Orientador: Glaucia Maria Pastore / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-08T05:10:05Z (GMT). No. of bitstreams: 1 Santos_Rosangelados_M.pdf: 814194 bytes, checksum: 40aac8890d5062af1c1b42f9e06c6d35 (MD5) Previous issue date: 2006 / Resumo: Os galactooligossacarídeos (GOS), um grupo de oligossacarídeos, são carboidratos não digeríveis (NDOs), resistentes à hidrólise das enzimas digestivas do intestino, tem os efeitos fisiológicos similares ao da fibra dietética. Sua ingestão aumenta a proliferação das Bifidobacterium e dos Lactobacilus no intestino. Eles são considerados como ingredientes prebióticos. Devido aos possíveis benefícios à saúde, associados com o consumo destes compostos, seu uso como Alimento Funcional tem crescido rapidamente, particularmente no Japão e Europa. Este trabalho teve como objetivo a extração da enzima _-galactosidase a partir do microrganismo Scopulariopsis sp, de avaliar o efeito da temperatura, tempo de reação, concentração de enzima e lactose, de modo a obter condições mais eficientes para a produção de GOS, além disso, comparar esses resultados com os GOS produzidos com a enzima comercial obtida por Aspergillus oryzae. Como resultado, observamos que a linhagem de Scopulariopsis sp sintetizou grandes quantidades de b-galactosidase por fermentação semi-sólida, que foi produzida constitutivamente. A enzima semi-purificada foi precipitada de etanol 70%. O extrato foi caracterizado bioquimicamente, mostrando ter pH ótimo de 5,0 e a melhor temperatura a 45ºC, para atividade de transgalactosilação. A enzima obtida a partir de Scopulariopsis sp converteu 20% de lactose em oligossacarídeos (80.8mg/mL de 4¿galactosyl-lactose), comparando com a _- galactosidase de Aspergillus oryzae, que converteu 6% de lactose em oligossacarídeos (25.6mg/mL de 4¿galactosyl-lactose), utilizando lactose 40% (p/v), à 45ºC, pH 5.0 e 10U/mL e o melhor tempo foi o de 12h de reação. Também houve a produção de outro galactooligossacarídeo de estrutura não identificada ainda, sendo necessário estudos adicionais que elucidem a estrutura e atividade destes GOS / Abstract: The galactooligosaccharides (GOS), a group of oligosaccharide, are not digerible carbohydrates (NDOs), they are resistant to hydrolysis by digestive enzymes from intestine and have similar dietary fiber physiological effect. Its ingestion increases the Bifidobacterium and Lactobacillus proliferation in the intestine. They are considered prebiotic ingredients. Due to the possible health benefits associated with the consumption of these compounds, their use as functional ingredients has grown quickly, particularly in Japan and Europe. They aim of this work was to extract the _-galactosidase from Scopulariopsis sp, and to evaluate the temperature conditions, reaction time, lactose and enzyme concentration, to improve the GOS production, beyond that, to make a comparison with the GOS obtained by commercial enzyme produced by Aspergillus oryzae. As result the Scopulariopsis sp strain had, accumulated great amounts of _- galactosidase on semisolid fermentation and the enzyme was produced continuosly. The enzyme solution was precipitated by using 70% ethanol. The extract was biochemically characterized showing the otimum pH of 5.0, and the best temperature was 45ºC, for transgalactosylation activity. The enzyme isolated from Scopulariopsis sp has converted 20% of lactose into oligosacharides (80.8mg/mL of 4'galactosyl-lactose), comparing with _-galactosidase from Aspergillus oryzae, a commercial enzyme, that converted 6% of lactose into oligosacharides (25.6 mg/mL of 4'galactosyl-lactose), using lactose 40% (p/v), at 45ºC, pH 5.0 and 10U/mL, and the best reaction time was at 12h of reaction. We also observed the production of other galactooligosaccharide with nonidentified structure, been necessary additional research to discover their structure and activity / Mestrado / Mestre em Ciência de Alimentos

No description available.

