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Subject: search.filters.subject.Lactobacillus reuteri

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    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.
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    The biotherapeutic potential of Lactobacillus reuteri DPC16 and bovine lactoferrin in controlling some pathogens, genotoxicity and inflammation in the gut: a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Engineering and Advanced Technology at Massey University, Auckland, New Zealand
    (Massey University, 2013) Tian, Hong
    This study investigated the effects of the probiotic bacterium, L. reuteri DPC16, alone and in combination with bovine lactoferrin, on intestinal pathogens, intestinal inflammation and carcinogenesis. Human and animal cellular model systems were designed and applied to this evaluation. The identity of the L. reuteri DPC16 strain was confirmed using 16S rRNA analysis and its ability to produce the antibacterial compound, reuterin. It was able to tolerate pH 2 and physiological concentrations of bile salts in a simulated gastrointestinal tract environment in the presence of protective nutrients. It was able to adhere to a Caco-2 human epithelial monolayer (modelling the human GI tract) and it did not degrade mucin. Both bovine lactoferrin and L. reuteri DPC16 inhibited the growth of the intestinal pathogens Listeria monocytogenes, Staphylococcus aureus, Salmonella typhimurium and Escherichia coli O157:H7, with much less effect on tested probiotic bacteria. Together, L. reuteri DPC16 and bovine lactoferrin showed synergistic inhibitory effects. L. reuteri DPC16 was also able to remove indole from faecal water. Using human/animal cellular model systems, combined with the use of E. coli endotoxin and genotoxic factors present in faecal water, bovine lactoferrin was shown to down-regulate inflammation by affecting the signalling pathway on immune receptors that recognize the endotoxin, while both bovine lactoferrin and strain DPC16 were shown to have the potential to prevent epithelial cell DNA damage. The study has demonstrated several significant properties of L. reuteri DPC16 and bovine lactoferrin, including antibacterial, antigenotoxic, and anti-inflammatory activities, and possible mechanisms for these activities have been proposed. Based on the information obtained from this work, a combination of the probiotic L. reuteri DPC16 and bovine lactoferrin could possibly be developed as a novel probiotic formula for human consumption, to maintain beneficial bacteria while controlling harmful bacteria in the GI tract. However, advanced in vitro model systems and in vivo studies are suggested to confirm these findings in order to consider the feasibility of commercialisation.
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    An immobilised cell system for the delivery of functional Lactobacillus reuteri DPC16 cells to their target site in a simulated gastrointestinal tract : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Science at Massey University, Albany, New Zealand
    (Massey University, 2012) Zhao, Qian
    The objective of this study was to design and produce calcium alginate beads that can deliver immobilised cells of Lactobacillus reuteri DPC16 to a target site of the colon in the gastrointestinal (GI) tract, without any diminution of their important physiological characteristics. Several factors that might affect the effectiveness of calcium alginate beads for the cell delivery were investigated, using an in vitro GI tract model to simulate the conditions within the tract. Firstly, by varying the concentration of alginate at a constant concentration of CaCl2, and combining the system with gelatin, chitosan or skim milk, the survival of immobilised DPC16 cells in simulated gastric fluid (SGF) was observed. Secondly, the physical stability of calcium alginate beads containing skim milk was observed during sequential incubation in the GI fluids using optimal concentrations of alginate. Finally, the survival of DPC16 cells immobilised within alginate beads containing skim milk was monitored when the beads were incubated for different times during sequential exposure to the simulated fluids. The results demonstrated that non-encapsulated DPC16 cells were sensitive to an acidic environment, and no viable cells were detected after 90 min exposure in SGF (pH 1.2). After appropriate experimentation, an alginate concentration of 3% (w/v) was deemed to be the optimum value and was used in subsequent investigations. When skim milk (8% (w/v)) was added to the alginate solution, the cell survival in SGF was improved markedly. The optimal concentration of calcium chloride was 0.3 M, based on the beads maintaining their integrity in SGF and simulated intestinal fluid (SIF) while disintegrating in simulated colonic fluid (SCF) to release viable cells. Hence, the beads made from 3% alginate, 8% skim milk and 0.3 M CaCl2 proved to be an effective delivery and release system for DPC16 cells. L. reuteri DPC16 has strong antimicrobial activities against pathogens, due mainly to its ability to produce reuterin. Hence this and other functional properties of the bacterial cells were studied before and after passage through the GI tract. The cells that were recovered after release from the alginate beads in the SCF showed no diminution in functional properties, including their growth kinetics, ability to adhere to epithelial cells and ability to inhibit the adhesion of E. coli to epithelial cells. However, the bacteriostatic and bactericidal properties of the recovered cells against some pathogens were significantly greater (P<0.05) than those of the original cells. Production of reuterin by the recovered cells was significantly greater (P<0.05) than that of the original cells when cultured in MRS medium in the absence of its metabolic precursor, glycerol. The results demonstrate significant (P<0.05) consequences for the application of the encapsulation technique to protect and/or enhance the functional properties of the probiotic cells. Subsequently, an investigation was carried out to find the reason for the antimicrobial activity enhancement. By recovering cells from different stages of the immobilisation and delivery process and examining them for their antimicrobial properties, it was found that it was the immobilisation process per se, rather than passage through the simulated gastrointestinal fluids, that caused the enhancement of antimicrobial activity, and that this was related to increased activity of the enzyme (diol dehydratase) that is responsible for reuterin production from glycerol. Finally, it was demonstrated that freeze-drying of the alginate beads was not an appropriate storage technique as it resulted in a significant (P<0.05) diminution of the antimicrobial activities. Based on these findings it is confirmed that the alginate-skim milk-CaCl2 immobilisation system is an effective and efficient method, not only for protecting the viability of DPC16 cells, but also for maintaining the physiological characteristics.
