Research was undertaken to examine different <i>in vitro</i> characteristics of probiotic bacteria, including <i>Lactobacillus acidophilus</i> ATCC® 11975, <i>Bifidobacterium infantis</i> ATCC 15697D, <i>Bifidobacterium catenulatum</i> ATCC® 27675 and <i>Bifidobacterium adolescentis</i> ATCC® 15703 in order to identify suitable strain(s) for encapsulation. Under simulated gastric conditions (pH 2.0), <i>L. acidophilus</i> was the most acid-tolerant strain (D-value 10.2 ± 0.8 min), and was able to survive for 30 min; whereas, the other tested probiotics underwent a rapid (within the first 5 min at pH 2.0) 4-5 log colony forming units (cfu)/mL loss in viability. All probiotics tested were able to survive 5 h exposure to 0.3% Oxgall bile at pH 5.8. The relative ranking of probiotic adherence to Caco-2 cells was determined to be: <i>L. acidophilus</i> > <i>B. catenulatum</i> > <i>B. adolescentis</i> > <i>B. infantis</i>, which correlated with 4.5 104, 3.1 103, 2.6 101, and 1.5 101 cfu/mL associated with Caco-2 cell monolayers, respectively. The most hydrophobic probiotics included <i>L. acidophilus</i> (46.5 ± 6.1%) and B. catenulatum (65.5 ± 5.2%); their hydrophobicity were positively correlated with auto-aggregation ability. Addition of divalent cations, EDTA, and bile salts were found to affect hydrophobicity as well; for example, 0.5 mM MgCl2 resulted in a 20% increase in cell surface hydrophobicity of <i>L. acidophilus</i> from baseline levels; whereas, the addition of 0.1 and 0.5% bile salts decreased <i>L. acidophilus</i> hydrophobicity from control levels by 60 and 90%, respectively. Cell free culture supernatant of <i>L. acidophilus</i> effectively inhibited the growth of <i>Escherichia coli</i> O157:H7, and <i>Clostridium sordelli</i>. Bactericidal activity of <i>L. acidophilus</i> cell-free supernatant (the lethal factor was determined to be both heat and trypsin-resistant) against Escherichia coli O157:H7 and <i>Clostridium sordelli</i> ATCC 9714 over 24 h resulted in reductions of 5.5 and 3.5 log cfu/mL, respectively. Further examination of probiotics revealed varying degrees of resistance to the iv antimicrobial agents ciprofloxacin (4 ìg/mL), naladixic acid (32 ìg/mL), kanamycin (64 ìg/mL) and sulfisoxazone (256 ìg/mL). Determination of carbon source utilization patterns indicated that <i>B. catenulatum</i> utilized a number of carbohydrates including -methyl-D-glucoside, D-xylose, D-cellobiose, and -D-lactose; whereas,<i>L. acidophilus, B. infantis</i>, and <i>B. adolescentis</i> utilized D-xylose. <i>Lactobacillus acidophilus</i> was ultimately selected for encapsulation in a 3 mm diameter pea protein-alginate matrix followed by <i>in vitro</i> challenge to simulated gastric conditions (pH 2.0). Encapsulation of <i>L. acidophilus</i> demonstrated a significant (P < 0.05) protective effect during the 2 h exposure to simulated acidic stomach conditions; within capsules, there was approximately 1 log cfu/mL loss in cell viability, whereas unprotected cells experienced > 6 log/mL loss in cell viability over the same period.