Die vorliegende Arbeit hatte vorrangig zum Ziel, die enzymatische Synthese von prebiotischen Galactooligosacchariden (GOS) in Süß- und Sauermolke unter Nutzung verschiedener Enzymquellen zu evaluieren. Aufgrund des tendenziell weltweit steigenden Molkenaufkommens besteht großes Interesse, eine möglichst ganzheitliche Wertschöpfung dieses teilweise ungenutzten Rohstoffs zu generieren. Eine Möglichkeit, um dies zu realisieren, wäre die Herstellung einer GOS-haltigen Molke und eine entsprechende Weiterverarbeitung zu GOS-haltigen Lebensmitteln. Durch die generell hohe Temperatur- und pH-Toleranz von GOS sind vielfältige Applikationsmöglichkeiten gegeben. Neben den kommerziell verfügbaren β-Galactosidasen aus Aspergillus oryzae und Kluyveromyces lactis wurden Enzyme aus Lactobacillus delbrueckii subsp. bulgaricus (kurz: L. bulgaricus) und Cryptococcus laurentii im Labormaßstab hergestellt und charakterisiert. Mit beiden Enzymen konnten höhere GOS-Ausbeuten in niedrig konzentrierten Lactoselösungen (Puffer als auch Molke) erhalten werden als mit den beiden kommerziellen Enzymen. Während die β-Galactosidase aus K. lactis stark vom umgebenden Medium beeinflusst wird, so zeigen alle anderen Enzyme eine gute Übertragbarkeit der Ergebnisse von Puffer auf das Substrat Süß- bzw. Sauermolke. Die höchste Transgalactosylierungsaktivität weist das Enzym aus C. laurentii mit Ausbeuten von ca. 50 % (inkl. GOS-Disaccharide) auf. Bei Verwendung der L. bulgaricus-Lactase können die Gesamtausbeute als auch die GOS-Zusammensetzung in Abhängigkeit von der gewählten Synthesetemperatur gezielt beeinflusst werden. Des Weiteren wurde der Einfluss einer parallel zur Synthese stattfindenden Glucose-Entfernung durch enzymatische Oxidation untersucht. Trotz tendenziell begünstigter Synthese von Tri- und höheren Oligosacchariden konnte die Ausbeute mit A. oryzae nicht signifikant gesteigert werden. Die Kopplung mit K. lactis führte zu einer signifikant verringerten Synthese von GOS-Disacchariden, wodurch die Ausbeute insgesamt sank. Der Einsatz von Glucose-Oxidase und Katalase ist demnach nur bei β-Galactosidasen empfehlenswert, welche vorrangig Tri- und kaum Disaccharide synthetisieren. Der Einsatz mehrerer β-Galactosidase-Enzyme stellte sich als vielversprechend heraus. In Abhängigkeit von den jeweils kombinierten Enzymen konnte die Ausbeute teilweise gesteigert werden. Positiv erwies sich eine sequentielle Kombination von A. oryzae und K. lactis im Sinne der Steigerung der Gesamtausbeute und der parallele Einsatz von A. oryzae und C. laurentii im Sinne der Erhöhung der Strukturdiversität der GOS-Mischung.:Inhaltsverzeichnis ABKÜRZUNGS- UND SYMBOLVERZEICHNIS IV ABBILDUNGSVERZEICHNIS VIII TABELLENVERZEICHNIS XIII 1 EINLEITUNG UND ZIELSTELLUNG 1 2 STAND DES WISSENS 4 2.1 Molke: Aufkommen, Inhaltsstoffe und Verwertungsmöglichkeiten 4 2.2 β-Galactosidasen 7 2.2.1 Aufbau und Eigenschaften 7 2.2.2 Inhibitoren und Aktivatoren in Milch und Molke 8 2.2.3 Einfluss von Glucose und Galactose 9 2.2.4 Kommerzielle β-Galactosidase-Präparate 11 2.3 Galactooligosaccharide 12 2.3.1 Definition und Syntheseweg 12 2.3.2 Einflussgrößen auf die Reaktion 14 2.3.2.1 Enzymquelle 14 2.3.2.2 Lactosekonzentration 16 2.3.2.3 Reaktionsbedingungen 17 2.3.2.4 Einfluss von Glucose und Galactose 21 2.3.3 GOS-Synthese in Milch und Molke 22 2.3.4 GOS-Synthese mit Lactobacillus sp. 26 2.3.5 GOS-Synthese mit Cryptococcus laurentii 26 2.3.6 GOS-Synthese mit mehreren β-Galactosidasen 27 2.3.7 Möglichkeiten der Glucose-Entfernung 27 2.3.8 Kommerziell erhältliche GOS-Produkte 28 2.3.9 Eigenschaften und Anwendung 30 2.3.10 Modifizierte GOS-Strukturen und GOS-Alternativen 32 3 MATERIAL UND METHODEN 40 3.1 Materialien 40 3.1.1 Verwendete Enzyme 40 3.1.2 Verwendete Mikroorganismen 40 3.1.3 Verwendete Molkeproben 40 