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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    Lipoproteins Contribute to the Anti-inflammatory Capacity of Lactobacillus plantarum WCFS1
    Lee, Chiao ; Swam, Iris I. van; Boeren, Sjef ; Vervoort, Jacques ; Meijerink, Marjolein ; Taverne, Nico ; Starrenburg, Marjo ; Bron, Peter A. ; Kleerebezem, Michiel - \ 2020
    Frontiers in Microbiology 11 (2020). - ISSN 1664-302X
    human immune system - Lactobacillus - Lactobacillus plantarum - LGT - lipoproteins - probiotics - prolipoprotein diacylglyceryl transferase - proteomics

    Bacterial lipoproteins are well-recognized microorganism-associated molecular patterns, which interact with Toll-like receptor (TLR) 2, an important pattern recognition receptor of the host innate immune system. Lipoproteins are conjugated with two- or three-acyl chains (di- or tri-acyl), which is essential for appropriate anchoring in the cell membrane as well as for the interaction with TLR2. Lipoproteins have mostly been studied in pathogens and have established roles in various biological processes, such as nutrient import, cell wall cross-linking and remodeling, and host-cell interaction. By contrast, information on the role of lipoproteins in the physiology and host interaction of probiotic bacteria is scarce. By deletion of lgt, encoding prolipoprotein diacylglyceryl transferase, responsible for lipidation of lipoprotein precursors, we investigated the roles of the collective group of lipoproteins in the physiology of the probiotic model strain Lactobacillus plantarum WCFS1 using proteomic analysis of secreted proteins. To investigate the consequences of the lgt mutation in host-cell interaction, the capacity of mutant and wild-type bacteria to stimulate TLR2 signaling and inflammatory responses was compared using (reporter-) cell-based models. These experiments exemplified the critical contribution of the acyl chains of lipoproteins in immunomodulation. To the best of our knowledge, this is the first study that investigated collective lipoprotein functions in a model strain for probiotic lactobacilli, and we show that the lipoproteins in L. plantarum WCFS1 are critical drivers of anti-inflammatory host responses toward this strain.

    Gut dysbacteriosis and intestinal disease: mechanism and treatment
    Meng, X. ; Zhang, G. ; Cao, H. ; Yu, D. ; Fang, X. ; Vos, W.M. de; Wu, H. - \ 2020
    Journal of Applied Microbiology (2020). - ISSN 1364-5072
    gut microbiome - immune response - intestinal diseases - prebiotics - probiotics

    The gut microbiome functions like an endocrine organ, generating bioactive metabolites, enzymes or small molecules that can impact host physiology. Gut dysbacteriosis is associated with many intestinal diseases including (but not limited to) inflammatory bowel disease, primary sclerosing cholangitis-IBD, irritable bowel syndrome, chronic constipation, osmotic diarrhoea and colorectal cancer. The potential pathogenic mechanism of gut dysbacteriosis associated with intestinal diseases includes the alteration of composition of gut microbiota as well as the gut microbiota–derived signalling molecules. The many correlations between the latter and the susceptibility for intestinal diseases has placed a spotlight on the gut microbiome as a potential novel target for therapeutics. Currently, faecal microbial transplantation, dietary interventions, use of probiotics, prebiotics and drugs are the major therapeutic tools utilized to impact dysbacteriosis and associated intestinal diseases. In this review, we systematically summarized the role of intestinal microbiome in the occurrence and development of intestinal diseases. The potential mechanism of the complex interplay between gut dysbacteriosis and intestinal diseases, and the treatment methods are also highlighted.

    Feed, Microbiota, and Gut Immunity: Using the Zebrafish Model to Understand Fish Health
    López Nadal, Adrià ; Ikeda-Ohtsubo, Wakako ; Sipkema, Detmer ; Peggs, David ; McGurk, Charles ; Forlenza, Maria ; Wiegertjes, Geert F. ; Brugman, Sylvia - \ 2020
    Frontiers in Immunology 11 (2020). - ISSN 1664-3224
    gut - immunity - intestine - microbiota - prebiotics - probiotics - zebrafish

    Aquafeed companies aim to provide solutions to the various challenges related to nutrition and health in aquaculture. Solutions to promote feed efficiency and growth, as well as improving the fish health or protect the fish gut from inflammation may include dietary additives such as prebiotics and probiotics. The general assumption is that feed additives can alter the fish microbiota which, in turn, interacts with the host immune system. However, the exact mechanisms by which feed influences host-microbe-immune interactions in fish still remain largely unexplored. Zebrafish rapidly have become a well-recognized animal model to study host-microbe-immune interactions because of the diverse set of research tools available for these small cyprinids. Genome editing technologies can create specific gene-deficient zebrafish that may contribute to our understanding of immune functions. Zebrafish larvae are optically transparent, which allows for in vivo imaging of specific (immune) cell populations in whole transgenic organisms. Germ-free individuals can be reared to study host-microbe interactions. Altogether, these unique zebrafish features may help shed light on the mechanisms by which feed influences host-microbe-immune interactions and ultimately fish health. In this review, we first describe the anatomy and function of the zebrafish gut: the main surface where feed influences host-microbe-immune interactions. Then, we further describe what is currently known about the molecular pathways that underlie this interaction in the zebrafish gut. Finally, we summarize and critically review most of the recent research on prebiotics and probiotics in relation to alterations of zebrafish microbiota and immune responses. We discuss the advantages and disadvantages of the zebrafish as an animal model for other fish species to study feed effects on host-microbe-immune interactions.

