No description available.

Oral administration of live probiotics such as Lactobacillus and Bifidobacterium spp. possess numerous beneficial effects. However, delivering viable probiotics to the host intestine has been a challenge due to poor survival of these bacteria during the gastric transit. An improved oral delivery system (modified alginate microcapsules) was developed in this study for targeted release of viable probiotics to the host intestine. Effect of various encapsulation parameters such as capsule size, alginate concentration, calcium chloride concentration, gelling/hardening time of microcapsules, addition of prebiotics and polymer coating, were individually investigated for improving the stability of microcapsules under simulated gastrointestinal (GI) conditions. Ability of microcapsules in protecting the viability of encapsulated bacteria improved significantly (p<0.05) in improving the stability of microcapsules. Optimisation of encapsulation parameters significantly improved the viability of encapsulated probiotics under simulated GI conditions. Furthermore, co-encapsulation of probiotics with complementary prebiotics (such as Hi-Maize starch) and chitosan coating provided additional protection to the encapsulated bacteria under simulated GI conditions. Release profile of chitosan-coated alginate-starch (CCAS) encapsulated bacteria was investigated in the GI tracts of different animal models. Addition of CCAS encapsulated bacteria to porcine GI contents (ex vivo) resulted in complete release of microencapsulated bacteria in the ileal contents within 8 h, while there was no significant release (p>0.05) of encapsulated bacteria in the gastric contents even after 24 h of incubation. In another experiment, CCAS microcapsules containing Lactobacillus casei Shirota (LCS) was orally administered to mice and the release profile of encapsulated bacteria was monitored throughout the murine GI tract for 24 h. Partial release of microencapsulated LCS was observed in duodenal and jejunal regions, while no significant (p>0.05) release of microencapsulated bacteria was observed in the stomach during the 24 h monitoring period. However, a significant release (nearly complete release) of microencapsulated bacteria was observed in ileal and colon of murine GI tract after 24 h. Elevated counts of LCS in ileum and colon indicated the most favorable site for the release of CCAS encapsulated bacteria. Further studies investigated the immunomodulatory activity of microencapsulated probiotic bacteria in a murine model. Lactobacillus casei Shirota was orally administered to mice either as microencapsulated or as free bacteria (non encapsulated) for two weeks. On day 14, the splenocytes from different experimental groups were harvested and assessed for ConA induced cytokine levels. A significant increase (p>0.05) in IFN-γ levels was observed in the activated splenocytes of groups treated with microencapsulated and free (non-encapsulated) LCS, compared to the control group (no LCS treatment). However, there was no significant difference (p<0.05) in the IFN-γ concentration between the groups treated with microencapsulated and free (non-encapsulated) LCS. No significant difference (p<0.05) in the IL-10 concentration was observed in the activated splenocytes of groups treated with microencapsulated and free (non-encapsulated) LCS. Finally, the stability of microencapsulated probiotics in different dairy products was investigated. CCAS microcapsules significantly protected the viability of probiotic bacteria in set and stirred yoghurts over 6-week refrigerated storage conditions compared to free (non-encapsulated) probiotics. Overall, chitosan-coated alginate-starch microcapsules developed in this study effectively protected the viability of probiotics from adverse gastric conditions and released the bacteria in the host intestine without detrimentally affecting its immunomodulatory properties. / Doctor of Philosophy

