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Metabolism of Caprine Milk Carbohydrates by Probiotic Bacteria and Caco-2:HT29⁻MTX Epithelial Co-Cultures and Their Impact on Intestinal Barrier Integrity
(MDPI (Basel, Switzerland), 2018-07-23) Barnett AM; Roy NC; Cookson AL; McNabb WC
The development and maturation of the neonatal intestine is generally influenced by diet and commensal bacteria, the composition of which, in turn, can be influenced by the diet. Colonisation of the neonatal intestine by probiotic Lactobacillus strains can strengthen, preserve, and improve barrier integrity, and adherence of probiotics to the intestinal epithelium can be influenced by the available carbon sources. The goal of the present study was to examine the role of probiotic lactobacilli strains alone or together with a carbohydrate fraction (CF) from caprine milk on barrier integrity of a co-culture model of the small intestinal epithelium. Barrier integrity (as measured by trans epithelial electrical resistance (TEER)), was enhanced by three bacteria/CF combinations (Lactobacillus rhamnosus HN001, L. plantarum 299v, and L. casei Shirota) to a greater extent than CF or bacteria alone. Levels of occludin mRNA were increased for all treatments compared to untreated co-cultures, and L. plantarum 299v in combination with CF had increased mRNA levels of MUC4, MUC2 and MUC5AC mucins and MUC4 protein abundance. These results indicate that three out of the four probiotic bacteria tested, in combination with CF, were able to elicit a greater increase in barrier integrity of a co-culture model of the small intestinal epithelium compared to that for either component alone. This study provides additional insight into the individual or combined roles of microbe⁻diet interactions in the small intestine and their beneficial contribution to the intestinal barrier.
Phenotypic and genotypic characterisation of Lactobacillus and yeast isolates from a traditional New Zealand Māori potato starter culture
(Elsevier BV, 2022-08-26) Sun J; Silander O; Rutherfurd-Markwick K; Wen D; Davy TP-P; Mutukumira AN
Parāroa Rēwena is a traditional Māori sourdough produced by fermentation using a potato starter culture. The microbial composition of the starter culture is not well characterised, despite the long history of this product. The morphological, physiological, biochemical and genetic tests were conducted to characterise 26 lactic acid bacteria (LAB) and 15 yeast isolates from a Parāroa Rēwena potato starter culture. The results of sugar fermentation tests, API 50 CHL tests, and API ID 32 C tests suggest the presence of four different LAB phenotypes and five different yeast phenotypes. 16S rRNA and 26S rRNA sequencing identified the LAB as Lacticaseibacillus paracasei and the yeast isolates as Saccharomyces cerevisiae, respectively. Multilocus sequence typing (MLST) of the L. paracasei isolates indicated that they had identical genotypes at the MLST loci, to L. paracasei subsp. paracasei IBB 3423 or L. paracasei subsp. paracasei F19. This study provides new insights into the microbial composition of the traditional sourdough Parāroa Rēwena starter culture.
Understanding the mechanism of action of the glycosylated bacteriocin glycocin F : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Manawatu, New Zealand
(Massey University, 2019) Bisset, Sean William
With the increasing threat posed by antibiotic-resistant bacteria, eﬀorts must be made to ﬁnd new antimicrobial agents. One growing area of promise is the bacteriocins, which are a diverse group of antimicrobial peptides produced by bacteria. This thesis focuses on determining the mechanism of action of one of these peptides, glycocin F (GccF). GccF is produced by the bacterium Lactobacillus plantarum and is modiﬁed with two N-acetyl glucosamine (GlcNAc) sugar moieties, one located on an interhelical loop region, and the other at the end of a ﬂexible C-terminal ‘tail’. It has also been shown to exhibit a unique eﬀect on susceptible bacteria, putting them into a reversible state of hybernation as opposed to outright killing them. However, little is known about the roles of the structural features of GccF, how it triggers bacteriostasis in target cells, or even what part(s) of bacterial cells it targets. This work addresses these questions using three main approaches: studying the structure-function relationship of diﬀerent parts of GccF with chemically synthesised analogues; looking at the transcriptional response of a pathogenic bacteria, Enterococcus faecalis, to GccF; and trying to identify binding partners of GccF and its respective immunity protein, GccH. The results presented here highlight diﬀerent roles of the GlcNAcs attached to GccF, with both the interhelical loop and presence of GlcNAc on this loop being vital for activity, while the sugar at the C-terminal position is important, but not crucial for the peptide’s activity. Additionally, a role of the GlcNAc phosphotransferase system on the mechanism of GccF is strongly indicated, with evidence from both the transcriptional studies and the protein interaction studies of GccF’s immunity protein. Taken together, the results allow for two theoretical models of GccF’s mechanism of action to be proposed. These models presented here should serve to increase the understanding of other glycocin-class bacteriocins and their mechanisms of action, and possibly contribute towards the creation of a blueprint for a new class of antimicrobial agents.
