Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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    Impact of growth conditions and role of sigB on Listeria monocytogenes fitness in single and mixed biofilms cultured with Lactobacillus plantarum
    Saa Ibusquiza, P. ; Nierop Groot, M.N. ; Deban Valles, A. ; Abee, T. ; Besten, H.M.W. den - \ 2015
    Food Research International 71 (2015). - ISSN 0963-9969 - p. 140 - 145.
    lactic-acid bacteria - oxidative stress resistance - gram-positive bacteria - superoxide-dismutase - tolerance response - arginine deiminase - species biofilms - stainless-steel - sodium-chloride - sigma(b)
    The role of sigB, a major transcriptional regulator of stress response genes, was assessed in formation of single and mixed species biofilms of Listeria monocytogenes EGD-e and Lactobacillus plantarum WCFS1 as secondary species at 20 °C and 30 °C using different medium compositions (nutrient-rich medium with and without supplementary manganese, glucose and salt). L. monocytogenes showed significant biofilm development at both temperatures and in all media tested although less biofilm was formed when glucose was supplemented only. The contribution of L. monocytogenes to the mixed species biofilm declined especially at higher temperature in glucose-rich medium in the absence and presence of manganese, due to lactic acid formation with concomitant decrease in culture pH below the pHmin of L. monocytogenes. Using an in-frame sigB deletion mutant and a complementation mutant we showed that sigB contributed to survival under these acid stress conditions. Notably, the additional presence of salt protected L. monocytogenes in the acidic mixed species biofilms resulting in an increase of around 2–3 log10 cfu/ml and this phenomenon showed to be sigB-dependent.
    Characterisation of biofilms formed by Lactobacillus plantarum WCFS1 and food spoilage isolates
    Fernández Ramírez, M.D. ; Smid, E.J. ; Abee, T. ; Nierop Groot, M.N. - \ 2015
    International Journal of Food Microbiology 207 (2015). - ISSN 0168-1605 - p. 23 - 29.
    lactic-acid bacteria - enterococcal surface protein - listeria-monocytogenes - pseudomonas-putida - bacillus-subtilis - starter cultures - genetic-analysis - rhamnosus gg - resistance - industry
    Lactobacillus plantarum has been associated with food spoilage in a wide range of products and the biofilm growth mode has been implicated as a possible source of contamination. In this study we analysed the biofilm forming capacity of L. plantarum WCFS1 and six food spoilage isolates. Biofilm formation as quantified by crystal violet staining and colony forming units was largely affected by the medium composition, growth temperature and maturation time and by strain specific features. All strains showed highest biofilm formation in Brain Heart Infusion medium supplemented with manganese and glucose. For L. plantarum biofilms the crystal violet (CV) assay, that is routinely used to quantify total biofilm formation, correlates poorly with the number of culturable cells in the biofilm. This can in part be explained by cell death and lysis resulting in CV stainable material, conceivably extracellular DNA (eDNA), contributing to the extracellular matrix. The strain to strain variation may in part be explained by differences in levels of eDNA, likely as result of differences in lysis behaviour. In line with this, biofilms of all strains tested, except for one spoilage isolate, were sensitive to DNase treatment. In addition, biofilms were highly sensitive to treatment with Proteinase K suggesting a role for proteins and/or proteinaceous material in surface colonisation. This study shows the impact of a range of environmental factors and enzyme treatments on biofilm formation capacity for selected L. plantarum isolates associated with food spoilage, and may provide clues for disinfection strategies in food industry.
    The relationship between fermented food intake and mortality risk in the European Prospective Investigation into Cancer and Nutrition-Netherlands cohort
    Praagman, J. ; Dalmeijer, G.W. ; Schouw, Y.T. van der; Soedamah-Muthu, S.S. ; Verschuren, W.M.M. ; Bueno-de Mesquita, H.B. ; Geleijnse, J.M. ; Beulens, J.W.J. - \ 2015
    The British journal of nutrition 113 (2015). - ISSN 0007-1145 - p. 498 - 506.
    coronary-heart-disease - lactic-acid bacteria - dairy-products - colorectal-cancer - consumption - stroke - metaanalysis - questionnaire - menaquinone - men
    The objective of the present study was to investigate the relationship between total and subtypes of bacterial fermented food intake (dairy products, cheese, vegetables and meat) and mortality due to all causes, total cancer and CVD. From the European Prospective Investigation into Cancer and Nutrition-Netherlands cohort, 34 409 Dutch men and women, aged 20–70 years who were free from CVD or cancer at baseline, were included. Baseline intakes of total and subtypes of fermented foods were measured with a validated FFQ. Data on the incidence and causes of death were obtained from the national mortality register. Cox proportional hazards models were used to analyse mortality in relation to the quartiles of fermented food intake. After a mean follow-up of 15 (sd 2·5) years, 2436 deaths occurred (1216 from cancer and 727 from CVD). After adjustment for age, sex, total energy intake, physical activity, education level, hypertension, smoking habit, BMI, and intakes of fruit, vegetables and alcohol, total fermented food intake was not found to be associated with mortality due to all causes (hazard ratio upper v. lowest quartile (HRQ4 v. Q1) 1·00, 95 % CI 0·88, 1·13), cancer (HRQ4 v. Q1 1·02, 95 % CI 0·86, 1·21) or CVD (HRQ4 v. Q1 1·04, 95 % CI 0·83, 1·30). Bacterial fermented foods mainly consisted of fermented dairy foods (78 %) and cheese (16 %). None of the subtypes of fermented foods was consistently related to mortality, except for cheese which was moderately inversely associated with CVD mortality, and particularly stroke mortality (HRQ4 v. Q1 0·59, 95 % CI 0·38, 0·92, Ptrend= 0·046). In conclusion, the present study provides no strong evidence that intake of fermented foods, particularly fermented dairy foods, is associated with mortality.
    Effect of sublethal preculturing on the survival of probiotics and metabolite formation in set-yoghurt
    Settachaimongkon, S. ; Valenberg, H.J.F. van; Winata, V. ; Wang, X. ; Nout, M.J.R. ; Hooijdonk, A.C.M. van; Zwietering, M.H. ; Smid, E.J. - \ 2015
    Food Microbiology 49 (2015). - ISSN 0740-0020 - p. 104 - 115.
    lactic-acid bacteria - fermented milks - tolerance response - functional foods - stress responses - bifidobacteria - lactobacillus - cultures - strains - microorganisms
    The objective of this study was to investigate the effect of preculturing of Lactobacillus rhamnosus GG and Bifidobacterium animalis subsp. lactis BB12 under sublethal stress conditions on their survival and metabolite formation in set-yoghurt. Prior to co-cultivation with yoghurt starters in milk, the two probiotic strains were precultured under sublethal stress conditions (combinations of elevated NaCl and low pH) in a batch fermentor. The activity of sublethally precultured probiotics was evaluated during fermentation and refrigerated storage by monitoring bacterial population dynamics, milk acidification and changes in volatile and non-volatile metabolite profiles of set-yoghurt. The results demonstrated adaptive stress responses of the two probiotic strains resulting in their viability improvement without adverse influence on milk acidification. A complementary metabolomic approach using SPME-GC/MS and 1H-NMR resulted in the identification of 35 volatiles and 43 non-volatile polar metabolites, respectively. Principal component analysis revealed substantial impact of the activity of sublethally precultured probiotics on metabolite formation demonstrated by distinctive volatile and non-volatile metabolite profiles of set-yoghurt. Changes in relative abundance of various aroma compounds suggest that incorporation of stress-adapted probiotics considerably influences the organoleptic quality of product. This study provides new information on the application of stress-adapted probiotics in an actual food-carrier environment
    Construction and validation of a mCherry protein vector for promoter analysis in Lactobacillus acidophilus
    Mohedano, M.L. ; Garcia-Cayuela, T. ; Perez-Ramos, A. ; Gaiser, R.A. ; Requena, T. ; Lopez, P. - \ 2015
    Journal of Industrial Microbiology and Biotechnology 42 (2015)2. - ISSN 1367-5435 - p. 247 - 253.
    lactic-acid bacteria - controlled gene-expression - streptococcus-pneumoniae - lactococcus-lactis - plasmid - cloning
    Lactobacilli are widespread in natural environments and are increasingly being investigated as potential health modulators. In this study, we have adapted the broad-host-range vector pNZ8048 to express the mCherry protein (pRCR) to expand the usage of the mCherry protein for analysis of gene expression in Lactobacillus. This vector is also able to replicate in Streptococcus pneumoniae and Escherichia coli. The usage of pRCR as a promoter probe was validated in Lactobacillus acidophilus by characterizing the regulation of lactacin B expression. The results show that the regulation is exerted at the transcriptional level, with lbaB gene expression being specifically induced by co-culture of the L. acidophilus bacteriocin producer and the S. thermophilus STY-31 inducer bacterium.
    Spoilage evaluation, shelf-life prediction, and potential spoilage organisms of tropical brackish water shrimp (Penaeus notialis) at different storage temperatures
    Dabade, D.S. ; Besten, H.M.W. den; Azokpota, P. ; Nout, M.J.R. ; Hounhouigan, D.J. ; Zwietering, M.H. - \ 2015
    Food Microbiology 48 (2015). - ISSN 0740-0020 - p. 8 - 16.
    lactic-acid bacteria - cold-smoked salmon - modified atmosphere - parapenaeus-longirostris - pandalus-borealis - chilled storage - chemical characteristics - indole production - hydrogen-sulfide - microbial-flora
    Maintaining the freshness of shrimp is a concern to shrimp stakeholders. To improve shrimp quality management, it is of importance to evaluate shrimp spoilage characteristics. Therefore, microbiological, sensory, and chemical changes of naturally contaminated tropical brackish water shrimp (Penaeus notialis) during storage at 28 °C, 7 °C and 0 °C were assessed. H2S-producing bacteria were the dominant group of microorganisms at 28 °C and 7 °C whereas Pseudomonas spp. were dominant at 0 °C. Total volatile basic nitrogen and trimethylamine correlated well (R2 > 0.90) with the sensory scores. An empirical model to predict the shelf-life of naturally contaminated tropical shrimp as a function of storage temperature was developed. Specific groups of organisms were isolated at the sensory rejection times and assessed for spoilage potential in shrimps of which the endogenous flora was heat inactivated. Isolates capable of producing strong off-odor identified by 16S rRNA sequencing were mainly lactic acid bacteria (LAB) and Enterobacteriaceae at 28 °C or 7 °C and Pseudomonas spp. and LAB at 0 °C. The study contributes to the knowledge about tropical shrimp spoilage and provides a basis for the development of methods and tools to improve shrimp quality management. Keywords: Shrimp quality; Microbiological change; TVBN; Sensory rejection; Shelf-life prediction
    Interactions between formulation and spray drying conditions related to survival of Lactobacillus plantarum WCFS1
    Perdana, J.A. ; Fox, M.B. ; Siwei, C. ; Boom, R.M. ; Schutyser, M.A.I. - \ 2014
    Food Research International 56 (2014). - ISSN 0963-9969 - p. 9 - 17.
    glass-transition temperature - membrane phase-behavior - lactic-acid bacteria - flow-cytometry - industrial applications - dairy ingredients - osmotic-stress - water activity - rhamnosus gg - gel phase
    Protective solid carriers are commonly added to probiotic cultures prior to drying. Their formulation is not trivial and depends on the drying conditions applied. In this study, we systematically investigated the influence of formulation parameters on the survival of Lactobacillus plantarum WCFS1 after drying. Low molecular weight carbohydrates (less than 2 kDa) with high glass transition temperatures provided the highest level of protection at both low (25 degrees C) and high (50 degrees C or higher) drying temperatures. Low molecular weight carbohydrates may provide stabilization by closely interacting with the lipid bilayer of the cell membranes. Meanwhile, carbohydrates with high glass transition temperatures probably provide stabilization via fixation of the cells in a glassy powder. Furthermore, adequate amounts of solid carrier are required to sufficiently stabilize the cells during drying. During drying, crystallization of solid carriers may occur. Depending on the crystal geometry, crystallization can be either beneficial (e.g. with mannitol or sorbitol) or detrimental (e.g. with lactose) to cell survival. Finally, the effect of formulation on cell viability during storage was studied. A decimal reduction time of approximately 300 days was observed when spray dried L. plantarum WCFS1 was stored at temperatures below 40 degrees C. The outcome of this study was used as a basis to construct a generalized diagram to indicate the combinations of formulation and drying conditions to maximally retain viability and operate dryers at high efficiency. (C) 2013 Elsevier Ltd. All rights reserved.
    H2O2 Production in Species of the Lactobacillus acidophilus Group: a Central Role for a Novel NADH-Dependent Flavin Reductase
    Hertzberger, R. ; Arents, J. ; Dekker, H.L. ; Pridmore, R.D. ; Gysler, C. ; Kleerebezem, M. ; Mattos, M.J.T. de - \ 2014
    Applied and Environmental Microbiology 80 (2014)7. - ISSN 0099-2240 - p. 2229 - 2239.
    hydrogen-peroxide production - alkyl hydroperoxide reductase - activated-receptor-gamma - lactic-acid bacteria - escherichia-coli - streptococcus-pneumoniae - amphibacillus-xylanus - pseudomonas-putida - johnsonii ncc-533 - crystal-structure
    Hydrogen peroxide production is a well-known trait of many bacterial species associated with the human body. In the presence of oxygen, the probiotic lactic acid bacterium Lactobacillus johnsonii NCC 533 excretes up to 1 mM H2O2, inducing growth stagnation and cell death. Disruption of genes commonly assumed to be involved in H2O2 production (e.g., pyruvate oxidase, NADH oxidase, and lactate oxidase) did not affect this. Here we describe the purification of a novel NADH-dependent flavin reductase encoded by two highly similar genes (LJ_0548 and LJ_0549) that are conserved in lactobacilli belonging to the Lactobacillus acidophilus group. The genes are predicted to encode two 20-kDa proteins containing flavin mononucleotide (FMN) reductase conserved domains. Reductase activity requires FMN, flavin adenine dinucleotide (FAD), or riboflavin and is specific for NADH and not NADPH. The K-m for FMN is 30 +/- 8 mu M, in accordance with its proposed in vivo role in H2O2 production. Deletion of the encoding genes in L. johnsonii led to a 40-fold reduction of hydrogen peroxide formation. H2O2 production in this mutant could only be restored by in trans complementation of both genes. Our work identifies a novel, conserved NADH-dependent flavin reductase that is prominently involved in H2O2 production in L. johnsonii.
    Development of the recombinase-based in vivo expression technology in Streptococcus thermophilus and validation using the lactose operon promoter
    Junjua, M. ; Galia, W. ; Gaci, N. ; Uriot, O. ; Genay, M. ; Bachmann, H. ; Kleerebezem, M. ; Dary, A. ; Roussel, Y. - \ 2014
    Journal of Applied Microbiology 116 (2014)3. - ISSN 1364-5072 - p. 620 - 631.
    lactic-acid bacteria - gene-expression - lactococcus-lactis - human gut - yogurt - system - mice - identification - transformation - proteinase


    To construct and validate the recombinase-based in vivo expression technology (R-IVET) tool in Streptococcus thermophilus (ST).

    Methods and Results

    The R-IVET system we constructed in the LMD-9 strain includes the plasmid pULNcreB allowing transcriptional fusion with the gene of the site-specific recombinase Cre and the chromosomal cassette containing a spectinomycin resistance gene flanked by two loxP sites. When tested in M17 medium, promoters of the genes encoding the protease PrtS, the heat-shock protein Hsp16 and of the lactose operon triggered deletion of the cassette, indicating promoter activity in these conditions. The lactose operon promoter was also found to be activated during the transit in the murine gastrointestinal tract.


    The R-IVET system developed in ST is relatively stable, functional, very sensitive and can be used to assay activity of promoters, which are specifically active in in vivo conditions.

    Significance and Impact of the Study

    This first adaptation of R-IVET to ST provides a highly valuable tool allowing an exploration of the physiological state of ST in the GIT of mammals, fermentation processes or dairy products.
    Functional characterization of probiotic surface layer protein-carrying Lactobacillus amylovorus strains
    Hynönen, U. ; Kant, R. ; Lähteinen, T. ; Pietilä, T.E. ; Beganovic, J. ; Smidt, H. ; Uroic, K. ; Åvall-Jääskeläinen, S. ; Palva, A. - \ 2014
    BMC Microbiology 14 (2014). - ISSN 1471-2180 - 16 p.
    enterotoxigenic escherichia-coli - human intestinal mucus - lactic-acid bacteria - human dendritic cells - s-layer - in-vitro - acidophilus ncfm - aeromonas-salmonicida - epithelial-cells - immune-system
    Background - Adhesiveness to intestinal epithelium, beneficial immunomodulating effects and the production of pathogen-inhibitory compounds are generally considered as beneficial characteristics of probiotic organisms. We showed the potential health-promoting properties and the mechanisms of probiotic action of seven swine intestinal Lactobacillus amylovorus isolates plus the type strain (DSM 20531T) by investigating their adherence to porcine intestinal epithelial cells (IPEC-1) and mucus as well as the capacities of the strains to i) inhibit the adherence of Escherichia coli to IPEC-1 cells, ii) to produce soluble inhibitors against intestinal pathogens and iii) to induce immune signaling in dendritic cells (DCs). Moreover, the role of the L. amylovorus surface (S) –layers - symmetric, porous arrays of identical protein subunits present as the outermost layer of the cell envelope - in adherence to IPEC-1 cells was assessed using a novel approach which utilized purified cell wall fragments of the strains as carriers for the recombinantly produced S-layer proteins. Results - Three of the L. amylovorus strains studied adhered to IPEC-1 cells, while four strains inhibited the adherence of E. coli, indicating additional mechanisms other than competition for binding sites being involved in the inhibition. None of the strains bound to porcine mucus. The culture supernatants of all of the strains exerted inhibitory effects on the growth of E. coli, Salmonella, Listeria and Yersinia, and a variable, strain-dependent induction was observed of both pro- and anti-inflammatory cytokines in human DCs. L. amylovorus DSM 16698 was shown to carry two S-layer-like proteins on its surface in addition to the major S-layer protein SlpA. In contrast to expectations, none of the major S-layer proteins of the IPEC-1 -adhering strains mediated bacterial adherence. Conclusions - We demonstrated adhesive and significant pathogen inhibitory efficacies among the swine intestinal L. amylovorus strains studied, pointing to their potential use as probiotic feed supplements, but no independent role could be demonstrated for the major S-layer proteins in adherence to epithelial cells. The results indicate that many intestinal bacteria may coexist with and confer benefits to the host by mechanisms not attributable to adhesion to epithelial cells or mucus.
    Functional implications of the microbial community structure of undefined mesophilic starter cultures
    Smid, E.J. ; Erkus, O. ; Spus, M. ; Wolkers-Rooijackers, J.C.M. ; Alexeeva, S.V. ; Kleerebezem, M. - \ 2014
    Microbial Cell Factories 13 (2014)suppl. 1. - ISSN 1475-2859
    lactic-acid bacteria - lactococcus-lactis - subsp lactis - listeria-monocytogenes - biovar diacetylactis - metabolic models - cheddar cheese - diversity - cremoris - dairy
    This review describes the recent advances made in the studies of the microbial community of complex and undefined cheese starter cultures. We report on work related to the composition of the cultures at the level of genetic lineages, on the presence and activity of bacteriophages and on the population dynamics during cheese making and during starter culture propagation. Furthermore, the link between starter composition and starter functionality will be discussed. Finally, recent advances in predictive metabolic modelling of the multi-strain cultures will be discussed in the context of microbe-microbe interactions.
    GtfA and GtfB are both required for protein O-glycosylation in Lactobacillus plantarum
    Lee, I.C. ; Swam, I.I. van; Tomita, S. ; Morsomme, P. ; Rolain, T. ; Hols, P. ; Kleerebezem, M. ; Bron, P.A. - \ 2014
    Journal of Bacteriology 196 (2014)9. - ISSN 0021-9193 - p. 1671 - 1682.
    complete genome sequence - lactic-acid bacteria - escherichia-coli - campylobacter-jejuni - acidophilus ncfm - epithelial-cells - surface protein - rhamnosus gg - glycoproteins - binding
    Acm2, the major autolysin of Lactobacillus plantarum WCFS1, was recently found to be O-glycosylated with N-acetylhexosamine, likely N-acetylglucosamine (GlcNAc). In this study, we set out to identify the glycosylation machinery by employing a comparative genomics approach to identify Gtf1 homologues, which are involved in fimbria-associated protein 1 (Fap1) glycosylation in Streptococcus parasanguinis. This in silico approach resulted in the identification of 6 candidate L. plantarum WCFS1 genes with significant homology to Gtf1, namely, tagE1 to tagE6. These candidate genes were targeted by systematic gene deletion, followed by assessment of the consequences on glycosylation of Acm2. We observed a changed mobility of Acm2 on SDS-PAGE in the tagE5E6 deletion strain, while deletion of other tagE genes resulted in Acm2 mobility comparable to that of the wild type. Subsequent mass spectrometry analysis of excised and in-gel-digested Acm2 confirmed the loss of glycosylation on Acm2 in the tagE5E6 deletion mutant, whereas a lectin blot using GlcNAc-specific succinylated wheat germ agglutinin (sWGA) revealed that besides Acm2, tagE5E6 deletion also abolished all but one other sWGA-reactive, protease-sensitive signal. Only complementation of both tagE5 and tagE6 restored those sWGA lectin signals, establishing that TagE5 and TagE6 are both required for the glycosylation of Acm2 as well as the vast majority of other sWGA-reactive proteins. Finally, sWGA lectin blotting experiments using a panel of 8 other L. plantarum strains revealed that protein glycosylation is a common feature in L. plantarum strains. With the establishment of these enzymes as protein glycosyltransferases, we propose to rename TagE5 and TagE6 as GtfA and GtfB, respectively.
