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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 - 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 p.
lactobacillus plantarum - molecules - probiotics - immunomodulatory properties - lipoproteins - interactions - molecular interactions - host pathogen interactions - 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 p.
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 - 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 - raw milk - goat milk - 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 - 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.
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.
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 - 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.
Single droplet analysis for spray drying of foods
Perdana, J.A. - \ 2013
Wageningen University. Promotor(en): Remko Boom, co-promotor(en): Maarten Schutyser; M.B. Fox. - S.l. : s.n. - ISBN 9789461735874 - 254
druppels - druppelgrootte - sproeidroging - probiotica - lactobacillus plantarum - modelleren - hitteresistentie - droplets - droplet size - spray drying - probiotics - modeling - heat resistance
Many food ingredients, such as enzymes and probiotics, are spray dried to provide shelf-life. Major hurdle to apply spray drying is the lack of scientific insight on the inactivation mechanisms of components and the extensive optimization required for formulation and drying conditions to obtain powders of acceptable quality. This thesis reports on the development of an alternative approach to study drying behaviour involving single droplet experimentation in combination with predictive modelling. During single droplet drying, conditions such as drying air temperature, drying time, air flow rate, and particle size could be well controlled, which enabled systematic investigation of inactivation kinetics of heat sensitive components. Experiments were carried out with the heat sensitive enzyme (b-galactosidase) and the probiotic microorganism (Lactobacillus plantarum WCFS1). A novel viability enumeration method was developed to measure survival of probiotic bacteria, involving rehydration of (semi-) dried particles on a micro-porous chip in combination with fluorescence-based viability enumeration. The drying history of single droplets was modelled using an effective diffusion model. The diffusivity in this model was measured and modelled as function of moisture content and temperature on the basis of gravimetric analysis of thin film drying in a dynamic vapour sorption analyser (DVS). Subsequently, models were developed to describe inactivation kinetics as function of particle drying history. Finally, the obtained insight and approach were validated by comparison of predictions to results obtained during lab-scale spray drying experiments. Moreover, the single droplet approach can be used as a tool for screening optimal spray drying conditions
Molecular analysis of candidate probiotic effector molecules of Lactobacillus plantarum
Remus, D.M. - \ 2012
Wageningen University. Promotor(en): Michiel Kleerebezem, co-promotor(en): P.A. Bron. - S.l. : s.n. - ISBN 9789461733733 - 187
lactobacillus plantarum - probiotica - genomica - gastheer-pathogeen interacties - probiotics - genomics - host pathogen interactions
Probiotics are health-promoting microorganisms that exert their beneficial effects in several ways. While it is known that probiotic bacteria interact with cells of the host gastrointestinal tractand modulate cell-signaling responses by which they might promote health, the underlying molecular mechanism and probiotic “effector” molecules that are responsible for these effects remain largely unexplored. In this thesis, effector molecules of the lactic acid bacterium Lactobacillus plantarum were discovered that interact with diverse host cell types and manipulate cell-associated signaling pathways. This work represents crucial steps to better understand the exact mode of probiotic action, which is a prerequisite for their controlled, safe, purpose-directed, and person-specific applications in the context of health improvement and disease prevention.
Probiotics, calcium and acute diarrhea : a randomized trial in Indonesian children
Agustina, R. - \ 2012
Wageningen University. Promotor(en): Frans Kok; A. Firmansyah, co-promotor(en): I.M.J. Bovee-Oudenhoven. - S.l. : s.n. - ISBN 9789461733726 - 174
probiotica - calcium - diarree - ademhalingsziekten - peuters en kleuters - indonesië - voedingstoestand - probiotics - diarrhoea - respiratory diseases - preschool children - indonesia - nutritional state
Interactions of lactobacilli with the host immune system
Meijerink, M. - \ 2011
Wageningen University. Promotor(en): Jerry Wells; Huub Savelkoul, co-promotor(en): J. Bilsen. - [S.l.] : S.n. - ISBN 9789461730442 - 223
probiotica - immunomodulerende eigenschappen - lactobacillus plantarum - spijsverteringskanaal - voedselallergieën - vaccinatie - probiotics - immunomodulatory properties - digestive tract - food allergies - vaccination
The aim of this thesis was to better understand the molecular mechanism of host res-ponses to probiotics. Probiotics can be used to stimulate or regulate immune responses in epithelial and immune cells of the intestinal mucosa and generate beneficial effects on the immune system. Carefully selected probiotics are able to steer the activity of the immune response in a predetermined manner by increasing or decreasing the activity of different aspects of the immune system (e.g. development and activity of T helper subsets). Beneficial effects of 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. Here in vitro molecular studies and in vivo mechanistic studies were combined in different mouse models to generate new insights into the beneficial mechanisms of selected lactobacilli and identify novel bacterial genes influencing the immune response. A further aim was to investigate the predictive value of in vitro immune assays for the effects of probiotics in vivo.
