In vitro and in vivo inhibition of Helicobacter pylori by Lactobacillus casei strain Shirota

We studied the potential inhibitory effect of Lactobacillus casei strain Shirota (from the fermented milk product Yakult [Yakult Ltd., Tokyo, Japan]) on Helicobacter pylori by using (i) in vitro inhibition assays with H. pylori SS1 (Sydney strain 1) and nine H. pylori clinical isolates and (ii) the in vivo H. pylori SS1 mouse model of infection over a period of 9 months. In vitro activity against H. pylori SS1 and all of the clinical isolates was observed in the presence of viable L. casei strain Shirota cells but not in the cell-free culture supernatant, although there was profound inhibition of urease activity. In vivo experiments were performed by oral administration of L. casei strain Shirota in the water supply over a period of 9 months to 6-week-old C57BL/6 mice previously infected with H. pylori SS1 (study group; n = 25). Appropriate control groups of H. pylori-infected but untreated animals (n = 25) and uninfected animals given L. casei strain Shirota (n = 25) also were included in the study. H. pylori colonization and development of gastritis were assessed at 1, 2, 3, 6, and 9 months postinfection. A significant reduction in the levels of H. pylori colonization was observed in the antrum and body mucosa in vivo in the lactobacillus-treated study group, as assessed by viable cultures, compared to the levels in the H. pylori-infected control group. This reduction was accompanied by a significant decline in the associated chronic and active gastric mucosal inflammation observed at each time point throughout the observation period. A trend toward a decrease in the anti-H. pylori immunoglobulin G response was measured in the serum of the animals treated with lactobacillus, although this decrease was not significant.     

Heterogeneity of Lactobacillus plantarum isolates from feta cheese throughout ripening

Thirty-two Lactobacillus plantarum strains isolated from Feta cheese throughout ripening were studied for their phenotypic characteristics, protein profile of cell-free extracts, enzyme profiles, plasmid profiles, proteolytic and acidifying abilities and ability to grow at low pH and in the presence of bile. Results showed that some biotechnologically important characteristics, such as acidifying and proteolytic activities, can differ between strains. In addition, different plasmid profiles suggest the presence of different Lact. plantarum strains in Feta cheese throughout ripening. The results suggest the possibility of choosing strains with specific biotechnologically interesting properties.     

Interactions between Saccharomyces cerevisiae and lactic acid bacteria in sourdough

The aim of this study was to assess the interactions between Saccharomyces cerevisiae and lactic acid bacteria that either form a stable consortium in Greek wheat sourdoughs (i.e. Lactobacillus sanfranciscensis and L. brevis) or occasionally constitute the secondary microbiota (i.e. Weissella cibaria, L. paralimentarius, Pediococcus pentosaceus and Enterococcus faecium). For this purpose, wheat dough was prepared by using strains of the above mentioned species either as single starters, or in combination of the yeast with each of the lactic acid bacteria strains. The determination of the metabolic products in sourdough samples was performed by HPLC analysis. Presence of lactic acid bacteria had no effect on S. cerevisiae final cell yield but affected negatively the maximum specific growth rate. Ethanol production was primarily affected negatively while the co-culture had a variable effect on glycerol production. On the other hand, the presence of S. cerevisiae favoured mannitol and acetic acid production, had a species-dependent effect on maximum specific growth rate and had no effect on final cfu/g sourdough and lactic acid production by the lactic acid bacteria and at the same time caused the depletion of glucose, fructose and maltose.     

Probiotic Features of Lactic Acid Bacteria Isolated from a Diverse Pool of Traditional Greek Dairy Products Regarding Specific Strain-Host Interactions

Probiotics and Antimicrobial Proteins - The increased consumers’ interest on the positive role of food in wellbeing and health underscores the need to determine new probiotic microorganisms....     

Acid Tolerance of Streptococcus macedonicus as Assessed by Flow Cytometry and Single-Cell Sorting

An in situ flow cytometric viability assay employing carboxyfluorescein diacetate and propidium iodide was used to identify Streptococcus macedonicus acid tolerance phenotypes. The logarithmic-phase acid tolerance response (L-ATR) was evident when cells were (i) left to autoacidify unbuffered medium, (ii) transiently exposed to nonlethal acidic pH, or (iii) systematically grown under suboptimal acidic conditions (acid habituation). Stationary-phase ATR was also detected; this phenotype was gradually degenerated while cells resided at this phase. Single-cell analysis of S. macedonicus during induction of L-ATR revealed heterogeneity in both the ability and the rate of tolerance acquisition within clonal populations. L-ATR was found to be partially dependent on de novo protein synthesis and compositional changes of the cell envelope. Interestingly, acid-habituated cells were interlaced in lengthier chains and exhibited an irregular pattern of active peptidoglycan biosynthesis sites when probed with BODIPY FL vancomycin. L-ATR caused cells to retain their membrane potential after lethal challenge, as judged by ratiometric analysis with oxonol [DiBAC₄(3)]. Furthermore, F-ATPase was important during the induction of L-ATR, but in the case of a fully launched response, inhibition of F-ATPase affected acid resistance only partially. Activities of both F-ATPase and the glucose-specific phosphoenolpyruvate-dependent phosphotransferase system were increased after L-ATR induction, distinguishing S. macedonicus from oral streptococci. Finally, the in situ viability assessment was compared to medium-based recovery after single-cell sorting, revealing that the culturability of subpopulations with identical fluorescence characteristics is dependent on the treatments imposed to the cells prior to acid challenge.     