    Functional Identification of Conserved Residues Involved in Lactobacillus rhamnosus Strain GG Sortase Specificity and Pilus Biogenesis
    Douillard, F.P. ; Rasinkangas, P. ; Ossowski, I. von; Reunanen, J. ; Palva, A. ; Vos, W.M. de - \ 2014
    Journal of Biological Chemistry 289 (2014)22. - ISSN 0021-9258 - p. 15764 - 15775.
    complete genome sequence - gram-positive bacteria - enterococcus-faecium isolate - group-b streptococcus - lactic-acid bacteria - lactococcus-lactis - corynebacterium-diphtheriae - bacillus-anthracis - pilin subunit - reveals pili
    In Gram-positive bacteria, sortase-dependent pili mediate the adhesion of bacteria to host epithelial cells and play a pivotal role in colonization, host signaling, and biofilm formation. Lactobacillus rhamnosus strain GG, a well known probiotic bacterium, also displays on its cell surface mucus-binding pilus structures, along with other LPXTG surface proteins, which are processed by sortases upon specific recognition of a highly conserved LPXTG motif. Bioinformatic analysis of all predicted LPXTG proteins encoded by the L. rhamnosus GG genome revealed a remarkable conservation of glycine residues juxtaposed to the canonical LPXTG motif. Here, we investigated and defined the role of this so-called triple glycine (TG) motif in determining sortase specificity during the pilus assembly and anchoring. Mutagenesis of the TG motif resulted in a lack or an alteration of the L. rhamnosus GG pilus structures, indicating that the TG motif is critical in pilus assembly and that they govern the pilin-specific and housekeeping sortase specificity. This allowed us to propose a regulatory model of the L. rhamnosus GG pilus biogenesis. Remarkably, the TG motif was identified in multiple pilus gene clusters of other Gram-positive bacteria, suggesting that similar signaling mechanisms occur in other, mainly pathogenic, species.
    The impact of selected strains of probiotic bacteria on metabolite formation in set yoghurt
    Settachaimongkon, S. ; Nout, M.J.R. ; Antunes Fernandes, E.C. ; Hooijdonk, A.C.M. van; Zwietering, M.H. ; Smid, E.J. ; Valenberg, H.J.F. van - \ 2014
    International Dairy Journal 38 (2014)1. - ISSN 0958-6946 - p. 1 - 10.
    nuclear-magnetic-resonance - delbrueckii subsp bulgaricus - lactic-acid bacteria - streptococcus-thermophilus - fermented milks - lactobacillus-acidophilus - functional foods - starter cultures - flavor compounds - dairy-products
    The influence of Lactobacillus rhamnosus GG and Bifidobacterium animalis subsp. lactis BB12 in cofermentation with traditional starters on metabolite formation in set yoghurt was evaluated. Microbial activity during fermentation and refrigerated storage was investigated by monitoring bacterial population dynamics, milk acidification and overall changes in yoghurt metabolite profiles. A complementary metabolomics approach using solid-phase microextraction-gas chromatography/mass spectrometry and 1H nuclear magnetic resonance resulted in the identification of 37 volatile and 43 non-volatile metabolites, respectively. Results demonstrated that the two probiotic strains did not influence acidity and the key-aroma volatile metabolites of set yoghurt. However, a contribution by the presence of L. rhamnosus GG on the non-volatile metabolite profile of yoghurt was specifically noticed during storage. Multivariate analysis allowed yoghurts fermented by different starter combinations and different durations of storage to be differentiated according to their metabolite profiles. This provides new insights regarding the impact of probiotics on the metabolome of yoghurt.
    Influence of different proteolytic strains of Streptococcus thermophilus in co-culture with Lactobacillus delbrueckii subsp. bulgaricus on the metabolite profile of set-yoghurt
    Settachaimongkon, S. ; Nout, M.J.R. ; Antunes Fernandes, E.C. ; Hettinga, K.A. ; Vervoort, J.J.M. ; Hooijdonk, A.C.M. van; Zwietering, M.H. ; Smid, E.J. ; Valenberg, H.J.F. van - \ 2014
    International Journal of Food Microbiology 177 (2014). - ISSN 0168-1605 - p. 29 - 36.
    lactic-acid bacteria - nuclear-magnetic-resonance - food fermentations - volatile compounds - functional foods - flavor formation - fermented milks - dairy-cows - shelf-life - metabolomics
    Proto-cooperation between Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus is one of the key factors that determine the fermentation process and final quality of yoghurt. In this study, the interaction between different proteolytic strains of S. thermophilus and L. delbrueckii subsp. bulgaricuswas investigated in terms of microbial growth, acidification and changes in the biochemical composition of milk during set-yoghurt fermentation. A complementary metabolomics approach was applied for global characterization of volatile and non-volatile polar metabolite profiles of yoghurt associated with proteolytic activity of the individual strains in the starter cultures. The results demonstrated that only non-proteolytic S. thermophilus (Prt-) strain performed proto-cooperation with L. delbrueckii subsp. bulgaricus. The proto-cooperation resulted in significant higher populations of the two species, faster milk acidification, significant abundance of aroma volatiles and non-volatile metabolites desirable for a good organoleptic quality of yoghurt. Headspace SPME-GC/MS and 1H NMR resulted in the identification of 35 volatiles and 43 non-volatile polar metabolites, respectively. Furthermore, multivariate statistical analysis allows discriminating set-yoghurts fermented by different types of starter cultures according to their metabolite profiles. Our finding underlines that selection of suitable strain combinations in yoghurt starters is important for achieving the best technological performance regarding the quality of product.
    Stability of (Bio)Functionalized Porous Aluminum Oxide
    Debrassi, A. ; Ribbera, A. ; Vos, W.M. de; Wennekes, T. ; Zuilhof, H. - \ 2014
    Langmuir 30 (2014). - ISSN 0743-7463 - p. 1311 - 1320.
    self-assembled monolayers - lactic-acid bacteria - lactobacillus-plantarum - nanoporous alumina - click chemistry - anodic alumina - surfaces - adsorption - membrane - carbohydrate
    Porous aluminum oxide (PAO), a nanostructured support for, among others, culturing microorganisms, was chemically modified in order to attach biomolecules that can selectively interact with target bacteria. We present the first comprehensive study of monolayer-modified PAO using conditions that are relevant to microbial growth with a range of functional groups (carboxylic acid, a-hydroxycarboxylic acid, alkyne, alkene, phosphonic acid, and silane). Their stability was initially assessed in phosphate-buffered saline (pH 7.0) at room temperature. The most stable combination (PAO with phosphonic acids) was further studied over a range of physiological pHs (4–8) and temperatures (up to 80 °C). Varying the pH had no significant effect on the stability, but it gradually decreased with increasing temperature. The stability of phosphonic acid-modified PAO surfaces was shown to depend strongly on the other terminal group of the monolayer structure: in general, hydrophilic monolayers were less stable than hydrophobic monolayers. Finally, an alkyne-terminated PAO surface was reacted with an azide-linked mannose derivative. The resulting mannose-presenting PAO surface showed the clearly increased adherence of a mannose-binding bacterium, Lactobacillus plantarum, and also allowed for bacterial outgrowth.
    The effect of dietary hydroxyproline and dietary oxalate on urinary oxalate excretion in cats
    Dijcker, J.C. ; Plantinga, E.A. ; Thomas, D.G. ; Queau, Y. ; Biourge, V.C. ; Hendriks, W.H. - \ 2014
    Journal of Animal Science 92 (2014). - ISSN 0021-8812 - p. 577 - 584.
    lactic-acid bacteria - oxalobacter-formigenes - primary hyperoxaluria - feline uroliths - adult cats - calcium - rats - dogs - metabolism - canine
    In humans and rodents, dietary hydroxyproline (hyp) and oxalate intake affect urinary oxalate (Uox) excretion. Whether Uox excretion occurs in cats was tested by feeding diets containing low oxalate (13 mg/100g DM) with high (Hhyp-Lox), moderate (Mhyp-Lox), and low hyp (Lhyp-Lox) concentrations (3.8, 2.0 and 0.2 g/100g DM, respectively), and low hyp with high oxalate (93 mg/100g DM; Lhyp-Hox) to 8 adult, female cats in a 48-d study using a Latin square design. Cats were randomly allocated to 1 of the four 12-d treatment periods and fed according to individual energy needs. Feces and urine were collected quantitatively using modified litter boxes during the final 5 d of each period. Feces were analyzed for oxalate and Ca, and urine for specific density, pH, oxalate, Ca, P, Mg, Na, K, ammonia, citrate, urate, sulphate, and creatinine. Increasing hyp intake (0.2, 2.0, and 3.8 g/100g DM) resulted in increased Uox excretion (Lhyp-Lox vs. Mhyp-Lox vs. Hhyp-Lox, P <0.05), and the linear dose-response equation was: Uox (mg ¿d(-1)) = 5.62 + 2.10 x g hyp intake/d (r(2) = 0.56; P <0.001). Increasing oxalate intake from 13 to 93 mg/100g DM did not affect Uox excretion, but resulted in an increase in fecal oxalate output (P <0.001) and positive oxalate balance (32.20 ± 2.06 mg¿d(-1)). The results indicate that the intestinal absorption of the supplemental oxalate, and thereby its contribution to Uox, was low (5.90 ± 5.24%). Relevant increases in endogenous Uox excretion were achieved by increasing dietary hyp intake. The hyp-containing protein sources should be minimized in Ca ox urolith preventative diets until their effect on Uox excretion is tested. The oxalate content (up to 93 mg/100g DM) in a diet with moderate Ca content does not contribute to Uox content.
    In vitro selection and characterization of putative probiotics isolated from the gut of Acipenser baerii (Brandt, 1869)
    Geraylou, Z. ; Vanhove, M.P.M. ; Souffreau, C. ; Rurangwa, E. ; Buyse, J. ; Ollevier, F. - \ 2014
    Aquaculture Research 45 (2014)2. - ISSN 1355-557X - p. 341 - 352.
    lactic-acid bacteria - gastrointestinal-tract - fish pathogens - intestinal microbiota - growth-performance - aquaculture - marine - lactobacillus - prevention - tolerance
    To select and characterize potential probiotic bacteria from the gut microbiota of Siberian sturgeon (Acipenser baerii), 129 strains isolated from the hindgut were screened for antagonistic activity against five fish pathogens. Ten isolates showed antagonism towards three or more pathogens. Nine of these isolates were Gram-positive, belonging to Lactococcus (seven) and Bacillus (two), and a single strain belonging to the Gram-negative Citrobacter. These inhibitory isolates were identified using genetic, phentotypic and biochemical traits, and further characterized by in vitro tests assessing the adhesion and growth in mucus and resistance to gastric and intestinal fluids. The candidate probiotics were determined to be non-pathogenic through an in vivo study. Based on these assays, Lactococcus lactis ssp. lactis STG45 and STG81 showed the broadest inhibitory potential, a high viability in simulated gastrointestinal juice and the highest adhesion capacity to mucus. They were therefore selected as the most promising candidate probiotics. This is the first study screening probiotics among the gut microflora of Siberian sturgeon.
    Human milk: a source of more life than we imagine
    Jeurink, P.V. ; Bergenhenegouwen, J. van; Jimenez, E. ; Knippels, L.M.J. ; Fernandez, L. ; Garssen, J. ; Knol, J. ; Rodriguez, J.M. ; Martin, R. - \ 2013
    Beneficial Microbes 4 (2013)1. - ISSN 1876-2883 - p. 17 - 30.
    lactic-acid bacteria - human breast-milk - fragment-length-polymorphism - human skin microbiome - healthy women - infectious mastitis - dendritic cells - infant gut - staphylococcus-epidermidis - intestinal microbiota
    The presence of bacteria in human milk has been acknowledged since the seventies. For a long time, microbiological analysis of human milk was only performed in case of infections and therefore the presence of non-pathogenic bacteria was yet unknown. During the last decades, the use of more sophisticated culture-dependent and -independent techniques, and the steady development of the -omic approaches are opening up the new concept of the 'milk microbiome', a complex ecosystem with a greater diversity than previously anticipated. In this review, possible mechanisms by which bacteria can reach the mammary gland (contamination versus active migration) are discussed. In addition, the potential roles of human milk for both infant and maternal health are summarised. A better understanding of the link between the milk microbiome and health benefit, the potential factors influencing this relationship and whether or not it can be influenced by nutrition is required to open new avenues in the field of pregnancy and lactation.
    Microbial Community Structure of Three Traditional Zambian Fermented Products: Mabisi, Chibwantu and Munkoyo
    Schoustra, S.E. ; Kasase, C. ; Toarta, C. ; Kassen, R. ; Poulain, A.J. - \ 2013
    PLoS ONE 8 (2013)5. - ISSN 1932-6203
    lactic-acid bacteria - adaptive radiation - ecology - foods - diversity - microorganisms - fermentations - systems - africa - safety
    Around the world, raw materials are converted into fermented food products through microbial and enzymatic activity. Products are typically produced using a process known as batch culture, where small volumes of an old culture are used to initiate a fresh culture. Repeated over many years, and provided samples are not shared among producers, batch culture techniques allow for the natural evolution of independent microbial ecosystems. While these products form an important part of the diets of many people because of their nutritional, organoleptic and food safety properties, for many traditional African fermented products the microbial communities responsible for fermentation are largely unknown. Here we describe the microbial composition of three traditional fermented non-alcoholic beverages that are widely consumed across Zambia: the milk based product Mabisi and the cereal based products Munkoyo and Chibwantu. Using culture and non-culture based techniques, we found that six to eight lactic acid bacteria predominate in all products. We then used this data to investigate in more detail the factors affecting community structure. We found that products made from similar raw materials do not harbor microbial communities that are more similar to each other than those made from different raw materials. We also found that samples from the same product taken at the same location were as different from each other in terms of microbial community structure and composition, as those from geographically very distant locations. These results suggest that microbial community structure in these products is neither a simple consequence of the raw materials used, nor the particular suite of microbes available in the environment but that anthropogenic variables (e. g., competition among sellers or organoleptic preferences by different tribes) are important in shaping the microbial community structures.
    Comparative genome analysis of Lactobacillus casei strains isolated from Actimel and Yakult products reveals marked similarities and points to a common origin
    Douillard, F.P. ; Kant, R. ; Ritari, J. ; Paulin, L. ; Palva, A. ; Vos, W.M. de - \ 2013
    Microbial Biotechnology 6 (2013)5. - ISSN 1751-7907 - p. 576 - 587.
    lactic-acid bacteria - gram-positive bacteria - rhamnosus gg - functional-analysis - cell-wall - surface-proteins - staphylococcus-aureus - controlled-trial - binding-protein - sequence
    The members of the Lactobacillus genus are widely used in the food and feed industry and show a remarkable ecological adaptability. Several Lactobacillus strains have been marketed as probiotics as they possess health-promoting properties for the host. In the present study, we used two complementary next-generation sequencing technologies to deduce the genome sequences of two Lactobacillus casei strains LcA and LcY, which were isolated from the products Actimel and Yakult, commercialized as probiotics. The LcA and LcY draft genomes have, respectively, an estimated size of 3067 and 3082 Mb and a G+ C content of 46.3%. Both strains are close to identical to each other and differ by no more than minor chromosomal re-arrangements, substitutions, insertions and deletions, as evident from the verified presence of one insertion-deletion (InDel) and only 29 single-nucleotide polymorphisms (SNPs). In terms of coding capacity, LcA and LcY are predicted to encode a comparable exoproteome, indicating that LcA and LcY are likely to establish similar interactions with human intestinal cells. Moreover, both L. casei LcA and LcY harboured a 59.6 kb plasmid that shared high similarities with plasmids found in other L. casei strains, such as W56 and BD-II. Further analysis revealed that the L. casei plasmids constitute a good evolution marker within the L. casei species. The plasmids of the LcA and LcY strains are almost identical, as testified by the presence of only three verified SNPs, and share a 3.5 kb region encoding a remnant of a lactose PTS system that is absent from the plasmids of W56 and BD-II but conserved in another smaller L. casei plasmid (pLC2W). Our observations imply that the results obtained in animal and human experiments performed with the Actimel and Yakult strains can be compared with each other as these strains share a very recent common ancestor.
    Transcriptome-based characterization of interactions between Saccharomyces cerevisiae and Lactobacillus delbrueckii subsp. bulgaricus in lactose-grown chemostat cocultures
    Mendes, F. ; Sieuwerts, S. ; Hulster, E. de; Almering, M.J. ; Luttik, M.A.H. ; Pronk, J.T. ; Smid, E.J. ; Baron, P.A. ; Daran-Lapujade, P. - \ 2013
    Applied and Environmental Microbiology 79 (2013)19. - ISSN 0099-2240 - p. 5949 - 5961.
    lactic-acid bacteria - streptococcus-thermophilus - mixed-culture - kefiran production - oenococcus-oeni - fermentation - reveals - yeasts - carbon - temperature
    Mixed populations of Saccharomyces cerevisiae yeasts and lactic acid bacteria occur in many dairy, food, and beverage fermentations, but knowledge about their interactions is incomplete. In the present study, interactions between Saccharomyces cerevisiae and Lactobacillus delbrueckii subsp. bulgaricus, two microorganisms that co-occur in kefir fermentations, were studied during anaerobic growth on lactose. By combining physiological and transcriptome analysis of the two strains in the cocultures, five mechanisms of interaction were identified. (i) Lb. delbrueckii subsp. bulgaricus hydrolyzes lactose, which cannot be metabolized by S. cerevisiae, to galactose and glucose. Subsequently, galactose, which cannot be metabolized by Lb. delbrueckii subsp. bulgaricus, is excreted and provides a carbon source for yeast. (ii) In pure cultures, Lb. delbrueckii subsp. bulgaricus grows only in the presence of increased CO2 concentrations. In anaerobic mixed cultures, the yeast provides this CO2 via alcoholic fermentation. (iii) Analysis of amino acid consumption from the defined medium indicated that S. cerevisiae supplied alanine to the bacterium. (iv) A mild but significant low-iron response in the yeast transcriptome, identified by DNA microarray analysis, was consistent with the chelation of iron by the lactate produced by Lb. delbrueckii subsp. bulgaricus. (v) Transcriptome analysis of Lb. delbrueckii subsp. bulgaricus in mixed cultures showed an overrepresentation of transcripts involved in lipid metabolism, suggesting either a competition of the two microorganisms for fatty acids or a response to the ethanol produced by S. cerevisiae. This study demonstrates that chemostat-based transcriptome analysis is a powerful tool to investigate microbial interactions in mixed populations.
    Multifactorial diversity sustains microbial community stability
    Erkus, O. ; Jager, V.C.L. de; Spus, M. ; Alen-Boerrigter, I.J. van; Rijswijck, I.M.H. van; Hazelwood, L. ; Janssen, P.W. ; Hijum, S.A.F.T. van; Kleerebezem, M. ; Smid, E.J. - \ 2013
    ISME Journal 7 (2013)11. - ISSN 1751-7362 - p. 2126 - 2136.
    lactic-acid bacteria - complete genome sequence - lactococcus-lactis - dairy environment - subsp lactis - raw-milk - cremoris - plasmids - cheese - identification
    Maintenance of a high degree of biodiversity in homogeneous environments is poorly understood. A complex cheese starter culture with a long history of use was characterized as a model system to study simple microbial communities. Eight distinct genetic lineages were identified, encompassing two species: Lactococcus lactis and Leuconostoc mesenteroides. The genetic lineages were found to be collections of strains with variable plasmid content and phage sensitivities. Kill-the-winner hypothesis explaining the suppression of the fittest strains by density-dependent phage predation was operational at the strain level. This prevents the eradication of entire genetic lineages from the community during propagation regimes (back-slopping), stabilizing the genetic heterogeneity in the starter culture against environmental uncertainty
    Comparative genomic and functional analysis of 100 Lactobacillus rhamnosus strains and their comparison with strain GG
    Douillard, F.P. ; Ribbera, A. ; Kant, R. ; Pietilä, T.E. ; Järvinen, H.M. ; Messing, M. ; Randazzo, C.L. ; Paulin, L. ; Laine, P.K. ; Ritari, J. ; Caggia, C. ; Lähteinen, T. ; Brouns, S.J.J. ; Satokari, R.M. ; Ossowski, I. von; Reunanen, J. ; Palva, A. ; Vos, W.M. de - \ 2013
    Plos Genetics 9 (2013)8. - ISSN 1553-7404
    lactic-acid bacteria - intestinal epithelial-cells - placebo-controlled trial - streptococcus-thermophilus - species identification - salmonella-typhimurium - gastrointestinal-tract - adaptive immunity - atopic disease - in-vitro
    Lactobacillus rhamnosus is a lactic acid bacterium that is found in a large variety of ecological habitats, including artisanal and industrial dairy products, the oral cavity, intestinal tract or vagina. To gain insights into the genetic complexity and ecological versatility of the species L. rhamnosus, we examined the genomes and phenotypes of 100 L. rhamnosus strains isolated from diverse sources. The genomes of 100 L. rhamnosus strains were mapped onto the L. rhamnosus GG reference genome. These strains were phenotypically characterized for a wide range of metabolic, antagonistic, signalling and functional properties. Phylogenomic analysis showed multiple groupings of the species that could partly be associated with their ecological niches. We identified 17 highly variable regions that encode functions related to lifestyle, i.e. carbohydrate transport and metabolism, production of mucus-binding pili, bile salt resistance, prophages and CRISPR adaptive immunity. Integration of the phenotypic and genomic data revealed that some L. rhamnosus strains possibly resided in multiple niches, illustrating the dynamics of bacterial habitats. The present study showed two distinctive geno-phenotypes in the L. rhamnosus species. The geno-phenotype A suggests an adaptation to stable nutrient-rich niches, i.e. milk-derivative products, reflected by the alteration or loss of biological functions associated with antimicrobial activity spectrum, stress resistance, adaptability and fitness to a distinctive range of habitats. In contrast, the geno-phenotype B displays adequate traits to a variable environment, such as the intestinal tract, in terms of nutrient resources, bacterial population density and host effects
    Transcriptome signatures of class I and III stress response deregulation in Lactobacillus plantarum reveal pleiotropic adaptation
    Bokhorst-van de Veen, H. van; Bongers, R.S. ; Wels, M. ; Bron, P.A. ; Kleerebezem, M. - \ 2013
    Microbial Cell Factories 12 (2013)1. - ISSN 1475-2859 - 15 p.
    gram-positive bacteria - heat-shock response - lactic-acid bacteria - bacillus-subtilis - listeria-monocytogenes - gastrointestinal-tract - low gc - streptococcus-pneumoniae - comparative genomics - helicobacter-pylori
    Background - To cope with environmental challenges bacteria possess sophisticated defense mechanisms that involve stress-induced adaptive responses. The canonical stress regulators CtsR and HrcA play a central role in the adaptations to a plethora of stresses in a variety of organisms. Here, we determined the CtsR and HrcA regulons of the lactic acid bacterium Lactobacillus plantarum WCFS1 grown under reference (28°C) and elevated (40°C) temperatures, using ctsR, hrcA, and ctsR-hrcA deletion mutants. Results - While the maximum specific growth rates of the mutants and the parental strain were similar at both temperatures (0.33¿±¿0.02 h-1 and 0.34¿±¿0.03 h-1, respectively), DNA microarray analyses revealed that the CtsR or HrcA deficient strains displayed altered transcription patterns of genes encoding functions involved in transport and binding of sugars and other compounds, primary metabolism, transcription regulation, capsular polysaccharide biosynthesis, as well as fatty acid metabolism. These transcriptional signatures enabled the refinement of the gene repertoire that is directly or indirectly controlled by CtsR and HrcA of L. plantarum. Deletion of both regulators, elicited transcriptional changes of a large variety of additional genes in a temperature-dependent manner, including genes encoding functions involved in cell-envelope remodeling. Moreover, phenotypic assays revealed that both transcription regulators contribute to regulation of resistance to hydrogen peroxide stress. The integration of these results allowed the reconstruction of CtsR and HrcA regulatory networks in L. plantarum, highlighting the significant intertwinement of class I and III stress regulons. Conclusions - Taken together, our results enabled the refinement of the CtsR and HrcA regulatory networks in L. plantarum, illustrating the complex nature of adaptive stress responses in this bacterium
    Probiotics can generate FoxP3 T-cell responses in the small intestine and simultaneously inducing CD4 and CD8 T cell activation in the large intestine.