Chapter 1and chapter 2 describe the current knowledge and understanding of the immunomodulatory effects of different probiotic species and strains on mucosal immune system, dendritic cells (DCs) and the adaptive immune system. The relevance and the implications of in vitro studies for clinical trials or mechanistic research in animal mo-dels are discussed.
Chapter 3and chapter 4 present new insights gained from research on the strain-dependent factors involved in probiotic immune modulation. Extensive variation was observed in the immune responses to 42 L. plantarum strains. These results were used to identify genetic loci that correlated with levels of induced cytokines (such as IL-10 or IL-12) following co-culture with DCs (chapter 3) or peripheral blood mononuclear cells (PBMCs) (chapter 4). This in silico “gene-trait matching” approach led to the identification of several candidate genes in the L. plantarum genome that might modulate the immune cytokine response to L. plantarum. Selective gene deletions mutants were constructed for the candidate genes in L. plantarum WCFS1 and compared to the wild-type strain in immune assays with PBMCs and DCs. The predicted phenotype of the genetic knock-out was confirmed for most of the candidate loci including genes encoding an N-acetyl-glucosamine/galactosamine phosphotransferase system, the LamBDCA quorum sensing system, a predicted transcriptional regulator gene (lp_2991) and components of the plantaricin (bacteriocin) biosynthesis and transport pathway. Transcriptome analysis and qPCR data showed that transcript level of gtcA3, which is predicted to be involved in the glycosylation of cell wall teichoic acids, was substantially increased in the lp_2991 deletion mutant (44- and 29-fold respectively).
In vitroassays for pre-screening of candidate probiotics would benefit from standar-dized methods and cryopreservation techniques for immature DCs (iDCs) or precursor monocytes. Literature on the effects of cryopreservation and thawing of monocytes or monocyte-derived iDCs suggested that this strategy might be useful although bacteria had not been previously used as a stimulus. Thus in chapter 5 we investigated the effects of cryopreservation and thawing of precursor monocytes and iDCs on the maturation and immune response of DCs to potential probiotic strains and bacterial TLR agonists. Surface markers CD83 and CD86 were expressed at similar levels on iDCs generated from cryopreserved or freshly isolated monocytes. Cryopreservation of iDCs led to slightly decreased expression of CD86 and CD83 compared to freshly generated iDCs prepared from unfrozen cells but this did not affect the capacity of DCs to acquire fully mature characteristics after stimulation. In contrast the cytokine response to lipoteichoic acid and bacterial stimulation was altered by cryopreservation of monocytes or iDCs, particularly for IL-12 which was decreased up to 250 fold or even not detected at all. Cryopreservation also decreased TNF-α and IL-1β production in stimulated iDCs but to a lesser extent than for IL-12, depending on the maturation factors used. The amounts of IL-10 produced by stimulated iDCs were increased up to 3.6 fold when iDCs were cryopreserved, but decreased up to 90 fold when generated from cryopreserved monocytes. Immature DCs are often used to investigate the immunomodulatory properties of probiotics and here we showed for the first time that cryopreserved monocytes and cryopreserved iDCs have a skewed cytokine response to microbial stimulation. Therefore we consider that standardization of probiotic screening assays by the use of cryopreservation methods is currently not applicable. The detailed method for generating human monocyte derived DC described in chapter 5 may however be useful for developing standardized immune assays.
In chapter 6 we screened the immunomodulatory properties of 28 commercially available bacterial strains in vitro using human PBMCs and investigated selected strains for their in vivo immunomodulatory potential in an established mouse peanut allergy model. The 28 probiotic strains induced highly variable cytokine profiles in PBMCs. L. salivarius HMI001 (HMI001), L. casei Shirota (LCS) and L. plantarum WCFS1 (WCFS1) were selected for further investigation due to their distinct patterns of IL-10, IL-12 and IFN-γ induction. Prophylactic treatment with both HMI001 and LCS attenuated the Th2 phenotype in the mouse model (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 the case of HMI001 (inducing a high IL-10/IL-12 ratio) and LCS (inducing high amounts of IFN-γ and IL-12) partial protection was seen in a mouse peanut allergy model. However, certain strains may worsen the allergic reaction as shown in the case of WCFS1. This approach indicated that pre-selection of candidate probiotics using in vitro immune assays is useful for selecting strains for translational research in humans.