3.1.4 Verwendete Geräte 40 3.2 Bestimmung der Inhaltsstoffe von Molke 40 3.3 Mikroorganismenkultivierung und Enzymgewinnung 41 3.3.1 Biochemische Analysenmethoden 41 3.3.2 Herstellung der Rohextrakte aus Lactobacillus sp. 41 3.3.3 Kultivierung im Labormaßstab 42 3.3.3.1 Lactobacillus bulgaricus LB4 42 3.3.3.2 Cryptococcus laurentii 43 3.3.4 Kultivierung im Fermentormaßstab (L. bulgaricus LB4) 43 3.4 Assays zur Bestimmung der Enzymaktivität 43 3.4.1 β-Galactosidase 43 3.4.2 Glucose-Oxidase 44 3.4.3 Katalase 44 3.4.4 Berechnung der Enzymaktivität 45 3.5 Enzymcharakterisierung 45 3.5.1 Temperatur- und pH-Optimum 45 3.5.2 Bestimmung von Aktivatoren und Inhibitoren 46 3.5.3 Enzymstabilität 46 3.6 Galactooligosaccharid-Synthese 47 3.6.1 Verwendung von kommerziellen β-Galactosidasen 47 3.6.2 Verwendung von β-Galactosidase aus Lactobacillus bulgaricus LB4 47 3.6.3 Verwendung von Cryptococcus laurentii-Zellen 47 3.6.4 Kopplung mit Glucose-Oxidase und Katalase 48 3.6.5 Kopplung mehrerer β-Galactosidasen 48 3.7 Galactooligosaccharid-Analytik 49 3.8 Statistische Auswertung 50 4 ERGEBNISSE UND DISKUSSION 51 4.1 Zusammensetzung der Molken 51 4.2 Charakterisierung der β-Galactosidasen 52 4.2.1 Temperatur- und pH-Optimum 52 4.2.2 Inhibitoren und Aktivatoren 55 4.2.3 Stabilität in Puffer und Molke 57 4.3 Synthese von Galactooligosacchariden 61 4.3.1 Synthese mit β-Galactosidase aus K. lactis 61 4.3.1.1 Synthese in Puffer 61 4.3.1.2 Synthese in Süßmolke 64 4.3.1.3 Synthese in Sauermolke 66 4.3.2 Synthese mit β-Galactosidase aus A. oryzae 67 4.3.2.1 Synthese in Puffer 67 4.3.2.2 Synthese in Süßmolke 69 4.3.2.3 Synthese in Sauermolke 70 4.3.3 Synthese mit β-Galactosidase aus L. bulgaricus LB4 73 4.3.3.1 Synthese in Puffer 73 4.3.3.2 Synthese in Süßmolke 76 4.3.4 Synthese mit C. laurentii-Zellen 78 4.3.4.1 Synthese in Puffer 78 4.3.4.2 Synthese in Sauermolke 80 4.3.5 Vergleich der untersuchen Enzyme und Substrate 83 4.3.6 Kopplung mit Glucose-Oxidase und Katalase 88 4.3.6.1 Charakterisierung von Glucose-Oxidase und Katalase 88 4.3.6.2 Einfluss simultaner Glucose-Entfernung auf die GOS-Ausbeute 90 4.3.6.3 Schlussfolgerungen 95 4.3.7 Kombination mehrerer β-Galactosidasen 95 4.3.7.1 Kopplung von A. oryzae und K. lactis 95 4.3.7.2 Kopplung von A. oryzae und C. laurentii 100 4.3.7.3 Schlussfolgerungen 105 4.4 Untersuchungen zur β-Galactosidase-Synthese mit L. bulgaricus LB4 106 4.4.1 Nährmedienumstellung auf koscher-/halal-zertifizierte Bestandteile 106 4.4.2 Kultivierung im Fermentormaßstab 111 5 ZUSAMMENFASSUNG UND AUSBLICK 114 6 LITERATURVERZEICHNIS 118 7 ANHANG 140 7.1 Anhang zu Kapitel 2 140 7.2 Anhang zu Kapitel 3 165 7.2.1 Verwendete Geräte 165 7.2.2 Zusammensetzung MRS-Medium 166 7.2.3 Wachstum von C. laurentii 167 7.2.4 Anfangslactosekonzentrationen 167 7.2.5 Katalaseaktivität 168 7.2.6 Vergleich GOS-Disaccharidanalytik 169 7.3 Anhang zu Kapitel 4 170 7.3.1 Temperaturabhängigkeit von verschiedenen β-Galactosidasen 170 7.3.2 Aktivatoren und Inhibitoren 171 7.3.3 Grafiken zur β-Galactosidase-Stabilität 173 7.3.4 Katalase-Stabilität 182 7.3.5 Aminosäuresequenz von β-Galactosidase aus L. bulgaricus LB4 183 7.3.6 Grafiken zur GOS-Synthese mit kommerziellen β-Galactosidasen 184 7.3.7 Grafiken zur GOS-Synthese mit β-Galactosidase aus L. bulgaricus LB4 188 7.3.8 Daten zur GOS-Synthese mit β-Galactosidase aus C. laurentii 190 7.3.9 Vergleich der maximalen GOS-Ausbeute in Puffer und Molke 191 7.3.10 Grafik zur GOS-Synthese mit β-Galactosidase/GOX/KAT 192 7.3.11 Grafiken zur GOS-Synthese mit mehreren β-Galactosidasen 193 7.3.12 Grafiken zur Kultivierung von L. bulgaricus LB4 194 / The prior aim of the present work was to study the synthesis of prebiotic galactooligosaccharides (GOS) in sweet and acid whey by using various enzyme origins. Worldwide, an increase in whey