    Pro-biotics for a healthy gut
    Belzer, C. - \ 2019
    probiotics - intestinal microorganisms - health foods - nutrition and health
    Have you ever noticed the little bottles in your local grocery store that contain living bacteria? When a microorganims is still alive in food and after consumption can have health benefits, it is called “a probiotic”. In this video we will explain how probiotics can promote gut health.
    This lesson is part of the WageningenX MOOC called 'Nutrition and Health: Human Microbiome'
    Effectiveness of Probiotics in Children with Functional Abdominal Pain Disorders and Functional Constipation A Systematic Review
    Wegh, Carrie A.M. ; Benninga, Marc A. ; Tabbers, Merit M. - \ 2018
    Journal of Clinical Gastroenterology 52 (2018)supp. 1. - ISSN 0192-0790 - p. S10 - S26.
    children - functional gastrointestinal disorders - gut microbiota - probiotics

    Objective: The objective of this study was to investigate the effect of probiotics on functional abdominal pain disorders (FAPD) and functional constipation (FC). Methods: A systematic review was conducted, searching PubMed and Cochrane databases from inception to January 2018 for randomized controlled trials (RCTs) investigating the efficacy of probiotics in children aged 4 to 18 years with FAPD or children aged 0 to 18 years with FC. Results: A total of 657 citations were identified. Finally, 11 RCTs for FAPD and 6 RCTs for FC were included. Some evidence exists for Lactobacillus rhamnosus GG (n=3) in reducing frequency and intensity of abdominal pain in children with irritable bowel syndrome. There is no evidence to recommend L. reuteri DSM 17938 (n=5), a mix of Bifidobacterium infantis, Bifidobacterium breve and Bifidobacterium longum (n=1), Bifidobacterium lactis (n=1) or VSL#3 (n=1) for children with FAPD. No evidence exists to support the use of Lactobacillus casei rhamnosus LCR35 (n=1), B. lactis DN173 010 (n=1), B. longum (n=1), L. reuteri DSM 17938 (n=1), a mix of B. infantis, B. breve and B. longum (n=1), or Protexin mix (n=1) for children with FC. In general, studies had an unclear or high risk of bias. Conclusions: Insufficient evidence exists for the use of probiotics in FAPD and FC, only L. rhamnosus GG seems to reduce frequency and intensity of abdominal pain but only in children with irritable bowel syndrome. A better understanding of differences in gut microbiota in health and disease might lead to better probiotic strategies to treat disease.

    Akkermansia muciniphila induces gut microbiota remodelling and controls islet autoimmunity in NOD mice
    Hänninen, Arno ; Toivonen, Raine ; Pöysti, Sakari ; Belzer, Clara ; Plovier, Hubert ; Ouwerkerk, Janneke P. ; Emani, Rohini ; Cani, Patrice D. ; Vos, Willem M. de - \ 2018
    Gut 67 (2018)8. - ISSN 0017-5749 - p. 1445 - 1453.
    autoimmunity - bacterial interactions - diabetes mellitus - gut immunology - probiotics

    Objective Intestinal microbiota is implicated in the pathogenesis of autoimmune type 1 diabetes in humans and in non-obese diabetic (NOD) mice, but evidence on its causality and on the role of individual microbiota members is limited. We investigated if different diabetes incidence in two NOD colonies was due to microbiota differences and aimed to identify individual microbiota members with potential significance. Design We profiled intestinal microbiota between two NOD mouse colonies showing high or low diabetes incidence by 16S ribosomal RNA gene sequencing and colonised the high-incidence colony with the microbiota of the low-incidence colony. Based on unaltered incidence, we identified a few taxa which were not effectively transferred and thereafter, transferred experimentally one of these to test its potential significance. Results Although the high-incidence colony adopted most microbial taxa present in the low-incidence colony, diabetes incidence remained unaltered. Among the few taxa which were not transferred, Akkermansia muciniphila was identified. As A. muciniphila abundancy is inversely correlated to the risk of developing type 1 diabetes-related autoantibodies, we transferred A. muciniphila experimentally to the high-incidence colony. A. muciniphila transfer promoted mucus production and increased expression of antimicrobial peptide Reg3γ, outcompeted Ruminococcus torques from the microbiota, lowered serum endotoxin levels and islet toll-like receptor expression, promoted regulatory immunity and delayed diabetes development. Conclusion Transfer of the whole microbiota may not reduce diabetes incidence despite a major change in gut microbiota, but single symbionts such as A. muciniphila with beneficial metabolic and immune signalling effects may reduce diabetes incidence when administered as a probiotic.

    Effect of Synbiotic on the Gut Microbiota of Cesarean Delivered Infants : A Randomized, Double-blind, Multicenter Study
    Chua, Mei Chin ; Ben-Amor, Kaouther ; Lay, Christophe ; Neo, Anne G.E. ; Chiang, Wei Chin ; Rao, Rajeshwar ; Chew, Charmaine ; Chaithongwongwatthana, Surasith ; Khemapech, Nipon ; Knol, Jan ; Chongsrisawat, Voranush - \ 2017
    Journal of Pediatric Gastroenterology and Nutrition 65 (2017). - ISSN 0277-2116 - p. 102 - 106.
    Bifidobacterium breve M-16V - C-section - gut microbiota - prebiotics - probiotics - synbiotics

    We determined the effect of short-chain galacto-oligosaccharides (scGOS), long-chain fructo-oligosaccharides (lcFOS) and Bifidobacterium breve M-16V on the gut microbiota of cesarean-born infants. Infants were randomized to receive a standard formula (control), the same with scGOS/lcFOS and B. breve M-16V (synbiotic), or with scGOS/lcFOS (prebiotic) from birth until week 16, 30 subjects born vaginally were included as a reference group. Synbiotic supplementation resulted in a higher bifidobacteria proportion from day 3/5 (P<0.0001) until week 8 (P=0.041), a reduction of Enterobacteriaceae from day 3/5 (P=0.002) till week 12 (P=0.016) compared to controls. This was accompanied with a lower fecal pH and higher acetate. In the synbiotic group, B. breve M-16V was detected 6 weeks postintervention in 38.7% of the infants. This synbiotic concept supported the early modulation of Bifidobacterium in C-section born infants that was associated with the emulation of the gut physiological environment observed in vaginally delivered infants.

