Controlling pathogenic presence in broilers has become a priority in the poultry industry to prevent economic losses due to disease and infection, as well as the possible contamination of chicken products. The use of antibiotics reduces the incidence of infections; however, their removal from production initiated the search for suitable alternatives. Probiotic ineed supplements have been widely evaluated as alternatives. Probiotic use has improved broiler performance, reduced pathogenic loads, and stimulated the immune system at later life stages. However, there is still a gap in protection during the first weeks after the chick hatches. The in ovo supplementation of probiotics has the potential of promoting early health benefits and protect the chick against pathogens after hatch. In the present study, the in ovo inoculation of different probiotic species was evaluated. It was determined that the inoculation of higher concentrations of E. faecium (107 cfu/50µL) into the egg improves growth performance and intestinal morphology compared to lower doses (105 and 106 cfu/50µL). It was also determined that not all B. subtilis serotypes are safe for in ovo inoculation, even if recognized as safe for use in feed, due to a high reduction in hatchability. However, certain B. subtilis are safe for in ovo inoculation and regulate the gut microflora through modulations in coliforms and aerobic bacteria after hatch. Lastly, the in ovo inoculation of different Lactobacillus strains does not affect hatchability or growth performance. However, different Lactobacillus species stimulated cytokine production even during the first week of hatch. The bursa of Fabricius morphology was modulated through an increase in follicular area, which could possibly induce higher antibody production against incoming pathogenic challenges. These results indicate that the in ovo inoculation of probiotic bacteria can induce earlier benefits to broiler health through early changes in gut microflora, as well as early stimulation in the immune system. The early protection provided through the in ovo inoculation of probiotics combined with the protection obtained through the administration of probiotics in feed could potentially result in overall healthier broilers and therefore improved performance.

Incorporation of probiotic cultures in products in order to replenish or supplement the normal gastrointestinal microflora is a well known and accepted practice. However survival of these cultures is a problem due to a number of reasons including effects of storage conditions. Various researchers from different countries around the world have reported probiotic product instability. Microencapsulation has been used in an attempt to solve this problem. However, most methods involve the use of organic solvents which is not ideal because their toxicity may cause destruction of the microbial cells. A novel encapsulation method for probiotics, which excludes the use of organic solvents, was developed by the Council for Scientific and Industrial Research (CSIR) (US Patent Application no. 20050112205). This thesis investigated the efficiency/potential of this new method for increasing stability of sensitive probiotic cultures, specifically bifidobacteria. Early studies using both culture dependent and culture independent techniques showed reduced numbers of viable cultures in probiotic products, mainly yoghurts, from all around the world. These results were confirmed in this study for similar products sold in South Africa. Most of the product labels did not specify viable numbers of probiotics nor the identity (genus and species names) of the microorganisms incorporated. Successful encapsulation of bifidobacteria was achieved using the CSIR patented method. Complete encapsulation was indicated by absence of cells on surfaces of the encapsulated particles and production of a product with an acceptable particle size distribution was obtained. It was also demonstrated that the encapsulation process produced no visible morphological changes to the bacterial cells nor did it have a negative effect on cell viability over time. The potential of interpolymer complex formation in scCO2 for the encapsulation of sensitive probiotic cultures was demonstrated for the first time. Once ingested, probiotic cultures are exposed to unfavourable acidic conditions in the upper gastrointestinal tract. It is desired that these cultures be protected from this in order to increase the viability of the probiotics for efficient colonization. Interpolymer complex encapsulated B. longum Bb-46 cells were therefore exposed to simulated gastric fluid (SGF) and subsequently to simulated intestinal fluid (SIF). It was found that the interpolymer complex protected bifidobacteria from gastric acidity, displaying pH-responsive release properties, with little to no release in SGF and substantial release in SIF. Thus the interpolymer complex demonstrated desirable characteristics retaining the encapsulated bacteria inside when conditions were unfavourable and only releasing them under favourable conditions. Survival was improved by the incorporation of glyceryl monostearate (GMS) in the matrix and by use of gelatine capsules. Protection efficiency of the interpolymer matrix was better when higher loading of GMS was used. Use of polycaprolactone (PCL) as an alternative to poly (vinylpyrrolidone) (PVP) and incorporation of ethylene