Studies on the stability of probiotic bacteria during long term storage : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Palmerston North, New Zealand
(Massey University, 2019) Nag, Arup
According to the Food and Agriculture Organization (FAO) and the World Health Organization (WHO), probiotics are defined as ‘‘live microorganisms which when administered in adequate amounts confer a health benefit for the host’’ (FAO/WHO, 2001). Lactobacilli and bifidobacteria are two major group of organisms considered to have probiotic properties. The primary objective of this project was to develop a novel stabilization technology for probiotic bacteria, through which a range of probiotic bacterial strains could potentially be delivered to the host through shelf stable dry and intermediate moisture foods. For preliminary experiments (reported in Chapter 4.0), Lactobacillus casei 431, a commercial strain from Chr Hansen, Denmark, was chosen as the experimental strain and milk powders (both skimmed and full-fat) were chosen as the principal supporting agent while stabilizing the bacterial cells. Stabilization efficiency in terms of long term ambient temperature storage viability was compared using freeze and fluidized bed drying techniques. Fluidized bed drying was able to retain 2.5 log cfu/g higher viability after 52 weeks of storage at 25 °C. A combination of fluidized bed drying and osmotic stress adaptation to the probiotic cells yielded further improvement of 0.83 log cfu/g higher viability compared to the unstressed cells. The findings were validated with other two lactobacilli and two bifidobacterium strains with probiotic characteristics and significant improvements in storage stability over freeze-dried samples were observed. Fortification of vitamin E in the stabilization matrix as an antioxidant improved the stability by 0.18 log cfu/g during 20 weeks storage period at 25 °C, whereas any similar benefit of fortifying inulin as a prebiotic was not observed. Incubation in simulated gastric fluid and intestinal fluid (in vitro) revealed that the L. casei 431 cells were better protected within the stabilized matrix than in the free form. The survival of the stabilized cells were 5.0 and 2.1 log cycles higher than free cells in gastric juice and bile salt solution respectively. Physical characterization of the probiotic ingredient showed very good flow-ability and solubility, with 470 Kg/m3 bulk density, water activity of 0.27 and agglomerated particles of 125.6 μm mean diameter. Thereafter, the project aimed to understand the underlying mechanism of the processes responsible for gradual decay in cell viability of another probiotic strain (Lactobacillus reuteri LR6) during long term storage at 37 °C (Chapter 5.0 onwards). Vacuum drying of sorbitol- or xylitol-coated Lactobacillus reuteri LR6 cells and fluidized bed drying of the same coated cells with different excipients were compared for the cell viability post drying. LR6 cells coated with xylitol and desiccated in unsupported form or together with skim milk powder as an excipient were found to be better protected when exposed to moderate as well as high drying temperatures. In Chapter 6.0, a closer examination of the protein and polypeptide components of the cell envelopes (amide regions) via Fourier transform infrared spectroscopy revealed different degrees of structural deformation in individual samples, which correlated well with the residual cell viability. It was also important to understand the underlying mechanisms responsible for the loss of viability of stabilized probiotic cells when stored at non-refrigerated temperatures. In Chapter 7.0, the stabilized Lactobacillus reuteri LR6 cells were stored at 37 °C and at two water activity (aw) levels. Superior storage stability was recorded in a lower aw environment, supported by a stronger glassy matrix when skim milk powder was used as the excipient. Fourier transform infrared spectroscopic examination of the cell envelopes revealed substantial dissimilarities between samples at the beginning and at the end of the storage period. In milk powder-based matrices, adjusting the aw to 0.30 resulted in a weaker or no glassy state whereas the same matrices had a high glass transition temperature at aw 0.11. This strong glassy matrix and low aw combination was found to enhance the bacterial stability at the storage temperature of 37 °C. During storage of the stabilized cells for 121 days at 37 °C, the measured Tg for all the samples was slightly lower than what was recorded at the beginning. Scanning electron microscopy revealed the formation of corrugated surfaces and blister-type deformations on the cell envelopes during the stabilization process whereas the freshly harvested cells were found to be with a smooth surface and undamaged membrane. Inspection of the cell bodies via transmission electron microscopy showed freshly harvested cells with normal shapes with no damage in the inner membrane structure. An almost intact but slightly waved outer membrane structure was observed. The findings emphasize the importance of protecting the integrity of the membrane of probiotic cells by using suitable protecting agents to enhance their stability during long term storage. The stabilized cell matrix samples were segregated into 4 groups based on the average particle diameter by passing through sieves of different mesh sizes. The degree of agglomeration had a very important role in offering physical protections to the LR6 cells during the desiccation process. The viable cell populations in the higher particle size groups (above 500μm and 1000μm) were between 9.5 to 9.9 log cfu/g whereas the same for the lower particle size (below 500μm but above 250μm) group was only 7.8 log cfu/g. The minimum viable cell concentration was recorded (7.3 log cfu/g) in the finer particles having less than 250μm diameter but having the maximum mass fraction. In case of stored samples, it was found that the bacterial cells adhered to the finest particles suffered the maximum loss in viability (41.4%) whereas the minimum loss (14.9%) was within the particles with average diameter above 500μm. In order to assess the effect of stabilization and storage (12 weeks, 37 °C) on the common probiotic attributes of the LR6 cells, an in vitro study on acid, bile salts tolerance and surface hydrophobicity was conducted. The results showed considerable reductions in cell viability for the desiccated as well as stored cells when incubated in simulated gastric (acid tolerance) and intestinal (bile salts tolerance) environments. A coating of xylitol over the cell bodies during desiccation was found to be marginally protective against these stresses. High aw storage was found to be more detrimental to the cells in terms of their ability to survive in the acid or bile environments. The cell surface hydrophobicity towards various hydrocarbons was also found to be adversely affected due to desiccation and non-refrigerated storage. Considerable degradation in hydrophobicity was found to be occurring in the cells stored at aw 0.30, a trend similar to the acid and bile resistance properties.