    Microbial interactions in the fish gut
    Giatsis, Christos - \ 2016
    Wageningen University. Promotor(en): Johan Verreth, co-promotor(en): Marc Verdegem; Detmer Sipkema. - Wageningen : Wageningen University - ISBN 9789462578777 - 196
    fishes - tilapia - larvae - microbial interactions - intestinal microorganisms - intestines - dynamics - fish feeding - probiotics - fish culture - aquaculture - vissen - tilapia - larven - microbiële interacties - darmmicro-organismen - darmen - dynamica - visvoeding - probiotica - visteelt - aquacultuur

    Aquaculture has realized considerable growth over the past years while the world demand on seafood has been increasing. As aquaculture intensifies, the production sector needs to tackle major bottlenecks such as suboptimal growth and high and unpredictable mortality, especially in larval cultures. Fish-microbe interactions are closely related to overall fish health. To obtain a healthy and resilient microbial community (MC), it is important to understand the underlying mechanisms of microbial colonization in the fish gut.

    The goal of this thesis was to investigate the role of water and feed microbial communities on shaping gut communities during early development of Nile tilapia.

    To determine the contribution of stochasticity to overall variation, we first characterized the spatio-temporal variation in MC composition between individuals reared within the same or in replicate recirculating or active suspension systems (RAS vs. AS). Highly similar MCs developed in the gut when larvae shared the same water and diet. Rearing larvae in replicate production systems resulted in significantly different gut communities indicating that compositional replication of the MCs of an ecosystem is not fully predictable. We found that mainly water MCs, and to a lesser degree feed MCs, were associated with changes in MCs. Thus, we could conclude that steering gut MCs can be possible through water MC management tailored on the specifications of the rearing system in use.

    Next, the possibility of early life steering of gut communities via microbial manipulations of feed MCs was explored. We hypothesized that gut microbial composition is strongly shaped by selective pressures in the gut and by the MCs present in the water. Thus similar MCs should develop between treatments regardless of the dietary treatments. Fish larvae were fed either a control feed or the control feed containing MCs derived from aerobic, methanogenic or denitrifying sludge reactors. We found that gut microbiota shared a much higher number of operational taxonomic units (OTUs) with microbiota in sludge-based feeds than with water, resulting in distinct gut MCs between treatments. Our findings suggest that Nile tilapia gut MC has a certain plasticity, which makes it amenable to interventions through proper feed microbial management.

    Subsequently, we tested the imprinting effect of early exposure to the probiotic Bacillus subtilis on shaping gut MC composition even after the administration of the probiotic discontinues. For this, we constrained the initial contact with microbes from the environment by producing axenic tilapia larvae, which were then exposed to normal husbandry conditions. Early life probiotic exposure affected gut MC composition during B. subtilis administration but also within the first two weeks after its administration stopped, thus indicating that early exposure to the probiotic strain via the water had a sustained impact on gut MC composition.

    Finally, overall conclusions and practical implications of our results for aquaculture production were presented. A meta-analysis was also performed to examine (1) the phylogenetic similarity among gut MCs of the same and different fish species reared in different habitats, fed different diets and at different developmental stages and (2) the factors primarily shaping gut MCs. We showed that the selective pressure responsible in shaping gut MC composition highly depends on the host as gut communities clustered primarily together by host and to a lesser extent reflected differences in habitat and diet. The phylogenetic analysis of gut communities revealed a clear clustering by study thus indicating that manipulation of gut communities is conceivable. Study-to-study variation could be attributed to the methodology used for MC analysis highlighting also the importance of methodological uniformity when comparisons between studies are made.

    Overall, this thesis provided fundamental knowledge on MC composition and development in aquaculture rearing systems. Although the insights generated by this thesis are still premature to fully explain, predict or steer MC composition, and though additional studies are needed, we believe that, in the long run, this approach will facilitate the development of safe and effective methods for manipulating gut microbial composition to promote fish health in aquaculture rearing systems.

    Host-interaction effector molecules of Lactobacillus plantarum WCFS1
    Lee, I.C. - \ 2016
    Wageningen University. Promotor(en): Michiel Kleerebezem, co-promotor(en): P.A. Bron. - Wageningen : Wageningen University - ISBN 9789462576858 - 183
    lactobacillus plantarum - molecules - probiotics - immunomodulatory properties - lipoproteins - interactions - molecular interactions - host pathogen interactions - lactobacillus plantarum - moleculen - probiotica - immunomodulerende eigenschappen - lipoproteïnen - interacties - moleculaire interacties - gastheer-pathogeen interacties