    Smelt, M.J. ; Haan, B.J. de; Bron, P.A. ; Swam, I. van; Meijerink, M. ; Wells, J. ; Faas, M.M. ; Vos, P. de - \ 2013
    PLoS ONE 8 (2013)7. - ISSN 1932-6203
    inflammatory-bowel-disease - influenza-virus infection - cd103(+) dendritic cells - complete genome sequence - lactic-acid bacteria - lactobacillus-plantarum - double-blind - in-vitro - maintaining remission - ulcerative-colitis
    Most studies on probiotics aim to restore intestinal homeostasis to reduce immune-pathology in disease. Of equal importance are studies on how probiotics might prevent or delay disease in healthy individuals. However, knowledge on mechanisms of probiotic actions in healthy individuals is scarce. To gain more insight in how different bacterial strains may modulate the healthy intestinal immune system, we investigated the effect of the food derived bacterial strains L. plantarum WCFS1, L. salivarius UCC118, and L. lactis MG1363, on the intestinal regulatory immune phenotype in healthy mice. All three bacterial strains induced an upregulation of activity and numbers of CD11c(+) MHCII(+) DCs in the immune-sampling Peyer's Patches. Only L. salivarius UCC118 skewed towards an immune regulatory phenotype in the small intestinal lamina propria (SILP). The effects were different in the large intestine lamina propria. L. salivarius UCC118 induced activation in both CD4 and CD8 positive T-cells while L. plantarum WCFS1 induced a more regulatory phenotype. Moreover, L. plantarum WCFS1 decreased the Th1/Th2 ratio in the SILP. Also L. lactis MG1363 had immunomodulatory effects. L. lactis MG1363 decreased the expression of the GATA-3 and T-bet in the SILP. As our data show that contradictory effects may occur in different parts of the gut, it is recommended to study effects of probiotic in different sites in the intestine. Our strain-specific results suggest that unspecified application of probiotics may not be very effective. Our data also indicate that selection of specific probiotic strain activities on the basis of responses in healthy mice may be a promising strategy to specifically stimulate or suppress immunity in specific parts of the intestine
    Glucosinolate content of blanched cabbage (Brassica oleracea var. capitata) fermented by the probiotic strain Lactobacillus paracasei LMG-P22043
    Kruse, I. ; Valerio, F. ; Lonigro, S.L. ; Candia, S. de; Verkerk, R. ; Dekker, M. ; Lavermicocca, P. - \ 2013
    Food Research International 54 (2013)1. - ISSN 0963-9969 - p. 706 - 710.
    plant-derived biomolecules - lactic-acid bacteria - white cabbage - isothiocyanates - vegetables - health - cancer - food - components - pathogens
    Conventional fermentation of cabbage like in sauerkraut production leads to a complete elimination of glucosinolates (GSs). In order to retain GSs in fermented cabbage, the effect of a thermal treatment (blanching) followed by fermentation (4% brine at 25 °C) by the probiotic strain Lactobacillus paracasei LMG P22043, was investigated. After 71 h fermentation the probiotic blanched cabbage still contained 27.2 ± 2.3 µmol/100 g GSs, corresponding to the 35% of the total GSs before fermentation. A final count of L. paracasei of 8.26 ± 1.2 log10 CFU/g, and a final pH of 4.12 ± 0.1 were reached. After 30 days of refrigerated vacuum packed storage, 23.7 ± 1.5 µmol/100 g of GSs still persisted. In the control cabbage (blanched and not inoculated with L. paracasei) no fermentation occurred and as a result final pH was 6.10 ± 0.21, leading to a product not suitable for storage and consumption. Compared to traditional sauerkraut the final product has the advantage of containing a high content of phytochemicals in combination with a high count of live probiotic bacterial cells.
    Challenges in translational research on probiotic lactobacilli: from in vitro assays to clinical trials
    Meijerink, M. ; Mercenier, A.M.E. ; Wells, J. - \ 2013
    Beneficial Microbes 4 (2013)1. - ISSN 1876-2883 - p. 83 - 100.
    placebo-controlled trial - lactic-acid bacteria - regulatory t-cells - blood mononuclear-cells - intestinal epithelial-cells - irritable-bowel-syndrome - influenza-virus infection - randomized controlled-trial - tight junction proteins - host-microbiota dialog
    Beneficial effects of certain probiotic strains have been established in the treatment and prevention of various immune and intestinal disorders in humans, including allergic diseases, chronic inflammatory diseases and diarrhoea. The proposed mechanisms underlying the immunomodulatory effects of probiotics in humans are not understood in precise detail but include enhancement of intestinal barrier function, altered epithelial signalling, competition with pathogens and effects on immune cells and immunity depending on the probiotic strain. The publication of controversial or inconclusive probiotic studies in humans highlights the need for a better understanding of the mechanisms and improved strain selection criteria. This review focuses on the immunomodulatory properties of lactobacilli and bifidobacteria in vitro and in vivo, current knowledge concerning the mechanisms in vivo and challenges in translational research on probiotics. A better understanding of the molecular mechanisms of probiotics, the effect of probiotic mixtures versus single strains, the effect of formulation of probiotics and the fate of ingested probiotics should help to clarify the value of immune assays as selection criteria for probiotics.
    Effects of dietary arabinoxylan-oligosaccharides (AXOS) and endogenous probiotics on the growth performance, non-specific immunity and gut micrbiota of juvenile Siberian sturgeon (Acipenser baerii)
    Geraylou, Z. ; Souffreau, C. ; Rurangwa, E. ; Meester, L. de; Courtin, C.M. ; Delcour, J.A. ; Buyse, J. ; Ollevier, F. - \ 2013
    Fish and Shellfish Immunology 35 (2013)3. - ISSN 1050-4648 - p. 766 - 775.
    lactic-acid bacteria - bacillus-subtilis - lactococcus-lactis - disease resistance - intestinal microbiota - scophthalmus-maximus - labeo-rohita - in-vitro - fish - supplementation
    We investigated the effects of administration of putative endogenous probiotics Lactococcus lactis spp. lactis or Bacillus circulans, alone and in combination with arabinoxylan-oligosaccharides (AXOS), a new class of candidate prebiotics, in juvenile Siberian sturgeon (Acipenser baerii). Eight experimental diets were tested: basal diet (Diet 1), basal diet supplemented with 2% AXOS (Diet 2), or L. lactis ST G81 (Diet 3), L. lactis ST G45 (Diet 4), B. circulans ST M53 (Diet 5), L. lactis ST G81 + 2% AXOS (Diet 6), L. lactis ST G45 + 2% AXOS (Diet 7), B. circulans ST M53 + 2% AXOS (Diet 8). After four weeks, growth performance and feed conversion ratio significantly improved in fish fed diet 7. Innate immune responses of fish were boosted with both AXOS and probiotic diets, however synergistic effects of AXOS and probiotic diets were only observed for phagocytic and alternative complement activity. Phagocytic and respiratory burst activity of fish macrophage increased in fish fed diet 2 and 7, while humoral immune responses only increased in fish fed diet 7. Pyrosequencing analysis (16S rDNA) of the hindgut microbiota demonstrated that AXOS improved the colonization or/and growth capacity of L. lactis, as a higher relative abundance of L. lactis was observed in fish receiving diet 7. However, no observable colonization of B. circulans was found in the hindgut of fish fed diet 5 or 8, containing this bacterium. The dietary L. lactis ST G45 + 2% AXOS caused significant alterations in the intestinal microbiota by significantly decreasing in bacterial diversity, demonstrated by the fall in richness and Shannon diversity, and improved growth performance and boosted immune responses of Siberian sturgeon.
    Using recombinant Lactococci as an approach to dissect the immunomodulating capacity of surface piliation in probiotic Lactobacillus rhamnosus GG
    Ossowski, I. von; Pietilä, T.E. ; Rintahaka, J. ; Nummenmaa, E. ; Mäkinen, V.M. ; Reunanen, J. ; Satokari, R.M. ; Vos, W.M. de; Palva, I. ; Palva, A. - \ 2013
    PLoS ONE 8 (2013)5. - ISSN 1932-6203
    lactic-acid bacteria - intestinal epithelial-cells - functional-analysis - dendritic cells - gastrointestinal-tract - dependent mechanism - protein-production - oral consumption - adhesion - pili
    Primarily arising from their well understood beneficial health effects, many lactobacilli strains are considered good candidates for use as probiotics in humans and animals. Lactobacillar probiosis can itself be best typified by the Lactobacillus rhamnosus GG strain, which, with its well-documented clinical benefits, has emerged as one of the most widely used probiotics in the food and health-supplement industries. Even so, many facets of its molecular mechanisms and limitations as a beneficial commensal bacterium still remain to be thoroughly explored and dissected. Because L. rhamnosus GG is one of only a few such strains exhibiting surface piliation (called SpaCBA), we sought to examine whether this particular type of cell-surface appendage has a discernible immunomodulating capacity and is able to trigger targeted responses in human immune-related cells. Thus, presented herein for this study, we recombinantly engineered Lactococcus lactis to produce native (and pilin-deleted) SpaCBA pili that were assembled in a structurally authentic form and anchored to the cell surface, and which had retained mucus-binding functionality. By using these recombinant lactococcal constructs, we were able to demonstrate that the SpaCBA pilus can be a contributory factor in the activation of Toll-like receptor 2-dependent signaling in HEK cells as well as in the modulation of pro- and anti-inflammatory cytokine (TNF-a, IL-6, IL-10, and IL-12) production in human monocyte-derived dendritic cells. From these data, we suggest that the recombinant-expressed and surface-anchored SpaCBA pilus, given its projected functioning in the gut environment, might be viewed as a new microbe-associated molecular pattern (MAMP)-like modulator of innate immunity. Accordingly, our study has brought some new insight to the molecular immunogenicity of the SpaCBA pilus, thus opening the way to a better understanding of its possible role in the multifaceted nature of L. rhamnosus GG probiosis within the human gut
    Microbe-microbe interactions in mixed culture food fermentations
    Smid, E.J. ; Lacroix, C. - \ 2013
    Current Opinion in Biotechnology 24 (2013)2. - ISSN 0958-1669 - p. 148 - 154.
    lactic-acid bacteria - cheese - genomics - growth - propionibacteria - communication - lactobacilli - probiotics - consortia - milk
    Most known natural and industrial food fermentation processes are driven by either simple or complex communities of microorganisms. Obviously, these fermenting microbes will not only interact with the fermentable substrate but also with each other. These microbe–microbe interactions are complex but thought to be crucial for obtaining the desired product characteristics. Microbial interactions are mediated through a variety of molecular and physiological mechanisms. Examples of interaction mechanisms which have an impact on the outcome of food fermentation processes will be discussed. Finally, the technological and scientific challenges associated with the production and propagation of complex mixed starter cultures are briefly addressed. Research on the composition and functionality of complex microbial consortia is gaining momentum and will open new avenues for controlling and improving food fermentation processes, and developing new applications for mixed cultures.
    Comparative Genomic and Functional Analysis of Lactobacillus casei and Lactobacillus rhamnosus Strains Marketed as Probiotics
    Douillard, F.P. ; Ribbera, A. ; Järvinen, H.M. ; Kant, R. ; Pietilä, T.E. ; Randazzo, C.L. ; Paulin, L. ; Laine, P.K. ; Caggia, C. ; Ossowski, I. von; Reunanen, J. ; Satokari, R. ; Salminen, S. ; Palva, A. ; Vos, W.M. de - \ 2013
    Applied and Environmental Microbiology 79 (2013)6. - ISSN 0099-2240 - p. 1923 - 1933.
    lactic-acid bacteria - binding-protein - in-vitro - carbohydrate-metabolism - sequence - gg - adhesion - bl23 - stress - mucus
    Four Lactobacillus strains were isolated from marketed probiotic products, including L. rhamnosus strains from Vifit (Friesland Campina) and Idoform (Ferrosan) and L. casei strains from Actimel (Danone) and Yakult (Yakult Honsa Co.). Their genomes and phenotypes were characterized and compared in detail with L. casei strain BL23 and L. rhamnosus strain GG. Phenotypic analysis of the new isolates indicated differences in carbohydrate utilization between L. casei and L. rhamnosus strains, which could be linked to their genotypes. The two isolated L. rhamnosus strains had genomes that were virtually identical to that of L. rhamnosus GG, testifying to their genomic stability and integrity in food products. The L. casei strains showed much greater genomic heterogeneity. Remarkably, all strains contained an intact spaCBA pilus gene cluster. However, only the L. rhamnosus strains produced mucus-binding SpaCBA pili under the conditions tested. Transcription initiation mapping demonstrated that the insertion of an iso-IS30 element upstream of the pilus gene cluster in L. rhamnosus strains but absent in L. casei strains had constituted a functional promoter driving pilus gene expression. All L. rhamnosus strains triggered an NF-¿B response via Toll-like receptor 2 (TLR2) in a reporter cell line, whereas the L. casei strains did not or did so to a much lesser extent. This study demonstrates that the two L. rhamnosus strains isolated from probiotic products are virtually identical to L. rhamnosus GG and further highlights the differences between these and L. casei strains widely marketed as probiotics, in terms of genome content, mucus-binding and metabolic capacities, and host signaling capabilities
    Metabolic shifts: a fitness perspective for microbial cell factories
    Goel, A. ; Wortel, M.T. ; Molenaar, D. ; Teusink, B. - \ 2012
    Biotechnology Letters 34 (2012)12. - ISSN 0141-5492 - p. 2147 - 2160.
    lactic-acid bacteria - heterologous protein secretion - escherichia-coli - saccharomyces-cerevisiae - lactococcus-lactis - bacillus-subtilis - pyruvate metabolism - overflow metabolism - aerobic glycolysis - glucose-metabolism
    Performance of industrial microorganisms as cell factories is limited by the capacity to channel nutrients to desired products, of which optimal production usually requires careful manipulation of process conditions, or strain improvement. The focus in process improvement is often on understanding and manipulating the regulation of metabolism. Nonetheless, one encounters situations where organisms are remarkably resilient to further optimization or their properties become unstable. Therefore it is important to understand the origin of these apparent limitations to find whether and how they can be improved. We argue that by considering fitness effects of regulation, a more generic explanation for certain behaviour can be obtained. In this view, apparent process limitations arise from trade-offs that cells faced as they evolved to improve fitness. A deeper understanding of such trade-offs using a systems biology approach can ultimately enhance performance of cell factories.
    Complete resequencing and reannotation of the Lactobacillus plantarum WCFS1 genome
    Siezen, R.J. ; Francke, C. ; Renckens, B. ; Boekhorst, L.J.S. ; Wels, M. ; Kleerebezem, M. ; Hijum, S.A.F.T. van - \ 2012
    Journal of Bacteriology 194 (2012)1. - ISSN 0021-9193 - p. 195 - 196.
    lactic-acid bacteria - gastrointestinal-tract - identification - fermentation - database - artemis - growth - genes - mice - carbohydrate
    There is growing interest in the beneficial effects of Lactobacillus plantarum on human health. The genome of L. plantarum WCFS1, first sequenced in 2001, was resequenced using Solexa technology. We identified 116 nucleotide corrections and improved function prediction for nearly 1,200 proteins, with a focus on metabolic functions and cell surface-associated proteins.
    Genome sequence of the naturally plasmid-free Lactobacillus plantarum strain NC8 (CCUG 61730)
    Axelsson, L. ; Rud, I. ; Naterstad, K. ; Blom, H. ; Renckens, B. ; Boekhorst, L.J.S. ; Kleerebezem, M. ; Hijum, S.A.F.T. van; Siezen, R.J. - \ 2012
    Journal of Bacteriology 194 (2012)9. - ISSN 0021-9193 - p. 2391 - 2392.
    inducible gene-expression - lactic-acid bacteria - sakei - fermentations - diversity - vectors - healthy - mucosa - wcfs1
    Lactobacillus plantarum is a highly versatile lactic acid bacterium found in various ecological niches, such as fermented vegetable, meat, and dairy products and the gastrointestinal tract. We sequenced the genome of L. plantarum NC8, a naturally plasmid-free strain, which has been used as a model strain in many laboratories worldwide.
    Transcriptomes reveal genetic signatures underlying physiological variations imposed by different fermentation conditions in Lactobacillus plantarum
    Bron, P.A. ; Wels, M. ; Bongers, R.S. ; Bokhorst-van de Veen, H. van; Wiersma, A. ; Overmars, L. ; Marco, M.L. ; Kleerebezem, M. - \ 2012
    PLoS ONE 7 (2012)7. - ISSN 1932-6203
    lactic-acid bacteria - complete genome sequence - lactococcus-lactis - ribonucleotide reductase - escherichia-coli - stationary-phase - microarray data - aerobic growth - diversity - pathways
    Lactic acid bacteria (LAB) are utilized widely for the fermentation of foods. In the current post-genomic era, tools have been developed that explore genetic diversity among LAB strains aiming to link these variations to differential phenotypes observed in the strains investigated. However, these genotype-phenotype matching approaches fail to assess the role of conserved genes in the determination of physiological characteristics of cultures by environmental conditions. This manuscript describes a complementary approach in which Lactobacillus plantarum WCFS1 was fermented under a variety of conditions that differ in temperature, pH, as well as NaCl, amino acid, and O2 levels. Samples derived from these fermentations were analyzed by full-genome transcriptomics, paralleled by the assessment of physiological characteristics, e.g., maximum growth rate, yield, and organic acid profiles. A data-storage and -mining suite designated FermDB was constructed and exploited to identify correlations between fermentation conditions and industrially relevant physiological characteristics of L. plantarum, as well as the associated transcriptome signatures. Finally, integration of the specific fermentation variables with the transcriptomes enabled the reconstruction of the gene-regulatory networks involved. The fermentation-genomics platform presented here is a valuable complementary approach to earlier described genotype-phenotype matching strategies which allows the identification of transcriptome signatures underlying physiological variations imposed by different fermentation conditions.
    Impact of 4 Lactobacillus plantarum capsular polysaccharide clusters on surface glycan composition and host cell signaling
    Remus, D.M. ; Kranenburg, R. van; Swam, I.I. van; Taverne, N. ; Bongers, R.S. ; Wels, M. ; Wells, J. ; Bron, P.A. ; Kleerebezem, M. - \ 2012
    Microbial Cell Factories 11 (2012)1. - ISSN 1475-2859 - 10 p.
    lactic-acid bacteria - gene-expression omnibus - streptococcus-pneumoniae - lactococcus-lactis - exopolysaccharide biosynthesis - microarray data - subsp cremoris - rhamnosus gg - identification - strains
    Background - Bacterial cell surface-associated polysaccharides are involved in the interactions of bacteria with their environment and play an important role in the communication between pathogenic bacteria and their host organisms. Cell surface polysaccharides of probiotic species are far less well described. Therefore, improved knowledge on these molecules is potentially of great importance to understand the strain-specific and proposed beneficial modes of probiotic action. Results - The Lactobacillus plantarum WCFS1 genome encodes 4 clusters of genes that are associated with surface polysaccharide production. Two of these clusters appear to encode all functions required for capsular polysaccharide formation (cps2A-J and cps4A-J), while the remaining clusters are predicted to lack genes encoding chain-length control functions and a priming glycosyl-transferase (cps1A-I and cps3A-J). We constructed L. plantarum WCFS1 gene deletion mutants that lack individual (¿cps1A-I, ¿cps2A-J, ¿cps3A-J and ¿cps4A-J) or combinations of cps clusters (¿cps1A-3J and ¿cps1A-3I, ¿cps4A-J) and assessed the genome wide impact of these mutations by transcriptome analysis. The cps cluster deletions influenced the expression of variable gene sets in the individual cps cluster mutants, but also considerable numbers of up- and down-regulated genes were shared between mutants in cps cluster 1 and 2, as well as between mutant in cps clusters 3 and 4. Additionally, the composition of overall cell surface polysaccharide fractions was altered in each mutant strain, implying that despite the apparent incompleteness of cps1A-I and cps3A-J, all clusters are active and functional in L. plantarum. The ¿cps1A-I strain produced surface polysaccharides in equal amounts as compared to the wild-type strain, while the polysaccharides were characterized by a reduced molar mass and the lack of rhamnose. The mutants that lacked functional copies of cps2A-J, cps3A-J or cps4A-J produced decreased levels of surface polysaccharides, whereas the molar mass and the composition of polysaccharides was not affected by these cluster mutations. In the quadruple mutant, the amount of surface polysaccharides was strongly reduced. The impact of the cps cluster mutations on toll-like receptor (TLR)-mediated human nuclear factor (NF)-¿B activation in host cells was evaluated using a TLR2 reporter cell line. In comparison to a L. plantarum wild-type derivative, TLR2 activation remained unaffected by the ¿cps1A-I and ¿cps3A-J mutants but appeared slightly increased after stimulation with the ¿cps2A-J and ¿cps4A-J mutants, while the ¿cps1A-3J and ¿cps1A-3J, ¿cps4A-J mutants elicited the strongest responses and clearly displayed enhanced TLR2 signaling. Conclusions - Our study reveals that modulation of surface glycan characteristics in L. plantarum highlights the role of these molecules in shielding of cell envelope embedded host receptor ligands. Although the apparently complete cps clusters (cps2A-J and cps4A-J) contributed individually to this shielding, the removal of all cps clusters led to the strongest signaling enhancement. Our findings provide new insights into cell surface glycan biosynthesis in L. plantarum, which bears relevance in the context of host-cell signaling by probiotic bacteria.