Stabilisation of dried Lactobacillus rhamnosus against temperature-related storage stresses : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Manawatū, New Zealand
(Massey University, 2019) Priour, Sarah
In the past few years, research has established a link between gut health and overall health and wellbeing. A diverse microbiome is a major step towards a healthy gut. Probiotics could help by improving the gut microbiome diversity and thus, are being added to a wide range of food products. However, maintaining them in a viable state within these food products is a considerable challenge. In order to increase the shelf-life of probiotics, numerous encapsulation systems have been developed to help protect them. Techniques such as emulsification, coacervation, or drying methods have all been employed with varying levels of success. While the final encapsulated bacteria may have enhanced protection and stability, a range of stresses are imposed on the bacterial cells during the actual encapsulation process, including mechanical, physical and chemical. Drying is the technique that confers the most protection to the probiotics, potentially stabilising them for up to several years. However, water plays a structural role and upon its removal, forces appear between cell components leading to the denaturation of proteins or the phase transition of the phospholipids membrane. Thus, bacterial cells need to be dried in the presence protectants that can prevent detrimental events from occurring and damaging the cells. It is thought that there are three main mechanisms by which protectants will confer superior stability. Firstly, the protective matrix can form a glassy system preventing further chemical reactions from happening, and thus protecting the bacteria. Secondly, if protectants are introduced for a period prior to drying, they can interact with the cellular biomolecules, replacing the structural role of the water, and maintaining the biomolecules in their native state when the water is removed from the system. Finally, the protectants can increase the free energy of water, maintaining it in the vicinity of the biomolecules, so that when the water is removed, the biomolecules are still hydrated and in their native state. Therefore, it is obvious that the role of protectants during the drying step is critical. The question that has remained largely unanswered, however, is how long and under what conditions should the protectants be introduced, and what type of protectants work best? Once the probiotics are successfully dehydrated, storage stresses may impair their stability on the shelf. Among these stresses, high temperatures of the surrounding environment is one that has been well documented to be detrimental to the cells and generally leads to a rapid drop in shelf stability. These temperatures can be experienced not only during the life of the product on the supermarket shelves, but also during transport of these consumables around the globe. The effect of changes in temperature on bacterial cell viability is an area which has not been explored in great depth, and the impact that encapsulation may have on the viability under these conditions even less so. Once again, like in the case of the protectants, the materials used to encapsulate the bacteria will be critical to final stability. Materials such as ‘phase change materials’ (PCM), which can absorb and release heat over different temperature ranges could be the key to protecting bacteria under extreme conditions. The aim of this thesis was thus to stabilise a model probiotic: Lactobacillus rhamnosus HN001 to high temperatures occurring during storage and transport. In order to do so, the study was separated into four principal research questions. Firstly, can a pre-drying step (for example the uptake of protectants) help the stability/viability of the bacteria during storage? Secondly, what are the best protectants for long-term storage of Lb. rhamnosus HN001, and why? Thirdly, is it possible that combinations of the most suitable protectants act in synergy, bringing increased storage stability compared to either protectant on its own? Finally, can the inclusion of PCM in the encapsulation matrix give extra protection to the cells during storage? This question would