    Lactobacillus plantarum is found in various environmental habitats, including fermentation products and the mammalian gastrointestinal tract, and specific strains are marketed as probiotics, which are defined as ‘live microorganisms which when administered in adequate amounts confer a health benefit on the host’. Throughout the studies of the mechanisms underlying probiotic activity, it became apparent that the probiotic effects are often species and/or strain specific. This situation has led more researchers to focus on the molecular characteristics of probiotic strains intending to link specific molecular structures to specific probiotic functions, and thereby deduce the mechanisms of molecular communication of probiotics. This thesis focuses on potential cell envelope effector molecules involved in interaction with the mammalian host cells, including lipoteichoic acid (LTA), lipo- and glyco-proteins, and extracellular polysaccharides (EPS), of L. plantarum WCFS1, a model strain for probiotic lactobacilli with a well-annotated genome sequences and sophisticated genetic engineering tools. First, existing research regarding the potential roles in probiotic functionality of Lactobacillus surface molecules in terms of their biosynthesis pathways and structure variations as well as interaction with host Pattern Recognition Receptors (PRRs) and immunomodulatory properties of these molecules are summarized and compared to provide an overview of the state-of-the-art in probiotic effector molecule research. Subsequently, specific molecules that reside in the cell envelope of L. plantarum WCFS1 were study for their role in bacterial physiology, as well as their role as ligands in Toll-like receptor (TLR) 2 signaling and immunomodulatory properties using human-cell co-incubation models. Our results showed that the deficiency of LTA had a drastic impact on cell division, cell morphology and growth in L. plantarum WCFS1, while LTA-deficient cells also elicited more pro-inflammatory responses in PBMCs rather than the expected loss of pro-inflammatory capacity as was observed with similar mutants of Lactobacillus acidophilus NCFM. Further studies on the signaling capacity of the purified LTA from L. plantarum WCFS1 revealed that these molecules are poor TLR2 activators, which is in clear contrast to the highly potent TLR2 stimulatory capacity of LTA obtained from Bacillus subtilis, implying that structural differences of the LTA produced by different bacteria are prominent determinants of their TLR2 signaling capacity and immunomodulatory properties. Lipoproteins of L. plantarum WCFS1 were studied using a derivative strain that is deficient in prolipoprotein diacylglyceryltransferase (Lgt), which transfers acyl chain moieties onto lipoproteins. The lipid moiety was shown to be important for proper anchoring of lipoproteins and TLR1/2 signaling capacity, but did not affect TLR2/6 signaling, suggesting that lipoproteins of L. plantarum WCFS1 are predominantly (if not exclusively) triacylated. The Lgt deficient strain elicited more pro-inflammatory responses in PBMCs as compared to the wild type, indicating that the native lipoproteins could play a role in dampening inflammation upon host-probiotic interaction. In addition, we explored the protein glycosylation machinery in L. plantarum WCFS1, responsible for the glycosylation of the major autolysin (Acm2) of this bacterium, which was previously shown to be O-glycosylated with N-acetylhexosamine conjugates. Using sequence similarity searches in combination with a lectin-based glycan detection and mass spectrometry analysis, two glycosyl-transferases, GtfA and GtfB (formerly annotated as TagE5 and TagE6, respectively), were shown to be required for the glycosylation of Acm2 and other unidentified L. plantarum WCFS1 glycosylated proteins. These results provide the first example of a general protein-glycosylation machinery in a Lactobacillus species. Finally, extracellular polysaccharides (EPS) in L. plantarum were studied in two strains that produce large amounts of EPS: L. plantarum SF2A35B and Lp90, in comparison to the lowly producing model strain WCFS1. Based on genome sequence comparison, both of the high producer strains were found to possess strain-specific and unique polysaccharide gene clusters. These gene clusters were deleted and the mutants were shown to have lost the capacity to produce large amounts of EPS, and were studied in relation to their properties in host-bacteria interaction. The results illustrate strain-specific and variable impacts of the removal of the EPS in the background of individual L. plantarum strains, supporting the importance of EPS in L. plantarum strains as a strain-specific determinant in host interaction. Overall, this thesis showed that surface molecules not only play important roles in bacterial physiology, but also in the interaction with the host mucosa through pattern recognition receptors expressed by the host cells. With the growing amount of evidence of structural variations in surface molecules, which are influenced by genetic background, physiological status, environmental factors, and other biological processes, these molecules form a unique signature associated with each strain that as a consequence elicits a strain-specific response when interacting with host cells.

    Phenotypic and genetic diversity of the species Lactobacillus rhamnosus
    Ceapa, C.D. - \ 2016
    Wageningen University. Promotor(en): Michiel Kleerebezem; Jan Knol; J. Lambert. - Wageningen : Wageningen University - ISBN 9789462576285 - 195
    fermentation products - probiotics - intestinal microorganisms - lactic acid bacteria - strains - medicinal properties - genomics - nutrition and health - fermentatieproducten - probiotica - darmmicro-organismen - melkzuurbacteriën - stammen (biologisch) - medicinale eigenschappen - genomica - voeding en gezondheid

    The thesis explores the diversity of Lactobacillus rhamnosus, a species from which strains are studied for their anti-inflammatory, anti-allergic, and diarrhea preventing effects. The work combines observations on the behavior of the bacteria in a simplified laboratory setting (use of carbohydrates, immune modulation effects, anti-pathogenic effects) with genomic information obtained by sequencing, with the aim to pinpoint genes that could be relevant for bacterial survival and metabolic capacities. Phenotypic and genotypic profiling analyses congruently revealed that carbohydrate metabolism and transport is essential for this species’ adaptation to the environment. Genotype–phenotype correlation analysis enabled us to predict and then experimentally verify genes responsible for the utilization of L-Sorbose, L-Fucose α-D-Methyl Glycoside.

    Mucus and gut barrier in health and disease
    Sovran, B. - \ 2015
    Wageningen University. Promotor(en): Jerry Wells; P. de Vos, co-promotor(en): J. Dekker. - Wageningen : Wageningen University - ISBN 9789462574892 - 233
    slijm - spijsverteringskanaal - darmen - muizen - probiotica - eilandjes van peyer - colitis - transcriptomen - immunohistologie - veroudering - geslacht (sex) - homeostase - gezondheid - ziekten - mucus - digestive tract - intestines - mice - probiotics - peyer patches - colitis - transcriptomes - immunohistology - senescence - sex - homeostasis - health - diseases

    This publication describes his work as a PhD student in the Host-Microbe Interactomics Chair group at Wageningen University within the Gastrointestinal Health theme. It has been completed under the supervision of Prof. Dr Jerry M Wells, Dr Jan Dekker and the TIFN project leader, Prof. Dr Paul de Vos.