Research was undertaken to examine different <i>in vitro</i> characteristics of probiotic bacteria, including <i>Lactobacillus acidophilus</i> ATCC® 11975, <i>Bifidobacterium infantis</i> ATCC 15697D, <i>Bifidobacterium catenulatum</i> ATCC® 27675 and <i>Bifidobacterium adolescentis</i> ATCC® 15703 in order to identify suitable strain(s) for encapsulation. Under simulated gastric conditions (pH 2.0), <i>L. acidophilus</i> was the most acid-tolerant strain (D-value 10.2 ± 0.8 min), and was able to survive for 30 min; whereas, the other tested probiotics underwent a rapid (within the first 5 min at pH 2.0) 4-5 log colony forming units (cfu)/mL loss in viability. All probiotics tested were able to survive 5 h exposure to 0.3% Oxgall bile at pH 5.8. The relative ranking of probiotic adherence to Caco-2 cells was determined to be: <i>L. acidophilus</i> > <i>B. catenulatum</i> > <i>B. adolescentis</i> > <i>B. infantis</i>, which correlated with 4.5 104, 3.1 103, 2.6 101, and 1.5 101 cfu/mL associated with Caco-2 cell monolayers, respectively. The most hydrophobic probiotics included <i>L. acidophilus</i> (46.5 ± 6.1%) and B. catenulatum (65.5 ± 5.2%); their hydrophobicity were positively correlated with auto-aggregation ability. Addition of divalent cations, EDTA, and bile salts were found to affect hydrophobicity as well; for example, 0.5 mM MgCl2 resulted in a 20% increase in cell surface hydrophobicity of <i>L. acidophilus</i> from baseline levels; whereas, the addition of 0.1 and 0.5% bile salts decreased <i>L. acidophilus</i> hydrophobicity from control levels by 60 and 90%, respectively. Cell free culture supernatant of <i>L. acidophilus</i> effectively inhibited the growth of <i>Escherichia coli</i> O157:H7, and <i>Clostridium sordelli</i>. Bactericidal activity of <i>L. acidophilus</i> cell-free supernatant (the lethal factor was determined to be both heat and trypsin-resistant) against Escherichia coli O157:H7 and <i>Clostridium sordelli</i> ATCC 9714 over 24 h resulted in reductions of 5.5 and 3.5 log cfu/mL, respectively. Further examination of probiotics revealed varying degrees of resistance to the iv antimicrobial agents ciprofloxacin (4 ìg/mL), naladixic acid (32 ìg/mL), kanamycin (64 ìg/mL) and sulfisoxazone (256 ìg/mL). Determination of carbon source utilization patterns indicated that <i>B. catenulatum</i> utilized a number of carbohydrates including -methyl-D-glucoside, D-xylose, D-cellobiose, and -D-lactose; whereas,<i>L. acidophilus, B. infantis</i>, and <i>B. adolescentis</i> utilized D-xylose. <i>Lactobacillus acidophilus</i> was ultimately selected for encapsulation in a 3 mm diameter pea protein-alginate matrix followed by <i>in vitro</i> challenge to simulated gastric conditions (pH 2.0). Encapsulation of <i>L. acidophilus</i> demonstrated a significant (P < 0.05) protective effect during the 2 h exposure to simulated acidic stomach conditions; within capsules, there was approximately 1 log cfu/mL loss in cell viability, whereas unprotected cells experienced > 6 log/mL loss in cell viability over the same period.