oxide-propylene oxide triblock copolymer (PEO-PPO-PEO) affected the interpolymer complex negatively, rendering it swellable in the low pH environment exposing the bifidobacteria to gastric acidity. The use of beeswax seemed to have a more protective effect though results were inconclusive. Probiotic cultures must also remain viable in products during storage. Encapsulated bacteria were either harvested from the reactor after 2 h of equilibration followed by depressurization, and then ground to a fine powder or after 2 h of equilibration the liquefied product was sprayed through a capillary tube with a heated nozzle at the end, into the product chamber. Encapsulated bacteria were stored in either sterile plastic bags or glass bottles under different conditions and then viable counts were determined over time. Survival of bacteria was generally better when the products were stored in glass bottles than in plastic bags. Bacteria encapsulated in an interpolymer complex formed between PVP and vinyl acetate-crotonic acid copolymer (VA-CA), (PVP:VA-CA) survived better than non-encapsulated bacteria under all storage conditions when the product was recovered from the reaction chamber. When the product was recovered from the product chamber, numbers of viable non-encapsulated bacteria were higher than the encapsulated bacteria for all interpolymer complex formulations. This was probably due to some exposure to high shear during spraying into the product chamber. The interpolymer complex between PCL and VA-CA i.e. PCL:VA-CA seemed weaker than the PVP:VA-CA nterpolymer complex as viable counts of bacteria released from it were lower than those from the latter complex. Addition of PEO-PPO-PEO to both the PVP:VA-CA and PCL:VA-CA complexes decreased the protection efficiency. However, results indicated that sufficient release of encapsulated bacteria from the interpolymer complexes was obtained when the encapsulated material was incubated in SIF rather than in Ringer’s solution. When SIF was used for release of encapsulated bacteria, the shelf life of B. longum Bb-46 was doubled. Encapsulation in an interpolymer complex therefore provided protection for encapsulated cells and thus has potential for improving shelf life of probiotic cultures in products. Further studies will investigate the effects of encapsulating probiotics together with prebiotics in the interpolymer complex as well as effects of encapsulating combinations of different probiotic strains together, both on survival in simulated gastrointestinal tract and during storage. The unique particles produced using the patented encapsulation technique increased the stability of probiotic cultures. This technique may find significant application in industries manufacturing probiotic products, especially food and pharmaceuticals, thereby improving the well being of consumers. / Thesis (PhD(Microbiology))--University of Pretoria, 2008. / Microbiology and Plant Pathology / PhD / unrestricted

Whole yellow pea flour (WPF) and fractionated yellow pea flour (FPF) are novel functional food ingredients that vary in nutritional composition. Consequently, the health benefits of WPF and FPF remain undefined. The purpose of this research was to identify the effects of WPF and FPF on risk factors and morbidities associated with cardiovascular disease, diabetes and obesity as well as the gastrointestinal microbiome. Using USDA recommended dosages of WPF and FPF, clinical endpoints and the colonic microbiome were investigated using a human clinical trial engaging a cross-over design and a diet and energy controlled paradigm. Humans were also utilized to investigate post-prandial glycemic responses and sensory characteristics of novel functional foods formulated with WPF. Finally, Golden Syrian hamsters were used to assess the impact of high doses of WPF and FPF on clinical endpoints and caecal microbial abundance. Results reveal that USDA recommended dosages of WPF and FPF in humans decreased (p<0.05) fasting insulin and estimates of insulin resistance compared to white wheat flour (WF). Android-to-gynoid fat ratios in women were lower (p=0.027) in the WPF group compared to the WF group. FPF decreased (p<0.05) post-prandial energy expenditure alongside a tendency (p<0.075) to reduce carbohydrate oxidation. Novel biscotti and banana bread formulated with WPF induced low post-prandial glycemic responses which were similar to boiled whole yellow peas and significantly lower (p<0.05) than white bread. Sensory analysis of novel WPF biscotti and banana bread demonstrated that WPF-based food products are palatable and acceptable for human consumption. Hamsters consuming diets containing 10% WPF and FPF induced similar reductions (p<0.05) in fasting insulin levels compared to controls. However, animals consuming WPF increased (p<0.05) oxygen consumption while FPF decreased (p<0.05) fasting glucose levels. In addition, terminal restriction fragment length polymorphism analysis revealed that WPF and FPF induced distinct shifts in caecal microbial populations within the phyla Firmicutes. Finally, pyrosequencing analysis of human fecal microbiota demonstrated that FPF and WPF induced shifts in bacterial genera, primarily within Bacteroidetes and Firmicutes. In conclusion, whole and fractionated yellow pea flours are functional food ingredients and can be utilized to manage risk factors for lifestyle-related diseases in humans.