be of particular significance when examining the effect of the fluctuating temperatures experienced during the transport of the probiotics. The first study, therefore, consisted of establishing a protocol to prepare the cells for drying, by finding the early stationary phase where cells are known to be most stable to stress, and then optimising the exposure of the cells to potentially protective solutions of glucose and sucrose at 4 and 20°C. The uptake of the solutes was explored using HPLC, before drying the cells and evaluating the effect that their uptake had on the shelf-life stability of freeze-dried cells. In order to try and understand any interactions between the intracellular biomolecules and the protectants, the Nano DSC was used. Results showed that when cells were exposed to glucose at 20°C, metabolisation took place, and the longer the exposure, the lower the stability of the cells after drying and over storage. Overall, the study revealed that cells exposed to sucrose at 20°C for 4 hours presented best stability indicating that both the type of protectant, and exposure settings are critical to a successful outcome. The results from the Nano DSC showed that sucrose interacted with some of the cell biomolecules, rendering them more stable. The exposure temperature for the rest of the experiments was thus set at 4°C to avoid metabolisation, and the time was set at one hour so that exposure settings would be adapted for both sugars. In the second part of the study, a range of nine protectants (glucose, fructose, galactose, sucrose, lactose, trehalose, betaine, monosodium glutamate (MSG) and sorbitol) were compared for their ability to stabilise freeze-dried Lb. rhamnosus at 30°C for 6 months. Inulin was used as a carrier. The impact of galactose, sucrose, betaine, MSG and sorbitol was studied using a Nano DSC to again try and establish links between biomolecule interaction and stability during storage. Interestingly, MSG led to the best stability overall with a cell loss of 0.19 /month, even though it had the highest water activity of all the samples following freeze-drying. This is contradictory to general thought on how water activity affects bacterial cell stability, with higher water activity generally resulting in increased cell death over time. It was shown, using the Nano DSC, that MSG interacted with most of the cell biomolecules rendering them more stable. MSG was thus selected for further study. Three additional protectants were selected (galactose, sucrose and sorbitol) to look for potential synergistic effects with MSG in terms of protecting the bacteria during storage. The study followed a mixture design of experiment (DoE) in order to obtain an optimal protective matrix. The powder structure was also studied at this point by microscopy along with analysis using the DSC to try and comprehend the importance of the powder structure on the stability of the dried cells. Multivariate analysis was used to link all factors and their relative impact on the cell death rate together. Interestingly, it was found that neither a high glass transition temperature (Tg) nor a low water activity helped to stabilise the bacteria. Instead, the amount of MSG was clearly shown to improve the shelf-life, and a synergy was found between sorbitol and MSG. Microscopy showed that this powder led to a unique structure that most likely collapsed during drying resulting in the shrinkage of the cake and the loss of the porous structure, thus lowering the exposure of the bacteria to oxygen. In addition, a small amount of the sorbitol present in the matrix seemed to help in stabilising additional biomolecules as shown by the Nano DSC. The slowest death rate results obtained were 0.04 /month when MSG alone was mixed with inulin, but the model predicted an even lower death rate due to the synergy occurring between MSG and sorbitol. Finally, this optimised stabilisation matrix was used to study the impact of further protection, in the form of an encapsulate containing a PCM, on the stability of the bacteria. Powders with two different structures were compared using freeze-drying and spray drying techniques. The viability of the resulting powders was assessed during two separate storage studies designed to test the cells against