The regulatory standards set by food authorities globally for probiotic foods such as Cheddar cheese makes it essential to have reliable enumeration media that will accurately monitor the survival of probiotic bacteria over the shelf life of Cheddar cheese. This study therefore investigated various selective and differential media for reliable enumeration of Lactobacillus acidophilus, Bifidobacterium spp., starter lactic acid bacteria (SLAB) and non-starter lactic acid bacteria (NSALB) from Cheddar cheese using pure cultures and commercial probiotic Cheddar cheese. All media showed variation in counts and selectivity. Some reported selective media failed to inhibit SLAB and NSLAB. The media that were reliable and also gave good recovery were, Reinforced Clostridium Agar with Bromocresol green and Clindamycin (RCABC), which was selective for L. acidophilus spp. and Reinforced Clostridium Agar with Aniline blue and Dicloxacillin (RCAAD), which was differential for Bifidobacterium spp. and SLAB. Reinforced Clostridium Agar with Bromocresol green and Vancomycin (RCABV) was found suitable for NSLAB. Additionally, an enzyme based colorimetric assay was modified successfully and used as a confirmatory test to check the presence of bifidobacterial colonies on enumeration media. Six batches of probiotic Cheddar cheese were manufactured with the incorporation of LAFTI L10 (L. acidophilus) and LAFTI B94 (B. lactis). The survival of probiotic bacteria, SLAB and NSLAB were monitored over a six-month ripening period using the selected media. The survival of free probiotic bacteria throughout the ripening process decreased consistently in all the six batches. In order to enhance the survival of probiotic bacteria, the effect of microencapsulation on the viability of LAFTI L10 and LAFTI B94 in Cheddar cheese was studied. Six batches of Cheddar cheese were manufactured with the incorporation of alginate-starch encapsulated and free cells of LAFTI L10 and LAFTI B94. The survival of both the encapsulated and free probiotic bacteria was studied over a six month ripening period. The survival of encapsulated LAFTI L10 and LAFTI B94 (107 cfu/g) was found to be significantly better than that of free bacteria (105 cfu/g) at the end of six months of ripening period in Cheddar cheese. / Master of Applied Science (M. App. Sci.) (Biotechnology)

Among 29 strains of bifidobacteria studied as sources of beta-galactosidase enzyme, Bifidobacterium infantis HL96 showed the highest hydrolytic and transgalactosylic activities. This strain grew well in a MRS medium containing various sugars including lactose, and produced three beta-galactosidases (termed beta-Gal I, II, III). / Two genes, beta-galI and beta-galIII, located on 4.6 and 4.4 kb DNA fragments respectively, were cloned into E. coli, and the nucleotide sequences were determined. The 3,069 by-long beta-galI, encoded a polypeptide with a Mr of 113 kDa. A putative ribosome-binding site and a promoter sequence were recognized at the 5' flanking region of beta-galI. A partial sequence of an ORF transcribing divergently from beta-galI resembled a lactose permease gene. The beta-galIII gene, which is 2,076 bp long, encoded a polypeptide with a Mr of 76 kDa. A rho-independent, transcription terminator-like sequence was found 25 bp downstream of the termination codon. / The amino acid sequences of beta-GalI and beta-GalIII were homologous to those in the LacZ and LacG families, respectively. The acid-base, nucleophilic, and substrate recognition sites conserved in the LacZ family were found in beta-GalI, and a possible acid-base site proposed for the LacG family was located in beta-GalIII, containing a glutamate at residue 160. beta-GalI and beta-GalIII were over-expressed 35 and 96 times respectively in E. coli by using a pET expression system. / Both beta-GalI and beta-GalIII were specific for beta-D -anomeric linked galactosides, but beta-GalI showed more hydrolytic and synthetic activities toward lactose than beta-GalIII. The galacto-oligosaccharides (GaOS) production mediated by beta-GalI at 37°C in 20% (w/v) lactose was 130 mg/ml, which is six times higher than that of beta-GalIII. The yield of GaOS further increased to 190 mg/ml in 30% (w/v) lactose. A major tri-saccharide produced by beta-GalI was characterized as O-beta- D-galactopyranosyl-(1-3)-O-beta-D-galactopyranosyl-(1-4)- D-glucopyranose. / beta-GalI was purified by ammonium sulphate precipitation, and anion-exchange (Mono-Q) and gel filtration (Superose 12) chromatographic steps. The enzyme appeared to be a tetramer, with a Mr of 470 kDa as estimated by native PAGE and gel-filtration chromatography. The optimum temperature and pH for ONPG and lactose as substrates were 60°C, pH 7.5, and 50°C, pH 7.5, respectively. The enzyme was stable over the pH range of 5~8.5, and was particularly active at 50°C for more than 80 min. The enzyme was significantly activated by reducing agents, especially glutathione, as well as by Na+ and K+ cations. Maximal activity required both Na+ and K+ at a concentration of 10 mM. The enzyme was strongly inhibited by p-chloromercuribenzoic acid, and by most bivalent metal ions. Hydrolytic activity using 20 mM lactose as substrate was significantly inhibited by 10 mM galactose. The Km and Vmax values for ONPG and lactose were 2.6 mM, 262 U/mg, and 73.8 mM, 1.28 U/mg, respectively. / The objectives of this research were to characterize beta-galactosidases of B. infantis HL96 at the molecular and biochemical levels, and to over-express the enzymes in Escherichia coli. Two beta-galactosidase isoenzymes with unique properties were genetically characterized for the first time. beta-GalI properties included a neutral pH optimum, relatively higher temperature stability and a high transgalactosylic activity that makes it very competitive for GaOS synthesis. The results were also important for the advancement of knowledge on the catalytic mechanism and the evolutionary aspect of this enzyme.