    Mucus serves as a protective layer between the intestinal content and the intestinal wall. It facilitates the passage of the luminal content through the intestine, reducing the risk of mechanical damage to the intestinal epithelium. The overarching goal of this thesis was to investigate the role of mucus in the maintenance of the intestinal immune barrier and the effects of ageing and gender differences on mucus production and the gut barrier.

    We found by using a mouse model that decreased mucus production leads to changes in microbiota and mucosal stress responses, without the appearance of pathology, demonstrating the importance of mucus in intestinal homeostasis. The mucus barrier was shown to deteriorate during aging but this could be prevented with specific probiotics. Furthermore gender-specific differences in the effects of ageing on the mucosal barrier were found. Increased knowledge on these mechanisms might contribute significantly to disease prevention and treatment, for instance by optimizing gender-specific dietary and pharmacological requirements.

    The study presented in this thesis was performed within the framework of Top Institute Food and Nutrition, within the GH002 project.

    Development of probiotic mutandabota, a locally sustainable functional food incorporating Lactobacillus rhamnosus
    Mpofu, A. - \ 2015
    Wageningen University. Promotor(en): Marcel Zwietering; Eddy Smid, co-promotor(en): Anita Linnemann. - Wageningen : Wageningen University - ISBN 9789462574731 - 175
    adansonia digitata - adansonia - rauwe melk - geitenmelk - lactobacillus rhamnosus - probiotica - voedselveiligheid - kwalitatieve analyse - kwantitatieve analyse - adansonia digitata - adansonia - raw milk - goat milk - lactobacillus rhamnosus - probiotics - food safety - qualitative analysis - quantitative analysis

    Development of probiotic mutandabota, a locally sustainable functional food incorporating Lactobacillus rhamnosus

    Mutandabota or umlondo is an indigenous food that is consumed in Southern Africa on a daily basis. The product is made by mixing raw cow’s or goat’s milk with 14 % (wt/vol) dry pulp of the baobab fruit (Adansonia digitata L.) and 7 % sugar. Mutandabota has a high protein content, and is rich in vitamin C and minerals. It also provides fibre to the diet, which evidently has potential health benefits in preventing diabetes, cardiovascular diseases, some cancers and constipation. Predominant microorganisms were isolated from mutandabota and identified. This indicated that different species of bacteria and yeast survive the acidity and low pH of 3.4±0.1 in mutandabota. While no pathogens were isolated, the identified microorganisms are capable of spoiling the product. Preparation of mutandabota is a gendered activity dominated by women.

    A probiotic dairy product was then developed at village level on the basis of mutandabota to enable resource-poor populations in Southern Africa to accrue health benefits from a functional food. Raw cow’s milk was pasteurised and dry baobab fruit pulp was added to the milk at a concentration of 4 % (wt/vol). This mixture was inoculated with the probiotic Lactobacillus rhamnosus yoba, an isolate of Lactobacillus rhamnosus GG, and left to ferment for 24 h. Baobab fruit pulp at 4% promoted growth of L. rhamnosus yoba. More pulp and sugar were then added to produce yoba mutandabota with 14 % (wt/vol) baobab fruit pulp and 7 % sugar. The final pH of yoba mutandabota was pH 3.5, which ensured the microbiological safety of the product. Viable plate count of L. rhamnosus yoba was 8.8 ± 0.4 log cfu/mL at the moment of consumption, thereby meeting the criterion to have a viable count of the probiotic bacterium in excess of 6 log cfu/mL in the product.

    There was no significant difference (p=0.31) in consumers’ preference between traditional and yoba mutandabota, despite a significant difference (p<0.001) in sensorial properties of the two products. Challenge tests to evaluate the impact of L. rhamnosus yoba on competing pathogens in mutandabota were done. In traditional mutandabota (pH 3.4±0.1) some food-borne pathogens survived and withstood the acids and low pH of the product. However, yoba mutandabota (pH 3.4±0.1) inactivated all tested food-borne bacterial pathogens during the 24 h potential consumption time. This demonstrated that yoba mutandabota can be safer stored than traditional mutandabota. The L. rhamnosus yoba showed robustness and grew from 5.5 log cfu/mL to 9.0 log cfu/mL within 24 h in the presence of pathogens in yoba mutandabota.

    The outcome of this work was a safe, healthy, optimum-quality product of relevant nutritional value. Although this work focused on growth of L. rhamnosus yoba in mutandabota, the potential exists to apply this approach to other traditional foods worldwide as a low-cost method to improve dietary quality and gastro-intestinal health of consumers. Yoba mutandabota processing and trading may ameliorate the well-being of rural households through improvements in health status and livelihoods.

    Emissiereductie door verneveling van probiotica over leefoppervlak : literatuurstudie en metingen bij vleesvarkens
    Ellen, H.H. ; Groenestein, K. ; Hol, J.M.G. ; Ogink, N.W.M. ; Pas, L. - \ 2015
    Wageningen : Wageningen UR Livestock Research (Livestock Research rapport 809) - 39
    varkens - slachtdieren - afmesten - probiotica - ammoniakemissie - geurstoffen - fijn stof - varkenshouderij - pigs - meat animals - finishing - probiotics - ammonia emission - odours - particulate matter - pig farming
    In twee identieke afdelingen met vleesvarkens is het effect gemeten van het aanbrengen van probiotica (PIP) via het vernevelen er van in de afdeling. Uit de gemeten waarden blijkt er geen significant effect te zijn van het toepassen van probiotica op de emissies van ammoniak (NH3), geur en fijnstof (PM10) en de concentratie van ammoniak.
    Host immunostimulation and substrate utilization of the gut symbiont Akkermansia muciniphila
    Ottman, N.A. - \ 2015
    Wageningen University. Promotor(en): Willem de Vos; Hauke Smidt, co-promotor(en): Clara Belzer. - Wageningen : Wageningen University - ISBN 9789462574564 - 208
    akkermansia muciniphila - akkermansia - microbiota van het spijsverteringskanaal - darmmicro-organismen - probiotica - immunostimulerende eigenschappen - immunostimulerende middelen - moedermelk - metabolische studies - akkermansia muciniphila - akkermansia - gastrointestinal microbiota - intestinal microorganisms - probiotics - immunostimulatory properties - immunostimulants - human milk - metabolic studies