Probiotics and prebiotic are becoming increasingly important to consumers to alleviate issues surrounding gut health, despite the lack of definitive efficacy studies to support health claims. The addition of both probiotics and prebiotics to foods is challenging due to the harsh environmental conditions within the food itself and during transit through the gastrointestinal (GI) tract. To circumvent these challenges encapsulation technology is being explored to protect sensitive ingredients and to control their release within the lower intestines thereby maximizing the health benefiting effects. The overall goal of this research was to design a protein delivery capsule using phase separated pea protein isolate (PPI)-alginate (AL) mixtures for the entrapment of the synbiot which includes the probiotics, Bifidobacterium adolescentis, and the prebiotic, fructooligosaccharides (FOS), such that the capsule design provides highly effective protection and release within the GI tract. Research was carried out in three studies.<p> In study 1, PPIn (native isolate) and AL interactions were studied in dilute aqueous solutions as a function of pH and biopolymer mixing ratio. Turbidimetric analysis and electrophoretic mobility during an acid titration was used to determine conditions where phase separation occurred. Critical structure forming events associated with the formation of soluble and insoluble complexes in a 1:1 PPIn-AL mixture were found to occur at pH 5.00 and 2.98, respectively, with optimal interactions occurring at pH 2.10. As the PPIn-AL ratio increased, critical pH values shifted towards higher pH until a mixing ratio between 4:1 and 8:1was reached, above which structure formation became independent of the ratios through to ratios of 20:1. Electrophoretic mobility measurements showed a similar trend, where the isoelectric point (pI) shifted from pH 4.00 (homogeneous PPIn) to pH 1.55 (1:1 PPIn-AL). As the ratio increased towards 8:1 PPIn-AL, net neutrality values shifted to higher pHs (~3.80) before becoming constant at higher ratios. Maximum coacervate formation occurred at a mixing ratio of 4:1. Based on these findings, capsule design by segregative phase separation was only used in future studies, due to the acidic nature associated with associative phase separation.<p> In study 2, capsule formation using a native and commercial PPI was studied, and showed no difference between the two formulations during challenge experiments in simulated gastric juice (SGJ). As a result study 3 focused on optimization and characterization of capsules prepared using the commercial PPI. Capsule designs were investigated as a function of protein concentration, prebiotic level, and extrusion conditions (20 vs. 27 G needle) in order to determine protective ability for B. adolescentis within SGJ. Capsule designs were also measured in terms of protein and prebiotic retention during the encapsulation process, geometric mean diameter and size distribution, swelling behaviour and release characteristics within simulated intestinal fluids (SIF). All capsules provided adequate protection over the 2 h duration within SGJ. Capsule breakdown and release was similar for all designs within SIF, with a release mechanism believed to be tied to enzymatic degradation of the PPI material within the wall matrix and/or the amount of excessive Na+ present in the SIF. Capsule size was found to be dependent only on the needle gauge used in the extrusion process. Swelling behaviour of the capsules with SGJ was also found to be dependent only on the protein concentration, where capsules shrank once immersed in SGJ.<p> A 2.0% PPI-0.5% AL capsule without FOS and extruded through a 20 G needle represents the best and most cost effective design for entrapping, protecting and delivering probiotic bacteria. Future work to establish the role FOS could play post-release as the entrapping probiotics colonize the GI tract, and the protective effect of the capsules wall on FOS structure during transit is recommended.