Three different types (A, B, and C) of bile salt hydrolase from different Bifidobacterium strains revealed during the purification study showed the type-specific characteristics in their electrophoretic migration and elution profiles from anion exchange and hydrophobic interaction chromatographic columns. The subunit molecular mass estimated by SDS-PAGE was around 35 kDa and the native molecular mass in all types of BSH was estimated to be between 130 and 150 kDa by gel filtration chromatography, indicating that all BSH enzymes have tetrameric structure. From the isoelectric focusing, pI value of 4.45 was obtained with type B, but type A and C BSHs showed similar pI values of around 4.65. While the N-terminal amino acid sequences of types A and B were highly homologous (19/20), six out of twenty amino acid residues were different in the N-terminal sequences of types A and C. / As the type A bsh gene was cloned from a strain of B. longum and the nucleotide sequence became available from the GenBank, our study has focused on the cloning and characterization of the type B and C bsh genes from Bifidobacterium strains. / The type B bsh gene was cloned from B. bifidum ATCC11863 and the DNA flanking the bsh gene was sequenced. The 951 by-long bsh gene encoded a 316-amino-acid protein with a molecular mass of 35 kDa and a pI of 4.48. For the first time in the genus Bifidobacterium, the transcriptional start point of the bsh gene was identified by primer extension analysis. Furthermore, Northern blot analysis revealed that B. bifidum bsh was transcribed as a monocistronic unit, contrary to that of B. longum bsh. Despite a high level of sequence similarity among the bsh genes, a BSH type-specific primer set based on the variable regions of bsh genes was designed in order to differentiate B. bifidum strains from the other species of Bifidobacterium commonly detected in the human gut. / The type C bsh gene was cloned from a bile salt tolerant strain of Bifidobacterium and the DNA flanking the bsh gene was further identified by a thermal asymmetric interlaced PCR (TAIL-PCR) technique. The 945 by-long bsh gene encoded a 314-amino-acid protein with a molecular mass of 35 kDa and a pI of 4.71. A predicted BSH promoter (Pbsh) sequence was experimentally verified and the transcriptional start point of the type C bsh gene was determined by primer extension analysis. An operonic structure including the type C bsh gene and two more ORFs, which were found within a complete set of a promoter and a transcription terminator, was identified in this study for the first time in the genus Bifidobacterium, and the polycistronic bsh transcript was revealed by RT-PCR and Northern blot analysis.