Whole yellow pea flour (WPF) and fractionated yellow pea flour (FPF) are novel functional food ingredients that vary in nutritional composition. Consequently, the health benefits of WPF and FPF remain undefined. The purpose of this research was to identify the effects of WPF and FPF on risk factors and morbidities associated with cardiovascular disease, diabetes and obesity as well as the gastrointestinal microbiome. Using USDA recommended dosages of WPF and FPF, clinical endpoints and the colonic microbiome were investigated using a human clinical trial engaging a cross-over design and a diet and energy controlled paradigm. Humans were also utilized to investigate post-prandial glycemic responses and sensory characteristics of novel functional foods formulated with WPF. Finally, Golden Syrian hamsters were used to assess the impact of high doses of WPF and FPF on clinical endpoints and caecal microbial abundance. Results reveal that USDA recommended dosages of WPF and FPF in humans decreased (p<0.05) fasting insulin and estimates of insulin resistance compared to white wheat flour (WF). Android-to-gynoid fat ratios in women were lower (p=0.027) in the WPF group compared to the WF group. FPF decreased (p<0.05) post-prandial energy expenditure alongside a tendency (p<0.075) to reduce carbohydrate oxidation. Novel biscotti and banana bread formulated with WPF induced low post-prandial glycemic responses which were similar to boiled whole yellow peas and significantly lower (p<0.05) than white bread. Sensory analysis of novel WPF biscotti and banana bread demonstrated that WPF-based food products are palatable and acceptable for human consumption. Hamsters consuming diets containing 10% WPF and FPF induced similar reductions (p<0.05) in fasting insulin levels compared to controls. However, animals consuming WPF increased (p<0.05) oxygen consumption while FPF decreased (p<0.05) fasting glucose levels. In addition, terminal restriction fragment length polymorphism analysis revealed that WPF and FPF induced distinct shifts in caecal microbial populations within the phyla Firmicutes. Finally, pyrosequencing analysis of human fecal microbiota demonstrated that FPF and WPF induced shifts in bacterial genera, primarily within Bacteroidetes and Firmicutes. In conclusion, whole and fractionated yellow pea flours are functional food ingredients and can be utilized to manage risk factors for lifestyle-related diseases in humans.