Comparative and evolutionary analysis of plasmid pREN isolated from Lactobacillus rennini, a novel member of the theta-replicating pUCL287 family

Here, we describe plasmid pREN of Lactobacillus rennini ACA-DC 1534, isolated from traditional Kopanisti cheese. pREN is a circular molecule of 4371 bp. Orf calling revealed a novel repA-orf2 operon with the deduced product of orf2 showing no similarity to other known proteins. Downstream of this operon, a gene cluster encoding different mobilization proteins, namely mobC, mobA1, mobA2 and mobB, was detected. Based on the sequence of the origin of replication (ori) and the similarity pattern of RepA, pREN was placed in the pUCL287 family of theta-replicating plasmids. Multiple sequence alignment demonstrated for the first time the degree of conservation in the pUCL287 oris. Our analysis supported that the identified conserved repeats could drive similar secondary structures in the oris of all plasmids. Furthermore, comparative mapping of pREN with its related plasmids (i.e. pLB925A03 and pLJ42) showed that they retain a unique combination in the architecture of their replication and mobilization elements within the pUCL287 family. Phylogenetic analysis also established that these plasmids have undergone a modular evolutionary process in order to acquire their mob genes. Research on plasmids from uncommon lactic acid bacteria will expand our appreciation for their divergence and will aid their rational selection for biotechnological applications.     

The biodiversity of lactic acid bacteria in Greek traditional wheat sourdoughs is reflected in both composition and metabolite formation

Lactic acid bacteria (LAB) were isolated from Greek traditional wheat sourdoughs manufactured without the addition of baker's yeast. Application of sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total cell protein, randomly amplified polymorphic DNA-PCR, DNA-DNA hybridization, and 16S ribosomal DNA sequence analysis, in combination with physiological traits such as fructose fermentation and mannitol production, allowed us to classify the isolated bacteria into the species Lactobacillus sanfranciscensis, Lactobacillus brevis, Lactobacillus paralimentarius, and Weissella cibaria. This consortium seems to be unique for the Greek traditional wheat sourdoughs studied. Strains of the species W. cibaria have not been isolated from sourdoughs previously. No Lactobacillus pontis or Lactobacillus panis strains were found. An L. brevis-like isolate (ACA-DC 3411 t1) could not be identified properly and might be a new sourdough LAB species. In addition, fermentation capabilities associated with the LAB detected have been studied. During laboratory fermentations, all heterofermentative sourdough LAB strains produced lactic acid, acetic acid, and ethanol. Mannitol was produced from fructose that served as an additional electron acceptor. In addition to glucose, almost all of the LAB isolates fermented maltose, while fructose as the sole carbohydrate source was fermented by all sourdough LAB tested except L. sanfranciscensis. Two of the L. paralimentarius isolates tested did not ferment maltose; all strains were homofermentative. In the presence of both maltose and fructose in the medium, induction of hexokinase activity occurred in all sourdough LAB species mentioned above, explaining why no glucose accumulation was found extracellularly. No maltose phosphorylase activity was found either. These data produced a variable fermentation coefficient and a unique sourdough metabolite composition.     

Purification and partial characterization of an intracellular aminopeptidase from Streptococcus salivarius subsp. thermophilus strain ACA-DC 114

E. TSAKALIDOU AND G. KALANTZOPOULOS. 1992. An intracellular aminopeptidase from Streptococcus salivarius subsp. thermophilus strain ACA-DC 114, isolated from traditional Greek yoghurt, was purified by chromatography on DEAE-cellulose and Sephadex G-100. The enzyme had a molecular weight of 89 000. It was active over a pH range 4.5-9.5 and had optimum activity on L-lysyl-4-nitroanilide at pH 6.5 and 35°C with K _m= 1.80 mmol/l; above 55°C the enzyme activity declined rapidly. The aminopeptidase was capable of degrading substrates by hydrolysis of the N -terminal amino acid; it had very low endopeptidase and no carboxypeptidase activity. The enzyme was strongly inactivated by EDTA. Serine and sulphydryl group reagents had no effect on enzyme activity.     

Macedocin,a food-grade lantibiotic produced by Streptococcus macedonicus ACA-DC 198

Streptococcus macedonicus ACA-DC 198, a strain isolated from Greek Kasseri cheese, produces a food-grade lantibiotic named macedocin. Macedocin has a molecular mass of 2,794.76 +/- 0.42 Da, as determined by electrospray mass spectrometry. Partial N-terminal sequence analysis revealed 22 amino acid residues that correspond with the amino acid sequence of the lantibiotics SA-FF22 and SA-M49, both of which were isolated from the pathogen Streptococcus pyogenes. Macedocin inhibits a broad spectrum of lactic acid bacteria, as well as several food spoilage and pathogenic bacteria, including Clostridium tyrobutyricum. It displays a bactericidal effect towards the most sensitive indicator strain, Lactobacillus sakei subsp. sakei LMG 13558(T), while the producer strain itself displays autoinhibition when it is grown under conditions that do not favor bacteriocin production. Macedocin is active at pHs between 4.0 and 9.0, and it retains activity even after incubation for 20 min at 121 degrees C with 1 atm of overpressure. Inhibition of macedocin by proteolytic enzymes is variable.     

Polyphasic identification of wild yeast strains isolated from Greek sourdoughs

A total of forty-five wild yeast strains were isolated from five traditional Greek wheat sourdoughs. Strains were identified using the classical identification technique along with the sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole cell proteins (SDS-PAGE), Fourier transform-infrared spectroscopy (FT-IR) and the randomly amplified polymorphic DNA-polymerase chain reaction analysis (RAPD-PCR). The latter methods confirmed the classical identification. According to the results obtained, fourteen strains were identified as Saccharomyces cerevisiae strains, twenty-five as Pichia membranaefaciens strains and six as Yarrowia lipolytica.