Bifidobacterium infantis (BI) and other probiotics are non-pathogenic living organisms that have recently gained attention for their possible therapeutic implications on the health of the digestive tract. The mechanisms by which probiotics exert their effects are largely unknown. This study explored the protective and regulatory effect of oral BI on the enteric nervous system (ENS) in the TNBS-induced colitis rats. Electrical field stimulation and chemical stimulation by serotonin (5-HT) were used to elicit changes in the short-circuit current (Isc) response of the colonic rat tissue. BI-fed colitis rats expressed trends of higher secretomotor activity and revealed signs of decreased macroscopic inflammatory damage when compared to sham-fed colitis rats, suggesting a protective and preventative role of oral BI. These findings may provide additional insights for understanding the prophylactic and therapeutic value of specific probiotics in intestinal inflammatory disorders, offering the possibility of a noninvasive alternative to toxic and immune-compromising drugs. / Access to thesis permanently restricted to Ball State community only / Department of Physiology and Health Science

La detecció de l’orgien de la contaminació fecal a l’aigua perment millorar la gestió del medi i mitigar la contaminació en l’origen recuperant amb més eficiència les zones afectades; a més a més permet estimar el risc sanitari que porta associat; prendre les mesures legals oportunes i establir responsabilitat econòmiques. Fins a l’actualitat s’han descrit un elevat número de tècniques i marcadors que indiquen la presència de contaminació i permeten conèixer el seu origen. Abans de la implementació d’aquestes metodologies cal conèixer altres factors. Aquests mètodes s’han definit en una determinada àrea demogràfica i per una determinada població, per tant, cal conèixer si existeix variació geogràfica i temporal, saber-ne la prevalència i persistència al medi, valorar la distribució entre espècies, materialitzar procediments stàndars que puguin ser utilitzats en qualsevol laboratori. A més a més, estudis previs han suggerit que cap dels indicadors de l’origen de la contaminació fecal presenta un 100% d’eficiència i especifitat, així que és necessària la combinació de varis d’ells per tal d’incrementar la discriminació entre diferents oríigens. Aquesta treball consisteix en tres parts diferenciades que volen aprofundir ens les mancances dels mètodes per a determinar l’origen de la contaminació. En la primera part (Capítol 2) s’ha valorat 10 marcadors diferents descrits prèviament: dues espècies del gènere Bifidobacterium marcadores de contaminació humana: Bif. adolescentis i Bif. dentium, dos marcadors específics d’humans (HF183 i HF134) i dos de rumiants (CF128 i CF193) que detecten espècies no cultivades del grup Bacteroidetes, un marcador del gen esp de soques d’Enterococcus faecium específiques d’humans i 3 marcadors d’ADN mitocondrial de cèl•lules humanes, bovines i porcines. Tot i que alguns dels marcadors moleculars analitzats presenten una elevada sensibilitat i especificitat, cap d’ells assegura un 100% d’encert. Això ha fer que es valorés la possibilitat de desenvolupar models predictius amb combinacions d’ells. D’aquesta manera s’ha obtingut una discriminació del 79,6% entre 4 oríigens diferents: humà, boví, porcí i avícola i d’un 90,1% distingint entre contaminació d’origen humà o no humà. A la segona part, veient la importància de Bif. adolescentis i Bif. dentium com a marcadors de contaminació fecal humana, s’ha utilitzat la tècnica de DGGE per tal d’aprofundir en l’ecologia del gènere. S’ha analitzat la distribució d’espècies en mostres d’aigua residual de diferents orígens. Bif. adolescentis s’ha confirmat com una de les espècies majoritàries en mostres d’origen humà. A la vegada, s’han definit espècies majoritàries en mostres d’origen boví com Bif. pseudolongum ssp. pseudolongum i Bif. thermophilum. Aquesta espècie també ha estat majoritària en porcs juntament amb una espècie que no identificada prèviament. En aus s’ha detectat una sola espècie identificada com Bif. saeculare. En els últims anys s’han dissenyat un elevat nombre de PCRs i q-PCRs específiques d’espècies no cultivades del grup Bacteroidetes per discriminar entre diferents hostes. En la tercera part, s’ha volgut aprofundir en el coneixement de la persistència del gènere Bacteroides i conèixer els factors ambientals que afecten la seva supervivència. Mitjançant experiments in situ al riu Llobregat s’ha observat la persistència de les soques tipus Bact. fragilis i Bact. thetaiotaomicron, majoritàries en aparell digestiu humà i espècies ambientals de l’aigua residual. S’ha realitzat el seguiment de la supervivència mitjançant tècniques de cultiu i tècniques moleculars. S’ha observat una major supervivència de Bact. fragilis a baixes temperatures que altes. Bact. thetaiotaomicron ha mostrat el contrari, major supervivència a l’estiu quan s’ha registrat una menor concentració d’oxigen dissolt a l’aigua. En