Immunostimulatory activity of potential probiotic yeast strains in the dorsal air pouch system and the gut mucosa.

AIMS: To determine the immunostimulatory activity of 15 presumptive probiotic yeast strains in the dorsal air pouch system in comparison with their activity in the gut mucosa. METHODS AND RESULTS: Presumptive probiotic yeast strains previously isolated from human gastrointestinal tract and Feta cheese were further characterized genotypically and biochemically. The Saccharomyces cerevisiae 982, Saccharomyces boulardii KK1 and Kluyveromyces lactis 630 strains exhibited in the air pouch increased polymorphonuclear cell influx and phagocytic activity as well as cytokine production with similar potency as the probiotics Ultra levure S. boulardii and Lactobacillus acidophilus NCFB 1748. Oral administration of these strains in mice results in differential activation of small intestine immune responses concerning IgA and cytokine production as well as Toll-like receptor expression. CONCLUSION: Besides the Saccharomyces strains 982 and KK1, the K. lactis 630 strain could also be considered as a candidate probiotic. SIGNIFICANCE AND IMPACT OF THE STUDY: The air pouch model may be used as an alternative and rapid method for the discrimination and selection of potential probiotic yeast strains.

Validation of the dorsal air pouch model to predict and examine immunostimulatory responses in the gut.

AIMS: To validate the use of the air pouch system to predict and examine early immune responses induced by the presumptive probiotics Lactobacillus paracasei subsp. paracasei B112, DC205, DC215 and DC412 strains in the gut mucosa. METHODS AND RESULTS: Only the DC412 strain interacted strongly with the cells forming the air pouch lining tissue and induced early innate immune responses such as polymorphonuclear (PMN) cell recruitment, phagocytosis and tumour necrosis factor alpha (TNF-alpha) production that equal the respective responses induced by the probiotic Lactobacillus acidophilus NCFB 1748. The strains exhibiting strong immunoregulatory activity in the air pouch also interacted strongly with the gut-associated lymphoid tissue (GALT). The strain DC412 exerts its effect on the intestine through stimulation of Toll-like receptor (TLR)2/TLR4-mediated signalling events leading to secretion of a certain profile of cytokines in which gamma interferon (IFN-gamma), TNF-alpha, interleukin (IL)-6 and IL-10 are included. The probiotic Lact. acidophilus NCFB 1748 induces the same cytokine profile in addition to IL-12B, and this response is potentially mediated by the synergy of TLR2 and TLR9. CONCLUSION: The strain DC412 possesses the in vitro and in vivo characteristics of a probiotic micro-organism. SIGNIFICANCE AND IMPACT OF THE STUDY: The dorsal mouse or rat air pouch may be used as an alternative and rapid method for the initial discrimination and selection of potential probiotic Lactobacillus strains.

Changes in numbers and kinds of bacteria during a chickpea submerged fermentation used as a leavening agent for bread production.

The microflora developed during a submerged fermentation of coarsely ground chickpea (Cicer arietinum L.) in water (primary starter) and during raising a dough from wheat flour (adapted starter) was studied. In the fermenting liquid, only populations of Bacillus and Clostridium developed. Bacilli increased their loads significantly (p<0.05) during fermentation for 8-12 h and then remained constant. Clostridia developed (p<0.05) subsequently to levels of 10(7) cfu/ml at 18 h, when the pH of the fermenting liquid had decreased (p<0.05) to approximately 5.4. It also seems that the rise of the adapted starter within 2 h was caused by enzymes present in the primary starter and those liberated after cell death by the declining populations of bacilli and clostridia. The principal groups of isolates in all fermentation experiments (with chickpea seeds from five different areas) seemed to have the phenotypic characteristics of Bacillus cereus group and Clostridium perfringens. SDS-PAGE of whole-cell proteins elucidated the taxonomic position of the B. cereus group of strains as B. cereus and Bacillus thuringiensis and confirmed the phenotypic allocation of C. perfringens isolates. Strains phenotypically characterized as Bacillus licheniformis and Clostridium beijerinckii were also found to belong to these same species by SDS-PAGE. In addition, results showed that the fermenting broth was not toxic to mice when inoculated intraperitoneally and the product can thus be considered as safe for consumption.

