Effie Tsakalidou

In Vitro and In Vivo Inhibition of Helicobacter pylori by Lactobacillus casei Strain Shirota

ABSTRACT 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.

The Biodiversity of Lactic Acid Bacteria in Greek Traditional Wheat Sourdoughs Is Reflected in Both Composition and Metabolite Formation

ABSTRACT Lactic acid bacteria (LAB) were isolated from Greek traditional wheat sourdoughs manufactured without the addition of bakers 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.

Biochemical properties of enterococci relevant to their technological performance

A total of 129 E. faecium, E. faecalis and E. durans strains of food, veterinary and human origin were screened for biochemical properties relevant to their technological performance. Strains exhibited low milk acidifying ability and low extracellular proteolytic activity, with food origin and E. faecalis strains being generally more active. Their peptidase activities were low and mainly specific against glycine-proline- and glutamate-4-nitroanilide, while only food origin and E. durans strains showed broader substrate specificity. In contrast, their lipolytic activities were relatively higher; food and veterinary origin and E. faecalis strains were the most lipolytic. The post-electrophoretic detection of esterase activities showed that the esterolytic system of enterococci was rather complex. All species showed strain-to-strain variation in their ability to metabolise citrate and pyruvate, with E. faecalis strains being generally more active. The main volatile compounds produced in milk were acetaldehyde, ethanol and acetoin; generally, E. faecalis strains produced the highest concentrations. None of the strains decarboxylated histidine, lysine and ornithine, but the majority produced tyramine from tyrosine, independently of origin and species. In respect of most biochemical properties considered in this study, E. faecalis strains were generally more active compared to E. faecium and E. durans. This was also the case for the isolates of food origin compared to those of veterinary and human origin. Results obtained allow the selection of enterococci strains to be used as adjunct starters in food fermentations. However, a final selection should take into account the potential virulence factors of enterococci.

Effect of Enterococcus faecium on microbiological, physicochemical and sensory characteristics of Greek Feta cheese.

Greek Feta cheese was prepared using as adjunct starter cultures Enterococcus faecium FAIR-E 198, E. faecium FAIR-E 243, and their combination. Numbers of enterococci in the control and in the batches containing E. faecium strains as adjunct starters rapidly increased until day 15 of ripening, and then remained constant. Both E. faecium strains positively affected the counts of non-starter lactic acid bacteria (NSLAB), micrococci and coliforms, while thermophilic cocci were not influenced. Moreover. E. faecium FAIR-E 243 enhanced the growth of mesophilic cocci and thermophilic bacilli. Physicochemical characteristics, such as pH, moisture, ash, salt in moisture and fat in dry matter (FDM) were not influenced by the addition of the E. faecium strains. The most pronounced effect was observed in the case of proteolysis. Both E. faecium strains, either as sole adjunct starter or in combination, increased the proteolytic index and the free amino groups concentration, and enhanced degradation of alpha(s1)- and beta-caseins in comparison to the control. Furthermore, the reverse-phase (RP)-HPLC peptide profiles of the water-soluble nitrogen (WSN) fractions were significantly affected by the addition of enterococci. The main volatile compounds produced were ethanol, acetate, acetone, acetaldehyde, acetoin and diacetyl, with highest amounts determined for ethanol, followed by acetate. Both E. faecium strains positively affected taste, aroma, colour and structure of the full-ripened cheeses, as well as the overall sensory profile. The present work emphasizes the technological significance of E. faecium strains and supports their use as adjunct cultures in the manufacture of Feta cheese.

Identification of streptococci from Greek Kasseri cheese and description of Streptococcus macedonicus sp. nov.

Taxonomic studies were performed on some Streptococcus-like organisms isolated from naturally fermented Greek Kasseri cheese. By SDS-PAGE analysis of whole-cell proteins the group was found to be quite different from Streptococcus thermophilus. Comparative 16S and 23S rRNA sequence analyses showed that the isolates represent a new species within the genus Streptococcus, where they are most closely related to the Streptococcus bovis cluster. On the basis of these phylogenetic results and some phenotypic differences, a new species, Streptococcus macedonicus, is proposed. The type strain is ACA-DC 206.

Functional properties of novel protective lactic acid bacteria and application in raw chicken meat against Listeria monocytogenes and Salmonella enteritidis

In this study 635 lactic acid bacteria of food origin were evaluated for their potential application as protective cultures in foods. A stepwise selection method was used to obtain the most appropriate strains for application as protective cultures in chicken meat. Specifically, all strains were examined for antimicrobial activity against various Gram positive and Gram negative pathogenic and spoilage bacteria. Strains exhibiting anti-bacterial activity were subsequently examined for survival in simulated food processing and gastrointestinal tract conditions, such as high temperatures, low pH, starvation and the presence of NaCl and bile salts. Selected strains where then examined for basic safety properties such as antibiotic resistance and haemolytic potential, while their antimicrobial activity was further investigated by PCR screening for possession of known bacteriocin genes. Two chosen strains were then applied on raw chicken meat to evaluate their protective ability against two common food pathogens, Listeria monocytogenes and Salmonella enteritidis, but also to identify potential spoilage effects by the application of the protective cultures on the food matrix. Antimicrobial activity in vitro was evident against Gram positive indicators, mainly Listeria and Brochothrix spp., while no antibacterial activity was obtained against any of the Gram negative bacteria tested. The antimicrobial activity was of a proteinaceous nature while strains with anti-listerial activity were found to possess one or more bacteriocin genes, mainly enterocins. Strains generally exhibited sensitivity to pH 2.0, but good survival at 45 degrees C, in the presence of bile salts and NaCl as well as during starvation, while variable survival rates were obtained at 55 degrees C. None of the strains was found to be haemolytic while variable antibiotic resistance profiles were obtained. Finally, when the selected strains Enterococcus faecium PCD71 and Lactobacillus fermentum ACA-DC179 were applied as protective cultures in chicken meat against L. monocytogenes and S. enteritidis respectively, a significantly reduced growth of these pathogenic bacteria was observed. In addition, these two strains did not appear to have any detrimental effect on biochemical parameters related to spoilage of the chicken meat.