    Identification of key peptidoglycan hydrolases for morphogenesis, autolysis, and peptidoglycan composition of Lactobacillus plantarum WCFS1.
    Rolain, T. ; Bernard, E. ; Courtin, P. ; Bron, P.A. ; Kleerebezem, M. ; Chapot-Chartier, M.P. ; Hols, P. - \ 2012
    Microbial Cell Factories 11 (2012). - ISSN 1475-2859
    lactic-acid bacteria - lactococcus-lactis - n-acetylglucosaminidase - cell-wall - staphylococcus-aureus - bacillus-subtilis - murein hydrolase - gene - genome - electroporation
    Background - Lactobacillus plantarum is commonly used in industrial fermentation processes. Selected strains are also marketed as probiotics for their health beneficial effects. Although the functional role of peptidoglycan-degrading enzymes is increasingly documented to be important for a range of bacterial processes and host-microbe interactions, little is known about their functional roles in lactobacilli. This knowledge holds important potential for developing more robust strains resistant to autolysis under stress conditions as well as peptidoglycan engineering for a better understanding of the contribution of released muramyl-peptides as probiotic immunomodulators. Results - Here, we explored the functional role of the predicted peptidoglycan hydrolase (PGH) complement encoded in the genome of L. plantarum by systematic gene deletion. From twelve predicted PGH-encoding genes, nine could be individually inactivated and their corresponding mutant strains were characterized regarding their cell morphology, growth, and autolysis under various conditions. From this analysis, we identified two PGHs, the predicted N-acetylglucosaminidase Acm2 and NplC/P60 D,L-endopeptidase LytA, as key determinants in the morphology of L. plantarum. Acm2 was demonstrated to be required for the ultimate step of cell separation of daughter cells, whereas LytA appeared to be required for cell shape maintenance and cell-wall integrity. We also showed by autolysis experiments that both PGHs are involved in the global autolytic process with a dominant role for Acm2 in all tested conditions, identifying Acm2 as the major autolysin of L. plantarum WCFS1. In addition, Acm2 and the putative N-acetylmuramidase Lys2 were shown to play redundant roles in both cell separation and autolysis under stress conditions. Finally, the analysis of the peptidoglycan composition of Acm2- and LytA-deficient derivatives revealed their potential hydrolytic activities by the disappearance of specific cleavage products. Conclusion - In this study, we showed that two PGHs of L. plantarum have a predominant physiological role in a range of growth conditions. We demonstrate that the N-acetylglucosaminidase Acm2 is the major autolysin whereas the D,L-endopeptidase LytA is a key morphogenic determinant. In addition, both PGHs have a direct impact on PG structure by generating a higher diversity of cleavage products that could be of importance for interaction with the innate immune system.
    B-glucans are involved in immune-modulation of THP-1
    Chanput, W. ; Reitsma, M. ; Kleinjans, L. ; Mes, J.J. ; Savelkoul, H.F.J. ; Wichers, H.J. - \ 2012
    Molecular Nutrition & Food Research 56 (2012)5. - ISSN 1613-4125 - p. 822 - 833.
    lactic-acid bacteria - factor-kappa-b - cell-line - cytokine production - interferon-gamma - dendritic cells - receptor - lentinan - mice - lipopolysaccharide
    Scope We aimed to examine different immunological aspects of ß-glucans derived from different food sources (oat, barley and shiitake) on phorbol myristate acetate (PMA)-differentiated THP-1 macrophages. Commercially purified barley ß-glucan (commercial BG) and lentinan were included to compare ß-glucans from the same origin but different degree of purity and processing. Methods and results Chemical composition and molecular weight distribution of ß-glucan samples were determined. Inflammation-related gene expression kinetics (IL-1ß, IL-8, nuclear factor kappa B [NF-¿B] and IL-10) after 3, 6 and 24 h of stimulation with 100 µg/mL ß-glucan were investigated. All tested ß-glucans mildly upregulated the observed inflammation-related genes with differential gene expression patterns. Similar gene expression kinetics, but different fold induction values, was found for the crude ß-glucan extracts and their corresponding commercial forms. Pre-incubation of THP-1 macrophages with ß-glucans prior to lipopolysaccharide (LPS) exposure decreased the induction of inflammation-related genes compared to LPS treatment. No production of nitric oxide (NO) and hydrogen peroxide (H2O2) was detected in ß-glucan stimulated THP-1 macrophages. Phagocytic activity was not different after stimulation by ß-glucan samples. Conclusion Based on these in vitro analyses, it can be concluded that the analysed ß-glucans have varying levels of immunomodulating properties, which are likely related to structure, molecular weight and compositional characteristic of ß-glucan
    Modulation of Lactobacillus plantarum gastrointestinal robustness by fermentation conditions enables identification of bacterial robustness markers
    Bokhorst-van de Veen, H. van; Lee, I. ; Marco, M.L. ; Bron, P.A. ; Kleerebezem, M. - \ 2012
    PLoS ONE 7 (2012)7. - ISSN 1932-6203 - 13 p.
    lactic-acid bacteria - stress-response - in-vitro - genetic-characterization - bile-salt - strains - wcfs1 - lactococcus - survival - reuteri
    Background - Lactic acid bacteria (LAB) are applied worldwide in the production of a variety of fermented food products. Additionally, specific Lactobacillus species are nowadays recognized for their health-promoting effects on the consumer. To optimally exert such beneficial effects, it is considered of great importance that these probiotic bacteria reach their target sites in the gut alive. Methodology/Principal Findings - In the accompanying manuscript by Bron et al. the probiotic model organism Lactobacillus plantarum WCFS1 was cultured under different fermentation conditions, which was complemented by the determination of the corresponding molecular responses by full-genome transcriptome analyses. Here, the gastrointestinal (GI) survival of the cultures produced was assessed in an in vitro assay. Variations in fermentation conditions led to dramatic differences in GI-tract survival (up to 7-log) and high robustness could be associated with low salt and low pH during the fermentations. Moreover, random forest correlation analyses allowed the identification of specific transcripts associated with robustness. Subsequently, the corresponding genes were targeted by genetic engineering, aiming to enhance robustness, which could be achieved for 3 of the genes that negatively correlated with robustness and where deletion derivatives displayed enhanced survival compared to the parental strain. Specifically, a role in GI-tract survival could be confirmed for the lp_1669-encoded AraC-family transcription regulator, involved in capsular polysaccharide remodeling, the penicillin-binding protein Pbp2A involved in peptidoglycan biosynthesis, and the Na+/H+ antiporter NapA3. Moreover, additional physiological analysis established a role for Pbp2A and NapA3 in bile salt and salt tolerance, respectively. Conclusion - Transcriptome trait matching enabled the identification of biomarkers for bacterial (gut-)robustness, which is important for our molecular understanding of GI-tract survival and could facilitate the design of culture conditions aimed to enhance probiotic culture robustness
    Immunomodulatory effects of potential probiotics in a mouse peanut sensitization model
    Meijerink, M. ; Wells, J. ; Taverne, N. ; Zeeuw Brouwer, M.L. de; Hilhorst, B. ; Venema, K. ; Bilsen, J. van - \ 2012
    FEMS Immunology and Medical Microbiology 65 (2012)3. - ISSN 0928-8244 - p. 488 - 496.
    lactic-acid bacteria - placebo-controlled trial - regulatory t-cells - blood mononuclear-cells - atopic-dermatitis - dendritic cells - food allergy - lactobacillus-rhamnosus - cytokine production - responses
    Peanut allergy accounts for the majority of severe food-related allergic reactions and there is a need for new prevention and treatment strategies. Probiotics may be considered for treatment on the basis of their immunomodulating properties. Cytokine profiles of probiotic strains were determined by in vitro co-culture with human PBMCs. Three strains were selected to investigate their prophylactic potential in a peanut sensitization model by analysing peanut-specific antibodies, mast cell degranulation and ex vivo cytokine production by splenocytes. The probiotic strains induced highly variable cytokine profiles in PBMCs. L. salivarius HMI001, L. casei Shirota (LCS) and L. plantarum WCFS1 were selected for further investigation owing to their distinct cytokine patterns. Prophylactic treatment with both HMI001 and LCS attenuated the Th2 phenotype (reduced mast cell responses and ex vivo IL-4 and/or IL-5 production). In contrast, WCFS1 augmented the Th2 phenotype (increased mast cell and antibody responses and ex vivo IL-4 production). In vitro PBMC screening was useful in selecting strains with anti-inflammatory and Th1 skewing properties. In case of HMI001 (high IL-10/IL-12 ratio) and LCS (high interferon- and IL-12), partial protection was seen in a mouse peanut allergy model. Strikingly, certain strains may worsen the allergic reaction as shown in the case of WCFS1.
    Complex microbiota of a Chinese "Fen" liquor fermentation starter (Fen-Daqu), revealed by culture-dependent and culture-independent methods
    Zheng, X. ; Zheng, Y. ; Han, B. ; Zwietering, M.H. ; Samson, R.A. ; Boekhout, T. ; Nout, M.J.R. - \ 2012
    Food Microbiology 31 (2012)2. - ISSN 0740-0020 - p. 293 - 300.
    gradient gel-electrophoresis - solid-state fermentation - lactic-acid bacteria - rice wine - saccharomycopsis-fibuligera - volatile metabolites - flavor liquor - identification - yeast - strains
    Daqu is a traditional fermentation starter that is used for Chinese liquor production. Although partly mechanized, its manufacturing process has remained traditional. We investigated the microbial diversity of Fen-Daqu, a starter for light-flavour liquor, using combined culture-dependent and culture-independent approaches (PCR–DGGE). A total of 190 microbial strains, comprising 109 bacteria and 81 yeasts and moulds, were isolated and identified on the basis of the sequences of their 16S rDNA (bacteria) and 26S rDNA and ITS regions (fungi). DGGE of DNA extracted from Daqu was used to complement the culture-dependent method in order to include non-culturable microbes. Both approaches revealed that Bacillus licheniformis was an abundant bacterial species, and Saccharomycopsis fibuligera, Wickerhamomyces anomalus, and Pichia kudriavzevii were the most common yeasts encountered in Fen-Daqu. Six genera of moulds (Absidia, Aspergillus, Mucor, Rhizopus, Rhizomucor and Penicillium) were found. The potential function of these microorganisms in starters for alcoholic fermentation is discussed. In general the culture-based findings overlapped with those obtained by DGGE by a large extent. However, Weissella cibaria, Weissella confusa, Staphylococcus saprophyticus, Enterobacter aerogenes, Lactobacillus sanfranciscensis, Lactobacillus lactis, and Bacillus megaterium were only revealed by DGGE
    Production of oat-based synbiotic beverage by two-stage fermentation with Rhizopus oryzae and Lactobacillus acidophilus
    Gao, F. ; Cai, S. ; Nout, M.J.R. ; Wang, Y. ; Xia, Y. ; Li, Y. ; Ji, B. - \ 2012
    Journal of Food, Agriculture & Environment 10 (2012)2. - ISSN 1459-0255 - p. 175 - 179.
    lactic-acid bacteria - beta-glucan - dietary fiber - in-vitro - survival - bifidobacterium - temperature - strains - health - model
    Many studies have reported that oats could effectively reduce the serum cholesterol levels in humans, and the ß-glucan in oat is believed to be responsible for this physiologic effect. Probiotics are live microorganisms that can confer a healthy benefit on the host when administered in adequate amounts. There is an increasing use of these microorganisms in food, aiming to balance intestinal microflora and alleviate dysfunction of the human gastrointestinal tract. However, a number of studies have shown that only 10-30% of these probiotic bacteria could survive after passing through the gastrointestinal (GI) tract. Lactobacillus acidophilus is used as a probiotic bacterium in many probiotic foods. However, L. acidophilus shows poor growth in cereal products due to its poor hydrolytic ability of protein and macromolecule carbohydrates. The aim of the present study was to combine Rhizopus oryzae-fermented oat mash and Lactobacillus acidophilus in an oat-based synbiotic beverage. Several factors, including starter culture concentration, R. oryzae-fermented oat mash and skim milk powder, were investigated. The nutritional contents in R. oryzae-fermented oat flour were just sufficient for survival but not growth of L. acidophilus. Adding sucrose (1% or 2%, w/v) did not improve the proliferation of L. acidophilus; however, L. acidophilus grew quickly when skim milk powder (1% or 2%, w/v) was added. When 5.5% R. oryzae-fermented oat mash with 2% added skim milk powder was used, the viable cell counts reached about 9.0 log cfu/ml at the end of 10 h fermentation. The concentration of ß-glucans (about 781 mg/l) was not significantly lowered during fermentation.
    Effect of respiration and manganese on oxidative stress resistance of Lactobacillus plantarum WCFS1
    Watanabe, M. ; Veen, S. van der; Nakajima, H. ; Abee, T. - \ 2012
    Microbiology 158 (2012)1. - ISSN 1350-0872 - p. 293 - 300.
    lactic-acid bacteria - lactococcus-lactis - electron-transport - hydrogen-peroxide - escherichia-coli - catalase - expression - survival - tolerance - toxicity
    Lactobacillus plantarum is a facultatively anaerobic bacterium that can perform respiration under aerobic conditions in the presence of haem, with vitamin K2 acting as a source of menaquinone. We investigated growth performance and oxidative stress resistance of Lb. plantarum WCFS1 cultures grown in de Man, Rogosa and Sharpe (MRS) medium without and with added manganese under fermentative, aerobic, aerobic with haem, and respiratory conditions. Previous studies showed that Lb. plantarum WCFS1 lacks a superoxide dismutase and requires high levels of manganese for optimum fermentative and aerobic growth. In this study, respiratory growth with added manganese resulted in significantly higher cell densities compared to the other growth conditions, while without manganese added, similar but lower cell densities were reached. Notably, cells derived from the respiratory cultures showed the highest hydrogen peroxide resistance in all conditions tested, although similar activity levels of haem-dependent catalase were detected in cells grown under aerobic conditions with haem. These results indicate that oxidative stress resistance of Lb. plantarum is affected by respiratory growth, growth phase, haem and manganese. As levels of haem and manganese can differ considerably in the raw materials used in fermentation processes, including those of milk, meat and vegetables, the insight gained here may provide tools to increase the performance and robustness of starter bacteria
    Antagonistic intestinal microflora produces antimicrobial substance inhibitory to pseudomonas species and other spoilage organisms
    Hatew, B. ; Delessa, T. ; Zakin, V. ; Gollop, N. - \ 2011
    Journal of Food Science 76 (2011)8. - ISSN 0022-1147 - p. M522 - M530.
    lactic-acid bacteria - listeria-monocytogenes - antibacterial activity - lactobacillus-reuteri - bacillus-cereus - shelf-life - pathogens - poultry - chicken - meat
    Chicken intestine harbors a vast number of bacterial strains. In the present study, antimicrobial substance produced by lactic acid bacteria (LAB) isolated from the gastrointestinal tract of healthy chicken was detected, characterized, and purified. Based on 16S rRNA sequencing, the bacteria were identified as Lactobacillus plantarum vN. The antimicrobial substance produced by this bacterium was designated vN-1 and exhibited a broad-spectrum of activity against many important pathogenic and spoilage microorganisms, including Pseudomonas aeruginosa, Staphylococcus aureus, Micrococcus luteus, Salmonella Typhimurium, and Erwinia amylovova. vN-1 was determined to be thermostable, insensitive to pH values ranging from 2.0 to 8.0, resistant to various organic solvents and to enzymatic inactivation. The inhibition kinetics displayed a bactericidal mode of action. This study revealed an antimicrobial substance with low molecular mass of less than 1 kDa as determined by ultrafiltration and having features not previously reported for LAB isolated from chicken intestines. The detection of this antimicrobial substance addresses an important aspect of biotechnological control agents of spoilage caused by Pseudomonas spp. and promises the possibility for preservation of refrigerated poultry meat.
    Immunomodulatory mechanisms of lactobacilli
    Wells, J. - \ 2011
    Microbial Cell Factories 10 (2011)supl. 1. - ISSN 1475-2859
    intestinal epithelial-cells - placebo-controlled trial - lactic-acid bacteria - blood mononuclear-cells - toll-like receptors - randomized controlled-trial - inflammatory-bowel-disease - tight junction proteins - necrosis-factor-alpha - in-vivo
    Over the past decade it has become clear that lactobacilli and other probiotic and commensal organisms can interact with mucosal immune cells or epithelial cells lining the mucosa to modulate specific functions of the mucosal immune system. The most well understood signalling mechanisms involve the innate pattern recognition receptors such as Toll-like receptors, nucleotide oligomerization domain-like receptors and C-type lectin receptors. Binding of microbe-associated molecular patterns with these receptors can activate antigen presenting cells and modulate their function through the expression of surface receptors, secreted cytokines and chemokines. In vitro the cytokine response of human peripheral blood mononuclear cells and dendritic cells to lactobacilli can be strikingly different depending on both the bacterial species and the strain. Several factors have been identified in lactobacilli that influence the immune response in vitro and in vivo including cell surface carbohydrates, enzymes modifying the structure of lipoteichoic acids and metabolites. In mice mechanistic studies point to a role for the homeostatic control of inducible T regulatory cells in the mucosal tissues as one possible immunomodulatory mechanism. Increasing evidence also suggests that induction of epithelial signalling by intestinal lactobacilli can modulate barrier functions, defensin production and regulate inflammatory signalling. Other probiotic mechanisms include modulation of the T cell effector subsets, enhancement of humoral immunity and interactions with the epithelial-associated dendritic cells and macrophages. A major challenge for the future will be to gain more knowledge about the interactions occurring between lactobacilli and the host in vivo and to understand the molecular basis of innate signalling in response to whole bacteria which trigger multiple signalling pathways.
    Probiotics - do they have a role in the pig industry?
    Kenny, M. ; Smidt, H. ; Mengheri, E. ; Miller, B. - \ 2011
    Animal 5 (2011)3. - ISSN 1751-7311 - p. 462 - 470.
    enterica serovar typhimurium - toll-like-receptor - cereus-var. toyoi - enterococcus-faecium ncimb-10415 - host-microbial interactions - human rotavirus infection - mucin gene-expression - lactic-acid bacteria - escherichia-coli k88 - growth-performance
    The delivery of certain living microorganisms in food has long been suggested as having positive health effects in humans. This practice has extended into food animal production, with a variety of microorganisms being used; lactic acid bacteria, various Bacillus species and the yeast Saccharomyces cerevisiae have been particularly used in the pig industry. The increased interest in probiotics is essentially due to the problem of microbial resistance to antibiotics and following the ban of the use of antibiotics in animal production, probiotics being considered an alternative means to reduce pathogen infection and improve animal health especially around the time of weaning. However, there is still a need to clarify the probiotic effectiveness in pigs, and the underlying mechanisms. When assessing the efficacy of probiotics one must consider the particular strain of organism being used and the production stage of the pigs being treated. The reproducible delivery of probiotics in industrial pig production is problematic as maintenance of viability is key to their beneficial activity, but difficult to achieve with commonly used feed processing technologies. One specific context where probiotics organisms may be reliably delivered is in systems utilising fermented liquid feeds. Liquid feed may be fermented by the activity of wild lactic acid bacteria or may be stimulated using specific isolates as 'starters'; the latter system has advantages in terms of reproducibility and speed of fermentation. The farm context in which the organism is used is likely to be critical; the use of probiotics is more likely to result in measurable economic gains in animals living in sub-optimal conditions rather than in those reared in the highest welfare and husbandry conditions. The establishment of a beneficial lactic acid bacteria population at birth may lead to healthier animals, this may be most effectively achieved by treating sows, which provide an amplification step and flood the neonatal pigs' environment with desirable bacterial strains. In contrast, it may be sufficient to provide a supportive, protective microbiota around the time of weaning as this is a time of major crisis with instability and loss of certain bacterial populations.
    An intimate tete-a-tete - How probiotic lactobacilli communicate with the host
    Remus, D.M. ; Kleerebezem, M. ; Bron, P.A. - \ 2011
    European Journal of Pharmacology 668 (2011)Suppl. 1. - ISSN 0014-2999 - p. S33 - S42.
    intestinal epithelial-cells - innate immune-system - peptidoglycan recognition proteins - inflammatory-bowel-disease - lactic-acid bacteria - toll-like receptor-2 - nf-kappa-b - rhamnosus gg - lipoteichoic acid - escherichia-coli
    Pharmaceutical agents are routinely used in the treatment of gastrointestinal disorders and their role as modulators of host cell responses is well characterized. In contrast, the understanding of the molecular mechanisms, which determine the role of probiotics, i.e. health-promoting bacteria, as host cell modulators is still in its infancy. Both in vitro and in vivo studies are just starting to reveal the capability of probiotic lactobacilli to modulate host cell-signaling networks and the associated influences on downstream regulatory pathways, including modulation of mucosal cytokine profiles that dictate host immune functions. The communication between probiotic lactobacilli and intestinal host cells is multifactorial and involves an integrative repertoire of receptors on the host side that recognize multiple effector molecules on the bacterial side, of which most have been found to be cell wall- or cell surface-associated compounds and proteins. This review describes the discovery of these bacterial effector molecules and their role in strain- and species-specific modulation of host signaling pathways. Unraveling the mechanisms responsible for probiotic-host interactions will progress this research field towards molecular science and will provide markers for probiotic product quality control as well as host-response efficacy. These developments can ultimately lead to a more dedicated, personalized application of probiotics with strong molecular and scientific support for health promotion.