Gastrointestinal microflora has been shown to have a bi-directional relationship with the host immune system. A variety of fermentable carbohydrate polymers largely pass through the small intestine, providing fermentable substrates for gut microflora. Dietary fibre supplementation may provide a strategy for manipulating the intestinal bacterial profile, changing the interaction with the mucosal immune system, thereby modulating the host immune system. We used a BBc rat animal model to evaluate the effects of oat bran and wheat bran dietary fibre on the immune system. Previous collaborative efforts have shown that these dietary fibres can change the intestinal microflora, with wheat bran fibre showing a greater ability to influence colonic microbial community diversity. We have shown that dietary wheat bran fibre led to reduced IL-4 levels in the liver and T lymphocyte numbers in the Mesenteric Lymph Node and may be involved in reduced IgA levels in the cecal contents. In addition, IgA in the cecal contents was decreased while MLN B cell numbers increased in response to dietary wheat bran fibre. It was observed that neither wheat bran or oat bran treatments exerted any pro-inflammatory effects, with oat bran actually improving antioxidant status. These results suggest that both oat and wheat bran fibre treatments induce changes in the intestinal microflora, and that the microflora changes due to wheat fibre are associated with immunomodulatory effects on the host. This type of dietary fibre supplementation could ultimately provide a potential strategy for promoting health through microflora-associated effects on the immune system.

O tratamento antimicrobiano pode destruir o equilíbrio da microbiota gastrintestinal, podendo induzir sintomas clínicos, principalmente a diarréia. A influência de Lactobacillus casei Shirota sobre a microbiota intestinal foi avaliada em um estudo prospectivo, randomizado, duplo-cego e controlado. Sessenta e três crianças hospitalizadas com idade de 2 a 14 anos, sob tratamento com antibióticos &#946;-lactâmicos, foram randomizadas para receber o leite fermentado por L. casei Shirota, 108-9 UFC/mL, ou o placebo, durante o tratamento antimicrobiano. As amostras de fezes foram colhidas antes da administração do leite fermentado, durante o tratamento antibiótico e uma semana após o término do tratamento com o antimicrobiano e a ingestão do leite fermentado. O número de L. casei Shirota aumentou significativamente (p<0,001) durante o período de ingestão do leite fermentado. Foi observado na microbiota do grupo que recebeu o placebo um aumento na contagem de Pseudomonas aeruginosa (p<0,05) e Clostridium sp (p<0,05), principalmente no último período da terapia antimicrobiana. A alteração da microbiota intestinal em decorrência do tratamento antibiótico foi constatada pela diminuição de acetato (p<0,05), butirato (p<0,05) e formato (p<0,05). Embora nenhuma criança deste estudo tenha apresentado diarréia, na avaliação geral, a microbiota daquelas que receberam o leite fermentado mostrou uma recuperação precoce da microbiota intestinal. Foi observado que a variação da contagem bacteriana realizada não foi significativa para as crianças do grupo que recebeu o leite fermentado, enquanto que no grupo placebo a contagem bacteriana ficou alterada, mostrando desequilíbrio da microbiota. Cerca de 50% das crianças ainda apresentaram L. casei Shirota nas fezes após uma semana da ingestão do leite fermentado. Este estudo mostrou que a ingestão do leite fermentado contendo L. casei Shirota promoveu um reequilíbrio mais rápido da microbiota intestinal quando comparada com a do grupo que ingeriu o placebo. / Antimicrobial treatment can destroy the balance of gastrointestinal microflora, which may induce clinical symptoms, mainly diarrhoea. The influence of Lactobacillus casei Shirota on the intestinal microflora was assessed in a prospective, randomised, double-blind controlled study. Sixty-three hospitalised children, with ages between 2 and 14 years, under treatment with &#946;-lactam antibiotics were randomised to receive milk fermented by L. casei Shirota, 108-9 CFU/mL, or placebo during the antimicrobial treatment. Stool samples were collected before the administration of fermented milk, during the antibiotic treatment, and one week after the end of treatment with the antimicrobial agent and the ingestion of fermented milk. The number of L. casei Shirota increased significantly (p<0.05) during the period in which fermented milk was ingested. An increase in the Pseudomonas aeruginosa (p<0.05) and Clostridium sp (p<0.05) count was observed in the microflora of the group that received placebo, mainly in the last period of antimicrobial therapy. The alteration of intestinal microflora as a result of antibiotic treatment was found by the reduction of acetate (p<0.05), butyrate (p<0.05) and formate (p<0.05). The variation in bacterial count proved not to be significant for the children under antimicrobial treatment who received fermented milk, while the placebo group showed imbalance of microflora with the result of the altered bacterial count. About 50% of the children still presented L. casei Shirota in their stools after interrupting the ingestion of fermented milk for one week. This study showed that ingestion of fermented milk containing L. casei Shirota promoted a much faster re-balance of the intestinal microflora when compared to the group that ingested a placebo.