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    A novel approach for controlling foodborne pathogens using modified atmosphere and Lactobacillus reuteri DPC16 : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Albany, New Zealand
    (Massey University, 2007) Lu, Guangjin
    The current trend of increasing demand for minimally processed food requires more effective preservation technologies than are presently used. In this study, an investigation has been made into a novel strategy to control some common foodborne pathogens, and therefore, to provide an alternative means for enhancing the safety and extending the shelf lives of food products. Modified atmosphere is able to extend the shelf life of seafood and meat products. In this study, a simulated controlled atmosphere (CA) broth system was used to investigate the potential of a modified atmosphere rich in CO2 at a concentration of 40%, supplemented with N2, to control common foodborne pathogens, such as Listeria monocytogenes, Escherichia coli O157:H7, Salmonella enterica serovar Typhimurium, Staphylococcus aureus and Vibrio parahaemolyticus. Controlled atmosphere significantly reduced the exponential growth rates of all tested pathogens, while the effects on other growth parameters (eg. lag phase duration and maximum population density) depended on the individual species and the specific growth conditions. The CA significantly extended the lag phase durations of S. aureus and V. parahaemolyticus at 20 degrees C at both pH 6.3 and 6.8, and that of L. monocytogenes at both 7 degrees C and 20 degrees C, and at both pH 6.3 and 6.8. The CA also significantly lowered the maximum population densities of S. aureus and V. parahaemolyticus at 20 degrees C, at pH 6.3 and 6.8, S. Typhimurium at pH 6.8, and L. monocytogenes at pH 6.3 and 7 degrees C. E. coli O157:H7 and S. Typhimurium were more resistant to the inhibitory effect of the CA, while S. aureus and V. parahaemolyticus were most sensitive. The inhibitory effect of CA was due mainly to the extensions of the lag phase duration and the reduction of the exponential growth rates of the test pathogens. This study confirms other studies that CA as a means for food preservation provides potential to control foodborne pathogens and therefore enhance the safety of a food product. The use of lactic acid bacteria (LAB) in controlling spoilage microorganisms and pathogens in foods has been a popular research theme worldwide. In this study, the antimicrobial effects of 18 lactic acid bacteria strains were evaluated in vitro, with emphasis on the most effective strain, the newly characterised Lactobacillus reuteri DPC16. The results demonstrated antagonistic effects of many strains against L. monocytogenes, E. coli O157:H7, S. Typhimurium and S. aureus. L. reuteri DPC16 showed the strongest antimicrobial activity against the tested pathogens including both Gram-positive and Gram-negative bacteria. Co-cultivation of L. reuteri DPC16, and co-incubation of its spent culture supernatant (DPC16-SCS), with the pathogens have demonstrated that the antimicrobial effect is bactericidal and valid at pH 4 - 6.5 and at a temperature as low as 10 degrees C. Further characterisation of the antimicrobial effect of L. reuteri DPC16 showed it to be mainly due to the presence of reuterin (ß-hydroxypropionaldehyde), although lactic acid may have also played a role. These characteristics of L. reuteri DPC16 and its metabolite reuterin make it an unique and potent candidate as a biopreservative to control both Gram-positive and Gram-negative bacteria in foods. The combination of L. reuteri DPC16 and CA was assessed for its inhibitory effect on L. monocytogenes using DPC16-SCS and the fermentative supernatant of L. reuteri DPC16 from a glycerol-water solution (DPC16-GFS). The results showed that both of these supernatants, at 25 AU/mL, in combination with CA (60% CO2:40% N2) had a combined inhibitory effect on L. monocytogenes which could not be achieved by any one of the individual factors alone. Analysis of the levels of expression of some stress response genes of L. monocytogenes, after growth in the presence of L. reuteri DPC16 supernatant and/or CA, showed that the expression of some genes was affected including genes betL, gbuA and opuCA responsible for osmosis adaptation and genes gadA, gadB and gadC responsible for acid tolerance. Induction of gbuA, gadB and gadC by the culture supernatant suggests activation of osmotic and acid adaptation and that these genes play a major role in the culture supernatant-induced stresses. An investigation was also carried out to determine if the changes in gene expression conferred a cross-protection to heat. The result showed that the survival of L. monocytogenes grown in the presence of the culture supernatant and CA was significantly increased after exposure to heat treatment at 56oC, suggesting that a cross-protection to thermal stress had been induced. Based on these findings it is proposed that a comprehensive novel strategy incorporating both L. reuteri DPC16 or its fermentative products and a modified atmosphere rich in CO2 could be developed to potentially control foodborne pathogens in food products.