    Cultivation independent analysis of the development of the Lactobacillus spp. Community in the intestinal tract of newborn piglets
    Yao, W. ; Zhu, W.Y. ; Smidt, H. ; Verstegen, M.W.A. - \ 2011
    Agricultural Sciences in China 10 (2011)3. - ISSN 1671-2927 - p. 438 - 447.
    gradient gel-electrophoresis - 16s ribosomal-rna - lactic-acid bacteria - weaning piglets - gastrointestinal-tract - weanling pigs - weight-gain - probiotics - microbiota - quantification
    Molecular diversity and development of the Lactobacillus community in the intestinal tract, as influenced by age and intestinal compartment, were studied in one litter of 12 conventionally raised piglets. Piglets were euthanized at each week (3 animals per time). Digesta and tissue samples from stomach, duodenum, jejunum, ileum, caecum, colon, and rectum were collected and analysed by using 16S ribosomal RNA-based methods. DGGE (denaturing gradient gel electrophoresis) profiles revealed that the Lactobacillus communities throughout the GI tract from duodenum to rectum showed good stability at same age. This indicates that fecal Lactobacillus communities can effectively represent the intestinal community. Two dominant bands were found in tissue samples of the small intestine, suggesting that the lactobacilli can adhere to the small intestinal wall. The Lactobacillus communities in different GI tract compartments developed over time. A successional change of Lactobacillus communities was observed from birth, through creep feeding to one week after weaning, showing a trend from simple to complex and back to simple. Furthermore, a clone library of Lactobacillus spp. 16S rRNA gene sequences were generated from jejunal and colonic chymes. Six dominant DGGE bands generated from jejunal chymes were matched with sequences that show 94-98% similarity to the bands derived from L. reuteri, L. delbrueckii, and L. crispatus. Seven dominant DGGE bands generated from colon chymes were matched with sequences that show 88-99% similarity to those derived from L. reuteri, L. delbrueckii, L. amylovorus/L. sobrius, and L. acidophilus. Amplicons related to L. reuteri were found in all DGGE fingerprints from jejunal digesta of age of weeks 1, 3, and 4. Amplicons related to L. amylovorus/L. sobrius were present in all DGGE fingerprints from colonic digesta of age of week 1, 3, and 4. Amplicons related to L. delbrueckii were found before weaning, L. crispatus after creep feeding before weaning and L. acidophilus after weaning. This indicates that L. reuteri and L. amylovorus/L. sobrius probably belong to the permanent composition, while L. delbruckii, L. acidophilus, and L. crispatus probably belong to the temporal groups of Lactobacillus communities in the GI tract of piglets.
    Short- and long-term adaptation to ethanol stress and its cross-protective consequences in Lactobacillus plantarum
    Bokhorst-van de Veen, H. van; Abee, T. ; Tempelaars, M.H. ; Bron, P.A. ; Kleerebezem, M. ; Marco, M.L. - \ 2011
    Applied and Environmental Microbiology 77 (2011)15. - ISSN 0099-2240 - p. 5247 - 5256.
    gram-positive bacteria - lactic-acid bacteria - heat-shock response - bacillus-subtilis - escherichia-coli - molecular characterization - citrate metabolism - oenococcus-oeni - lactococcus-lactis - gene-expression
    This paper describes the molecular responses of Lactobacillus plantarum WCFS1 toward ethanol exposure. Global transcriptome profiling using DNA microarrays demonstrated adaptation of the microorganism to the presence of 8% ethanol over short (10-min and 30-min) and long (24-h) time intervals. A total of 57 genes were differentially expressed at all time points. Expression levels of an additional 859 and 873 genes were modulated after 30 min and 24 h of exposure to the solvent, respectively. Ethanol exposure led to induced expression of genes involved in citrate metabolism and cell envelope architecture, as well as canonical stress response pathways controlled by the central stress regulators HrcA and CtsR. Correspondingly, cells grown for 24 h in medium containing 8% ethanol exhibited higher levels of citrate consumption and modified cell membrane fatty acid composition and showed invaginating septa compared with cells grown in liquid medium without ethanol. In addition, these physiological changes resulted in cross-protection against high temperatures but not against several other stresses tested. To evaluate the role of HrcA and CtsR in ethanol tolerance, ctsR and hrcA gene deletion mutants were constructed. The growth rate of the L. plantarum ¿ctsR::cat strain was impaired in de Man-Rogosa-Sharpe (MRS) medium containing 8% ethanol, whereas growth of the L. plantarum ¿hrcA::cat and ¿ctsR ¿hrcA::cat mutants was indistinguishable from that of wild-type cells. Overall, these results suggest that the induction of CtsR class III stress responses provides cross-protection against heat stress.
    Comparative genomics of Lactobacillus
    Kant, R. ; Siezen, R.J. ; Vos, W.M. de - \ 2011
    Microbial Biotechnology 4 (2011)3. - ISSN 1751-7907 - p. 323 - 332.
    lactic-acid bacteria - genus lactobacillus - sequence - plantarum - diversity - protein - tract
    The genus Lactobacillus includes a diverse group of bacteria consisting of many species that are associated with fermentations of plants, meat or milk. In addition, various lactobacilli are natural inhabitants of the intestinal tract of humans and other animals. Finally, several Lactobacillus strains are marketed as probiotics as their consumption can confer a health benefit to host. Presently, 154 Lactobacillus species are known and a growing fraction of these are subject to draft genome sequencing. However, complete genome sequences are needed to provide a platform for detailed genomic comparisons. Therefore, we selected a total of 20 genomes of various Lactobacillus strains for which complete genomic sequences have been reported. These genomes had sizes varying from 1.8 to 3.3 Mb and other characteristic features, such as G+C content that ranged from 33% to 51%. The Lactobacillus pan genome was found to consist of approximately 14 000 protein-encoding genes while all 20 genomes shared a total of 383 sets of orthologous genes that defined the Lactobacillus core genome (LCG). Based on advanced phylogeny of the proteins encoded by this LCG, we grouped the 20 strains into three main groups and defined core group genes present in all genomes of a single group, signature group genes shared in all genomes of one group but absent in all other Lactobacillus genomes, and Group-specific ORFans present in core group genes of one group and absent in all other complete genomes. The latter are of specific value in defining the different groups of genomes. The study provides a platform for present individual comparisons as well as future analysis of new Lactobacillus genomes
    Analysis of infant isolates of Bifidobacterium breve by comparative genome hybridization indicates the existence of new subspecies with marked infant specificity
    Boesten, R.J. ; Vos, W.M. de - \ 2011
    Research in Microbiology 162 (2011)7. - ISSN 0923-2508 - p. 664 - 670.
    lactic-acid bacteria - intestinal bifidobacteria - lactobacillus - ucc2003 - actinobacteria - identification - communities - expression - diversity - system
    A total of 20 Bifidobacterium strains were isolated from fecal samples of 4 breast- and bottle-fed infants and all were characterized as Bifidobacterium breve based on 16S rRNA gene sequence and metabolic analysis. These isolates were further characterized and compared to the type strains of B. breve and 7 other Bifidobacterium spp. by comparative genome hybridization. For this purpose, we constructed and used a DNA-based microarray containing over 2000 randomly cloned DNA fragments from B. breve type strain LMG13208. This molecular analysis revealed a high degree of genomic variation between the isolated strains and allowed the vast majority to be grouped into 4 clusters. One cluster contained a single isolate that was virtually indistinguishable from the B. breve type strain. The 3 other clusters included 19 B. breve strains that differed considerably from all type strains. Remarkably, each of the 4 clusters included strains that were isolated from a single infant, indicating that a niche adaptation may contribute to variation within the B. breve species. Based on genomic hybridization data, the new B. breve isolates were estimated to contain approximately 60-90% of the genes of the B. breve type strain, attesting to the existence of various subspecies within the species B. breve. Further bioinformatic analysis identified several hundred diagnostic clones specific to the genomic clustering of the B. breve isolates. Molecular analysis of representatives of these revealed that annotated genes from the conserved B. breve core encoded mainly housekeeping functions, while the strain-specific genes were predicted to code for functions related to life style, such as carbohydrate metabolism and transport. This is compatible with genetic adaptation of the strains to their niche, a combination of infants and diet
    Functional characterization of a mucus-specific LPXTG surface adhesin from probiotic Lactobacillus rhamnosus GG
    Ossowski, I. von; Vos, W.M. de; Palva, A. - \ 2011
    Applied and Environmental Microbiology 77 (2011)13. - ISSN 0099-2240 - p. 4465 - 4472.
    lactic-acid bacteria - cell-surface - listeria-monocytogenes - oral consumption - intestinal mucus - protein - pili - plantarum - mucin - identification
    In spite of the wealth of clinical evidence supporting the health benefits of Lactobacillus rhamnosus GG in humans, there is still a lack of understanding of the molecular mechanisms behind its probiosis. Current knowledge suggests that the health-promoting effects of this probiotic strain might be partly dependent on its persistence in the intestine and adhesion to mucosal surfaces. Moreover, L. rhamnosus GG contains mucus-binding pili that might also explain the occupation of its ecological niche as a comparatively less stringent allochthonous intestine-dwelling bacterium. To uncover additional surface proteins involved in mucosal adhesion, we investigated the adherence properties of the only predicted protein (LGG_02337) in L. rhamnosus GG that exhibits homology with a known mucus-binding domain. We cloned a recombinant form of the gene for this putative mucus adhesin and established that the purified protein readily adheres to human intestinal mucus. We also showed that this mucus adhesin is visibly distributed throughout the cell surface and participates in the adhesive interaction between L. rhamnosus GG and mucus, although less prominently than the mucus-binding pili in this strain. Based on primary structural comparisons, we concluded that the current annotation of the LGG_02337 protein likely does not accurately reflect its predicted properties, and we propose that this mucus-specific adhesin be called the mucus-binding factor (MBF). Finally, we interpret our results to mean that L. rhamnosus GG MBF, as an active mucus-specific surface adhesin with a presumed ancillary involvement in pilus-mediated mucosal adhesion, plays a part in the adherent mechanisms during intestinal colonization by this probiotic
    Lactobacillus strains differentially modulate cytokine production by hPBMC from pollen allergic patients
    Vissers, Y.M. ; Snel, J. ; Zuurendonk, P.F. ; Kleerebezem, M. ; Wichers, H.J. ; Savelkoul, H.F.J. - \ 2011
    FEMS Immunology and Medical Microbiology 61 (2011)1. - ISSN 0928-8244 - p. 28 - 40.
    blood mononuclear-cells - lactic-acid bacteria - regulatory t-cells - tumor-necrosis-factor - in-vitro - probiotic bacteria - intestinal microbiota - food allergy - immunomodulatory properties - mucosal immunology
    The objective of this study was to assess the potential immunomodulatory effect of six Lactobacillus strains on human peripheral blood mononuclear cells (hPBMC) isolated from allergic patients. hPBMC from patients allergic to birch pollen or grass pollen were cultured in vitro in the presence or absence of selective bacterial strains. Cultures were left unstimulated or stimulated with aCD3/aCD28 or Bet v 1. After 1, 4 and 8 days, cells and culture supernatants were harvested and the effect on cellular proliferation and the supernatant levels of several cytokines was assessed. All strains had the ability to repress IL-13 production but did show a differential effect on IFN-¿ induction. Both strains B223 and B1697 showed a lower IFN-¿, IL-12 and TNF-a induction as compared with the other tested strains. Strain B633 showed the best proliferation-suppressive properties in aCD3/aCD28-stimulated cells. Suppression of the T-helper type 2 (Th2) cytokine induction and induction of the Th1 cytokine production by specific strains might be beneficial for allergic patients having a disturbed Th1/Th2 immune balance. Furthermore, hPBMC of patients with seasonal allergy outside the pollen season can be used to determine the immunomodulatory activities of probiotic bacteria
    Antibiotic susceptibility of members of the Lactobacillus acidophilus group using broth microdilution and molecular identification of their resistance determinants
    Mayrhofer, S. ; Hoek, A.H.A.M. van; Mair, C. ; Huys, G. ; Aarts, H.J.M. ; Kneifel, W. ; Domig, K.J. - \ 2010
    International Journal of Food Microbiology 144 (2010)1. - ISSN 0168-1605 - p. 81 - 87.
    lactic-acid bacteria - antimicrobial susceptibility - safety - genes - food - bifidobacteria - probiotics - strains - origin - pediococcus
    The range of antibiotic susceptibility to 13 antibiotics in 101 strains of the Lactobacillus acidophilus group was examined using the lactic acid bacteria susceptibility test medium (LSM) and broth microdilution. Additionally, microarray analysis and PCR were applied to identify resistance genes responsible for the displayed resistant phenotypes in a selection of strains. In general, narrow as well as broad unimodal and bimodal MIC distributions were observed for the Lactobacillus acidophilus group and the tested antimicrobial agents. Atypically resistant strains could be determined by visual inspection of the obtained MIC ranges for ampicillin, chloramphenicol, clindamycin, erythromycin, quinupristin/dalfopristin, streptomycin and tetracycline. For most of these atypically resistant strains underlying resistance determinants were found. To our knowledge erm(A) was detected in lactobacilli for the first time within this study. Data derived from this study can be used as a basis for reviewing present microbiological breakpoints for categorization of susceptible and resistant strains within the Lactobacillus acidophilus group to assess the safety of microorganisms intended for use in food and feed applications
    The extracellular biology of the lactobacilli
    Kleerebezem, M. ; Hols, P. ; Bernard, E. ; Rolain, T. ; Zhou, M. ; Siezen, R.J. ; Bron, P.A. - \ 2010
    FEMS Microbiology Reviews 34 (2010)2. - ISSN 0168-6445 - p. 199 - 230.
    gram-positive bacteria - lactic-acid bacteria - s-layer protein - bile-salt hydrolase - mouse gastrointestinal-tract - complete genome sequence - johnsonii strain ncc533 - alanyl ester depletion - cell-surface protein - d-alanine ligase
    Lactobacilli belong to the lactic acid bacteria, which play a key role in industrial and artisan food raw-material fermentation, including a large variety of fermented dairy products. Next to their role in fermentation processes, specific strains of Lactobacillus are currently marketed as health-promoting cultures or probiotics. The last decade has witnessed the completion of a large number of Lactobacillus genome sequences, including the genome sequences of some of the probiotic species and strains. This development opens avenues to unravel the Lactobacillus-associated health-promoting activity at the molecular level. It is generally considered likely that an important part of the Lactobacillus effector molecules that participate in the proposed health-promoting interactions with the host (intestinal) system resides in the bacterial cell envelope. For this reason, it is important to accurately predict the Lactobacillus exoproteomes. Extensive annotation of these exoproteomes, combined with comparative analysis of species- or strain-specific exoproteomes, may identify candidate effector molecules, which may support specific effects on host physiology associated with particular Lactobacillus strains. Candidate health-promoting effector molecules of lactobacilli can then be validated via mutant approaches, which will allow for improved strain selection procedures, improved product quality control criteria and molecular science-based health claims
    Identification of Lactobacillus plantarum genes modulating the cytokine response of human peripheral blood mononuclear cells
    Hemert, S. van; Meijerink, M. ; Molenaar, D. ; Bron, P.A. ; Vos, P. de; Kleerebezem, M. ; Wells, J. ; Marco, M. - \ 2010
    BMC Microbiology 10 (2010). - ISSN 1471-2180
    lactic-acid bacteria - necrosis-factor-alpha - gastrointestinal-tract - immunomodulatory properties - transcriptome analysis - probiotic properties - growth-phase - in-vivo - strains - wcfs1
    Background Modulation of the immune system is one of the most plausible mechanisms underlying the beneficial effects of probiotic bacteria on human health. Presently, the specific probiotic cell products responsible for immunomodulation are largely unknown. In this study, the genetic and phenotypic diversity of strains of the Lactobacillus plantarum species were investigated to identify genes of L. plantarum with the potential to influence the amounts of cytokines interleukin 10 (IL-10) and IL-12 and the ratio of IL-10/IL-12 produced by peripheral blood mononuclear cells (PBMCs). Results A total of 42 Lactobacillus plantarum strains isolated from diverse environmental and human sources were evaluated for their capacity to stimulate cytokine production in PBMCs. The L. plantarum strains induced the secretion of the anti-inflammatory cytokine IL-10 over an average 14-fold range and secretion of the pro-inflammatory cytokine IL-12 over an average 16-fold range. Comparisons of the strain-specific cytokine responses of PBMCs to comparative genome hybridization profiles obtained with L. plantarum WCFS1 DNA microarrays (also termed gene-trait matching) resulted in the identification of 6 candidate genetic loci with immunomodulatory capacities. These loci included genes encoding an N-acetyl-glucosamine/galactosamine phosphotransferase system, the LamBDCA quorum sensing system, and components of the plantaricin (bacteriocin) biosynthesis and transport pathway. Deletion of these genes in L. plantarum WCFS1 resulted in growth phase-dependent changes in the PBMC IL-10 and IL-12 cytokine profiles compared with wild-type cells. Conclusions The altered PBMC cytokine profiles obtained with the L. plantarum WCFS1 mutants were in good agreement with the predictions made by gene-trait matching for the 42 L. plantarum strains. This study therefore resulted in the identification of genes present in certain strains of L. plantarum which might be responsible for the stimulation of anti- or pro-inflammatory immune responses in the gut
    The effects of iron fortification on the gut microbiota in African children: a randomized controlled trial in Côte d'Ivoire
    Zimmermann, M.B. ; Chassard, C. ; Rohner, F. ; N'goran, E.K. ; Nindjin, C. ; Dostal, A. ; Utzinger, J. ; Ghattas, H. ; Lacroix, C. ; Hurrell, R.F. - \ 2010
    American Journal of Clinical Nutrition 92 (2010)6. - ISSN 0002-9165 - p. 1406 - 1415.
    gradient gel-electrophoresis - 16s ribosomal-rna - routine prophylactic supplementation - inflammatory-bowel-disease - placebo-controlled trial - lactic-acid bacteria - fecal microbiota - fermented milk - folic-acid - pcr
    Background: Iron is essential for the growth and virulence of many pathogenic enterobacteria, whereas beneficial barrier bacteria, such as lactobacilli, do not require iron. Thus, increasing colonic iron could select gut microbiota for humans that are unfavorable to the host. Objective: The objective was to determine the effect of iron fortification on gut microbiota and gut inflammation in African children. Design: In a 6-mo, randomized, double-blind, controlled trial, 6–14-y-old Ivorian children (n = 139) received iron-fortified biscuits, which contained 20 mg Fe/d, 4 times/wk as electrolytic iron or nonfortified biscuits. We measured changes in hemoglobin concentrations, inflammation, iron status, helminths, diarrhea, fecal calprotectin concentrations, and microbiota diversity and composition (n = 60) and the prevalence of selected enteropathogens. Results: At baseline, there were greater numbers of fecal enterobacteria than of lactobacilli and bifidobacteria (P <0.02). Iron fortification was ineffective; there were no differences in iron status, anemia, or hookworm prevalence at 6 mo. The fecal microbiota was modified by iron fortification as shown by a significant increase in profile dissimilarity (P <0.0001) in the iron group as compared with the control group. There was a significant increase in the number of enterobacteria (P <0.005) and a decrease in lactobacilli (P <0.0001) in the iron group after 6 mo. In the iron group, there was an increase in the mean fecal calprotectin concentration (P <0.01), which is a marker of gut inflammation, that correlated with the increase in fecal enterobacteria (P <0.05). Conclusions: Anemic African children carry an unfavorable ratio of fecal enterobacteria to bifidobacteria and lactobacilli, which is increased by iron fortification. Thus, iron fortification in this population produces a potentially more pathogenic gut microbiota profile, and this profile is associated with increased gut inflammation. This trial was registered at as ISRCTN21782274.
    Physiological responses to folate overproduction in Lactobacillus plantarum WCFS1
    Wegkamp, A. ; Mars, A.E. ; Faijes, M. ; Molenaar, D. ; Vos, R.C.H. de; Klaus, M.J. ; Hanson, A.D. ; Vos, W.M. de; Smid, E.J. - \ 2010
    Microbial Cell Factories 9 (2010). - ISSN 1475-2859 - 14 p.
    lactic-acid bacteria - lactococcus-lactis - escherichia-coli - streptococcus-cremoris - growth-rate - folic-acid - expression - protein - biosynthesis - recombinant
    Background Using a functional genomics approach we addressed the impact of folate overproduction on metabolite formation and gene expression in Lactobacillus plantarum WCFS1. We focused specifically on the mechanism that reduces growth rates in folate-overproducing cells. Results Metabolite formation and gene expression were determined in a folate-overproducing- and wild-type strain. Differential metabolomics analysis of intracellular metabolite pools indicated that the pool sizes of 18 metabolites differed significantly between these strains. The gene expression profile was determined for both strains in pH-regulated chemostat culture and batch culture. Apart from the expected overexpression of the 6 genes of the folate gene cluster, no other genes were found to be differentially expressed both in continuous and batch cultures. The discrepancy between the low transcriptome and metabolome response and the 25% growth rate reduction of the folate overproducing strain was further investigated. Folate production per se could be ruled out as a contributing factor, since in the absence of folate production the growth rate of the overproducer was also reduced by 25%. The higher metabolic costs for DNA and RNA biosynthesis in the folate overproducing strain were also ruled out. However, it was demonstrated that folate-specific mRNAs and proteins constitute 8% and 4% of the total mRNA and protein pool, respectively. Conclusion Folate overproduction leads to very little change in metabolite levels or overall transcript profile, while at the same time the growth rate is reduced drastically. This shows that Lactobacillus plantarum WCFS1 is unable to respond to this growth rate reduction, most likely because the growth-related transcripts and proteins are diluted by the enormous amount of gratuitous folate-related transcripts and proteins.