Probiotics have been shown to increase the efficacy of different vaccines and can be easily consumed in food, and therefore probiotics might be useful in the improvement of current mucosal vaccines. In chapter 7 we have investigated the mechanisms behind the effect of lactobacilli on humoral responses to an intranasal vaccine. In addition to L. rhamnosus GG we selected 6 strains of Lactobacillus plantarum which have strikingly different immunomodulatory properties in vitro and TLR-2/6 activating properties. This selection was based on the approach outlined in chapter 3 and chapter 4 examining the in vitro immune responses of human monocyte derived DCs and PBMCs to 42 different L. plantarum strains. First we established an influenza vaccination model in Balb/c mice that would be sensitive to immunomodulation by lactobacilli, which allowed potential up- and down-regulation by the lactobacilli of the immune response. Strain WCFS1, that induced the lowest IL-10 to IL-12 cytokine ratio in DC co-culture significantly increased vaccine-specific antibody responses to the intranasal vaccine compared to the vaccine control group. Several Lactobacillus strains appeared to increase delayed-type hypersensitivity responses after vaccination compared to the vaccine control group indicating increased Th1-mediated vaccine responses. For strain LMG18021 this was also reflected in the significantly higher vaccine-specific IgG2a to IgG1 antibody ratio. LMG18021, CIP104448 and CIP104450 which have the highest IL-10 to IL-12 ratios of the strains tested, significantly enhanced the ex vivo vaccine-specific induction of IL-10, IL-17A, IL-6 and IL-4 in MLN cells. B1839 which was included as negative control, as it was a low cytokine inducer, did not enhance the vaccine-specific antibody or immune response indicating that the immune-stimulatory properties are important in mediating effects on the vaccine response. Further research is needed to demonstrate that these effects on the vaccine response impact on protection from influenza challenge and to validate the immunomodulatory mechanisms involved. Nevertheless, the in vivo studies described in this thesis support other publications proposing that in vitro immune assays can be useful for predicting which candidate probiotic strains will be most effective in vivo.
Chapter 8 completes this thesis with an overview of the most important findings of this thesis and discusses possible research limitations and future research perspectives. We stress the importance of proper strain selection using in vitro assays, and the use of strategies to identify novel immunomodulatory factors. The results described in this thesis support the rationale of using in vitro co-culture assays for selection of candidate probiotics for in vivo animal experiments or human trials.
Het effect van pre- en probiota op de groei en gezondheid van geitenlammeren
Dinteren, L. van; Haaij, A. de; Louis Bolk, - \ 2008
Driebergen : Louis Bolk Instituut (Biogeit rapport / Louis Bolk Instituut 14) - 41 p.
dierhouderij - biologische landbouw - geitenhouderij - diergezondheid - probiotica - coccidiose - geitenlamproductie - geiten - prebiotica - dierziektepreventie - animal husbandry - organic farming - goat keeping - animal health - probiotics - coccidiosis - kid production - goats - prebiotics - animal disease prevention
In de biologische dierhouderij is het gebruik van geneesmiddelen beperkt toegestaan. Daarom zijn dierziektepreventie en het op een zo natuurlijk mogelijke wijze de diergezondheid op peil houden belangrijke uitgangspunten. Het toedienen van weerstandsverhogende middelen (o.a. pre- en probiotica) past hier goed bij. Een veel voorkomend ziekteprobleem bij geitenlammeren is coccidiose rondom het spenen. Het inzetten van pre- en probiotica bij geitenlammeren in de melkperiode zou coccidiose beheersbaar kunnen maken en daarbij een positief effect kunnen hebben op de groei en de algemene gezondheid van geitenlammeren. In dit praktijkonderzoek kregen drie groepen geitenlammeren verschillende behandelingen, voornamelijk in de melkperiode. De weerstandverhogende middelen hadden niet het verwachte effect op groei en gezondheid van geitenlammeren.