production can be observed, thus a complete usage of this partially wasted resource is preferable. This could be implemented by producing a GOS containing whey and its subsequent processing to GOS containing foods. Due to the high temperature stability over a wide pH range, manifold food applications are possible. Besides the commercial available β-galactosidases from Aspergillus oryzae and Kluyveromyces lactis, enzymes from Lactobacillus delbrueckii subsp. bulgaricus (short: L. bulgaricus) and Cryptococcus laurentii were produced on the laboratory scale and characterized accordingly. With both enzymes, higher GOS yields in low lactose solutions (buffer as well as whey) were achieved compared to the two commercial enzymes. While the β-galactosidase from K. lactis was strongly influenced by the surrounding environment, all other tested enzymes showed a good transferability of the results from buffer to sweet and acid whey, respectively. The enzyme from C. laurentii exhibited the highest affinity to transgalactosylation, the yield being about 50 % (including GOS disaccharides). When using the L. bulgaricus lactase, total GOS yield as well as GOS composition can be adjusted via the synthesis temperature. Furthermore, the effect of glucose depletion during GOS synthesis using enzymatic oxidation was examined. Although a tendency towards the synthesis of tri- and higher oligosaccharides was observed, GOS yield by A. oryzae was not significantly enhanced. The combination with the K. lactis enzyme led to a significantly reduced synthesis of GOS disaccharides, resulting in a decreased total GOS yield. Thus, the use of glucose oxidase and catalase is only beneficial for β-galactosidases, which have a preference for synthesizing trisaccharides, but less disaccharides. The use of more than one β-galactosidase has shown to be promising. Depending on the respective enzyme combinations, it was partially possible to enhance the GOS yield. Positive results in terms of increasing total GOS yield were obtained using a consecutive coupling of A. oryzae and K. lactis, while in terms of enhancing the structural diversity of the GOS mixture, a simultaneous combination of A. oryzae und C. laurentii led to the best results.:Inhaltsverzeichnis ABKÜRZUNGS- UND SYMBOLVERZEICHNIS IV ABBILDUNGSVERZEICHNIS VIII TABELLENVERZEICHNIS XIII 1 EINLEITUNG UND ZIELSTELLUNG 1 2 STAND DES WISSENS 4 2.1 Molke: Aufkommen, Inhaltsstoffe und Verwertungsmöglichkeiten 4 2.2 β-Galactosidasen 7 2.2.1 Aufbau und Eigenschaften 7 2.2.2 Inhibitoren und Aktivatoren in Milch und Molke 8 2.2.3 Einfluss von Glucose und Galactose 9 2.2.4 Kommerzielle β-Galactosidase-Präparate 11 2.3 Galactooligosaccharide 12 2.3.1 Definition und Syntheseweg 12 2.3.2 Einflussgrößen auf die Reaktion 14 2.3.2.1 Enzymquelle 14 2.3.2.2 Lactosekonzentration 16 2.3.2.3 Reaktionsbedingungen 17 2.3.2.4 Einfluss von Glucose und Galactose 21 2.3.3 GOS-Synthese in Milch und Molke 22 2.3.4 GOS-Synthese mit Lactobacillus sp. 26 2.3.5 GOS-Synthese mit Cryptococcus laurentii 26 2.3.6 GOS-Synthese mit mehreren β-Galactosidasen 27 2.3.7 Möglichkeiten der Glucose-Entfernung 27 2.3.8 Kommerziell erhältliche GOS-Produkte 28 2.3.9 Eigenschaften und Anwendung 30 2.3.10 Modifizierte GOS-Strukturen und GOS-Alternativen 32 3 MATERIAL UND METHODEN 40 3.1 Materialien 40 3.1.1 Verwendete Enzyme 40 3.1.2 Verwendete Mikroorganismen 40 3.1.3 Verwendete Molkeproben 40 3.1.4 Verwendete Geräte 40 3.2 Bestimmung der Inhaltsstoffe von Molke 40 3.3 Mikroorganismenkultivierung und Enzymgewinnung 41 3.3.1 Biochemische Analysenmethoden 41 3.3.2 Herstellung der Rohextrakte aus Lactobacillus sp. 41 3.3.3 Kultivierung im Labormaßstab 42 3.3.3.1 Lactobacillus bulgaricus LB4 42 3.3.3.2 Cryptococcus laurentii 43 3.3.4 Kultivierung im Fermentormaßstab (L. bulgaricus LB4) 43 3.4 Assays zur Bestimmung der Enzymaktivität 43 3.4.1 β-Galactosidase 43 3.4.2 Glucose-Oxidase 44 3.4.3 Katalase 44 3.4.4 Berechnung der Enzymaktivität 45 3.5 Enzymcharakterisierung 45 3.5.1 Temperatur- und pH-Optimum 45 3.5.2 Bestimmung von Aktivatoren und Inhibitoren 46 3.5.3 Enzymstabilität 46 3.6 Galactooligosaccharid-Synthese 47 3.6.1 Verwendung von kommerziellen β-Galactosidasen 47 3.6.2 Verwendung von β-Galactosidase aus Lactobacillus bulgaricus LB4 47 3.6.3 Verwendung von Cryptococcus laurentii-Zellen 47 3.6.4 Kopplung mit Glucose-Oxidase und Katalase 48 3.6.5 Kopplung mehrerer β-Galactosidasen 48 3.7 Galactooligosaccharid-Analytik 49 3.8 Statistische Auswertung 50 4 ERGEBNISSE UND DISKUSSION 51 4.1 Zusammensetzung der Molken 51 4.2 Charakterisierung der β-Galactosidasen 52 4.2.1 Temperatur- und pH-Optimum 52 4.2.2 Inhibitoren und Aktivatoren 55 4.2.3 Stabilität in Puffer und Molke 57 4.3 Synthese von Galactooligosacchariden 61 4.3.1 Synthese mit β-Galactosidase aus K. lactis 61 4.3.1.1 Synthese in Puffer 61 4.3.1.2 Synthese in Süßmolke 64 4.3.1.3 Synthese in Sauermolke 66 4.3.2 Synthese mit β-Galactosidase aus A. oryzae 67 4.3.2.1 Synthese in Puffer 67 4.3.2.2 Synthese in Süßmolke 69 4.3.2.3 Synthese in Sauermolke 70 4.3.3 Synthese mit β-Galactosidase aus L. bulgaricus LB4 73 4.3.3.1 Synthese in Puffer 73 4.3.3.2 Synthese in Süßmolke 76 4.3.4 Synthese mit C. laurentii-Zellen 78 4.3.4.1 Synthese in Puffer 78 4.3.4.2 Synthese in Sauermolke 80 4.3.5 Vergleich der untersuchen Enzyme und Substrate 83 4.3.6 Kopplung mit Glucose-Oxidase und Katalase 88 4.3.6.1 Charakterisierung von Glucose-Oxidase und Katalase 88 4.3.6.2 Einfluss simultaner Glucose-Entfernung auf die GOS-Ausbeute 90 4.3.6.3 Schlussfolgerungen 95 4.3.7 Kombination mehrerer β-Galactosidasen 95 4.3.7.1 Kopplung von A. oryzae und K. lactis 95 4.3.7.2 Kopplung von A. oryzae und C. laurentii 100 4.3.7.3 Schlussfolgerungen 105 4.4 Untersuchungen zur β-Galactosidase-Synthese mit L. bulgaricus LB4 106 4.4.1 Nährmedienumstellung auf koscher-/halal-zertifizierte Bestandteile 106 4.4.2 Kultivierung im Fermentormaßstab 111 5 ZUSAMMENFASSUNG UND AUSBLICK 114 6 LITERATURVERZEICHNIS 118 7 ANHANG 140 7.1 Anhang zu Kapitel 2 140 7.2 Anhang zu Kapitel 3 165 7.2.1 Verwendete Geräte 165 7.2.2 Zusammensetzung MRS-Medium 166 7.2.3 Wachstum von C. laurentii 167 7.2.4 Anfangslactosekonzentrationen 167 7.2.5 Katalaseaktivität 168 7.2.6 Vergleich GOS-Disaccharidanalytik 169 7.3 Anhang zu Kapitel 4 170 7.3.1 Temperaturabhängigkeit von verschiedenen β-Galactosidasen 170 7.3.2 Aktivatoren und Inhibitoren 171 7.3.3 Grafiken zur β-Galactosidase-Stabilität 173 7.3.4 Katalase-Stabilität 182 7.3.5 Aminosäuresequenz von β-Galactosidase aus L. bulgaricus LB4 183 7.3.6 Grafiken zur GOS-Synthese mit kommerziellen β-Galactosidasen 184 7.3.7 Grafiken zur GOS-Synthese mit β-Galactosidase aus L. bulgaricus LB4 188 7.3.8 Daten zur GOS-Synthese mit β-Galactosidase aus C. laurentii 190 7.3.9 Vergleich der maximalen GOS-Ausbeute in Puffer und Molke 191 7.3.10 Grafik zur GOS-Synthese mit β-Galactosidase/GOX/KAT 192 7.3.11 Grafiken zur GOS-Synthese mit mehreren β-Galactosidasen 193 7.3.12 Grafiken zur Kultivierung von L. bulgaricus LB4 194

Orientador: Glaucia Maria Pastore / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-15T22:31:39Z (GMT). No. of bitstreams: 1 Santos_Rosangelados_D.pdf: 1329629 bytes, checksum: 3c51b0ca511ea2a0133affa7ea5d0039 (MD5) Previous issue date: 2010 / Resumo: A maioria dos estudos que envolvem prebióticos têm sido realizados comfrutooligossacarídeos (FOS) e galactooligossacarídeos (GOS) devido a segurança, estabilidade, propriedades sensoriais, resistência à digestão na parte superior do intestino e fermentabilidade no cólon, bem como suas capacidades em promover o