A beta-galactosidase gene from Bifidobacterium breve B24 which showed the higher hydrolytic and synthetic activity was cloned in E. coli. The complete beta-galactosidase gene contained 2076 bp nucleotides and encoded 691 amino acids which had a high homology to the other Bifidobacterium species. This beta-galactosidase was homologous to that of the LacA family. The galA gene was successfully over-expressed in E. coli ER2566. To observe any change in the recombinant enzyme, beta-galactosidases from Bifidobacterium breve B24 and recombinant E. coli ER2566 were purified to homogeneity by ion exchange chromatography (Mono-Q) and gel-filtration chromatography (Superose-12 and Superdex 200) columns. The molecular mass of both beta-galactosidases was estimated to be 75 kDa on SDS-PAGE. Activity staining on non-denaturing Native-PAGE and Superose-12 gel-filtration chromatography showed that the enzymes are composed of a dimer with a molecular mass of 150 kDa. / The optimum pHs of the native and recombinant enzymes for hydrolyzing O-nitrophenyl-beta-D-galactopyranose (ONPG) were pH 6.0 and 7.0, respectively, and they were stable over the pH range of 5-8 and 6-9, respectively. The optimum temperature of both enzymes for hydrolyzing ONPG was similar at 45 °C and they were stable over the temperature range of 20-45 °C. Both enzymes were stable up to 45 °C during 5 h of incubation at pH 6.5. The recombinant enzyme was slightly activated by bivalent metal ions, Mg2+, Mn2+, and Zn2+ at 1 mM but strongly inhibited by Hg2+ and p-chloromercuribenzoic acid (PCMB). The K m values of both native and recombinant beta-galactosidases for ONPG were 2.77 and 1.82 mM, respectively, and the Vmax values were 1.02 and 1.39 mM/min, respectively. / The two beta-galactosidase activities were also tested with lactose as substrate. The optimum pH of the native and recombinant enzymes for hydrolyzing lactose was similar at pH 6.0. Both enzymes had more than 80 % of their activity in the range of pH 6-8, indicating that the enzymes were stable at neutral pH. However, the native beta-galactosidase had around 40 % of its activity at pH 5.0, whereas the recombinant enzyme had no activity at this pH. On the other hand, the recombinant enzyme had over 50 % of its activity at pH 9.0, while the native beta-galactosidase showed lower than 5 % of its activity. The optimum temperature of both enzymes was at 45 °C. The profiles of both enzyme activities were very similar except at the temperature of 10 °C. The recombinant beta-galactosidase still had around 20 % of its enzyme activity at 10 °C, while no enzyme activity from the native enzyme was detected at this temperature.

The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on Oct. 6, 2009). Thesis advisor: Dr. Azlin Mustapha. Includes bibliographical references.