aquesta espècie s’ha descrit prèviament una menor tolerància a l’oxigen. La presència de predadors a l’aigua també afecta de manera important la supervivència del gènere. / Faecal pollution is one of the main causes of degradation of surface and coastal water quality. Faecal pollution can be attributed to different sources, for example urban sewage, sewer misconnections, slaughterhouse wastewaters, manure run-off and solid disposals. The principal faecal bacterial indicators E. coli and Enterococcus are used to show the presence of pollution as well as the faecal load in water. However, these indicators do not discriminate between the possible sources of pollution. During the last decade many authors have described different methodologies based on molecular tools to detect different Microbial Source Tracking (MST) markers. However there is a lack of knowledge about the behaviour of the markers when they appear in the environment. Usually these markers have been described in a specific geographical area. For this reason their geographical and temporal stability has to be tested. Other aspects such as their prevalence and persistence in the aquatic environment must be known and standard operating procedures must be determined. The current project aimed to address these questions. 144 samples of different human and animal sources around Catalonia were collected in order to evaluate the applicability of some described MST markers, e.g. Bifidobacterium adolescentis, Bif. dentium, Bacteroides species human and ruminant specifics, esp gene of Ent. faecium and mitochondrial DNA of human, bovine and swine cells. The specificity and sensitivity of the markers was determined. Each marker alone does not give 100% source discrimination and for that reason predictive models were developed. These models increased the capacity of source discrimination using the lowest possible number of markers. Bif. adolescentis and Bif. dentium have been described as human specific species. Denaturant gradient gel electrophoresis technique was used to broaden the knowledge of Bifidobacterium distribution among different animal sources and to describe new species specificity to enable their uses as new MST markers. In addition, Bacteroidetes species have shown themselves to be capable MST markers. How environmental factors such as temperature and dissolved oxygen affect their survival in the environment has been studied in order to predict the durability of the markers in these environments. For this purpose, molecular techniques such as PCR and Q-PCR and cultivable methodologies combined with colony hybridization were used.

Thesis (Ph.D.) -- University of Western Sydney, 2003. / "A thesis submitted for the degree of Doctor of Philosophy" Chief supervisor, Kaila Kailasapathy. Includes bibliography.

Thesis (M.S.) -- Mississippi State University. Department of Food Science, Nutrition, and Health Promotion. / Title from title screen. Includes bibliographical references.

Thesis (M.S.)--University of Missouri-Columbia, 2006. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on August 29, 2007) Includes bibliographical references.

In my study I was examining the influence of the probiotic Bifidobacterium sp. on the functional status of the cattle´s rumen. Two adult cows Aberdeen-angus were used in this experiment. They were treated with a permanent cannula, which served for daily dosing of probiotics Bifidobacterium sp.. Samples of rumen fluid were analyzed for the amount of volatile fatty acids, protozoans, pH and the quantity of ammonia. When we tested the effect of the probiotics on each variable, the fixed effect of the influence of an individual has not been proved. When we tested the data without the effect of the individual in a linear model, the variables best describing my data were the butyric and acetic acids. The amount of protozoans increased as there levels grew. There is a strong effect of the individual as only two individuals were used. My results indicate that the influence of the probiotics Bifidobacterium sp., on the functional status of the rumen is low. These results could be affected by the low number of experiment-replication as well as by small quantity of tested animals.