Genotypic and phenotypic diversity of Lactococcus lactis isolates from Batzos, a Greek PDO raw goat milk cheese.

The genotypic and phenotypic variability of 40 Lactococcus lactis isolates obtained from three cheese-making trials of Batzos cheese made one in each, winter, spring and summer was investigated. RAPD-PCR, plasmid profiling and PFGE were used to study the genetic variability and distinguish closely related isolates. Results showed a high degree of heterogeneity among strains. According to PFGE data, all strains except one were clustered together (at a similarity level of approximately 50%) with the L. lactis subsp. lactis reference strain and eleven groups of isolates consisting of 2-8 strains each were distinguished. Plasmid profiling results revealed that there were eight isolates lacking plasmids and nine having unique plasmids. Twenty-three isolates were allocated into six groups. There was an interesting similarity between the plasmid profiling groups and those formed according to PFGE. Clustering of strains according to RAPD-PCR was in agreement with results obtained by both plasmid profiling and PFGE for the majority of the strains. In addition, results obtained by molecular methods indicate a grouping of most of the strains according to the season of cheese production. All strains inhibited the growth of Escherichia coli O157:H7. Their ability to affect the growth of Yersinia enterocolitica, Staphylococcus aureus, Bacillus cereus, Listeria monocytogenes and Enterococcus faecalis was strain dependent. In 42.5% of the isolates high acidifying ability in milk after 24 h was recorded and these were isolates, mainly, from fresh cheese. The 75% of the isolates from winter cheese exhibited higher Lys- than Leu-aminopeptidase activity while the approximately 67% of the isolates from summer cheese showed higher Leu- than Lys-aminopeptidase activity. Their caseinolytic activity after growth in milk for 24 h was significant with preference for alpha(s)-casein degradation. The majority (90%) of the strains formed methanethiol from methionine and this ability was strain dependent. These results suggest that among the wild lactococcal population from Batzos cheese there are interesting strains appropriate to be used as starters for the dairy industry.

Genotypic and phenotypic heterogeneity in Enterococcus isolates from Batzos, a raw goat milk cheese.

This study investigated the genotypic and phenotypic diversity in 34 isolates of enterococci obtained during ripening of Batzos cheese from raw goat milk and characterized phenotypically as Enterococcus durans. RAPD-PCR, plasmid profiling and PFGE were used to study the genetic variability and distinguish closely related isolates. Species recognition by means of RAPD-PCR was in agreement with the phenotypic identification for 29 strains. One strain was characterized as Lactococcus lactis subsp. lactis by RAPD-PCR and four strains were grouped with the Enterococcus faecium reference strain. All strains were vancomycin sensitive, while 10 strains showed beta-haemolytic reaction on human blood and the majority of them (88.9%) showed decarboxylase activity on tyramine. All strains exhibited antagonistic activity against Bacillus cereus, Staphylococcus aureus, Escherichia coli and Listeria monocytogenes and the majority inhibited Enterococcus faecalis. Isolates displayed weak acidifying ability and low proteolytic activities when grown in milk for 24h. However, their caseinolytic activity after growth in milk for seven days was significant with preference for alphas-casein degradation.

Assimilation of cholesterol by yeast strains isolated from infant feces and Feta cheese.

Eight yeast strains isolated from infant feces and the traditional Greek Feta cheese, selected for their probiotic properties, were tested along with a commercially available strain of Saccharomyces boulardii for their ability to remove cholesterol from a growth medium (yeast extract glucose peptone broth) supplemented with 0.3% Oxgall. The amount of cholesterol removed during 72 h of growth at 37 degrees C revealed significant variations among the yeast strains examined. Two isolates from infant feces, namely Saccharomyces cerevisiae KK1 and Isaatchenkia orientalis KK5.Y.1 and one isolate from Feta cheese, namely S. cerevisiae 832, along with the commercial strain S. boulardii, were able to remove cholesterol from the growth medium after 48 h of incubation at 37 degrees C. However, Saccharomyces strains proved to be able to remove cholesterol even after 24 h of growth at 37 degrees C. The cholesterol removed from the growth medium was not metabolically degraded but was rather assimilated into the yeast cells. The ability to assimilate cholesterol in vitro and to tolerate low pH levels, gastric juice, and bile indicate that S. cerevisiae 832, and especially S. cerevisiae KK1 and I. orientalis KK5.Y.1 (being more bile and gastric juice tolerant because of their human origin) may be promising candidate strains for use as probiotics.