Bacteriocin production by Enterococcus faecium FAIR-E 198 in view of its application as adjunct starter in Greek Feta cheese making

Bacteriocin production by Enterococcus faecium FAIR-E 198, isolated from Greek Feta cheese, was studied in batch fermentations, under conditions simulating Feta cheese preparation. Maximum enterocin activity and growth rate was obtained in de Man-Rogosa-Sharpe (MRS) broth at 37 degrees C with controlled pH 6.5. The enterocin was produced throughout the growth phase of the microorganism, showing primary metabolite kinetics with a peak activity during the mid-exponential phase. The use of skimmed milk as substrate revealed low enterocin activity. When fermentations were performed in skimmed milk in the presence of rennet, CaCl2, and a mixed starter culture, no enterocin activity was observed, although the examined strain grew well under the above conditions. Finally, when E. faecium FAIR-E 198 was applied as adjunct starter in Feta cheese making, no enterocin activity was detected throughout ripening. Results obtained underline the frequently underestimated finding that in vitro production by novel bacteriocinogenic starter or co-cultures is no guarantee for in situ efficiency. It was concluded that the complex food environment thoroughly interferes with bacteriocin production levels.

Characterization of Pseudomonas spp. Associated with Spoilage of Gilt-Head Sea Bream Stored under Various Conditions

ABSTRACT The population dynamics of pseudomonads in gilt-head sea bream Mediterranean fish (Sparusaurata) stored under different conditions were studied. Phenotypic analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins were performed to identify a total of 106 Pseudomonas strains isolated from S.aurata stored under different temperatures (at 0, 10, and 20°C) and packaging conditions (air and a modified atmosphere of 40% CO2-30% N2-30% O2). Pseudomonas lundensis was the predominant species, followed by Pseudomonas fluorescens, while Pseudomonas fragi and Pseudomonas putida were detected less frequently. Fluorescent Pseudomonas strains dominated under air conditions, while proteolytic and less lipolytic strains dominated under modified-atmosphere packaging. Different storage conditions appear to govern the selection of pseudomonads in gilt-head sea bream fish.

Lactic acid bacteria efficiently protect human and animal intestinal epithelial and immune cells from enteric virus infection.

Abstract This study aimed to examine the potential antiviral activity of lactic acid bacteria (LAB) using animal and human intestinal and macrophage cell line models of non tumor origin. To this end, LAB strains selected on the basis of previous in vitro trials were co-incubated with cell line monolayers, which were subsequently challenged with rotavirus (RV) and transmissible gastroenteritis virus (TGEV). In order to elucidate the possible mechanism responsible for the antiviral activity, the induction of reactive oxygen species (ROS) release as well as the attachment ability of LAB on the cell lines was investigated. Various strains were found to exhibit moderate to complete monolayer protection against viral RV or TGEV disruption. Highest protection effects were recorded with the known probiotics Lactobacillus rhamnosus GG and Lactobacillus casei Shirota against both RV and TGEV, while notable antiviral activity was also attributed to Enterococcus faecium PCK38, Lactobacillus fermentum ACA-DC179, Lactobacillus pentosus PCA227 and Lactobacillus plantarum PCA236 and PCS22, depending on the cell line and virus combination used. A variable increase (of up to 50%) on the release of NO− and H2O2 (ROS) was obtained when LAB strains were co-incubated with the cell lines, but the results were found to be LAB strain and cell line specific, apart from a small number of strains which were able to induce strong ROS release in more than one cell line. In contrast, the ability of the examined LAB strains to attach to the cell line monolayers was LAB strain but not cell line specific. Highest attachment ability was observed with L. plantarum ACA-DC 146, L. paracasei subsp. tolerans ACA-DC 4037 and E. faecium PCD71. Clear indications on the nature of the antiviral effect were evident only in the case of the L. casei Shirota against TGEV and with L. plantarum PCA236 againt both RV and TGEV. In the rest of the cases, each interaction was LAB-cell line–virus specific, barring general conclusions. However, it is probable that more than one mechanism is involved in the antiviral effect described here. Further investigations are required to elucidate the underlying mode of action and to develop a cell line model as a system for selection of probiotic strains suited for farm animal applications.

Macedocin, a Food-Grade Lantibiotic Produced by Streptococcus macedonicus ACA-DC 198

ABSTRACT 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 13558T, 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°C with 1 atm of overpressure. Inhibition of macedocin by proteolytic enzymes is variable.