regulation of genes involved in purine nucleotide biosynthesis, indicating that several factors, among them trehalose and GTP, were implicated in bacterial adaptation to oxidative stress. Generally, transcriptional changes were more pronounced during fermentation of oxygen sparged milk. Genes up-regulated in response to oxygen depletion were implicated in biosynthesis and transport of pyrimidine nucleotides, branched chain amino acids and in arginine catabolic pathways; whereas genes involved in salvage of nucleotides and cysteine pathways were repressed. Expression pattern of genes involved in pyruvate metabolism indicated shifts towards mixed acid fermentation after oxygen depletion with production of specific end-products, depending on milk treatment. Differential expression of genes, involved in amino acid and pyruvate pathways, suggested that initial oxygen might influence the release of flavor compounds and, thereby, flavor development in dairy fermentations. The knowledge of molecular responses involved in adaptation of L. lactis to the shifts of redox state and pH during milk fermentations is important for the dairy industry to ensure better control of cheese production.

Survival of Listeria monocytogenes in simulated gastrointestinal system and transcriptional profiling of stress- and adhesion-related genes.

Food ingestion is the major route of exposure to the important human pathogen Listeria monocytogenes. An in vitro gastrointestinal model was used to (1) compare the survival rates of L. monocytogenes strains of serotypes 1/2a, 1/2c, and 4b; and (2) examine the transcription of stress- and adhesion-related genes after exposure to the conditions similar to those encountered in the mouth, stomach, and small intestine. None of the L. monocytogenes strains investigated could survive in the gastric juice at pH 2.5 or 3.0. Their survival increased at higher pH (3.5 and 4.0) in the gastric stress. Relative survival of L. monocytogenes serotypes 4b and 1/2a strains were higher than that of serotype 1/2c, suggesting that pathogenicity might be related to the viability in the gastrointestinal tract. The transcription levels of prfA and the general stress-related genes clpC, clpE, and clpP were upregulated after passing through the simulated gastrointestinal tract, whereas that of the adhesion-related gene ami was downregulated. Taken together, this study revealed that L. monocytogenes strains enhanced the expression of stress-related genes and decreased the transcription of adhesion-related gene in order to survive in the diverse microenvironments.

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.

Occurrence and growth of yeasts in processed meat products - Implications for potential spoilage.

Spoilage of meat products is in general attributed to bacteria but new processing and storage techniques inhibiting growth of bacteria may provide opportunities for yeasts to dominate the microflora and cause spoilage of the product. With the aim of obtaining a deeper understanding of the potential role of yeast in spoilage of five different processed meat products (bacon, ham, salami and two different liver patés), yeasts were isolated, enumerated and identified during processing, in the final product and in the final product at the end of shelf life. Yeasts were isolated along the bacon production line in numbers up to 4.2 log (CFU/g). Smoking of the bacon reduced the yeast counts to lower than 1.0 log (CFU/g) or non-detectable levels. In general, yeasts were only isolated in low numbers during the production of salami, cooked ham and liver paté. In the final products yeasts were detected in low numbers in a few samples (3 out of 30) samples, 1.0-1.3 log (CFU/g). By the end of storage, yeasts were only detected in 1 out of 25 investigated samples 1.8 log (CFU/g). A combination of phenotypic and genotypic methods was used to identify the yeast microflora present during production of the processed meat products. The yeast microflora was complex with 4-12 different species isolated from the different production sites. In general, Candida zeylanoides, Debaryomyces hansenii and the newly described Candida alimentaria were found to be the dominant yeast species. In addition, three putatively previously undescribed yeast species were isolated. Fourteen isolates, representing seven different species isolated during the production of the processed meat products and one species isolated from spoiled, modified atmosphere packed, sliced ham, were screened for their ability to grow in a meat model substrate under a low oxygen/high carbon-dioxide atmosphere (0.5% O(2), 20% CO(2), 79.5% N(2)) at two different temperatures (5 and 8°C). Eleven out of the tested 14 strains were able to grow in the meat model substrate with C. zeylanoides, D. hansenii, Pichia guilliermondii and Candida sake reaching levels of 10(5)-5×10(6) log (CFU/g), where sensoryical changes appear.

In vitro screening of probiotic properties of Saccharomyces cerevisiae var. boulardii and food-borne Saccharomyces cerevisiae strains.

The probiotic potential of 18 Saccharomyces cerevisiae strains used for production of foods or beverages or isolated from such, and eight strains of Saccharomyces cerevisiae var. boulardii, was investigated. All strains included were able to withstand pH 2.5 and 0.3% Oxgall. Adhesion to the nontumorigenic porcine jejunal epithelial cell line (IPEC-J2) was investigated by incorporation of 3H-methionine into the yeast cells and use of liquid scintillation counting. Only few of the food-borne S. cerevisiae strains exhibited noteworthy adhesiveness with the strongest levels of adhesion (13.6-16.8%) recorded for two isolates from blue veined cheeses. Merely 25% of the S. cerevisiae var. boulardii strains displayed good adhesive properties (16.2-28.0%). The expression of the proinflammatory cytokine IL-1alpha decreased strikingly in IPEC-J2 cells exposed to a Shiga-like toxin 2e producing Escherichia coli strain when the cells were pre- and coincubated with S. cerevisiae var. boulardii even though this yeast strain was low adhesive (5.4%), suggesting that adhesion is not a mandatory prerequisite for such a probiotic effect. A strain of S. cerevisiae isolated from West African sorghum beer exerted similar effects hence indicating that food-borne strains of S. cerevisiae may possess probiotic properties in spite of low adhesiveness.

Protein expression during lag phase and growth initiation in Saccharomyces cerevisiae.

In order to obtain a better understanding of the biochemical events taking place in Saccharomyces cerevisiae during the lag phase, the proteins expressed during the first hours after inoculation were investigated by two-dimensional (2-D) gel electrophoresis and compared to those expressed in late respiratory growth phase. The studies were performed on a haploid strain (S288C) grown in defined minimal medium. Some of the abundant proteins, whose expression relative to total protein expression was induced during the lag phase, were identified by MALDI MS, and the expression of the corresponding genes was assessed by Northern blotting. The rate of protein synthesis was found to increase strongly during the lag phase and the number of spots detected on 2-D gels increased from 502 spots just after inoculation to 1533 spots at the end of the lag phase. During the first 20 min, the number of detectable spots was considerably reduced compared to the number of spots detected from the yeast in respiratory growth just prior to harvest and inoculation (747 spots), indicating an immediate pausing or shutdown in synthesis of many proteins just after inoculation. In this period, the cells got rid of most of their buds. The MALDI MS-identified, lag phase-induced proteins were adenosine kinase (Ado1p), whose cellular role is presently uncertain, cytosolic acetaldehyde dehydrogenase (Ald6p) and (DL)-glycerol-3-phosphatase 1, both involved in carbohydrate metabolism, a ribosomal protein (Asc1p), a fragment of the 70-kDa heat shock protein Ssb1, and translationally controlled tumour protein homologue (Yk1056cp), all involved in translation, and S-adenosylmethionine synthetase I involved in biosynthesis reactions. The level of mRNA of the corresponding genes was found to increase strongly after inoculation. By pattern matching using previously published 2-D maps of yeast proteins, several other lag phase-induced proteins were identified. These were also proteins involved in carbohydrate metabolism, translation, and biosynthesis reactions. The identified proteins together with other, yet unidentified, lag phase-induced proteins are expected to be important for yeast growth initiation and could be valuable biological markers for yeast performance. Such markers would be highly beneficial in the control and optimisation of industrial fermentations.