Protease, peptidase and esterase activities by lactobacilli and yeast isolates from Feta cheese brine.

AIMS: The study of peptidase, esterase and caseinolytic activity of Lactobacillus paracasei subsp. paracasei, Debaryomyces hansenii and Sacchromyces cerevisiae isolates from Feta cheese brine. METHODS AND RESULTS: Cell-free extracts from four strains of Lact. paracasei subsp. paracasei, four strains of D. hansenii and three strains of S. cerevisiae, isolated from Feta cheese brine were tested for their proteolytic and esterase enzyme activities. Lactobacillus paracasei subsp. paracasei strains had intracellular aminopeptidase, dipeptidyl aminopeptidase, dipeptidase, endopeptidase and carboxypeptidase activities. Esterases were detected in three of four strains of lactobacilli and their activities were smaller with higher molecular weight fatty acids. The strains of yeasts did not exhibit endopeptidase as well as dipeptidase activities except on Pro-Leu. Their intracellular proteolytic activity was higher than that of lactobacilli. Esterases from yeasts preferentially degraded short chain fatty acids. Lactobacilli degraded preferentially beta-casein. Caseinolytic activity of yeasts was higher than that of lactobacilli. CONCLUSIONS: The results suggest that Lact. paracasei subsp. paracasei and yeasts may contribute to the development of flavour in Feta cheese. SIGNIFICANCE AND IMPACT OF THE STUDY: Selected strains could be used as adjunct starters to make high quality Feta cheese.

Characterization of lactic acid bacteria isolated from a Greek dry-fermented sausage in respect of their technological and probiotic properties.

A total of 147 lactic acid bacteria was isolated from two types of naturally fermented dry sausages at four different stages of the ripening process studied in order to select the most suitable strains according to their technological characteristics including probiotic properties and antimicrobial activity against food-borne pathogens. Identification of the isolates revealed that 90% were lactobacilli, 4% enterococci, 3% Pediococcus sp. and sporadic isolates of Weissella viridescens, Leuconostoc pseudomesenteroides, and Leuconostoc sp. The isolated strains of Lactobacillus sakei (49 isolates), Lactobacillus curvatus (24 isolates) and Lactobacillus plantarum (7 isolates) were further characterized. All strains could grow at 15 °C, whereas the majority of the strains was able to grow in the presence of 6.5% NaCl and on acetate agar. The enzymatic potential of the strains was evaluated using the API ZYM system. During in vitro investigations all strains exhibited high leucine and valine aminopeptidase activities and moderate acid phosphatase and phosphohydrolase activities. Some strains showed very weak lipolytic activity. The enzyme profiling is an important factor for selection of strains as starter cultures. A large majority of the strains tolerated 0.1% bile salts whereas 58% of Lactobacillus curvatus strains and all Lactobacillus plantarum strains were resistant to 0.3% bile salts. All Lactobacillus sakei strains and the majority of Lactobacillus curvatus and Lactobacillus plantarum strains exhibited an anti-listerial activity against three Listeria monocytogenes strains. A percentage of 75, 50 and 29% of Lactobacillus sakei, L. curvatus and L. plantarum strains, respectively, could inhibit two Staphylococcus aureus strains. The contribution of the selected strains to a possible inhibition of Listeria monocytogenes and S. aureus in situ on fermented meats would be of considerable interest to enhance the hygienic quality of these products.

Technologically important properties of lactic acid bacteria isolates from Beyaz cheese made from raw ewes' milk.