Mannose-specific interactions of Lactobacillus plantarum in the intestine : bacterial genes, molecular host responses and potential probiotic effects
Pretzer, G. - \ 2008
Wageningen University. Promotor(en): Mari Smits, co-promotor(en): T.A. Niewold; J. Snel. - S.l. : S.n. - ISBN 9789085049661 - 176
lactobacillus plantarum - darmmicro-organismen - darmen - mannose - probiotica - relaties tussen gastheer en gast - genetica - intestinal microorganisms - intestines - probiotics - host guest relations - genetics
One potential mechanism by which probiotic microorganisms may exert beneficial health effects to the host is the inhibition of intestinal infections by competitive exclusion of pathogenic bacteria. This concept may also be applicable for mannose-specific adhesion to the epithelial surface, which has been demonstrated for several pathogens as well as for the probiotic Lactobacillus plantarum. In this thesis, a complementary approach was performed to focus on mannose-specific host-microbe interactions of L. plantarum in the intestine. Firstly, the mannose-specific adhesin encoding gene (msa) of L. plantarum was identified using an in vitro biodiversity-based approach including genotype-phenotype matching and subsequent mutagenesis analysis. The domain composition of the encoded protein Msa was further characterized in selected L. plantarum strains, revealing strain-specific variations in domain composition. Secondly, intestinal host responses as reflected in differential gene expression patterns induced by L. plantarum 299v wildtype and its isogenic msa-deletion derivative were investigated using DNA microarrays. In an in situ Small Intestinal Segment Perfusion model in pigs, increased mRNA expression of pancreatitis-associated protein, a protein with antibacterial activity, was caused by the wildtype strain only. In vivo, transcriptome profiles induced by oral administration of L. plantarum 299v wildtype and the msa-mutant strain were found to be different in pigs, corroborating a role for Msa in host-microbe interactions. Finally, potential probiotic effects of L. plantarum 299v wildtype strain based on competitive exclusion of pathogens at mannose-specific binding sites on the intestinal epithelium were studied in different animal models. However, these studies did not provide clear evidence on measurable probiotic effects related to mannose adhesion. Apparently, mannose-specific interactions only play a limited role in competitive exclusion mechanisms and total persistence properties of L. plantarum in the gut. Nevertheless, since Msa was found to induce particular host responses in epithelial cells this protein seems to be one of the factors that mediate part of the complex host-bacterial interplay in the intestine by facilitating temporary contact with the epithelium. This thesis contributes to multi-dimensional investigations of host-probiotic interactions on a molecular level, which are needed for a better understanding of probiotic modes of action and an improved future development of next generation probiotics with targeted and optimized functionality for particular health benefits.
Managing the developing gut microbiota of growing piglets - novel probiotic and prebiotic strategies
Yao, W. - \ 2008
Wageningen University. Promotor(en): Martin Verstegen, co-promotor(en): Hauke Smidt; W.Y. Zhu. - [S.l.] : S.n. - ISBN 9789085048169 - 168
biggen - spenen - microbiële flora - darmen - spijsverteringskanaal - selectieve bijvoedering - probiotica - polymerase-kettingreactie - voedingsfysiologie - microbiota van het spijsverteringskanaal - prebiotica - piglets - weaning - microbial flora - intestines - digestive tract - creep feeding - probiotics - polymerase chain reaction - nutrition physiology - gastrointestinal microbiota - prebiotics
Keywords: creep feeding, weaning, intestinal microbiota, Lactobacillus sobrius, prebiotics, daidzein, probiotics, 16S rRNA, DGGE, cloning, real-time PCR
Nursing is a major critical period in the life of piglets. On one hand maternal antibodies are not able to cross the placenta, thus piglets are born without circulating antibodies and consequently lack maternal passive protection. On the other hand, creep-feeding and weaning increase susceptibility to gut disorders, infections and diarrhea. Therefore clarification of the composition and function of the normal gut microbiota of piglets is pivotal as a knowledge base for the design of innovative nutritional strategies based on pre- and probiotics to keep piglets healthy. The objectives of this study were to describe the composition and function of the intestinal microbiota of piglets during the nursing period through creep feeding and weaning, to in vivo and in vitro evaluate the effect of daidzein on composition and function of intestinal microbiota of nursing piglets in order to evaluate its prebiotic function, and to investigate the probiotic effect of Lactobacillus sobrius S1 on composition and function of intestinal microbiota of nursing piglets.