    Involvement of the mannose phosphotransferase system of Lactobacillus plantarum WCFS1 in peroxide stress tolerance
    Stevens, M.J.A. ; Molenaar, D. ; Jong, A. de; Vos, W.M. de; Kleerebezem, M. - \ 2010
    Applied and Environmental Microbiology 76 (2010)11. - ISSN 0099-2240 - p. 3748 - 3752.
    lactic-acid bacteria - lactate utilization - oxygen - mutants - glucose
    A Lactobacillus plantarum strain with a deletion in the gene rpoN, encoding the alternative sigma factor 54 (sigma(54)), displayed a 100-fold-higher sensitivity to peroxide than its parental strain. This feature could be due to sigma(54)-dependent regulation of genes involved in the peroxide stress response. However, transcriptome analyses of the wild type and the mutant strain during peroxide exposure did not support such a role for sigma(54). Subsequent experiments revealed that the impaired expression of the mannose phosphotransferase system (PTS) operon in the rpoN mutant caused the observed increased peroxide sensitivity
    Mixed-culture transcriptome analysis reveals the molecular basis of mixed-culture growth in Streptococcus thermophilus and Lactobacillus bulgaricus
    Sieuwerts, S. ; Molenaar, D. ; Hijum, S.A.F.T. van; Beerthuyzen, M. ; Stevens, M.J.A. ; Janssen, P.W. ; Ingham, C.J. ; Bok, F.A.M. de; Vos, W.M. de; Hylckama Vlieg, J.E.T. van - \ 2010
    Applied and Environmental Microbiology 76 (2010)33. - ISSN 0099-2240 - p. 7775 - 7784.
    lactic-acid bacteria - microarray data - milk - metabolism - plantarum - delbrueckii - pathways - yogurt - identification - proteinases
    Many food fermentations are performed using mixed cultures of lactic acid bacteria. Interactions between strains are of key importance for the performance of these fermentations. Yogurt fermentation by Streptococcus thermophilus and Lactobacillus bulgaricus (basonym, Lactobacillus delbrueckii subsp. bulgaricus) is one of the best-described mixed-culture fermentations. These species are believed to stimulate each other's growth by the exchange of metabolites such as folic acid and carbon dioxide. Recently, postgenomic studies revealed that an upregulation of biosynthesis pathways for nucleotides and sulfur-containing amino acids is part of the global physiological response to mixed-culture growth in S. thermophilus, but an in-depth molecular analysis of mixed-culture growth of both strains remains to be established. We report here the application of mixed-culture transcriptome profiling and a systematic analysis of the effect of interaction-related compounds on growth, which allowed us to unravel the molecular responses associated with batch mixed-culture growth in milk of S. thermophilus CNRZ1066 and L. bulgaricus ATCC BAA-365. The results indicate that interactions between these bacteria are primarily related to purine, amino acid, and long-chain fatty acid metabolism. The results support a model in which formic acid, folic acid, and fatty acids are provided by S. thermophilus. Proteolysis by L. bulgaricus supplies both strains with amino acids but is insufficient to meet the biosynthetic demands for sulfur and branched-chain amino acids, as becomes clear from the upregulation of genes associated with these amino acids in mixed culture. Moreover, genes involved in iron uptake in S. thermophilus are affected by mixed-culture growth, and genes coding for exopolysaccharide production were upregulated in both organisms in mixed culture compared to monocultures. The confirmation of previously identified responses in S. thermophilus using a different strain combination demonstrates their generic value. In addition, the postgenomic analysis of the responses of L. bulgaricus to mixed-culture growth allows a deeper understanding of the ecology and interactions of this important industrial food fermentation process
    Differential effects of Lactobacillus acidophilus and Lactobacillus plantarum strains on cytokine induction in human peripheral blood mononuclear cells
    Vissers, Y.M. ; Snel, J. ; Zuurendonk, P.F. ; Smit, B.A. ; Wichers, H.J. ; Savelkoul, H.F.J. - \ 2010
    FEMS Immunology and Medical Microbiology 59 (2010)1. - ISSN 0928-8244 - p. 60 - 70.
    lactic-acid bacteria - in-vitro - immunomodulatory properties - activation - probiotics - il-12 - interleukin-10 - tolerance - responses - mixture
    Lactic acid bacterial strains have received interest for their immunomodulating activities and potential use in probiotic products. A wide variety of strain-dependent properties have been reported, but comparative studies at the species level are scarce. The objective of this study was to assess the immunomodulatory effect of Lactobacillus species on the cytokine profiles and proliferative response of human peripheral blood mononuclear cells (hPBMC), and in particular, on the comparison between the species Lactobacillus acidophilus and Lactobacillus plantarum. hPBMC from healthy donors were stimulated in the presence or absence of the lactic acid bacteria, and cytokine production, surface marker staining, proliferation and cell death were determined after 1 and 4 days of culture. All Lactobacillus strains tested were capable of inducing the production of interleukin (IL)-1ß, IL-10, interferon-¿ (IFN-¿) and tumor necrosis factor-a (TNF-a). The bacterial strains did not differentially influence the amount of proliferating, viable, apoptotic and necrotic cells. Generally, L. plantarum showed a significantly higher induction capacity of IFN-¿, IL-12 and TNF-a compared with L. acidophilus. We conclude that the variation in immunomodulatory effects between species is even larger than the variation between the strains of the same species. In addition, we demonstrate that L. plantarum strains are most potent in skewing the T-cell differentiation toward a putative Th1 response.
    Identification of Genetic Loci in Lactobacillus plantarum That Modulate the Immune Response of Dendritic Cells Using Comparative Genome Hybridization
    Meijerink, M. ; Hemert, S. van; Taverne, N. ; Wels, M.W.W. ; Bron, P.A. ; Vos, P. de; Savelkoul, H.F.J. ; Bilsen, J.G.P.M. van; Kleerebeezem, M. ; Wells, J. - \ 2010
    PLoS ONE 5 (2010)5. - ISSN 1932-6203 - 12 p.
    lactic-acid bacteria - necrosis-factor-alpha - regulatory t-cells - intestinal inflammation - functional-analysis - probiotic bacteria - lipoteichoic acid - rhamnosus gg - immunomodulatory properties - gastrointestinal-tract
    Background - Probiotics can be used to stimulate or regulate epithelial and immune cells of the intestinal mucosa and generate beneficial mucosal immunomodulatory effects. Beneficial effects of specific strains of probiotics have been established in the treatment and prevention of various intestinal disorders, including allergic diseases and diarrhea. However, the precise molecular mechanisms and the strain-dependent factors involved are poorly understood. Methodology/Principal Findings - In this study, we aimed to identify gene loci in the model probiotic organism Lactobacillus plantarum WCFS1 that modulate the immune response of host dendritic cells. The amounts of IL-10 and IL-12 secreted by dendritic cells (DCs) after stimulation with 42 individual L. plantarum strains were measured and correlated with the strain-specific genomic composition using comparative genome hybridisation and the Random Forest algorithm. This in silico “gene-trait matching” approach led to the identification of eight candidate genes in the L. plantarum genome that might modulate the DC cytokine response to L. plantarum. Six of these genes were involved in bacteriocin production or secretion, one encoded a bile salt hydrolase and one encoded a transcription regulator of which the exact function is unknown. Subsequently, gene deletions mutants were constructed in L. plantarum WCFS1 and compared to the wild-type strain in DC stimulation assays. All three bacteriocin mutants as well as the transcription regulator (lp_2991) had the predicted effect on cytokine production confirming their immunomodulatory effect on the DC response to L. plantarum. Transcriptome analysis and qPCR data showed that transcript level of gtcA3, which is predicted to be involved in glycosylation of cell wall teichoic acids, was substantially increased in the lp_2991 deletion mutant (44 and 29 fold respectively). Conclusion - Comparative genome hybridization led to the identification of gene loci in L. plantarum WCFS1 that modulate the immune response of DCs
    Mucosal adhesion properties of the probiotic Lactobacillus rhamnosus GG SpaCBA and SpaFED pilin subunits
    Ossowski, I. von; Reunanen, J. ; Satokari, R.M. ; Vesterlund, S. ; Kankainen, M. ; Huhtinen, H. ; Tynkkynen, S. ; Salminen, S. ; Vos, W.M. de; Palva, A. - \ 2010
    Applied and Environmental Microbiology 76 (2010)7. - ISSN 0099-2240 - p. 2049 - 2057.
    human intestinal mucus - gram-positive bacteria - lactic-acid bacteria - cell-surface - streptococcus-agalactiae - gastrointestinal-tract - oral consumption - dodecyl-sulfate - protein - colonization
    Lactobacillus rhamnosus GG is a well-established Gram-positive probiotic strain, whose health-benefiting properties are in part dependent upon a prolonged residency in the gastrointestinal tract and likely dictated by an adherence to the intestinal mucosa. Previously, we identified two pilus gene clusters (spaCBA and spaFED) in the genome of this probiotic, each of which contained the predicted genes for three pilin subunits and a single sortase. We also confirmed the presence of SpaCBA pili on the cell surface and attributed an intestinal mucus-binding capacity to one of the pilin subunits (SpaC). Herein, we now report the cloning of the remaining pilin genes (spaA, spaB, spaD, spaE, and spaF) in Escherichia coli, production and purification of the recombinant proteins, and an assessment of their adherence to human intestinal mucus. Our findings indicate that the SpaB and SpaF pilin subunits also exhibit substantial mucus binding, which can be inhibited competitively in a dose-related manner. Moreover, the binding between SpaB pilin subunit and the mucosal substrate appears to operate through electrostatic contacts and is not related to a recognized mucus-binding domain. We conclude from these results that it is conceivable two pilin subunits (SpaB and SpaC) in the SpaCBA pilus fiber play a role in binding to intestinal mucus, but for the uncharacterized and putative SpaFED pilus fiber only a single pilin subunit (SpaF) is potentially responsible for mucus adhesion
    Indigenous and environmental modulation of mutation frequencies in Lactobacillus plantarum
    Machielsen, M.P. ; Alen-Boerrigter, I.J. van; Koole, L.A. ; Bongers, R.S. ; Kleerebezem, M. ; Hylckama-Vlieg, J.E.T. van - \ 2010
    Applied and Environmental Microbiology 76 (2010)5. - ISSN 0099-2240 - p. 1587 - 1595.
    controlled gene-expression - lactic-acid bacteria - site-specific recombination - repair protein muts - lactococcus-lactis - escherichia-coli - mismatch-repair - streptococcus-lactis - hydrogen-peroxide - oxidative stress
    The reliability of microbial (starter) strains in terms of quality, functional properties, growth performance and robustness is essential for industrial applications. In an industrial fermentation process, the bacterium should be able to successfully withstand various adverse conditions during processing such as acid, osmotic, temperature, and oxidative stress. Besides the evolved defense mechanisms, stress-induced mutations participate in adaptive evolution towards survival under these stress conditions. However, this may lead to the accumulation of mutant strains, which may be accompanied by loss of desired functional properties. Defining the effect of specific fermentation or processing conditions on the mutation frequencies is an important step towards preventing loss of genome integrity and maintaining the productivity of industrial strains. Therefore, a set of Lactobacillus plantarum mutator reporter strains suitable for qualitative and quantitative analysis of low-frequency mutation events was developed. The mutation reporter system constructed was validated by using chemical mutagenesis (NTG) and by controlled expression of endogenous candidate mutator genes (e.g. truncated derivative of the L. plantarum hexA gene). Growth at different temperatures, in low pH conditions, at high salt concentrations or in starvation conditions did not result in a significant effect on the mutation frequency. However, incubation with sublethal levels of hydrogen peroxide showed a 100-fold increase of the mutation frequency when compared to the background mutation frequency. Importantly, when cells of L. plantarum were adapted to 42 degrees C, prior to the treatment with sublethal levels of hydrogen peroxide, this induced a 10-fold increase in peroxide treatment survival, with a concomitant 50-fold decrease of the mutation frequency. These results show that specific environmental conditions encountered by bacteria may significantly influence the genetic stability of strains, while protection against mutagenic conditions may be achieved by pre-treatment of cultures with other, non-mutagenic stress conditions
    Phenotypic and genomic diversity of Lactobacillus plantarum strains isolated from various environmental niches
    Siezen, R.J. ; Tzeneva, V.A. ; Castioni, A. ; Wels, M.W.W. ; Phan, H.T. ; Rademaker, J.L.W. ; Starrenburg, M.J.C. ; Kleerebezem, M. ; Molenaar, D. ; Hylckama Vlieg, J.E.T. van - \ 2010
    Environmental Microbiology 12 (2010)3. - ISSN 1462-2912 - p. 758 - 773.
    lactic-acid bacteria - horizontal gene-transfer - streptococcus-thermophilus - starter cultures - sequence - identification - paraplantarum - evolution - pcr - differentiation
    Lactobacillus plantarum is a ubiquitous microorganism that is able to colonize several ecological niches, including vegetables, meat, dairy substrates and the gastro-intestinal tract. An extensive phenotypic and genomic diversity analysis was conducted to elucidate the molecular basis of the high flexibility and versatility of this species. First, 185 isolates from diverse environments were phenotypically characterized by evaluating their fermentation and growth characteristics. Strains clustered largely together within their particular food niche, but human fecal isolates were scattered throughout the food clusters, suggesting that they originate from the food eaten by the individuals. Based on distinct phenotypic profiles, 24 strains were selected and, together with a further 18 strains from an earlier low-resolution study, their genomic diversity was evaluated by comparative genome hybridization against the reference genome of L. plantarum WCFS1. Over 2000 genes were identified that constitute the core genome of the L. plantarum species, including 121 unique L. plantarum-marker genes that have not been found in other lactic acid bacteria. Over 50 genes unique for the reference strain WCFS1 were identified that were absent in the other L. plantarum strains. Strains of the L. plantarum subspecies argentoratensis were found to lack a common set of 24 genes, organized in seven gene clusters/operons, supporting their classification as a separate subspecies. The results provide a detailed view on phenotypic and genomic diversity of L. plantarum and lead to a better comprehension of niche adaptation and functionality of the organism
    Development of a minimal growth medium for Lactobacillus plantarum
    Wegkamp, H.B.A. ; Teusink, B. ; Vos, W.M. de; Smid, E.J. - \ 2010
    Letters in Applied Microbiology 50 (2010)1. - ISSN 0266-8254 - p. 57 - 64.
    lactic-acid bacteria - lactococcus-lactis - nutritional-requirements - streptococcus-cremoris - folate production - escherichia-coli - biosynthesis - transport - pathways - wcfs1
    Aim: A medium with minimal requirements for the growth of Lactobacillus plantarum WCFS was developed. The composition of the minimal medium was compared to a genome-scale metabolic model of L. plantarum. Methods and Results: By repetitive single omission experiments, two minimal media were developed: PMM5 (true minimal medium) and PMM7 [a pseudominimal medium, supporting proper biomass formation of 350 mg l(-1) dry weight (DW)]. The specific growth rate of L. plantarum on PMM7 was found to be 50% and 63% lower when compared to growth on established growth media (chemically defined medium and MRS, respectively). Using a genome-scale metabolic model of L. plantarum, it was predicted that PMM5 and PMM7 would not support the growth of L. plantarum. This is because the biosynthesis of para-aminobenzoic acid (pABA) was predicted to be essential for growth. The discrepancy in simulated growth and experimental growth on PMM7 was further investigated for pABA; a molecule which plays an important role in folate production. The growth performance and folate production were determined on PMM7 in the presence and absence of pABA. It was found that a 12 000-fold reduction in folate pools exerted no influence on formation of biomass or growth rate of L. plantarum cultures when grown in the absence of pABA. Conclusion: Largely reduced folate production pools do not have an effect on the growth of L. plantarum, showing that L. plantarum makes folate in a large excess. Significance and Impact of the study: These experiments illustrate the importance of combining genome-scale metabolic models with growth experiments on minimal media
    Factors influencing the incidence and prevalence of food allergy
    Cochrane, S. ; Beyer, K. ; Clausen, M. ; Wjst, M. ; Hiller, R. ; Nicoletti, C. ; Szepfalusi, Z. ; Savelkoul, H.F.J. ; Breiteneder, H. ; Manios, Y. ; Crittenden, R. ; Burney, P. - \ 2009
    Allergy 64 (2009)9. - ISSN 0105-4538 - p. 1246 - 1255.
    placebo-controlled trial - cows milk allergy - 1st 6 months - randomized controlled-trial - bovine beta-lactoglobulin - lactic-acid bacteria - koala birth cohort - breast-fed infants - life risk-factors - atopic-dermatitis
    Food allergy is an increasing problem in Europe and elsewhere and severe reactions to food are also becoming more common. As food allergy is usually associated with other forms of allergic sensitisation it is likely that many risk factors are common to all forms of allergy. However the potential severity of the disease and the specific public heath measures required for food allergy make it important to identify the specific risk factors for this condition. Food allergy is unusual in that it often manifests itself very early in life and commonly remits with the development of tolerance. Hypotheses that explain the distribution of food allergy include specific genetic polymorphisms, the nature of the allergens involved and the unique exposure to large quantities of allergen through the gut. Progress has been made in developing more specific and testable hypotheses but the evidence for any of these is still only preliminary. Further collaborative research is required to develop an appropriate public health response to this growing problem.
    Branched chain aldehydes: production and breakdown pathways and relevance for flavour in foods
    Smit, B.A. ; Engels, W.J.M. ; Smit, G. - \ 2009
    Applied Microbiology and Biotechnology 81 (2009)6. - ISSN 0175-7598 - p. 987 - 999.
    lactic-acid bacteria - dry-fermented sausages - alpha-keto acids - solid-phase microextraction - odor-active compounds - lactococcal aromatic aminotransferase - gas chromatography-olfactometry - freshly distilled calvados - fusel alcohol production - amino-acids
    Branched aldehydes, such as 2-methyl propanal and 2- and 3-methyl butanal, are important flavour compounds in many food products, both fermented and non-fermented (heat-treated) products. The production and degradation of these aldehydes from amino acids is described and reviewed extensively in literature. This paper reviews aspects influencing the formation of these aldehydes at the level of metabolic conversions, microbial and food composition. Special emphasis was on 3-methyl butanal and its presence in various food products. Knowledge gained about the generation pathways of these flavour compounds is essential for being able to control the formation of desired levels of these aldehydes.
    Lifestyle of Lactobacillus plantarum in the mouse caecum
    Marco, M. ; Peters, T.H.F. ; Bongers, R.S. ; Molenaar, D. ; Hemert, S. van; Sonnenburg, J.L. ; Gordon, J. ; Kleerebezem, M. - \ 2009
    Environmental Microbiology 11 (2009)10. - ISSN 1462-2912 - p. 2747 - 2757.
    johnsonii strain ncc533 - lactic-acid bacteria - gastrointestinal-tract - human gut - lactococcus-lactis - teichoic-acids - in-vivo - genes - identification - metabolism
    Lactobacillus plantarum is a common inhabitant of mammalian gastrointestinal tracts. Strains of L. plantarum are also marketed as probiotics intended to confer beneficial health effects upon delivery to the human gut. To understand how L. plantarum adapts to its gut habitat, we used whole genome transcriptional profiling to characterize the transcriptome of strain WCFS1 during colonization of the caeca of adult germ-free C57Bl/6 J mice fed a standard low-fat rodent chow diet rich in complex plant polysaccharides or a prototypic Western diet high in simple sugars and fat. Lactobacillus plantarum colonized the digestive tracts of these animals to high levels, although L. plantarum was found in 10-fold higher amounts in the caeca of mice fed the standard chow. Metabolic reconstructions based on the transcriptional data sets revealed that genes involved in carbohydrate transport and metabolism form the principal functional group that is upregulated in vivo compared with exponential phase cells grown in three different culture media, and that a Western diet provides a more nutritionally restricted, growth limiting milieu for the microbe in the distal gut. A set of bacterial genes encoding cell surface-related functions were differentially regulated in both groups of mice. This set included downregulated genes required for the d-alanylation of lipoteichoic acids, extracellular structures of L. plantarum that mediate interactions with the host immune system. These results, obtained in a reductionist gnotobiotic mouse model of the gut ecosystem, provide insights about the niches (professions) of this lactic acid bacterium, and a context for systematically testing features that affect epithelial and immune cell responses to this organism in the digestive tract
    A high-throughput cheese manufacturing model for effective cheese starter culture screening
    Bachmann, H. ; Kruijswijk, Z. ; Molenaar, D. ; Kleerebezem, M. ; Hylckama Vlieg, J.E.T. van - \ 2009
    Journal of Dairy Science 92 (2009)12. - ISSN 0022-0302 - p. 5868 - 5882.
    lactic-acid bacteria - lactococcus-lactis - flavor formation - cheddar cheese - amino-acids - aminotransferase - identification - diversity - strains - gene
    Cheese making is a process in which enzymatic coagulation of milk is followed by protein separation, carbohydrate removal, and an extended bacterial fermentation. The number of variables in this complex process that influence cheese quality is so large that the developments of new manufacturing protocols are cumbersome. To reduce screening costs, several models have been developed to miniaturize the cheese manufacturing process. However, these models are not able to accommodate the throughputs required for systematic screening programs. Here, we describe a protocol that allows the parallel manufacturing of approximately 600 cheeses in individual cheese vats each with individual process specifications. Protocols for the production of miniaturized Gouda- and Cheddar-type cheeses have been developed. Starting with as little as 1.7 mL of milk, miniature cheeses of about 170 mg can be produced and they closely resemble conventionally produced cheese in terms of acidification profiles, moisture and salt contents, proteolysis, flavor profiles, and microstructure. Flavor profiling of miniature cheeses manufactured with and without mixed-strain adjunct starter cultures allowed the distinguishing of the different cheeses. Moreover, single-strain adjunct starter cultures engineered to overexpress important flavor-related enzymes revealed effects similar to those described in industrial cheese. Benchmarking against industrial cheese produced from the same raw materials established a good correlation between their proteolytic degradation products and their flavor profiles. These miniature cheeses, referred to as microcheeses, open new possibilities to study many aspects of cheese production, which will not only accelerate product development but also allow a more systematic approach to investigate the complex biochemistry and microbiology of cheese making
    Rich nutrition from the poorest - Cereal fermentations in Africa and Asia
    Nout, M.J.R. - \ 2009
    Food Microbiology 26 (2009)7. - ISSN 0740-0020 - p. 685 - 692.
    lactic-acid bacteria - millet pennisetum-glaucum - maize dough fermentation - burkina-faso - ben-saalga - natural fermentation - process combinations - kenkey production - starter culture - energy density
    Cereal fermentations in Africa and Asia involve mainly the processing of maize, rice, sorghum and the millets. Lactic acid bacteria (Lactobacillus, Pediococcus), Enterobacter spp., yeasts (Candida, Debaryomyces, Endomycopsis, Hansenula, Pichia, Saccharomyces and Trichosporon spp.) and filamentous fungi (Amylomyces, Aspergillus, Mucor, and Rhizopus spp.) contribute to desirable modifications of taste, flavour, acidity, digestibility, and texture in non-alcoholic beverages (e.g., uji, and ben-saalga), porridges (e.g., mawè) and cooked gels (e.g., kenkey, idli, and mifen). In addition, alcoholic beverages (beers such as tchoukoutou and jnard; and spirits e.g. jiu) are obtained using malt, or using amylolytic mixed microbial starter cultures as generators of fermentable substrates. Wet processing, marketing of multi-purpose intermediate products, co-fermentation for texture and nutrition, and mixed culture fermentations as practiced in indigenous fermentation processes are of interest for industrial innovation and for better control of natural mixed culture fermentation systems. On the other hand, the nutritional properties of traditional cereal fermented products can be enhanced by increasing their nutrient and energy density, as well as by increasing their mineral status by combining mineral fortification and dephytinization
    Kinetics of Lactobacillus plantarum 44a in the faeces of tilapia (Oreochromis niloticus) after its intake in feed
    Bucio Galindo, A. ; Hartemink, R. ; Schrama, J.W. ; Verreth, J.A.J. ; Bucio Galindo, L. ; Zwietering, M.H. - \ 2009
    Journal of Applied Microbiology 107 (2009)6. - ISSN 1364-5072 - p. 1967 - 1975.
    lactic-acid bacteria - trout oncorhynchus-mykiss - cod gadus-morhua - salmo-salar l. - intestinal mucus - nile tilapia - fish - tract - rhamnosus - survival
    Aims: To study the kinetic passage of Lactobacillus plantarum 44a from feed to faeces of tilapia in order to calculate the number of Lactobacillus excreted. Methods and Results: In a single-dose experiment, duplicate lots of 26 fish devoid of intestinal lactobacilli were fed with diets containing c. 4·5 × 1011, 6·3 × 108, 6·0 × 105 and 0 CFU of Lact. plantarum 44a per single feed ration. In the multiple-dose experiment, duplicate lots of 30 fish each were supplied with a diet containing 1 × l011 CFU of Lact. plantarum 44a as follows: 14 times in 14 days, five times in 14 days, once in 14 days and zero times in 14 days. Faeces were periodically collected and analysed for their lactobacilli content by using a selective media. The kinetics of Lactobacillus in the faeces was described as a pulse signal defined by three parameters: ¿, Ao and the time. ¿, was identified as the time to reach the peak (x axis) and Ao was a constant. Ao divided by e, was identified as the height of the peak (y axis). The area below the curve Ao¿ allowed the calculation of the total number of lactobacilli excreted. The ability of the mathematical model to describe the actual values was tested by a linear regression analysis. In most of the cases, the equations showed an intercept close to zero (P > 0·05) and angular coefficients near one. Conclusions: Lactobacillus plantarum 44a was excreted in short pulse signals described by a mathematical model which allowed calculating the area below the curve and consequently the survival rate. Significance and Impact of the Study: This study provides a quantitative method to study the kinetics of excretion of a probiotic bacterium in the faeces.