crescimento das bactérias benéficas no trato gastrintestinal. Assim, esta pesquisa teve como objetivo estudar a produção e aplicação do prebiótico galactooligossacarídeo (GOS) em estudos in vitro e in vivo. A produção de GOS foi realizada utilizando a enzima ß-galactosidase, extraída de Scopulariopsis sp., na forma livre e imobilizada no suporte orgânico DEAE-celulose. Por meio do estudo in vitro avaliou-se o efeito do GOS sobre a produção de citocinas em culturas de células tumorais, utilizando interferon gama (INF-?) como marcador. Verificou-se também o efeito bifidogênico do GOS, utilizando as culturas probióticas Lactobacillus acidophillus (LA05) e Bifidobacterium animalis (Bb12), bem como a ação dos metabolitos, resultantes da fermentação por estes micro-organismos probióticos, em relação à atividade antiproliferativa em modelos de células tumorais. Foi realizado ainda o estudo in vivo com ratas adultas, com o objetivo de avaliar o impacto fisiológico dos prebióticos FOS e GOS no intestino (ceco) dos animais, através das análises de pH fecal e técnicas de morfometria intestinal. O rendimento da produção de GOS foi de 30% em 12 horas e 24% em 15 dias para a enzima livre e imobilizada, respectivamente. Nos estudos in vitro, a ação direta do GOS apresentou indícios de atividade antiproliferativa na linhagem HT-29 (adenocarcinoma de cólon), todavia, não foi capaz de reduzir em pelo menos 50% o crescimento das células tumorais. Não houve diferença significativa (p>0,05) entre as células tumorais estudadas em relação à concentração de IFN-?. O efeito bifidogênico do GOS foi demonstrado pelas diferenças observadas entre as contagens iniciais e finais ( logUFC.mL-1) utilizando o meio MRS+GOS, que foram de 3,98 logUFC.mL-1 para LA05 e de 4,6 logUFC.mL-1 para Bb12. Os metabólitos resultantes da fermentação da linhagem LA05 foram testados quanto à atividade antiproliferativa em linhagens tumorais humanas. Verificou-se que o meio sem dextrose (MRS-mínimo) apresentou atividade antiproliferativa para as linhagens UACC-62 (melanoma), MCF-7 (mama), NCI-ADR/RES (ovário), 786-0 (renal), NCI-H460 (pulmão) e HT-29 (cólon). O meio de cultura acrescido com GOS, por sua vez, apresentou efeito citostático apenas para duas linhagens (UACC-62 e HT-29). Os resultados obtidos sugerem que os metabólitos bacterianos provenientes da LA05 cultivada em meio sem GOS exercem efeito antiproliferativo e que o GOS reduz a produção desses compostos resultando em diminuição da atividade antiproliferativa. Quanto aos estudos in vivo, os animais que consumiram ração com os prebióticos tiveram um discreto aumento de peso, sem aumento no consumo de ração; além disso, não foi observado decréscimo relevante em relação ao pH do ceco. Os animais alimentados com FOS ou com GOS apresentaram aumento na altura das vilosidades intestinais em relação ao grupo controle (p<0.05). Foi verificado que apenas as vilosidades intestinais dos animais que consumiram FOS apresentavam-se mais largas, quando comparado com o grupo controle. Por outro lado, quanto à altura dos enterócitos, as ratas que consumiram GOS apresentaram média maior que o grupo padrão, enquanto as alimentadas com FOS não apresentaram diferença significativa (p<0,05). Embora os mecanismos envolvidos em relação à atividade metabólica do GOS não estejam totalmente esclarecidos, sugere-se que este oligossacarídeo tenha potencial de modulação no epitélio intestinal das células. Não é possível, no entanto, excluir o efeito mediado pelos ácidos graxos de cadeia curta, estimulados a partir da ingestão dos prebióticos, em relação à nutrição dos enterócitos / Abstract: Most studies involving prebiotic have been performed with fructooligossacarídeos (FOS) and galactooligosaccharides (GOS), due to security, stability, sensory properties, resistance