PORTELA, Masu Capistrano Camurça. Produção de sorvete dietético de leite de cabra, com característica simbiótica e avaliação do seu efeito funcional. 2015. 164f. Tese (Doutorado em Biotecnologia)- Universidade Federal do Ceará, Fortaleza, 2015. / Submitted by Weslayne Nunes de Sales (weslaynesales@ufc.br) on 2017-05-19T12:59:59Z No. of bitstreams: 1 2015_tese_mccportela.pdf: 3083384 bytes, checksum: 03138dcd593d91f95f39830715d9bba4 (MD5) / Approved for entry into archive by Jairo Viana (jairo@ufc.br) on 2017-05-19T20:09:48Z (GMT) No. of bitstreams: 1 2015_tese_mccportela.pdf: 3083384 bytes, checksum: 03138dcd593d91f95f39830715d9bba4 (MD5) / Made available in DSpace on 2017-05-19T20:09:48Z (GMT). No. of bitstreams: 1 2015_tese_mccportela.pdf: 3083384 bytes, checksum: 03138dcd593d91f95f39830715d9bba4 (MD5) Previous issue date: 2015 / The aim of the present study was to develop symbiotic ice creams formulations, goat milk using inulin and/or frutooligosaccharides (FOS), and Bifidobacterium animalis subsp. lactis (Bb12), and to evaluate the effect of the added prebiotics on their technological characteristics, probiotic viability, and sensory acceptability: overrun, instrumental hardness and melting profile. The resistance of the probiotic incorporated in the ice cream matrix to in vitro simulated gastrointestinal (GIT) conditions was evaluated. Additionally, the post prandial glicemic response of the most accepted ice-cream formulation, based on the sensory acceptability scores, was evaluated among healthy (control n = 8) and diabetic individuals (experimental n = 6). Four ice-cream formulations were manufactured, denoted: SC (control ice-cream, containing polydextrose), SF (with 6% FOS, and Bb12), SI (with 6% inulin, and Bb12), and SFI (with 3% FOS, 3% inulin, and Bb12). The ice-creams were stored at -18 °C for up to 84 days. At the end of the storage period, the SI formulation had its instrumental hardness, melting profile and overrun increased compared to the SF ice-cream. All the diet ice-cream formulations obtained good acceptability scores, mainly the SI, which was the one chosen for the sensory evaluation within a diabetic group. The diabetic group average scores for the SI ice cream were higher than the ones abtained among the healthy painellists. The probiotic Bb12 in vitro survival test was developed during the period of at 15 and 84 days.The populations of the probiotic Bb12 in the SI, SF and SFI ice-cream were higher than 8.0 log UFC/g during the whole studied storage period, and the pH values of all the ice-creams was mantained around 6.0. In average, the survival rate of the probiotic Bb12 after the exposition to GIT conditions was 74%, and the prebiotics inulin and FOS contributed to Bb12 resistance. The glicemic response of the control group showed a significant difference (p<0,01) in lowering the glicemic answer compared to glucose and the post prandial levels of the experimental group were lower after the ice-cream consumption, although there was not a significant difference compared to glucose (p>0,05). The addition of 6% of inulin to the diet ice-cream (SI) showed a hipoglicemic effect, especially among the control group. The developed ice-cream formulations showed to be good vehicles to Bb12, being in conformity to the Brazilian legislation for probiotic products and maintaining high populations after the exposure to in vitro simulated GIT conditions. This work corroborates the literature data showing the prebiotic inulin and FOS as potencially texturizing ingredients, adequate to the production of simbiotic diet ice-creams with a good sensory acceptability. The new product can contributes, if offered in large scale, for the availability of diet and functional ice-cream for consumers choice. / O objetivo desse trabalho foi desenvolver sorvete de leite de cabra adicionado de inulina e ou frutooligossacarídeos (FOS) e Bifidobacterium animalis subsp. lactis (Bb12) e avaliar o efeito da adição de inulina e FOS sobre as características tecnológicas e de aceitabilidade do sorvete dietético simbiótico: overrun, dureza instrumental e fração de derretimento. A sobrevivência da Bb12 no sorvete funcional durante o período de armazenamento e frente às condições encontradas no trato gastrointestinal simulados in vitro também foram estudadas. Adicionalmente, foi avaliada a resposta glicêmica pós-prandial do sorvete dietético simbiótico mais aceito com um grupo saudável (controle n = 8) e um grupo de diabéticos (experimental n = 6). Foram produzidas 4 formulações do sorvete: SC (denominado sorvete controle = + polidextrose, - inulina, - FOS, - Bb12); SF (+ 6%FOS, + Bb12), SI (+ 6%Inulina, + Bb12); SFI (+ 3%FOS, + 3%Inulina, + Bb12). Após seus processamentos, os sorvetes foram armazenados a -18 °C durante 84 dias. Ao final desse período os sorvetes contendo inulina aumentaram a dureza instrumental, teste de derretimento e overrun comparados ao SF. Todas as formulações de sorvetes dietéticos obtiveram boa aceitação sensorial, com destaque para o sorvete contendo inulina que foi a formulação selecionada para a sensorial dos diabéticos. As notas sensoriais dos diabéticos foram maiores em relação às notas descritas acima dos voluntários saudáveis. O ensaio in vitro de sobrevivência da Bb12 foi desenvolvido no período de 15 e 84 dias. A população de Bb12 nos sorvetes manteve-se superior a 8 log UFC/g durante o período de estocagem estudado e o pH de todos os sorvetes manteve-se em torno de 6,0. A inulina e os FOS contribuíram para a resistência in vitro em média de 74% de sobrevivência. A resposta glicêmica do grupo controle apresentou diferença significativa (p<0,01) na redução da resposta glicêmica comparado à glicose e do grupo experimental, os níveis de glicemia pós-prandiais foram menores após o consumo do sorvete, mas não obteve diferença significativa em relação à glicose (p>0,05). A adição de 6% de inulina no sorvete dietético (SI) demonstrou efeito hipoglicêmico, principalmente no grupo controle. Os sorvetes desenvolvidos mostraram-se bons veículos para a Bb12, atendendo à legislação brasileira para produtos probióticos e apresentaram habilidade de resistência in vitro às condições gastrointestinais simuladas. Este trabalho, confirma os dados da literatura que apontam os prebióticos inulina e FOS como ingredientes potenciais texturizantes, indicados na elaboração de sorvetes dietéticos simbióticos, e de boa aceitação sensorial. Este novo produto formulado pode contribuir, se ofertado em grande escala, na variedade da dieta dos diabéticos.

No description available.

No description available.