    Host immunostimulation and substrate utilization of the gut symbiont Akkermansia muciniphila

    Noora A. Ottman

    The human gastrointestinal tract is colonized by a complex community of micro-organisms, the gut microbiota. The majority of these are bacteria, which perform various functions involved in host energy metabolism and immune system stimulation. The field of gut microbiology is continuously expanding as novel species are isolated and high-throughput techniques are developed. The research focus is shifting from DNA-based techniques, looking at microbial community composition, to techniques relying on analysis of RNA and proteins, which reveal more about the activity and functionality of the microbiota.

    The mucosa-associated microbiota forms a distinct population in the gut, and is influenced by the close proximity of the epithelial layer and nutrients present in the mucus layer. One of the key players in this community is the mucus degrader Akkermansia muciniphila. This Gram-negative, anaerobic bacterium can use mucin, the main component of mucus, as the sole carbon and nitrogen source for growth. A. muciniphila belongs to the phylum Verrucomicrobia and is present in the majority of humans, starting from early life. Interestingly, the levels of A. muciniphila are negatively correlated with several disorders, including inflammatory bowel diseases and diabetes. A. muciniphila lives in a symbiosis with its host, harvesting energy from mucin; whether the relationship is mutualistic, and thereby also beneficial to the host, remains to be discovered. In this thesis, the ability of A. muciniphila to utilize the host-derived glycans mucin and human milk oligosaccharides was studied in detail. In addition, the host-bacterial interactions were examined by immunological assays, focusing especially on the effect of A. muciniphila outer membrane proteins on host immune response.

    The genome of A. muciniphila encodes numerous enzymes involved in mucin degradation. Transcriptome analysis comparing the gene expression of A. muciniphila grown on mucin or the non-mucin sugar glucose confirmed the activity of these genes and revealed most of them to be upregulated in the presence of mucin. This was also confirmed by a proteome analysis, reinforcing the adaptation of A. muciniphila to the mucosal environment. A genome-based metabolic model was constructed to test amino acid auxotrophy, vitamin biosynthesis, and sugar-degrading capacities of A. muciniphila. The model predicted A. muciniphila to be able to synthesize all the essential amino acids, with the exception of threonine, which was added to the mucin-free medium designed to test A. muciniphila growth on single sugars. A. muciniphila was able to individually metabolize all the main monomeric sugars present in mucin, albeit with limited efficiency in comparison to mucin. As mucin shares structural similarities with human milk oligosaccharides (HMOs), which stimulate the bacterial community colonizing the gut in early life, growth of A. muciniphila on human milk and its components was tested. A. muciniphila showed metabolic activity on human milk and one of the HMOs, 2’-fucosyllactose. Comparison of A. muciniphila activity during growth on human milk or mucin revealed that the expression of genes involved in mucin degradation was similar for both experimental conditions, suggesting that A. muciniphila might be capable of also using the corresponding gene products for utilization of human milk glycans. The capacity to survive in the early life environment by degrading and consuming human milk components would be beneficial for A. muciniphila during initial colonization before reaching the mucosal layer in the intestine.

    Several mouse studies have indicated that A. muciniphila is able to modulate the host immune system, possibly to the benefit of the host, but not much is known about its immunological mechanism of action. The cell envelope structures of bacteria can have a big influence on their immunostimulatory capacities, and therefore the outer membrane (OM) proteome of A. muciniphila was characterized. The membrane structure of A. muciniphila is also of interest because it belongs to the Planctomycetes-Verrucomicrobia-Chlamydiae superphylum, which contains bacteria with features that differentiate them from classical Gram-negative bacteria, including a complex endomembrane system. Mass spectrometry data, coupled with bioinformatics analysis, revealed the presence of highly abundant OM proteins involved in secretion, transport and biogenesis of the Gram-negative membranes, as well as proteins predicted to take part in formation of the fimbriae-like structures observed in A. muciniphila by electron microscopy. Live A. muciniphila and the identified OM proteins induced production of a wide range of cytokines and activated the intestinal Toll-like receptors 2 and 4. Moreover, a 30 kDa protein that was predicted to form a part of the fimbriae, increased transepithelial resistance, indicating it may be involved in improving gut barrier function.

    Based on the evidence from in vitro and in vivo studies, A. muciniphila is a promising candidate for a next-generation probiotic. However, further confirmation of causal relationships between disease development and presence of this species in the gut is required. The findings of this thesis provide valuable insight into the bacterial lifestyle and host interactions of the gut symbiont A. muciniphila.