to digestion in the upper bowel and fermentability in the colon as well as their ability to promote beneficial bacteria growth in the gastrointestinal tract. Thus, the aim of this research was to study the prebiotic galactooligosaccharide (GOS) production and application in vitro and in vivo. GOS production was performed using ß-galactosidase enzyme extracted from Scopulariopsis sp. in free form and immobilized on an organic DEAE-cellulose. The in vitro study GOS effect on cytokine production was evaluated on human cancer cell lines, using interferon gamma (INF-?) as a marker. GOS bifidogenic effect was determined using probiotic cultures Lactobacillus acidophillus (LA05) and Bifidobacterium animalis (Bb12) and the antiproliferative activity of metabolites resulting from these probiotics microorganisms fermentation was studied in human cancer cell lines. An in vivo study was also performed with adult female rats in order to evaluate the physiological impact of prebiotics FOS and GOS in the cecum of animals by analyzing fecal pH and intestinal morphology was done. The yield of GOS production was 30% at 12 hours and 24% at 15 days for free and immobilized enzyme, respectively. The direct action of GOS in vitro showed evidence of antiproliferative activity in HT-29 line (colon adenocarcinoma), however, it was not able to reduce at least 50% growth of tumor cells. There was no significant difference (p> 0.05) between tumor cells studied in IFN-? concentration. GOS bifidogenic effect was demonstrated by differences between initial and final scores ( logUFC.mL-1) when MRS + GOS was used. The difference was 3,98 logUFC.mL-1 for LA05 and 4,6 logUFC.mL-1 for Bb12. The metabolites resulting from LA05 fermentation were tested for antiproliferative activity on human cancer cell lines. The medium without dextrose (MRS-minimum) showed antiproliferative activity for the cell lines UACC-62 (melanoma), MCF-7 (breast), NCI-ADR/RES (ovary), 786-0 (renal) NCI-H460 (lung) and HT-29 (colon). The culture medium supplemented with GOS, in turn, showed only a cytostatic effect for two cell lines (UACC-62 and HT-29). The results suggest that bacterial metabolites from LA05 grown in medium without GOS exert antiproliferative effect while GOS supplementation propably reduced the active metabolites concentration. Animals fed with diet supplemented by prebiotics showed slight weight gain with no changes in fed intake and no significant decrease on cecal pH. Animals fed with FOS or GOS showed an increase in villus height in comparison with control group (p <0.05). It was found that only the villi of the animals fed with FOS were wider when compared with control group. Moreover, the enterocytes height was larger in rats that consumed GOS in comparison of control group, while those fed with FOS did not differ significantly (p<0.05). Although the mechanisms involved in GOS metabolic activity are not entirely clear, our results suggested that this oligosaccharide has a potential as intestinal epithelial cells modulator. It is not possible, however, to exclude the effect mediated by short-chain fatty acids, stimulated by prebiotics ingestion in enterocytes nutrition / Doutorado / Doutor em Ciência de Alimentos

Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2017-04-10T11:37:49Z No. of bitstreams: 1 2016 - Pedro Campinho Belsito.pdf: 1175009 bytes, checksum: 76927e794a55e13aa297bb3b657cc97b (MD5) / Made available in DSpace on 2017-04-10T11:37:49Z (GMT). No. of bitstreams: 1 2016 - Pedro Campinho Belsito.pdf: 1175009 bytes, checksum: 76927e794a55e13aa297bb3b657cc97b (MD5) Previous issue date: 2016-04-12 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / The aim of this study was to develop a prebiotic ?requeij?o cremoso? cheese with galactooligosaccharide, evaluating