    Proteome Analysis of Lactobacillus rhamnosus GG Using 2-D DIGE and Mass Spectrometry Shows Differential Protein Production in Laboratory and Industrial-Type Growth Media
    Koskenniemi, K. ; Koponen, J. ; Kankainen, M. ; Savijoki, K. ; Tynkkynen, S. ; Vos, W.M. de; Kalkkinen, N. ; Varmanen, P. - \ 2009
    Journal of Proteome Research 8 (2009)11. - ISSN 1535-3893 - p. 4993 - 5007.
    lactic-acid bacteria - delbrueckii subsp bulgaricus - placebo-controlled trial - long-term consumption - chain amino-acids - lactococcus-lactis - streptococcus-thermophilus - probiotic lactobacillus - stress responses - strain-gg
    Lactobacillus rhamnosus GG (LGG) is one of the most extensively studied and widely used probiotic bacteria. While the benefits of LGG treatment in gastrointestinal disorders and immunomodulation are well-documented, functional genomics research of this bacterium has only recently been initiated. In the present study, a 2-D DIGE approach was used for the quantitative analysis of growth media-dependent changes in LGG protein abundance. Proteins were isolated from cells grown in industrial-type whey-based medium or in rich laboratory medium for subsequent 2-D DIGE. The analysis revealed patterns of protein abundance unique to each growth condition. In total, 196 quantitatively altered protein spots (at least 1.5-fold change in relative abundance, p <0.05) representing approximately 13% of all protein spots in the gel were detected. From these protein spots, 157 were identified by mass spectrometry and were found to represent 100 distinct gene products. Collectively, these data show that growth of LGG in whey medium increased the relative abundance of proteins involved in purine biosynthesis, galactose metabolism, and fatty acid biosynthesis. In comparison, growth of LGG in laboratory medium resulted in an increase in the amount of proteins involved in translation and the general stress response, as well as pyrimidine and exopolysaccharide biosynthesis. Moreover, several enzymes of the proteolytic system of LGG demonstrated growth medium-dependent production. The present study demonstrates the fundamental effects of culture conditions on the proteome of LGG, which are likely to affect the functionality and characteristics of its use as a probiotic
    Comparative genomic analysis of Lactobacillus rhamnosus GG reveals pili containing a human- mucus binding protein
    Kankainen, M. ; Paulin, L. ; Tynkkynen, S. ; Ossowski, I. von; Reunanen, J. ; Partanen, P. ; Satokari, A. ; Vesterlund, S. ; Hendrickx, A.P. ; Lebeer, S. ; Keersmaecker, S.C. de; Vanderleyden, J. ; Hämäläinen, T. ; Laukkanen, S. ; Salovuori, N. ; Ritari, J. ; Alatalo, E. ; Korpela, R. ; Mattila-Sandholm, T. ; Lassig, A. ; Hatakka, K. ; Kinnunen, K.T. ; Karjalainen, H. ; Saxelin, M. ; Laakso, K. ; Surakka, A. ; Palva, A. ; Salusjärvi, T. ; Auvinen, P. ; Vos, W.M. de - \ 2009
    Proceedings of the National Academy of Sciences of the United States of America 106 (2009)40. - ISSN 0027-8424 - p. 17193 - 17198.
    lactic-acid bacteria - intestinal epithelial-cells - gram-positive bacteria - gene clusters - in-vitro - strains - long - exopolysaccharide - biosynthesis - colonization
    To unravel the biological function of the widely used probiotic bacterium Lactobacillus rhamnosus GG, we compared its 3.0-Mbp genome sequence with the similarly sized genome of L. rhamnosus LC705, an adjunct starter culture exhibiting reduced binding to mucus. Both genomes demonstrated high sequence identity and synteny. However, for both strains, genomic islands, 5 in GG and 4 in LC705, punctuated the colinearity. A significant number of strain-specific genes were predicted in these islands (80 in GG and 72 in LC705). The GG-specific islands included genes coding for bacteriophage components, sugar metabolism and transport, and exopolysaccharide biosynthesis. One island only found in L. rhamnosus GG contained genes for 3 secreted LPXTG-like pilins (spaCBA) and a pilin-dedicated sortase. Using anti-SpaC antibodies, the physical presence of cell wall-bound pili was confirmed by immunoblotting. Immunogold electron microscopy showed that the SpaC pilin is located at the pilus tip but also sporadically throughout the structure. Moreover, the adherence of strain GG to human intestinal mucus was blocked by SpaC antiserum and abolished in a mutant carrying an inactivated spaC gene. Similarly, binding to mucus was demonstrated for the purified SpaC protein. We conclude that the presence of SpaC is essential for the mucus interaction of L. rhamnosus GG and likely explains its ability to persist in the human intestinal tract longer than LC705 during an intervention trial. The presence of mucus-binding pili on the surface of a nonpathogenic Gram-positive bacterial strain reveals a previously undescribed mechanism for the interaction of selected probiotic lactobacilli with host tissues
    Probiotic and Gut Lactobacilli and Bifidobacteria: Molecular Approaches to Study Diversity and Activity
    Kleerebezem, M. ; Vaughan, E.E. - \ 2009
    Annual Review of Microbiology 63 (2009). - ISSN 0066-4227 - p. 269 - 290.
    lactic-acid bacteria - diet-induced obesity - gastrointestinal-tract microbiota - gradient gel-electrophoresis - intestinal epithelial-cells - johnsonii strain ncc533 - gram-positive bacteria - alanyl ester depletion - bile-salt hydrolase - formula-fed infants
    Lactobacilli and bifidobacteria have traditionally been recognized as potential health-promoting microbes in the human gastrointestinal tract, which is clearly reflected by the pre- and probiotic supplements on the market. Bacterial genomics of lactobacilli and bifidobacteria is initiating the identification and validation of specific effector molecules that mediate host health effects. Combined with advanced postgenomic mammalian host response analyses, elucidations of the molecular interactions and mechanisms that underlie the host-health effects observed are beginning to be gathered. These developments should be seen in the complexity of the microbiota-host relationships in the intestine, which through the new metagenomic era has regained momentum and will undoubtedly progress to functional microbiomics and host response analyses within the next decade. Taken together, these developments are anticipated to dramatically alter the scope and impact of the probiotic field, offering tremendous new opportunities with accompanying challenges for research and industrial application
    Activity of ethanol-stressed Oenococcus oeni cells: a flow cytometric approach
    Silveira, M.G. da; Abee, T. - \ 2009
    Journal of Applied Microbiology 106 (2009)5. - ISSN 1364-5072 - p. 1690 - 1696.
    electrogenic malate uptake - lactic-acid bacteria - leuconostoc-oenos - malolactic fermentation - lactococcus-lactis - fluorescent-probe - membrane - energy - glucose - growth
    Aims: To study the effect of ethanol on Oenococcus oeni activity at the single cell level. Methods and Results: The active extrusion of the fluorescent probe carboxy fluorescein (cF) was used to assess the metabolic activity of ethanol-stressed O. oeni cells. Subsequent flow cytometric analysis revealed that O. oeni cells extrude the accumulated cF upon energizing with l-malic acid. However, O. oeni cells exposed to 12% (v/v) ethanol for 1 h showed a decreased capacity for active extrusion of cF. Moreover, two subpopulations could be distinguished, one of which being able to extrude cF and the other one remaining cF fluorescent. Growing cells in the presence of 8% (v/v) ethanol resulted in robust cells that maintained the capacity to actively extrude cF after being exposed to 12% (v/v) ethanol, which in turn correlated with the high levels of ATP observed in these ethanol stressed, malolactic fermentation (MLF) performing cells. Conclusion: From our results, it becomes evident that active extrusion of cF can be used to assess malolactic activity in O. oeni. Significance and Impact of the Study: The present study provides information for the development of a rapid method to assess the malolactic activity of individual O. oeni cells performing MLF during wine production
    A survey on composition and microbiota of fresh and fermented yak milk at different Tibetan altitudes
    Wu, X.H. ; Luo, Z. ; Yu, L. ; Ren, F.Z. ; Han, B.Z. ; Nout, M.J.R. - \ 2009
    Dairy Science and Technology 89 (2009)2. - ISSN 1958-5586 - p. 201 - 209.
    lactic-acid bacteria - dairy-products - yeasts - identification - growth
    Yak milk is a type of milk that people are less familiar with due to its remote geographical location, the particular geographical environment and climatic conditions in Tibet, which may have significant effects on composition, microbiota and fermentation outcome. To investigate the chemical composition and microbiota of fresh and fermented yak milk, and to isolate and characterize the predominant microorganisms in the fermented milk, yak milk (24 fresh and 30 fermented milk samples) was collected from four areas of different altitudes in Tibet, and their microbiological profile and chemical composition were investigated. Yak milk had a higher fat, crude protein, lactose and dry matter content than cow milk. The fermented yak milk showed a great diversity in fat and dry matter levels due to the different ways of processing in different localities, and lower pH and higher lactic acid content compared with commercial cow milk yogurt. Fermented yak milk had a better sanitary quality than fresh yak milk. Three species of lactobacilli (Lactobacillus fermentum, Lactobacillus helveticus and Lactobacillus curvatus) and five species of yeast (Saccharomyces cerevisiae, Candida kefyr, Candida lambica, Candida famat and Candida holmii) were identified phenotypically and encountered as predominant fermentation microbiota. The predominant lactic species in fermented milk was L. fermentum
    The effects of selected probiotic strains on the development of eczema (The PandA study)
    Niers, L.E.M. ; Martin, R. ; Rijkers, G.T. ; Sengers, F. ; Timmerman, H.M. ; Uden, N.O. van; Smidt, H. ; Kimpen, J.L.L. ; Hoekstra, M.O. - \ 2009
    Allergy 64 (2009)9. - ISSN 0105-4538 - p. 1349 - 1358.
    placebo-controlled trial - lactic-acid bacteria - high-risk children - real-time pcr - atopic-dermatitis - double-blind - respiratory symptoms - primary prevention - mononuclear-cells - allergic diseases
    Background: Modification of the intestinal microbiota by administration of probiotic bacteria may be a potential approach to prevent allergic disease. We aimed to study primary prevention of allergic disease in high-risk children by pre- and postnatal supplementation of selected probiotic bacteria. Methods: In a double-blind, randomized, placebo-controlled trial, a mixture of probiotic bacteria selected by in-vitro experiments (Bifidobacterium bifidum, Bifidobacterium lactis, and Lactococcus lactis; Ecologic((R)) Panda) was prenatally administered to mothers of high-risk children (i.e. positive family history of allergic disease) and to their offspring for the first 12 months of life. Results: Parental-reported eczema during the first 3 months of life was significantly lower in the intervention group compared with placebo, 6/50 vs 15/52 (P = 0.035). After 3 months, the incidence of eczema was similar in both groups. Cumulative incidence of parental-reported eczema at 1 and 2 years was 23/50 (intervention) vs 31/48 (placebo) and 27 (intervention) vs 34 (placebo), respectively. The number needed to treat was 5.9 at age 3 and 12 months and 6.7 at age 2 years. The intervention group was significantly more frequently colonized with higher numbers of Lc. lactis. Furthermore, at age 3 months, in vitro production of IL-5 (146 pg/ml vs 72 pg/ml; P = 0.04) was decreased in the probiotic-group compared with the placebo-group. Conclusions: This particular combination of probiotic bacteria shows a preventive effect on the incidence of eczema in high-risk children, which seems to be sustained during the first 2 years of life. In addition to previous studies, the preventive effect appears to be established within the first 3 months of life
    Unraveling microbial interactions in food fermentations: from classical to genomics approaches
    Sieuwerts, S. ; Bok, F.A.M. de; Hugenholtz, J. ; Vlieg, J.E.T.V.H. - \ 2008
    Applied and Environmental Microbiology 74 (2008)16. - ISSN 0099-2240 - p. 4997 - 5007.
    lactic-acid bacteria - lactobacillus-plantarum wcfs1 - gram-positive bacteria - streptococcus-thermophilus - lactococcus-lactis - mixed cultures - listeria-monocytogenes - saccharomyces-cerevisiae - probiotic bacteria - escherichia-coli
    Biosafety assessment of probiotics used for human consumption: recommendations from the EU-PROSAFE project
    Vankerckhoven, V. ; Huys, G. ; Vancanneyt, M. ; Vael, C. ; Klare, I. ; Romond, M.B. ; Entenza, J.M. ; Moreillon, P. ; Wind, R.D. ; Knol, J. ; Wiertz, E. ; Pot, B. ; Vaughan, E.E. ; Kahlmeter, G. ; Goossens, H. - \ 2008
    Trends in Food Science and Technology 19 (2008)2. - ISSN 0924-2244 - p. 102 - 114.
    lactic-acid bacteria - urinary-tract infection - enterococcus-faecalis - in-vitro - bile-acids - experimental endocarditis - lactobacillus-plantarum - surface protein - gastrointestinal-tract - aggregation substance
    On June 26-27, 2006, 60 academic and industry scientists gathered during the PROSAFE workshop to discuss recommendations on taxonomy, antibiotic resistance, in vitro assessment of virulence and in vivo assessment of safety of probiotics used for human consumption. For identification of lactic acid bacteria (LAB) intended for probiotic use, it was recommended that conventional biochemical methods should be complemented with molecular methods and that these should be performed by an expert lab. Using the newly developed LAB Susceptibility test Medium (LSM), tentative epidemiological cut-off values were proposed. It was recommended that potentially probiotic strains not belonging to the wildtype distributions of relevant antimicrobials should not be developed as future products for human or animal consumption. Furthermore, it was recommended that the use of strains harbouring known and confirmed virulence genes should be avoided. Finally, for in vivo assessment of safety by investigating strain pathogenicity in animal models, the rat endocarditis model appeared to be the most reliable model tested in the PROSAFE project. Moreover, consensus was reached for approving the necessity of a human colonisation study in a randomised placebo-controlled double-blind design; however, further discussions are needed on the details of such as study.
    A standardized conjugation protocol to assess antibiotic resistance transfer between lactococcal species
    Lampkowska, J. ; Feld, L. ; Monaghan, A. ; Toomey, N. ; Schjørring, S. ; Jacobsen, B. ; Voet, H. van der; Andersen, S.R. ; Bolton, D. ; Aarts, H.J.M. ; Krogfelt, K.A. ; Wilcks, A. ; Bardowski, J.K. - \ 2008
    International Journal of Food Microbiology 127 (2008)1-2. - ISSN 0168-1605 - p. 172 - 175.
    lactic-acid bacteria - lactobacillus-plantarum - streptococcus-faecalis - enterococcus-faecalis - plasmid pam-beta-1 - reuteri
    Optimal conditions and a standardized method for conjugation between two model lactococcal strains, Lactococcus lactis SH4174 (pAMbeta1-containing, erythromycin resistant donor) and L. lactis Bu2-60 (plasmid-free, erythromycin sensitive recipient), were developed and tested in a inter-laboratory experiments involving five laboratories from different countries. The ultimate goal of the study was to assess the microbial potential of antibiotic resistance transfer among Lactic Acid Bacteria (LAB). The influence of culture age (various OD values) and ratios of donor and recipient cultures as well as filter, solid and liquid mating techniques, were examined in order to optimize the conjugation protocol. In the result of these studies, we concluded that the donor-to-recipient ratio appear to be important; the most efficient technique for conjugation was filter mating and the optimal conditions for gene transfer were observed when late logarithmic cultures of both donor and recipient were used. Comparison of conjugal transfer frequencies between five partner laboratories showed that results are sufficiently intra-laboratory repeatable and inter-laboratory comparable. This is the first study of this kind, in which a standardized protocol of conjugal mating for testing antibiotic resistance dissemination among LAB was established and validated
    Interactomics in the human intestine: Lactobacilli and Bifidobacteria make a difference
    Boesten, R.J. ; Vos, W.M. de - \ 2008
    Journal of Clinical Gastroenterology 42 (2008)Suppl 3, part 2. - ISSN 0192-0790 - p. S163 - S167.
    lactic-acid bacteria - human gastrointestinal-tract - gram-positive bacteria - plantarum wcfs1 - predicted secretome - salivarius ucc118 - genome sequence - diversity - proteins - strains
    Scientific evidence that supports a correlation between our intestinal microbiota and health status has caused significant interest in microbe-host interaction studies. It has generated a paradigm shift from analyzing pathogens to that involving commensal and probiotic bacteria. This review summarizes the interaction mechanisms described for Lactobacilli and Bifidobacteria based on recent omics-based developments. This information is expected to provide new avenues for further unravelling the set of interactions that includes the interactome of microbial and host cells
    A simple and fast method for determining colony forming units
    Sieuwerts, S. ; Bok, F.A.M. de; Mols, E. ; Vos, W.M. de; Hylckama Vlieg, J.E.T. van - \ 2008
    Letters in Applied Microbiology 47 (2008)4. - ISSN 0266-8254 - p. 275 - 278.
    lactic-acid bacteria - plate method - probes - wine
    Aims: To develop a flexible and fast colony forming unit quantification method that can be operated in a standard microbiology laboratory. Methods and Results: A miniaturized plating method is reported where droplets of bacterial cultures are spotted on agar plates. Subsequently, minicolony spots are imaged with a digital camera and quantified using a dedicated plug-in developed for the freeware program ImageJ. A comparison between conventional and minicolony plating of industrial micro-organisms including lactic acid bacteria, Eschericha coli and Saccharomyces cerevisiae showed that there was no significant difference in the results obtained with the methods. Conclusions: The presented method allows downscaling of plating by 100-fold, is flexible, easy-to-use and is more labour-efficient and cost-efficient than conventional plating methods. Significance and Impact of the Study: The method can be used for rapid assessment of viable counts of micro-organisms similar to conventional plating using standard laboratory equipment. It is faster and cheaper than conventional plating methods
    Functional analysis of four bile salt hydrolase and penicillin acylase family members in Lactobacillus plantarum WCFS1
    Lambert, J.M. ; Bongers, R.S. ; Vos, W.M. de; Kleerebezem, M. - \ 2008
    Applied and Environmental Microbiology 74 (2008)15. - ISSN 0099-2240 - p. 4719 - 4726.
    lactic-acid bacteria - lactococcus-lactis - escherichia-coli - clostridium-perfringens - listeria-monocytogenes - bifidobacterium-longum - gastrointestinal-tract - gene - strains - cloning
    Bile salts play an important role in the digestion of lipids in vertebrates and are synthesized and conjugated to either glycine or taurine in the liver. Following secretion of bile salts into the small intestine, intestinal microbes are capable of deconjugating the glycine or taurine from the bile salts, using an enzyme called bile salt hydrolase (Bsh). Intestinal lactobacilli are regarded as major contributors to bile salt hydrolysis in vivo. Since the bile salt-hydrolyzing strain Lactobacillus plantarum WCFS1 was predicted to carry four bsh genes (bsh1, bsh2, bsh3, and bsh4), the functionality of these bsh genes was explored using Lactococcus lactis heterologous overexpression and multiple bsh deletion strains. Thus, Bsh1 was shown to be responsible for the majority of Bsh activity in L. plantarum WCFS1. In addition, bsh1 of L. plantarum WCFS1 was shown to be involved in conferring tolerance to specific bile salts (i.e., glycocholic acid). Northern blot analysis established that bsh1, bsh2, bsh3, and bsh4 are all expressed in L. plantarum WCFS1 during the exponential growth phase. Following biodiversity analysis, bsh1 appeared to be the only bsh homologue that was variable among L. plantarum strains; furthermore, the presence of bsh1 correlated with the presence of Bsh activity, suggesting that Bsh1 is commonly responsible for Bsh activity in L. plantarum strains. The fact that bsh2, bsh3, and bsh4 genes appeared to be conserved among L. plantarum strains suggests an important role of these genes in the physiology and lifestyle of the species L. plantarum. Analysis of these additional bsh-like genes in L. plantarum WCFS1 suggests that they might encode penicillin acylase rather than Bsh activity, indicating their implication in the conversion of substrates other than bile acids in the natural habitat
    Recent advances in molecular techniques to study microbial communities in food-associated matrices and processes
    Justé, A. ; Thomma, B.P.H.J. ; Lievens, B. - \ 2008
    Food Microbiology 25 (2008)6. - ISSN 0740-0020 - p. 745 - 761.