    Simulating Welfare Effects of Europe’s Nutrition and Health Claims regulation: the Italian Yogurt Market
    Bonanno, A. ; Huang, R. ; Liu, Y. - \ 2015
    European Review of Agricultural Economics 42 (2015)3. - ISSN 0165-1587 - p. 499 - 533.
    discrete-choice models - product differentiation - functional foods - empirical-analysis - demand - information - quality - price - probiotics - valuation
    With the enactment of Regulation (EC) No. 1924/2006, 20 December 2006, ‘On nutrition and health claims made on foods’ several health claims can no longer be used on food products in European markets. We simulate the overall impact of the regulation on consumers and producers using the Italian yogurt market as a case study, and data prior to the introduction of the policy. We quantify welfare losses incurred if accepted claims were false, and simulate scenarios where rejected truthful health claims are removed, considering also the case where the products carrying them exit the market. We find that consumers can incur large welfare losses if approved claims are untruthful; if truthful claims are instead denied both consumers and producers may incur losses, with consumers being penalised more than producers.
    The genus Romboutsia : genomic and functional characterization of novel bacteria dedicated to life in the intestinal tract
    Gerritsen, J. - \ 2015
    Wageningen University. Promotor(en): Hauke Smidt; Willem de Vos, co-promotor(en): G.T. Rijkers. - Wageningen : Wageningen University - ISBN 9789462572423 - 280
    darmmicro-organismen - voeding en gezondheid - microbiota van het spijsverteringskanaal - darmziekten - moleculaire technieken - probiotica - intestinal microorganisms - nutrition and health - gastrointestinal microbiota - intestinal diseases - molecular techniques - probiotics
    The genus Romboutsia: genomic and functional characterization of novel bacteria dedicated to life in the intestinal tract

    PhD thesis Jacoline Gerritsen, 2015


    Humans, like other mammals, are not single-species organisms, but they constitute in fact very complex ecosystems. The extensive network of host-microbe and microbe-microbe interactions is tremendously important for our health, and we are just starting to unravel the mechanisms by which microbes contribute to host health and disease.

    Especially the intestinal tract of both humans and mammals contains an enormous diversity of microbial species of which many still remain to be cultured and characterized. There are numerous diseases for which aberrations in composition and diversity of the intestinal microbiota have been reported. Probiotic microorganism defined as “live microorganisms that, when administered in adequate amounts, confer a health benefit on the host” have the potential to modulate the intestinal microbiota and thereby contribute to health and well-being. To this end, the relative abundance of a specific bacterial phylotype, named CRIB, was found to be associated with probiotic-induced changes in gut microbiota and decreased severity of pancreatitis and associated sepsis in an experimental rat model for acute pancreatitis studies. Later, a representative of this phylotype (strain CRIB) was isolated, and characterized using a polyphasic taxonomic approach. The taxonomy of several closely related members of the family Peptostreptococcaceae was revised in order to provide a valid systematic name to the isolate, for which Romboutsia ilealis was chosen. It was found that the majority of Romboutsia-associated 16S rRNA gene sequences have an intestinal origin, however, the specific roles that Romboutsia species play in the intestinal tract are largely unknown. To gain more insight in metabolic and functional capabilities of members of the genus Romboutsia, efforts towards the isolation of additional representatives were undertaken. This ultimately led to the isolation of a human small intestine-derived representative (strain FRIFI) of another novel Romboutsia species which was given the name R. hominis. Characterization of both novel species of intestinal origin, i.e. R. ilealis and R. hominis, belonging to the genus Romboutsia at the genomic and functional level provided first insights into the genetic diversity within the genus Romboutsia and their adaptation to a life in the (upper) intestinal tract. To this end, Romboutsia species are flexible anaerobes that are adapted to a nutrient-rich environment in which carbohydrates and exogenous sources of amino acids and vitamins are abundantly available.

    Microbiomic approaches such as those employed in this study can be used to pinpoint specific commensal microbes that might have a beneficial effect on the health of the host. In addition, the combination of genomic and functional analyses with single organisms and complex communities can be used to identify microbial functionalities that are related to health and disease, which in turn can be used to select potential probiotic strains based on specific functional properties. Ultimately, these approaches will lead to the characterization of (new) beneficial commensal microbes that exert health-promoting effects, with the ultimate possibility for them to be exploited as next-generation probiotics.

    Genomic Characterization of Non-Mucus Adherent Derivatives of Lactobacillus rhamnosus GG Reveals Genes Affecting Pilus Biogenesis
    Rasinkangas, P. ; Reunanen, J. ; Douillard, F.P. ; Ritari, J. ; Uotinen, V. ; Palva, A. ; Vos, W.M. de - \ 2014
    Applied and Environmental Microbiology 80 (2014)22. - ISSN 0099-2240 - p. 7001 - 7009.
    intestinal epithelial-cells - placebo-controlled trial - gram-positive bacteria - functional-analysis - atopic disease - strain gg - adhesion - probiotics - surface - prevention
    Lactobacillus rhamnosus GG is one of the best-characterized lactic acid bacteria and can be considered a probiotic paradigm. Comparative and functional genome analysis showed that L. rhamnosus GG harbors a genomic island including the spaCBA-srtC1 gene cluster, encoding the cell surface-decorating host-interacting pili. Here, induced mutagenesis was used to study pilus biogenesis in L. rhamnosus GG. A combination of two powerful approaches, mutation selection and next-generation sequencing, was applied to L. rhamnosus GG for the selection of pilus-deficient mutants from an enriched population. The isolated mutants were first screened by immuno-dot blot analysis using antiserum against pilin proteins. Relevant mutants were selected, and the lack of pili was confirmed by immunoelectron microscopy. The pilosotype of 10 mutant strains was further characterized by analyzing pilin expression using Western blot, dot blot, and immunofluorescence methods. A mucus binding assay showed that the mutants did not adhere to porcine intestinal mucus. Comparative genome sequence analysis using the Illumina MiSeq platform allowed us to determine the nature of the mutations in the obtained pilus-deficient derivatives. Three major classes of mutants with unique genotypes were observed: class I, with mutations in the srtC1 gene; class II, with a deletion containing the spaCBA-srtC1 gene cluster; and class III, with mutations in the spaA gene. Only a limited number of collateral mutations were observed, and one of the pilus-deficient derivatives with a deficient srtC1 gene contained 24 other mutations. This strain, PB12, can be considered a candidate for human trials addressing the impact of the absence of pili.
    Simultaneous growth and metabolite production by yoghurt starters and probiotics: a metabolomics approach
    Settachaimongkon, S. - \ 2014
    Wageningen University. Promotor(en): Toon van Hooijdonk; Marcel Zwietering, co-promotor(en): Eddy Smid. - Wageningen : Wageningen University - ISBN 9789462570115 - 213
    yoghurt - melk starterculturen - probiotica - co-vergisting - metabolieten - yoghurt - cultured milk starters - probiotics - co-fermentation - metabolites