The porcine intestinal microbiota development and diversity, the in vivo and in vitro evaluation of prebiotic effect of daidzein and the investigation of probiotic effect of Lactobacillus sobrius S1 on composition and function of intestinal microbiota of piglets during nursing period through creep feeding and weaning was described using real-time PCR, and PCR analysis of 16S ribosomal RNA gene by denaturing gradient gel electrophoresis (DGGE) and cloning, in combination with analysis of gas production, lactate and VFA yield. The data obtained during the course of the study indicated that 1) Early creep-feeding stabilizes the microbiota of piglets around the weaning period. 2) Lactobacillus communities follow a successional change associated with piglet growth and diet shifting. Creep feeding stabilizes the Lactobacillus community of weaning piglets. Within the Lactobacillus community, some members like L. reuteri and L. amylovorus / L. sobrius might be permanent colonizers, while L. delbruckii, L. acidophilus and L. crispatus are more likely to be transient members of the Lactobacillus communities in the piglet´s GI tract. 3) Both in vitro and in vivo evaluations indicated that daidzein has the potential for use as a prebiotic additive in animal feed. 4) Lactobacillus sobrius S1 has the potential of promoting beneficial bacteria and inhibiting pathogens.
Kringloop als basis van bedrijfsvoering
Linde, A. van der - \ 2007
Boerderij/Veehouderij 92 (2007)15. - p. 16 - 17.
melkveehouderij - diergezondheid - alternatieve landbouw - kringlopen - probiotica - immuunsysteem - immuniteit - bedrijfssystemen - prebiotica - dairy farming - animal health - alternative farming - cycling - probiotics - immune system - immunity - farming systems - prebiotics
Gerard Keurentjes bekijkt zijn bedrijf als kringloop waarin grond en koeien de spil zijn. Het gebruik van microbiotica in plaats van antibiotica past daarin perfect. Een microbioticum bestaat uit een mengsel van acht kruiden opgelost in alcohol. Onderzoek toont aan dat het de productie van witte bloedcellen stimuleert en het immuunsysteem 6 keer beter gaat werken. De verhoogde weerstand van de koeien zijn een van de resultaten van het FIR-systeem dat de boer nu 5 jaar gebruikt
|Multispecies probiotics - composition and functionality
Timmerman, H.M. - \ 2006
Utrecht University. Promotor(en): A.C. Beynen; Frans Rombouts; L.M.A. Akkermans, co-promotor(en): G.T. Rijkers. - Utrecht : s.n. - ISBN 903934129X - 320
probiotica - dissertaties - probiotics - theses
AMGB's en coccidiostatica in pluimveevoeders: zijn er goede en veilige alternatieve toevoegingsmiddelen
Ban, E.C.D. van den; Aarts, H.J.M. ; Bokma-Bakker, M.H. ; Bouwmeester, H. ; Jansman, A.J.M. - \ 2005
Wageningen : Animal Sciences Group (Rapport nutrition and food / Animal Sciences Group 05/100649) - 61
anti-infectieuze middelen - coccidiostatica - pluimveevoeding - voedertoevoegingen - probiotica - diergezondheid - voedselveiligheid - antiinfective agents - coccidiostats - poultry feeding - feed additives - probiotics - animal health - food safety
De EU stelt een volledig verbod in op het gebruik van antimicrobiële groeibevorderaars (AMGB’s) als toevoegingsmiddel in diervoeders per 1 januari 2006. Daarnaast beoogt de EU een totaalverbod (uitfasering) op coccidiostatica (en histomonostatica) als toevoegingsmiddel voor 31 december 2012. Vanwege het aanstaande verbod op AMGB's wordt gezocht naar alternatieven om de verwachte (negatieve) effecten van het verbod op de productiviteit en gezondheid van landbouwhuisdieren tegen te gaan. Er is echter tot nu toe relatief weinig aandacht besteed aan de eventuele risico's van het gebruik van alternatieve additieven ten aanzien van volksgezondheid, voedselveiligheid, diergezondheid en productiviteit. Dit rapport richt zich op de veiligheid van alternatieve toevoegingsmiddelen voor AMGB’s en coccidiostatica in pluimveevoeders.
Effects of prebiotics, probiotics and synbiotics in the diet of young pigs
Shim, S.B. - \ 2005
Wageningen University. Promotor(en): Martin Verstegen, co-promotor(en): J.M.A.J. Verdonk. - s.l. : S.n. - ISBN 9085041937 - 178
biggen - probiotica - koolhydraten - anti-infectieuze middelen - voedertoevoegingen - microbiële ecologie - fermentatie - spijsverteringsstelsel - varkensvoeding - voer - diergezondheid - groei - voedingsfysiologie - piglets - probiotics - carbohydrates - antiinfective agents - feed additives - microbial ecology - fermentation - digestive system - pig feeding - feeds - animal health - growth - nutrition physiology