    16s ribosomal-rna - restriction-fragment-length - in-situ hybridization - gradient gel-electrophoresis - real-time pcr - strand-conformation polymorphism - lactic-acid bacteria - intergenic spacer analysis - culture-independent methods - single nucleotide polymorp
    In the last two decades major changes have occurred in how microbial ecologists study microbial communities. Limitations associated with traditional culture-based methods have pushed for the development of culture-independent techniques, which are primarily based on the analysis of nucleic acids. These methods are now increasingly applied in food microbiology as well. This review presents an overview of current community profiling techniques with their (potential) applications in food and food-related ecosystems. We critically assessed both the power and limitations of these techniques and present recent advances in the field of food microbiology attained by their application. It is unlikely that a single approach will be universally applicable for analyzing microbial communities in unknown matrices. However, when screening samples for well-defined species or functions, techniques such as DNA arrays and real-time PCR have the potential to overtake current culture-based methods. Most importantly, molecular methods will allow us to surpass our current culturing limitations, thus revealing the extent and importance of the `non-culturable¿ microbial flora that occurs in food matrices and production.
    High-Level folate production in fermented foods by the B12 producer Lactobacillus reuteri JCM1112
    Santos, F. dos; Wegkamp, A. ; Vos, W.M. de; Smid, E.J. ; Hugenholtz, J. - \ 2008
    Applied and Environmental Microbiology 74 (2008)10. - ISSN 0099-2240 - p. 3291 - 3294.
    lactic-acid bacteria - lactococcus-lactis - crl1098 - biosynthesis - plantarum - cobalamin - sequence - systems
    We observed that Lactobacillus reuteri JCM1112 produces B12 and folate. However, the folate/B12 mass ratio found was far below that desired for human consumption (170:1). We used metabolic engineering applying genetic and physiological approaches to improve this ratio and developed a generic and natural process that significantly increases folate production.
    Letter to the editor: Antimicrobial susceptibility profiles of 32 type strains of lactobacillus, Bifidobacterium, Lactococcus and streptococcus spp
    Flórez, A.B. ; Ammor, M.S. ; Mayo, B. ; Hoek, A.H.A.M. van; Aarts, H.J.M. ; Huys, G. - \ 2008
    International Journal of Antimicrobial Agents 31 (2008)5. - ISSN 0924-8579 - p. 484 - 486.
    lactic-acid bacteria - antibiotic-resistance
    Bifidobacterium carbohydrases-their role in breakdown and synthesis of (potential) prebiotics
    Broek, L.A.M. van den; Hinz, S.W.A. ; Beldman, G. ; Vincken, J.P. ; Voragen, A.G.J. - \ 2008
    Molecular Nutrition & Food Research 52 (2008)1. - ISSN 1613-4125 - p. 146 - 163.
    adolescentis dsm 20083 - intestinal anaerobic bacterium - sequence-based classification - alpha-l-arabinofuranosidase - glycoside hydrolase family - lactic-acid bacteria - beta-d-galactosidase - sucrose phosphorylase - molecular-cloning - escherichia-coli
    Abstract There is an increasing interest to positively influence the human intestinal microbiota through the diet by the use of prebiotics and/or probiotics. It is anticipated that this will balance the microbial composition in the gastrointestinal tract in favor of health promoting genera such as Bifidobacterium and Lactobacillus. Carbohydrates like non-digestible oligosaccharides are potential prebiotics. To understand how these bacteria can grow on these carbon sources, knowledge of the carbohydrate-modifying enzymes is needed. Little is known about the carbohydrate-modifying enzymes of bifidobacteria. The genome sequence of Bifidobacterium adolescentis and Bifidobacterium longum biotype longum has been completed and it was observed that for B. longum biotype longum more than 8% of the annotated genes were involved in carbohydrate metabolism. In addition more sequence data of individual carbohydrases from other Bifidobacterium spp. became available. Besides the degradation of (potential) prebiotics by bifidobacterial glycoside hydrolases, we will focus in this review on the possibilities to produce new classes of non-digestible oligosaccharides by showing the presence and (transglycosylation) activity of the most important carbohydrate modifying enzymes in bifidobacteria. Approaches to use and improve carbohydrate-modifying enzymes in prebiotic design will be discussed.
    Two Different Tetracycline Resistance Mechanisms, Plasmid-Carried tet(L) and Chromosomally Located Transposon-Associated tet(M), Coexist in Lactobacillus sakei Rits 9
    Ammor, M.S. ; Gueimonde, M. ; Danielsen, M. ; Zagorec, M. ; Hoek, A.H.A.M. van; Reyes-Gavilán, C.G. de los; Mayo, B. ; Margolles, A. - \ 2008
    Applied and Environmental Microbiology 74 (2008)5. - ISSN 0099-2240 - p. 1394 - 1401.
    lactic-acid bacteria - gram-positive bacteria - antibiotic-resistance - enterococcus-faecalis - nucleotide-sequence - dry sausage - genes - food - genome - meat
    Lactobacillus sakei is extensively used as functional starter culture in fermented meat products. One of the safety criteria of a starter culture is the absence of potentially transferable antibiotic resistance determinants. However, tetracycline-resistant L. sakei strains have already been observed. In this paper, we show that tetracycline resistance in L. sakei Rits 9, a strain isolated from Italian Sola cheese made from raw milk, is mediated by a transposon-associated tet(M) gene coding for a ribosomal protection protein and a plasmid-carried tet(L) gene coding for a tetracycline efflux pump. pLS55, the 5-kb plasmid carrying the tet(L) gene, is highly similar to the pMA67 plasmid recently described for Paenibacillus larvae, a species pathogenic to honeybees. pLS55 could be transferred by electroporation into the laboratory strain L. sakei 23K. While the L. sakei 23K transformant containing pLS55 displayed an intermediate tetracycline resistance level (MIC,
    Analysis of functional properties of Lactobacillus acidophilus
    Zhao, R.X. ; Sun, J.L. ; Mo, H.Z. ; Yang Zhu, Yang - \ 2007
    World Journal of Microbiology and Biotechnology 23 (2007)2. - ISSN 0959-3993 - p. 195 - 200.
    lactic-acid bacteria - diarrhea - humans - ingestion - culture - lactose - gg
    Metabolites from Lactobacillus acidophilus were analysed. The results showed that Lactobacillus acidophilus Ind-1 and Lactobacillus acidophilus Lakcid produced respectively 12.73 g and 13.33 g lactic acid l¿1 after incubating in skim milk at 37 °C for 36 h; and 2.229 unit and 1.808 unit ß-galactosidase l¿1 in an MRS medium. The proteolytic activity of Lactobacillus acidophilus was high and the content of 17 free amino acids in the fermented milk of Lactobacillus acidophilus Ind-1 and Lactobacillus acidophilus Lakcid was 394.4 mg l¿1 and 563.2 mg l¿1, respectively. Meantime, Lactobacillus acidophilus reduced cholesterol level in an MRS medium supplemented with cholesterol. Furthermore, Lactobacillus acidophilus Ind-1 and Lactobacillus acidophilus Lakcid showed antimicrobial activity against Bacillus anthracis and Escherichia coli
    The European LABDEL project and its relevance to the prevention and treatment of allergies
    Daniel, C. ; Repa, A. ; Mercenier, A.M.E. ; Wiedermann, U. ; Wells, J. - \ 2007
    Allergy 62 (2007)11. - ISSN 0105-4538 - p. 1237 - 1242.
    lactic-acid bacteria - birch pollen allergen - bovine beta-lactoglobulin - t-regulatory cells - immune-responses - immunoglobulin-e - murine model - in-vivo - disease - immunotherapy
    In March 2001, the European Commission funded a 3-year project (contract no. QLK3-CT-2000-00340) under the fifth Framework Programme to develop and test prototype products based on the oral delivery of vaccine and therapeutic agents using harmless lactic acid bacteria (LAB). The project, best known under its acronym LABDEL (for LAB delivery) also included research on LAB fermentation and technological innovations aimed at enhancing the efficiency of LAB delivery systems (1). One of the key scientific objectives was to investigate the possibility to prevent or treat a type I allergic disease using mucosal administration of LAB expressing the pollen allergen Bet v 1. The aim of this paper was to describe the background of the project with reference to a limited selection of articles and recent reviews as well as the results and major conclusions arising from this part of the project.
    Preservation of blue-jack mackerel (Trachurus picturatus Bowdich) silage by chemical and fermentative acidification
    Enes Dapkevicius, M.L.N. ; Nout, M.J.R. ; Rombouts, F.M. ; Houben, J.H. - \ 2007
    Journal of Food Processing and Preservation 31 (2007)4. - ISSN 0145-8892 - p. 454 - 468.
    lactic-acid bacteria - rainbow-trout - fish silage - products - glucose - viscera - offal
    We compared acidified and lactic acid fermented silage approaches for the preservation of blue-jack mackerel. Silages acidified with formic and propionic acids had stable pH (3.8) and low (19 mg/g N) levels of volatile nitrogen compounds (total volatile basic nitrogen, TVBN), but relatively high (82 g/100 g) final non-protein-nitrogen (NPN) values. The silage was fermented with Lactobacillus plantarum LU853, a homofermentative lactic acid bacterium with a high growth (0.51/h) and acidification rate at 37C (optimum temperature), able to grow in the presence of 40 g/L NaCl and to ferment sucrose and lactose. The silages at 37C reached safe pH <4.5 values within 48¿72 h, either (F2a) or not (F0), in combination with 20 g/kg salt addition; F2a acidified more rapidly, which may be an advantage for its microbiological stability. Proteolysis resulting in 53¿59 g NPN/100 g N was lower in fermented than in acidified silages; however, in fermented silages, the levels of TVBN were much higher (50¿80 mg TVBN/g N) than generally considered acceptable.
    Two Lactobacillus strains, isolated from breast milk, differently modulate the immune response
    Diaz-Ropero, M.P. ; Martin, R. ; Sierra, S. ; Lara-Villoslada, F. ; Rodriguez, J.M. ; Xaus, J. ; Olivares, M. - \ 2007
    Journal of Applied Microbiology 102 (2007)2. - ISSN 1364-5072 - p. 337 - 343.
    lactic-acid bacteria - dendritic cells - colonization - enhancement - expression - maturation - probiotics - rhamnosus - diarrhea - flora
    Aims: The ability of two different Lactobacillus strains (Lactobacillus salivarius CECT5713 and Lactobacillus fermentum CECT5716), isolated from human breast milk, to modulate the immune response was examined. Methods and Results: In rodent bone-marrow-derived macrophages (BMDM), the presence of Lact. fermentum CECT5716 induced pro-inflammatory cytokines, in contrast to the activation of IL-10 induced by Lact. salivarius CECT5713. Although both strains reduced the lipopolysaccharide (LPS)-induced inflammatory response in BMDM, the effect of Lact. salivarius CECT5713 was more efficient, probably because of the production of higher amounts of IL-10 cytokine. In vivo assays in mice showed similar results; the consumption of Lact. fermentum CECT5716 enhanced the production of Th1 cytokines by spleen cells and increased the IgA concentration in faeces. However, the consumption of Lact. salivarius CECT5713 induced IL-10 production by spleen cells. Conclusion: Therefore, in general, the effect of Lact. fermentum CECT5716 is immunostimulatory in contrast to the anti-inflammatory effect of Lact. salivarius CECT5713. Significance and Impact of the Study: The results of this study show that two Lactobacillus strains isolated from breast milk can exert different and even opposing effects on immune response demonstrating the specificity of each strain.
    Safety assessment of two probiotic strains, Lactobacillus coryniformis CECT5711 and Lactobacillus Gasseri CECT5714
    Lara-Villoslada, F. ; Sierra, S. ; Martin, R. ; Delgado, S. ; Rodriguez, J.M. ; Olivares, M. ; Xaus, J. - \ 2007
    Journal of Applied Microbiology 103 (2007)1. - ISSN 1364-5072 - p. 175 - 184.
    lactic-acid bacteria - glycopeptide resistance - susceptibility - vancomycin - diarrhea - sepsis - plasma - milk - pcr
    Aims: The object of the present study was to evaluate the oral toxicity of the recently isolated probiotic bacteria Lactobacillus coryniformis CECT5711 and Lactobacillus gasseri CECT5714. Methods and Results: Enzymatic activity and antibiotic resistance profile were evaluated in vitro. Then, the oral toxicity was analysed by an in vivo experiment using 20 Balb/C mice, which were orally treated with CECT5711 or CECT5714 (1010 CFU mouse¿1 day¿1) during 30 days. Results showed that CECT5711 and CECT5714 have no deleterious enzymatic activities and present intrinsic antibiotic resistance profile. Administration of both strains to mice had no adverse effects on body weight or food intake. No bacteraemia was present in liver or spleen and there was no treatment-associated bacterial translocation to these tissues. Liver glutathione content as well as plasma malondialdehide concentration were not statistically different in probiotic-treated mice when compared with control mice. Probiotic treatment did not cause changes in the biochemical and haematological parameters analysed. Conclusions: These results suggest that strains CECT5711 and CECT5714 are nonpathogenic and likely to be safe for human consumption. Significance and Impact of the Study: This study reveals the oral safety of two new lactobacilli strains that are aimed to be used as probiotics in food and pharmaceutical applications.
    Making sense of quorum sensing in lactobacilli: a special focus on Lactobacillus plantarum WCFS1
    Sturme, M.H.J. ; Francke, C. ; Siezen, R. ; Vos, W.M. de; Kleerebezem, M. - \ 2007
    Microbiology 153 (2007). - ISSN 1350-0872 - p. 3939 - 3947.
    gram-positive bacteria - signal-transduction systems - histidine protein-kinase - complete genome sequence - lactic-acid bacteria - transcriptional regulators - autoinducing peptide - identification - family - biosynthesis
    In silico identification criteria were defined to predict if genes encoding histidine protein kinases (HPKs) and response regulators (RRs) could be part of peptide-based quorum sensing (QS) two-component regulatory systems (QS-TCSs) in Firmicutes. These criteria were used to screen HPKs and RRs annotated on the completed genome sequences of Lactobacillus species, and several (putative) QS-TCSs were identified in this way. The five peptide-based QS-TCSs that were predicted on the Lactobacillus plantarum WCFS1 genome were further analysed to test their (QS) functionality. Four of these systems contained an upstream gene encoding a putative autoinducing peptide (AIP), of which two were preceded by a double-glycine-type leader peptide. One of these was identical to the plnABCD regulatory system of L. plantarum C11 and was shown to regulate plantaricin production in L. plantarum WCFS1. The third TCS was designated lamBDCA for Lactobacillus agr-like module, where the lamD gene was shown to encode a cyclic thiolactone peptide. The fourth TCS was paralogous to the lam system and contained a putative AIP-encoding gene but lacked the lamB gene. Finally, a genetically separated orphan HPK and RR that showed clear peptide-based QS characteristics could form a fifth peptide-based QS-TCS. The predicted presence of multiple (peptide-based) QS-TCSs in some lactobacilli and in particular in L. plantarum might be a reflection of the ability of these species to persist in a diverse range of ecological niches.
    Diversity of the Lactobacillus group in breast milk and vagina of healthy women and potential role in the colonization of the infant gut
    Martin, R. ; Heilig, G.H.J. ; Zoetendal, E.G. ; Smidt, H. ; Rodriguez, J.M. - \ 2007
    Journal of Applied Microbiology 103 (2007)6. - ISSN 1364-5072 - p. 2638 - 2644.
    16s ribosomal-rna - gel-electrophoresis analysis - lactic-acid bacteria - communities - microbiota - bifidobacteria - reveals
    Aims: To evaluate the diversity of the Lactobacillus group in breast milk and the vagina of healthy women and understand their potential role in the infant gut colonization using the 16S rRNA gene approaches. Methods and Results: Samples of breast milk, vaginal swabs and infant faeces were aseptically collected from five mothers whose neonates were born by vaginal delivery and another five that had their babies by caesarean section. After polymerase chain reaction (PCR) amplification using Lactobacillus group-specific primers, amplicons were analysed by denaturing gradient gel electrophoresis (DGGE). Clone libraries were constructed to describe the Lactobacillus group diversity. DGGE fingerprints were not related to the delivery method. None of the species detected in vaginal samples were found in breast milk-derived libraries and only few were detected in infant faeces. Conclusions: The bacterial composition of breast milk and infant faeces is not related to the delivery method. Significance and Impact of the Study: It has been suggested that neonates acquire lactobacilli by oral contamination with vaginal strains during delivery; subsequently, newborns would transmit such bacteria to the breast during breastfeeding. However, our findings confirm, at the molecular level that in contrast to the maternal vagina, breast milk seems to constitute a good source of lactobacilli to the infant gut.
    Comparison of quenching and extraction methodologies for metabolome analysis of Lactobacillus plantarum
    Faijes, M. ; Mars, A.E. ; Smid, E.J. - \ 2007
    Microbial Cell Factories 6 (2007). - ISSN 1475-2859 - 8 p.
    lactic-acid bacteria - saccharomyces-cerevisiae - lactococcus-lactis - intracellular metabolites - electrospray-ionization - escherichia-coli - microbial metabolomics - streptococcus-cremoris - mass-spectrometry - growth-rate
    Background A reliable quenching and metabolite extraction method has been developed for Lactobacillus plantarum. The energy charge value was used as a critical indicator for fixation of metabolism. Results Four different aqueous quenching solutions, all containing 60% of methanol, were compared for their efficiency. Only the solutions containing either 70 mM HEPES or 0.85% (w/v) ammonium carbonate (pH 5.5) caused less than 10% cell leakage and the energy charge of the quenched cells was high, indicating rapid inactivation of the metabolism. The efficiency of extraction of intracellular metabolites from cell cultures depends on the extraction methods, and is expected to vary between micro-organisms. For L. plantarum, we have compared five different extraction methodologies based on (i) cold methanol, (ii) perchloric acid, (iii) boiling ethanol, (iv) chloroform/methanol (1:1) and (v) chloroform/water (1:1). Quantification of representative intracellular metabolites showed that the best extraction efficiencies were achieved with cold methanol, boiling ethanol and perchloric acid. Conclusion The ammonium carbonate solution was selected as the most suitable quenching buffer for metabolomics studies in L. plantarum because (i) leakage is minimal, (ii) the energy charge indicates good fixation of metabolism, and (iii) all components are easily removed during freeze-drying. A modified procedure based on cold methanol extraction combined good extractability with mild extraction conditions and high enzymatic inactivation. These features make the combination of these quenching and extraction protocols very suitable for metabolomics studies with L. plantarum.
    Identification of prebiotic fructooligosaccharide metabolism in Lactobacillus plantarum WCFS1 through microarrays
    Saulnier, D.M. ; Molenaar, D. ; Vos, W.M. de; Gibson, G. ; Kolida, S. - \ 2007
    Applied and Environmental Microbiology 73 (2007)6. - ISSN 0099-2240 - p. 1753 - 1765.
    lactic-acid bacteria - beta-fructofuranosidase - gastrointestinal-tract - catabolite repression - glucose kinase - gene - bifidobacteria - acidophilus - modulation - probiotics
    Short-chain fructooligosaccharides (scFOS) and other prebiotics are used to selectively stimulate the growth and activity of lactobacilli and bifidobacteria in the colon. However, there is little information on the mechanisms whereby prebiotics exert their specific effects upon such microorganisms. To study the genomic basis of scFOS metabolism in Lactobacillus plantarum WCFS1, two-color microarrays were used to screen for differentially expressed genes when grown on scFOS compared to glucose (control). A significant up-regulation (8- to 60-fold) was observed with a set of only five genes located in a single locus and predicted to encode a sucrose phosphoenolpyruvate transport system (PTS), a ß-fructofuranosidase, a fructokinase, an -glucosidase, and a sucrose operon repressor. Several other genes were slightly overexpressed, including pyruvate dehydrogenase. For the latter, no detectable activity in L. plantarum under various growth conditions has been previously reported. A mannose-PTS likely to encode glucose uptake was 50-fold down-regulated as well as, to a lower extent, other PTSs. Chemical analysis of the different moieties of scFOS that were depleted in the growth medium revealed that the trisaccharide 1-kestose present in scFOS was preferentially utilized, in comparison with the tetrasaccharide nystose and the pentasaccharide fructofuranosylnystose. The main end products of scFOS fermentation were lactate and acetate. This is the first example in lactobacilli of the association of a sucrose PTS and a ß-fructofuranosidase that could be used for scFOS degradation.
    Spatial and temporal expression of Lactobacillus plantarum genes in the gastrointestinal tracts of mice
    Marco, M. ; Bongers, R.S. ; Vos, W.M. de; Kleerebezem, M. - \ 2007
    Applied and Environmental Microbiology 73 (2007)1. - ISSN 0099-2240 - p. 124 - 132.
    lactic-acid bacteria - 16s ribosomal-rna - protein-synthesis - human-microbiota - in-vivo - identification - promoters - transit - casei - diversity
    Lactobacillus plantarum is a common inhabitant of mammalian gastrointestinal tracts, and L. plantarum strain WCFS1 is a human isolate with a known genome sequence. L. plantarum WCFS1 survives intestinal passage in an active form, and its transit time and transcriptional activities were monitored in 15 BALB/c mice at 2, 4, 6, 8, and 24 h after being fed a single intragastric dose of this organism. Enumeration of viable cells isolated from fecal material revealed that the majority of the L. plantarum inoculum transited the mouse intestine within 4 h after ingestion. Three mice were sacrificed at each time point, and total RNA was isolated from the mouse intestinal compartments (stomach through colon). Quantification of L. plantarum 16S rRNA by quantitative real-time reverse-transcription-PCR revealed that L. plantarum was present at elevated levels in the stomach and small intestine for at least 4 h following ingestion and for over 8 h in the cecum and colon. We also examined the expression of 9 L. plantarum housekeeping genes and 15 L. plantarum in vivo-inducible (ivi) genes previously identified by recombination-based in vivo expression technology to be induced in the mouse gastrointestinal tract. The relative expression levels of the ivi genes increased up to 350-fold in the mouse intestine compared to levels observed for L. plantarum WCFS1 cells grown in a rich laboratory medium. Moreover, several genes displayed intestinal compartment-specific (small intestine versus colon) activities. These results confirm that L. plantarum displays specific and differential responses at various sites along the mammalian intestine.
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