fluctuating temperatures (20 to 50°C) and at constant temperature (35°C). The results showed that PCM appeared to have little impact on the overall stability of the powder. However, it was confirmed that a dense and smooth powder structure helped to maintain the bacteria in a viable state for a longer time than a more porous structure. This was most likely due to the lower surface-area ratio decreasing the exposure with the environment and preventing detrimental reaction such as oxidation. The bacteria in the optimised stabilisation matrix had the best stability, with a death rate of 0.07 /month at 35°C and 0.18 /month under fluctuating temperature from 20 to 50°C. In conclusion, it was found that the interaction of the protectants with cells is of paramount importance in maintaining the cells in a dried, viable state for longer periods at elevated temperatures. In addition, the structure of the powder should also be considered as one of the main mechanisms for protecting the bacteria, as it has a substantial impact on the shelf-life of the powder. Conversely, in this body of work it was shown that a high glass temperature did not enhance, or indeed help to maintain cell viability as has been suggested by many previous studies. A dense structure is, however, believed to protect the bacteria through preventing exchanges with the environment, especially with oxygen. If future work is to be done, it should follow the oxidation of the cells during storage and link it with measures of the powder porosity to gain further insight into the impact of the structure on oxidation stress.
Identification and functional characterisation of a novel surface protein complex of Lactobacillus rhamnosus : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology and Genetics at Massey University, Manawatu Campus, New Zealand
(Massey University, 2016) Wen, Wesley Xingli
Proteins are the most diverse structures on bacterial surfaces; hence they are candidates for species- and strain-specific interactions of bacteria with the host, environment and other microorganisms. In probiotic bacteria, some surface and secreted proteins mediate interactions with the host and may consequently contribute to the health-promoting effects. However, a limited fraction of surface-associated proteins from probiotic bacteria have been functionally characterised to date. A secreted protein of Lactobacillus rhamnosus HN001, SpcA, containing two bacterial immunoglobulin-like domains type 3 (Big-3) and a domain distantly related to plant pathogen response domain 1 (PR-1-like), was previously shown to bind to HN001 cells, however the nature of its ligand on the surface of the cells was unknown. In this study, a series of binding assays first demonstrated that SpcA binds to a cell wall anchored protein of HN001. Next, the SpcA-“docking” protein, named SpcB, was identified using phage display. SpcB is a 3275-residue cell-surface protein that has all the features of large glycosylated serine-rich adhesins/fibrils from Gram-positive bacteria, including the hallmark glycoprotein signal sequence motif KxYKxGKxW and the cell wall anchor motif LPxTG. The spcA and spcB genes are located in a gene cluster, spcBCDA, which is present in 94 out of 100 strains of L. rhamnosus species and some strains of L. casei and L. paracasei whose genome sequences have been determined, but was absent from other Lactobacillus clades. To confirm the role of SpcB as the SpcA anchor and investigate the roles of these two proteins in surface properties of probiotic L. rhamnosus strains HN001 and GG, stable double-crossover mutations of these two genes were constructed. Binding assays to L. rhamnosus mutant cells confirmed dependence on SpcB in both GG and HN001 strains. Comparison of the wild-type and mutant surface properties suggested that SpcB in GG interferes with biofilm formation and aggregation, while it might contribute to the protective effect against TNFa-mediated disruption of the polarised Caco-2 cell monolayer integrity. Deletion of HN001 spcB or spcA had no effect on functions other than the SpcA binding. Our findings indicate that the roles of a surface protein can vary considerably among the strains of a species, requiring functional data to validate the bioinformatics-based hypotheses.