O tratamento antimicrobiano pode destruir o equilíbrio da microbiota gastrintestinal, podendo induzir sintomas clínicos, principalmente a diarréia. A influência de Lactobacillus casei Shirota sobre a microbiota intestinal foi avaliada em um estudo prospectivo, randomizado, duplo-cego e controlado. Sessenta e três crianças hospitalizadas com idade de 2 a 14 anos, sob tratamento com antibióticos &#946;-lactâmicos, foram randomizadas para receber o leite fermentado por L. casei Shirota, 108-9 UFC/mL, ou o placebo, durante o tratamento antimicrobiano. As amostras de fezes foram colhidas antes da administração do leite fermentado, durante o tratamento antibiótico e uma semana após o término do tratamento com o antimicrobiano e a ingestão do leite fermentado. O número de L. casei Shirota aumentou significativamente (p<0,001) durante o período de ingestão do leite fermentado. Foi observado na microbiota do grupo que recebeu o placebo um aumento na contagem de Pseudomonas aeruginosa (p<0,05) e Clostridium sp (p<0,05), principalmente no último período da terapia antimicrobiana. A alteração da microbiota intestinal em decorrência do tratamento antibiótico foi constatada pela diminuição de acetato (p<0,05), butirato (p<0,05) e formato (p<0,05). Embora nenhuma criança deste estudo tenha apresentado diarréia, na avaliação geral, a microbiota daquelas que receberam o leite fermentado mostrou uma recuperação precoce da microbiota intestinal. Foi observado que a variação da contagem bacteriana realizada não foi significativa para as crianças do grupo que recebeu o leite fermentado, enquanto que no grupo placebo a contagem bacteriana ficou alterada, mostrando desequilíbrio da microbiota. Cerca de 50% das crianças ainda apresentaram L. casei Shirota nas fezes após uma semana da ingestão do leite fermentado. Este estudo mostrou que a ingestão do leite fermentado contendo L. casei Shirota promoveu um reequilíbrio mais rápido da microbiota intestinal quando comparada com a do grupo que ingeriu o placebo. / Antimicrobial treatment can destroy the balance of gastrointestinal microflora, which may induce clinical symptoms, mainly diarrhoea. The influence of Lactobacillus casei Shirota on the intestinal microflora was assessed in a prospective, randomised, double-blind controlled study. Sixty-three hospitalised children, with ages between 2 and 14 years, under treatment with &#946;-lactam antibiotics were randomised to receive milk fermented by L. casei Shirota, 108-9 CFU/mL, or placebo during the antimicrobial treatment. Stool samples were collected before the administration of fermented milk, during the antibiotic treatment, and one week after the end of treatment with the antimicrobial agent and the ingestion of fermented milk. The number of L. casei Shirota increased significantly (p<0.05) during the period in which fermented milk was ingested. An increase in the Pseudomonas aeruginosa (p<0.05) and Clostridium sp (p<0.05) count was observed in the microflora of the group that received placebo, mainly in the last period of antimicrobial therapy. The alteration of intestinal microflora as a result of antibiotic treatment was found by the reduction of acetate (p<0.05), butyrate (p<0.05) and formate (p<0.05). The variation in bacterial count proved not to be significant for the children under antimicrobial treatment who received fermented milk, while the placebo group showed imbalance of microflora with the result of the altered bacterial count. About 50% of the children still presented L. casei Shirota in their stools after interrupting the ingestion of fermented milk for one week. This study showed that ingestion of fermented milk containing L. casei Shirota promoted a much faster re-balance of the intestinal microflora when compared to the group that ingested a placebo.