    The main objective of this research was to investigate the simultaneous growth and metabolite production by yoghurt starters and different probiotic strains, i.e. Lactobacillus rhamnosus GG, Bifidobacterium animalis subsp. lactis BB12 and Lactobacillus plantarum WCFS1, during set-yoghurt fermentation and refrigerated storage. In this context, the microbial activity was evaluated in terms of bacterial population dynamics, milk acidification and formation of volatile and non-volatile metabolites in set-yoghurt. A complementary metabolomics approach using headspace SPME-GC/MS and 1H-NMR was applied for characterization of biochemical changes associated with the microbial metabolism during fermentation and storage. The results revealed that incorporation of the three probiotic strains did not significantly influence the acidity and concentrations of key-aroma volatile compounds of set-yoghurt. Still, the presence of probiotics substantially contributed to the formation of a large number of volatile and non-volatile metabolites detected at low concentration. Because many probiotic strains are not able to survive well in fermented milk, a strategy to enhance their survival was additionally applied by preculturing the three probiotic strains under sublethal salt and low pH stress conditions prior to inoculation in milk. The results revealed an improved survival of L. rhamnosus GG and B. animalis subsp. lactis BB12, specifically by preculturing at relatively low pH conditions. Moreover, incorporation of sublethally precultured L. plantarum WCFS1 significantly impaired the survival of L. delbrueckii subsp. bulgaricus, which consequently reduced the post-acidification of yoghurt. Metabolomics analyses revealed that the presence of stress-adapted probiotics induced significant changes in the overall metabolite profile of yoghurt. This finding is important, since variations in the relative abundance of various organic acids, aroma volatiles and proteolytic-derived compounds may directly influence the organoleptic quality of product. Finally, multivariate analysis enabled to distinguish yoghurts fermented by different types of starter combinations and different durations of storage according to their metabolite profiles. This research provides new information regarding the impact of probiotics on the metabolome of yoghurt and potential application of stress-adapted probiotics in an actual food-carrier environment.

    Triggered release kinetics of living cells from composite microcapsules
    Hamad, S.A. ; Stoyanov, S.D. ; Paunov, V.N. - \ 2013
    Physical Chemistry Chemical Physics 15 (2013)7. - ISSN 1463-9076 - p. 2337 - 2344.
    micro-rods - shellac - fabrication - probiotics
    We have developed a theoretical model for the kinetics of release of living cells from composite shellac-cell microcapsules. The model describes the kinetics of cell release from the microcapsules triggered by: (i) pH change, which dissolves the shellac and (ii) the growth of the encapsulated cells, when placed in culture media. For pH triggered release of cells from the composite microcapsules, the rate constant of cell release depends on the swelling/dissolution rate of the shellac matrix and varies with the pH of the aqueous media. The model links the microcapsules disintegration time with the cell release rate constant. For growth triggered release of cells from the composite microcapsules, the cell release rate constant depends on concentration of nutrients in the culture media and the volume fraction of cells in the microcapsules. In a complementary experimental study we compare the release rate constants of cells from shellac-cell microcapsules at different values of pH in the aqueous media. This study may allow fine-tuning of the rate of cell release in a variety of encapsulated cell products, including cell implants, probiotics, and live vaccines.
    Dehydration and thermal inactivation of Lactobacillus plantarum WCFS1: Comparing single droplet drying to spray and freeze drying
    Perdana, J.A. ; Bereschenko, L.A. ; Fox, M.B. ; Kuperus, J.H. ; Kleerebezem, M. ; Boom, R.M. ; Schutyser, M.A.I. - \ 2013
    Food Research International 54 (2013)2. - ISSN 0963-9969 - p. 1351 - 1359.
    desiccation tolerance - phase-transitions - probiotics - temperature - membranes - vitrification - survival - bacteria - injury - cells
    We demonstrated that viability loss during single droplet drying can be explained by the sum of dehydration and thermal inactivation. For Lactobacillus plantarum WCFS1, dehydration inactivation predominantly occurred at drying temperatures below 45 °C and only depended on the moisture content. Above 45 °C the inactivation was due to a combination of dehydration and thermal inactivation, which depended on the moisture content, temperature, and drying time. A Weibull model was successfully applied to describe the thermal and dehydration inactivation and enabled the prediction of residual viability of L. plantarum WCFS1 after single droplet drying. Subsequently, the model was evaluated to predict the viability loss during laboratory scale spray drying, showing a remarkable agreement if assumed that only thermal inactivation occurred. This indicated that very high drying rates in laboratory scale spray drying could induce instant fixation of the cell suspensions in a vitrified matrix and thereby preventing dehydration inactivation. Finally, the influence of drying rate on remaining viability was evaluated by comparing single droplet drying, freeze drying and laboratory scale spray drying of the same bacterial suspension. It was shown that slow drying leads to large dehydration inactivation, which diminished in fast drying processes such as laboratory scale spray drying where thermal inactivation appears to be the predominant mechanism of inactivation.
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