A comparative study of two Lactobacillus fermentum strains that show opposing effects on intestinal barrier integrity : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy, Massey University, Manawatu, New Zealand
(Massey University, 2014) Sengupta, Ranjita
The Lactobacillus species can exert health promoting effects in the gastrointestinal tract (GIT) of humans through several mechanisms, which include pathogen inhibition, maintenance of microbial balance, immunomodulation and enhancement of the GIT barrier function. However, different strains of lactobacilli can evoke different responses in the host and not all strains of the same species can be considered health promoting. Two strains identified as Lactobacillus fermentum, namely AGR1485 and AGR1487, isolated from human oral cavities, exhibit opposing effects on intestinal barrier integrity. Studies have shown that AGR1485 maintains trans-epithelial electric resistance (TEER), a measure of GIT barrier integrity, across Caco-2 cell monolayers, while AGR1487 decreases TEER by 12 hours. This work aimed to test the hypotheses that the varying effects shown by these two L. fermentum strains are related to phenotypic differences between the two strains and are mediated by the interaction of secreted and/or cell-associated bacterial components with the GIT epithelial layer. Differences in metabolic events that occur during the various phases of growth in bacteria can impact not only cellular structure and secreted molecules, but may also affect their interactions with the intestinal epithelial cells. TEER assays were conducted to investigate if variation in bacterial secreted molecules and cell wall components associated with various phases of microbial growth can affect Caco-2 cell TEER. The effect on Caco-2 cell TEER caused by both strains was independent of bacterial growth phase. To test the hypothesis that it is the bacterial structural and/or secreted components that influence Caco-2 cell TEER, assays were conducted with live versus UV-killed bacteria on Caco-2 cells. Results showed that for both strains of L. fermentum, dead bacteria have similar effects on Caco-2 cell TEER as live bacteria, implying that direct bacterial contact with Caco-2 cells is necessary for the effects. Analogous to TEER assays, live AGR1487 increased mannitol permeability while UV-killed AGR1487 did not, implying that AGR1487 uses both cell surface structures and/or metabolites through distinct mechanisms to modulate host barrier properties. Subsequent experiments conducted using secreted metabolites from bacteria, Caco-2 cells and bacteria-Caco-2 cell interactions indicated that they have no effect on Caco-2 cell TEER, strengthening the assumption that bacterial cell surface-associated components are involved in mediating these effects. The bacterial cells were subjected to ultrasonication followed by ultracentrifugation to isolate the bacterial cell wall extract. TEER assays conducted with the cell wall extracts from both strains resulted in decreasing Caco-2 cell TEER, although at high concentrations, further strengthening the role of bacterial cell surface components in influencing barrier integrity of the Caco-2 cells. To narrow down proteinaceous components of the cell wall extracts from both the strains that influence Caco-2 cell TEER, they were fractionated through size exclusion chromatography and the effects of these cell wall fractions on Caco-2 cell TEER were studied. One fraction of AGR1487 CW appeared to decrease Caco-2 cell TEER, although at a high concentration. However, the results could not be repeated when the same fraction was applied at concentrations that the proteins comprising this fraction would be found in live AGR1487. Even the high concentration tested previously did not decrease Caco-2 cell TEER and the discrepancy in results remains unexplained. The results reported in this dissertation have added to the knowledge that the two strains of L. fermentum AGR1485 and AGR1487 show differences in their genome size and in their phenotypic characteristics. In addition, these bacteria utilise both cell surface and/or secreted metabolites through multiple mechanisms to modulate host response. In the future, identification of specific bacterial effector molecules that influence host response will be a major step towards understanding strain-specific characteristics shown by Lactobacilli.