the physicochemical, rheological, optical an microstructural as well as the sensory acceptance of the processed products. The ?requeij?o cremoso? cheese was processed and added galactoligossaccharide (0, 1.5%, 3.0%, 4.0% w / w%, respectively) and subjected to physical and chemical analysis (pH, moisture, fat and protein), instrumental color (system L*, a, b), rheology (flow curve, and oscillatory tests), microstructure and sensory analysis ( hedonic test with 80 customers). The results show that there was no significant influence of the treatments with galactooligosaccharide in physicochemical parameters (p > 0.05) of ?requeij?es cremosos? cheeses, while regarding the optical characteristics the addition of GOS provided higher brightness (L*) compared to the added treatments of increasing concentrations of galactooligosaccharide as the highest whiteness index (WI). Regarding the rheological behavior was observed that all samples showed similar behavior, but the ?requeij?o cremoso? cheese control tended to higher shear stress values for shear rate and higher stiffness and storage modulus values . All samples showed curd behavior index (n) less than 1, indicating pseudoplastic behavior, in which the apparent viscosity decreased with increasing applied shear rate. There was an increase in the flow of cheese samples subjected to melting test. From microstructural point of view, the addition of GOS provided a denser and compact matrix protein and the number and size of fat globules reduced while the sensory analysis showed better results in all attributes in the analysis showing potential functional food option for marketing. / A suplementa??o com fibras prebi?ticas constitui-se uma potencial op??o de alimento funcional, dado que um produto com propriedade prebi?tica pode ter um impacto positivo na sa?de gastrointestinal. O objetivo deste trabalho foi o desenvolvimento do requeij?o cremoso adicionado de Galactooligossacar?deo (GOS), avaliando o produto bem como seu efeito nas caracter?sticas f?sico-qu?micas, reol?gicas, ?pticas e na aceita??o sensorial. Requeij?o cremoso foi processado e adicionado de galactoligossacar?deo (0, 1,5% 3,0% 4,0% % p/p, respectivamente) e submetidos a an?lises f?sico-qu?micas (pH , umidade, gordura e prote?na), cor instrumental ( sistema L*, a, b), reologia (curva de fluxo, e ensaios oscilat?rios), microestrutura e an?lise sensorial ( teste hed?nico com 80 consumidores). Os resultados obtidos mostram que n?o houve influ?ncia significativa dos tratamentos com galactooligossacar?deo nos requeij?es nos par?metros f?sico-qu?micos (p>0,05), enquanto com rela??o as caracter?sticas ?pticas a adi??o do GOS proporcionou maior luminosidade (L*) comparada aos tratamentos adicionados de concentra??es crescentes de galactooligossacar?deo assim como o maior ?ndice de brancura (WI). Com rela??o aos aspectos reol?gicos foi poss?vel observar que todas as amostras apresentaram comportamento similar, por?m o requeij?o controle apresentou tend?ncia a maiores valores de tens?o de cisalhamento por taxa de deforma??o e maiores valores de m?dulo de rigidez e estocagem. Todas as amostras de requeij?o apresentaram ?ndices de comportamento (n) menores que 1, indicando comportamento pseudopl?stico, em que a viscosidade aparente diminuiu com o aumento da taxa de deforma??o aplicada. Houve aumento do fluxo de queijo nas amostras submetidas ao teste de derretimento. Do ponto de vista microestrutural, a adi??o de GOS proporcionou uma matriz proteica mais densa e compacta e com o n?mero e tamanho dos gl?bulos de gordura diminu?dos enquanto que na an?lise sensorial foram observados resultados melhores em todos os atributos, mostrando potencial op??o de alimento funcional para a comercializa??o

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