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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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    The development of an enzyme-linked immunosorbent assay for bovine lactoferrin : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University
    (Massey University, 1988) Mock, Michele Kay
    Methods were established for the estimation of bovine lactoferrin by enzyme-linked immunosorbent assay (ELISA) on microtitre plates and nitrocellulose (dot assay). Mfinity-puri:fied antibodies to bovine lactoferrin were prepared and conjugated to horseradish peroxidase by the periodate method. Conjugates with enzymatic and immunological activity had an apparent molecular weight of 65 or 95 kd. Four methods of ELISA on microtitre plates and three dot assays were developed. ii Differences between the seven assays could be attributed to the absorption capacity of the solid phase and the types of conjugates and substrates used. The range of the two successful quantitative assays were 3-lOOOng lactoferrin/ml (sandwich) and 60-8000ng lactoferrin/ml (competitive), while the qualitative dot assays had a range of 1-lOOJ.lg lactoferrin/ml. More replicates would be required to reduce variability. Results from these assays generally corresponded to results from ROCKET electrophoresis. Dot assay on nitrocellulose has a greater potential for reproducible and quantitative assays than assay on microtitre plates, because of the greater adsorption capacity of the nitrocellulose. In addition, the dot assays are faster and lend themselves to more applications than either ROCKET electrophoresis or ELISA on microtitre plates. The ELISA developed in this project appear to be the first alternatives to radial immunodiffusion and ROCKET electrophoresis for the measurement of bovine lactoferrin.
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    Cloning and sequencing of the cDNA for bovine lactoferrin : this thesis is submitted to Massey University as partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry
    (Massey University, 1992) Mead, Paul Evan
    Bovine lactoferrin isolated from colostrum was partially sequenced by tryptic mapping and automated peptide sequencing. Homogeneous lactoferrin was used to raise polyclonal antibodies in rabbits. Specific anti-lactoferrin antibodies were isolated from the total rabbit gamma-globulin fraction by affinity chromatography on bovine lactoferrin Sepharose. These antibodies were used to quantify lactoferrin in various solutions (by electroimmuno-diffusion assay) and to demonstrate the de novo synthesis of lactoferrin in involuting bovine mammary tissue. RNA was isolated from mammary tissue biopsies that were synthesizing lactoferrin. The presence of lactoferrin messenger RNA was verified by northern blot analysis. Complementary DNA (cDNA) was prepared from RNA samples and ligated into either the bacteriophage vector λ-gt11 or the plasmid vector pGEM-2. Recombinant clones with cDNA inserts coding for bovine lactoferrin were identified by hybridisation to radiolabelled human lactoferrin cDNA. Several clones were isolated and characterised by restriction map analysis and DNA sequencing. The overlapping nucleotide sequence from these clones encoded most of the mature protein sequence for bovine lactoferrin. Nucleotide sequence encoding the 5' end of the lactoferrin messenger RNA was isolated by enzymatic amplification of homopolymeric-tailed first strand cDNA. Specific oligonucleotide primers were used to direct the synthesis of lactoferrin-specific sequences by the polymerase chain reaction (PCR). Double-stranded products were produced by the inclusion of an oligonucleotide that would prime DNA synthesis from the homopolymeric tract on the 3' end of the first strand cDNA. The nucleotide sequence of the PCR products overlapped the 5'-most sequence of the cDNA clones and extended to encode the initiation codon for bovine lactoferrin. The combined nucleotide sequence of the cDNA and PCR clones overlapped to encode the entire coding region for bovine lactoferrin and included 5' and 3' untranslated flanking sequences. The deduced amino acid sequence of the mature protein concurred with the amino acid sequence of the tryptic peptides prepared from bovine colostrum lactoferrin.