The search for Lactobacillus proteins that bind to host targets : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University, Turitea, New Zealand
(Massey University, 2014) Erridge, Zoe Amber
Interactions between microorganisms and host cells in the gastrointestinal tract are crucial to the host’s health. Probiotic bacteria, such as the lactobacilli provide numerous benefits to human health thought to be mediated by bacterial proteins called effectors. Lactobacillus rhamnosus HN001 (L. rhamnosus HN001) is a cheesefermenting isolate with probiotic characteristics and Lactobacillus reuteri 100-23 (L. reuteri 100-23) is a coloniser of the rodent forestomach. Whereas L. rhamnosus HN001 was shown to reduce eczema in children, L. reuteri 100-23 reduces inflammation in mice. The effector proteins for these strains are largely unknown. In this thesis, phage display technology was used to search for proteins that bind specific ligands. Shot-gun genomic phage display library of L. rhamnosus HN001 was affinity screened on fibronectin as bait, leading to enrichment of specific recombinant clones. Analysis of 10 candidate clones, however, determined that these are not genuine binders, but may have been selected due to a potential growth advantage during amplification steps of the library. The L. reuteri 100-23 genomic shot-gun phage display library was subjected to two affinity screens on two baits: fibronectin and murine stomach tissue. The aim of the screen on the murine stomach tissue was to identify keratin-binding proteins, as this strain naturally colonises the murine keratinous forestomach. Whereas no enrichment was detected in the screen on fibronectin as a bait, a strong enrichment of a phagemid displaying a short peptide, IGINS, derived from a cell-surface protease of L. reuteri 100-23 was identified. Identifying and characterising probiotic bacterial proteins that positively influence health will lead to a greater understanding of gastrointestinal tract interactions. Ultimately, this aids development of probiotic use as therapeutic agents.
The effects of rice fibre on probiotic fermentation : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology and Microbiology at Massey University, Palmerston North, New Zealand
(Massey University, 2011) Fernando, Warnakulasuriya Mary Ann Dipika Binosha; Fernando, Warnakulasuriya Mary Ann Dipika Binosha
The role of rice fibre in stimulating the growth and SCFA (Short Chain Fatty Acid) formation by human faecal micro-flora and individual probiotics and co-cultures was investigated. The effects of environmental factors on the adhesion of probiotics on rice fibre were also evaluated. Fibre fractions of rice enhanced the growth of human colon microflora (Bifidobacterium species and Lactobacillus species) with a corresponding increase in the quantity of SCFA produced. However, individual microorganisms showed different preferences for different rice varieties and specific fractions of rice fibre. Pure cultures of the genus Bifidobacterium and genus Lactobacillus fermented rice fibre fractions irrespective of the rice variety. However, the genus Bifidobacterium produced more SCFA than genus Lactobacillus. Co-cultures of Bifidobacteria and Lactobacilli showed a greater ability than pure cultures to digest fibre and form SCFA, indicating synergism. Co-cultures used the fibre fractions irrespective of the rice variety. All microflora from mixed faecal inocula, pure and combinations of probiotic cultures showed a preference for total dietary fibre than insoluble and soluble dietary fibre fractions based on fermentation and SCFA production. All cultures tested, including human faecal cultures, pure cultures and co-cultures, produced more acetate than propionate and butyrate. Pure cultures and co-cultures adhered to rice fibre. Adhesion was influenced by environmental factors and is believed to play a role in the fermentation of rice fibre. Rice fibre is a suitable substrate for probiotic microflora.

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