AIMS: The aim of the study was to characterize isolates of lactic acid bacteria from Beyaz cheese and to study some of their technologically important properties. METHODS AND RESULTS: Seventy-seven lactic acid bacteria isolated from Beyaz, a Turkish white-brined cheese made from raw ewes' milk without any starter, were classified by phenotypic criteria and sodium dodecyl sulphate-polyacrylamide gel electrophoresis of whole-cell proteins. Whole cells of Lactococcus lactis subsp. lactis and enterococci showed lipolytic and proteolytic activities. Strains were found that differed in terms of their acidifying and caseinolytic activity. Most of the enterococci isolates showed tyrosine decarboxylase activity; moreover, lactobacilli exhibited weak antibacterial activities against foodborne pathogens. CONCLUSIONS: Strains of lactic acid bacteria with interesting biotechnologically important properties may be found in Beyaz cheese. SIGNIFICANCE AND IMPACT OF THE STUDY: Lactic acid bacteria isolates with interesting biotechnological profiles may influence the quality and variety of dairy products, if they are used as starters, and their cheese-making characteristics seem promising.

Some probiotic properties of yeast isolates from infant faeces and Feta cheese.

Yeast isolates from infant faeces and Feta cheese were characterized to species level by phenotypic criteria, Randomly Amplified Polymorphic DNA (RAPD)-PCR and mitochondrial DNA (mt-DNA) restriction analysis. Results suggested that there is a good agreement between phenotypic characterization of yeasts and RAPD-PCR at species level; in addition, RAPD-PCR as well as mt-DNA restriction analysis provided good discrimination at strain level. Some technological and probiotic properties of selected strains were also investigated. The test strains exhibited lipolytic and proteolytic activities. They also tolerated low pH and survived satisfactory in gastric juice in vitro as well as in the presence of bile. In general, the isolates from faeces were more resistant to low pH and bile than those from Feta cheese. Selected strains could be used as starter supplements for industrial fermentations.

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.

Differentiation of Lactobacillus isolates from infant faeces by SDS-PAGE and rRNA-targeted oligonucleotide probes.

Isolates of lactobacilli from infant faeces phenotypically characterized as Lactobacillus paracasei subsp. paracasei (six strains), Lact. rhamnosus (six strains), Lact. gasseri (three strains), Lact. acidophilus (one strain) and Lact. fermentum/reuteri (three strains) according to recent classification systems were subjected to SDS-PAGE of whole cell proteins and rRNA-targeted oligonucleotide probe hybridization, in order to confirm the phenotypic characterization and elucidate the exact taxonomic position of the three strains that had properties between fermentum and reuteri. Results suggested a good agreement between the phenotypic characterization, SDS-PAGE and rRNA-targeted oligonucleotide probe hybridization for strains of all species except for the Lact. fermentum/reuteri strains. Results obtained by rRNA probes suggested a possible phylogenetic relatedness of the strains to Lact. reuteri. Isolates from infant faeces with interesting probiotic properties could be used as components of fermented milk products.

Microbiological characteristics of anevato: a traditional greek cheese

Nine batches of Anevato, raw goat milk cheese, were examined throughout a 60 day storage time at three different periods within the lactation season of the goat. High mean log counts per gram of cheese for aerobic bacteria (7.92-9.56), lactic acid bacteria (7. 78-9.32), Gram-negative organisms 5.64-9.67), psychrotrophs (7.90-11. 79) and proteolytic bacteria (7.57-9.36) were found. Enterobacteriaceae, coliforms and yeasts were considerably lower. Enterobacteriaceae and coliforms in the curd of cheese made in May were lower by approximately 3.0 log10 cfu g-1 than counts in curd made in January, and were lower by about 2.5 log10 cfu g-1 than those in cheese made in March. This coincided with lower pH and higher counts of lactic acid bacteria in cheese made in March and May. Yeast populations were affected by the season and were higher in May than March and/or January. Lactococci dominated in the cheese until 15 days, but lactobacilli became predominant after 30 days. Lactococcus lactis was the most abundant species of lactic acid bacteria found in Anevato cheese. Results suggest the need for improving milk quality and/or using heat-treated milk to produce Anevato cheese; the use of L. lactis as a starter would possibly eliminate or suppress the growth of undesirable organisms.