Atherosclerosis is the major cause of cardiovascular diseases, which constitute the top ten leading causes of death worldwide. Atherosclerotic plaque development initiates from the inflamed endothelium under an atherogenic environment – chronic low grade inflammation, hypercholesterolemia, endotoxemia, etc. The principal cause of such inflammation has yet to be defined – with growing evidence that microbial stimulants like lipopolysaccharide (LPS) and peptidoglycan (PGN), which can activate toll-like receptors (TLRs) and nuclear factor-kappaB (NFκB) signaling might be the plausible origins. The gastrointestinal tract is suggested to be the major site for absorption and translocation of such stimulants, where gut microbiota have been associated with systemic inflammation and is essential in generating atherogenic substances. Since probiotics have the potential to induce systemic anti-inflammatory effects and fortify gut barrier to reduce bacterial translocation, evaluation of whether probiotics can help reduce atherogenesis was done by feeding the disease model, ApoE-/- mice with high fat diet alone, with telmisartan (1 or 5 mg/kg/day, positive controls) or with probiotics VSL#3 (2.8 x 1011 CFU/day) and/or Lactobacillus rhamnosus strain GG (LGG) (1x108 CFU/day), or the combination of which for 12 weeks. All treatments reduced lesion size significantly; with some treatments reduced plasma endotoxin, cholesterol and various proinflammatory biomarkers. The gut microbiota assessed with PCR-amplified 16S rRNA gene sequences using 454 pyrosequencing and thereafter correlation studies revealed that at least 20 bacterial families that were significantly altered by high fat diet in apolipoprotein E knockout (ApoE-/-)mice correlated with atherosclerotic plaque size and related biomarkers including cholesterol, adipocyte fatty acidbinding protein (A-FABP), etc. Probiotics showed potential in improving atherogenic environment by immunomodulation and induction or inhibition of growth of bacteria correlated with the atherosclerotic plaque and biomarkers. The atherosclerotic condition was also improved by telmisartan, which correlated with the altered gut microbiota. The newly identified atherosclerosis-related gut bacteria will require further exploration into their properties and mechanisms, which will eventually lead to the potential of developing probiotics for the treatment or prevention of atherosclerosis, and thus may be used as an affordable and non-invasive alternative that brings health benefits worldwide. / published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

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Submitted in complete fulfillment for the Degree of Doctor of Philosophy in Biotechnology, Durban University of Technology, Durban, South Africa, 2016. / Colon cancer is the third most common cancer worldwide with a high morbidity and mortality rate. Therapies are less effective during metastasis, therefore prevention and earlier detection is key to reducing the risk of colon cancer. Increased dietary fibre and probiotic intake is known to lower the risk of colon cancer. Probiotics are defined as “live microorganisms which when administered orally in an adequate amount confer a health benefit on the host”. The International Dairy Federation recommends a viable minimum level of 6–7 log10cfu/g in a probiotic product being consumed. Different biopolymer matrices have been used for encapsulation of probiotics; however, loss of viability is still a major challenge. Citrus pectin is a dietary fibre polysaccharide broken down into smaller fragments to form modified citrus pectin (MCP). The unique bioactivity of MCP against carcinogenesisis is linked to its sugar β-galactose inhibiting the cell signalling protein marker, galectin-3 (gal-3), which is intimately involved in endothelial cell morphogenesis. The vascular endothelial growth factor (VEGF) signalling, which invariably drives angiogenesis can be activated when gal-3 binds to integrins. The bioactivity and uptake of MCP may be improved through a novel approach if conjoined with a supplement for example probiotic. Therefore, the synergistic inhibitory effect of modified citrus pectin alginate (MCPA) probiotic microbeads on gal-3 and VEGF in an azoxymethane (AOM) induced colon carcinogenesis Balb/c mouse model was investigated. A microencapsulation process was used to produce a MCPA microbead containing probiotic, Lactobacillus acidophilus ATCC 4356. Efficiency of the microbead was evaluated in vitro (simulated conditions) and in vivo (Balb/c mouse model). Genomic identification of faecal lactobacilli samples from the treated mice was analyzed. Optimization of AOM dose-time with 10 and 15 mg/kg AOM intraperitoneal (ip) administered to Balb/c mice for 2 and 4 weeks were performed. The optimal AOM dose was initiated prior to intake