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro Tecnológico, Programa de Pós-Graduação em Ciência dos Alimentos, Florianópolis, 2014. / Made available in DSpace on 2015-02-05T20:31:18Z (GMT). No. of bitstreams: 1 330909.pdf: 1456399 bytes, checksum: b6b137adf35fc650c8b0612f0e370a6d (MD5) Previous issue date: 2014 / Este trabalho visou elucidar o efeito da adição de Bifidobacterium BB-12 em um queijo tipo Minas Frescal do leite de búfala. Para avaliar a influência da adição das bifidobactérias no queijo foram realizadas a contagem de células viáveis e análises físico-químicas, de sinérese, de cor, reológicas e microestruturais, por 30 dias de armazenamento. Para o estudo das propriedades reológicas foram avaliados os seguintes modelos: os mecânicos de Maxwell e Maxwell generalizado e o modelo empírico de Peleg. O queijo adicionado de bifidobactérias foi submetido às condições gastrintestinais simuladas in vitro (boca, esôfago-estômago, duodeno e íleo continuamente) a fim de determinar o efeito dessa matriz sobre a sobrevivência das bifidobactérias. A contagem de células viáveis de Bifidobacterium BB-12 no queijo foi de 8,15 log UFC/g no dia 1 e 8,36 log UFC/g no dia 30. A partir dos resultados obtidos, pôde-se observar que ao final de 30 dias de armazenamento os queijos contendo as bifidobactérias foram classificados como probióticos. As amostras de queijo controle e probiótico apresentaram aumento nos valores da sinérese de 2,58±0,09 g/100g no dia 1 para 4,66±0,01 g/100g no dia 30 e de 2,16±0,16 g/100g no dia 1 para 4,04±0,05 g/100g no dia 30, respectivamente. Os demais parâmetros físicos permaneceram inalterados. Os queijos mostraram tendência à cor branca (L*=89). O modelo de Maxwell generalizado (R²>0,99) ajustou-se melhor aos dados experimentais do que os modelos de Maxwell (R²<0,96) e de Peleg (R²<0,95). A adição de bifidobactérias e o tempo de armazenamento dos queijos tipo Minas Frescal do leite de búfala não afetaram as suas propriedades reológicas. Estas propriedades indicaram uma tendência à obtenção de queijos rígidos e elásticos, mostrando também tendência a ser mais elástico do que viscoso. Já, o efeito protetor do queijo de leite de búfala sobre as bifidobactérias foi observado durante as condições gastrointestinais simuladas, inclusive quanto à recuperação das células injuriadas após a etapa do duodeno (109,55±2,39%). Desta forma, o queijo tipo Minas Frescal do leite de búfala apresentou efeito protetor para Bifidobacterium BB-12 durante a simulação gastrointestinal in vitro, tornando-se assim um promissor carreador desta bactéria probiótica.<br> / Abstract : This study aimed to elucidate the effect of the Bifidobacterium BB-12 addition in a buffalo Minas Frescal cheese. To evaluate the influence of the addition of bifidobacteria in cheese the following analyzes were performed: Viability, physicochemical properties, syneresis, color, rheological and microstructural properties, for 30 days of storage. To study the rheological properties three mathematical models were tested for fit the relaxation data, the mechanical Maxwell model and Generalized Maxwell model and the empirical Peleg model. Subsequently, the cheese added with bifidobacteria was submitted to in vitro simulated gastrointestinal conditions (mouth, esophagus-stomach, duodenum and ileum, continuously) to evaluate the effect of this matrix on the survival of the bacteria. The count of viable Bifidobacterium BB-12 cells in the cheese was equal to 8.15 log CFU/g on day 1 and 8.36 log CFU/g on the day 30. From the results obtained, we observed that at the end of the storage time the sample containing bifidobacteria was classified as probiotic. The control and probiotic cheese samples, showed an increase on the syneresis values from 2.58±0.09 g/100g on day 1 to 4.66±0.01 g/100g on day 30 and from 2.16±0.16 g/100g on day 1 to 4.04±0.05 g/100g on day 30, respectively. The other physical parameters remained unchanged. The cheeses also showed a tendency to white color (L*=89). The generalized Maxwell model (R²>0.99) was adjusted to the experimental data better than the models of Maxwell (R²<0.96) and Peleg (R²<0.95). As expected, the addition of bifidobacteria and the storage time of Buffalo Minas Frescal cheese did not affect their rheological properties. These properties indicated a tendency to obtain stiff and elastic cheeses, showing a tendency to be more elastic than viscous. Already, the protective effect of the Buffalo Minas Frescal cheese on bifidobacteria was observed during simulated gastrointestinal conditions, including the recovery of injured cells after the duodenum step (109.55±2.39%). Thus, Buffalo Minas Frescal cheese appeared as a protector for Bifidobacterium BB-12 during the in vitro gastrointestinal simulation, making it a promising carrier for this probiotic bacterium.