of MCPA, AP (alginate calcium) probiotic microbeads and MCP in Balb/c mice for 16 weeks; samples were analyzed for colon histopathology and immunohistochemistry. The MCPA probiotic microbeads significantly enhanced the viability of L. acidophilus ATCC 4356 compared to the AP microbeads in vitro (p< 0.05). Exposure of the MCPA probiotic microbeads to 3 h of simulated gastric juice (SGJ) resulted in 82.7% survival of L. acidophilus ATCC 4356. Also, the faecal lactobacilli count in the MCPA probiotic treated mice significantly increased after 28 days by 10.2% compared to the AP probiotic, MCP treated and control mice (p< 0.0001). A total of 4DNA encoding 16S rRNA gene closest to the genera namely Lactobacillus, Bacillus, Enterococcus and Bifidobacterium were identified from faecal samples of the colon cancer-induced Balb/c mice. Azoxymethane at 15 mg/kg for 4 weeks induced optimal gal-3 and VEGF immunoexpression. Furthermore, MCPA probiotic treatment significantly reduced gal-3 immunoexpression in the colon of AOM induced cancer Balb/c mice compared to the control mice (p< 0.0001). The immunoexpresion of VEGF in the MCPA and AP probiotic treated groups was weakly positive and significantly reduced when compared to the control group (p<0.05), while the MCP treated group was barely positive (p< 0.001). Modified citrus pectin alginate is a novel effective means of oral delivery of bacterial cells and bioactive compounds. It has a good biodegradability, inexpensive, non-toxic, proven efficiency, and stability at low temperatures warranting its use as a drug carrier by pharmaceuticals. Modified citrus pectin alginate probiotic microbeads increase bioactivity and chemoprevention against colon pre-cancerous lesions and adenocarcinoma through inhibition of gal-3 and VEGF in the mouse model. Modified citrus pectin alginate can be used in probiotic therapy, which may improve the prevention of colon cancer. / D

BACKGROUND: Cystic fibrosis (CF) patients grow poorly and tend to be malnourished. They frequently suffer from lung infections necessitating the repeated use of antibiotics. AIM: This study was conducted to determine whether supplementation with a probiotic Lactobacillus reuteri (L. reuteri) could reduce the incidence and duration of lung infections, and whether this would impact on their anthropometric data. The secondary purpose was to compare the nutritional status of the CF patients attending CF clinics in Kwazulu-Natal (KZN) with CF patients attending CF clinics in Cape Town (CT). METHODS: Twenty three CF patients 6-31 years of age from 2 CF clinics in Kwazulu-Natal started the study although only 16 patients completed it. The study was a randomized, double blind, placebo controlled crossover trial with six months on placebo and six months on probiotic. Weight, height, mid arm circumference (MAC), triceps skin fold thickness (TSF), forced expiratory volume in 1 second (FEV1) and forced vital capacity (FVC) were measured, sputum collected and a symptom diary completed over the 12 month period. Anthropometric data of CF patients attending CF clinics in CT was obtained from the publication by Westwood & Saitowitz (1999). RESULTS: Compliance with taking the L. reuteri was poor. Most took only 50% of the required daily dose. Probiotic supplementation showed a slight (non significant) trend to improve FEV1 and FVC, while no significant difference could be seen in the number and duration of the lung infections. Sputum analysis showed a non significant trend towards the probiotic reducing the number of bacteria in the sputum. There was a significant reduction of symptoms for fever, running nose, sore throat and ear ache while on placebo. There was a significant increase in weight gained off probiotic compared to the probiotic period. The changes in height, weight for age (WFA) percentiles, height for age (HFA) percentiles, WFA and HFA Z-scores, percentage expected weight for age and percentage expected height for age all showed no difference whether on or off probiotic. Over half the CF children in the KZN clinics were underweight for their actual height compared to one third in the CT clinics with a higher number of subjects below the 5th percentile for MAC and TSF readings compared to CT. CONCLUSION: Due to a small sample size and poor compliance no firm conclusions could be drawn. However a slight (non significant) improvement could be seen in favour of the probiotic for FEV1, FVC, and sputum analysis. Although all other findings were not significantly different it would be of benefit to carry out further investigation with improved compliance with the probiotic to see if the parameters set out above could be improved. The KZN and CT CF groups were comparable and the nutritional status of CF patients on KZN was well below that of the CT CF clinics and further monitoring would need to be carried out.