[EN] Human milk contains a large number of oligosaccharides, either free or bound to proteins and lipids, and their physiological role is mostly unknown. These oligosaccharides are resistant to host gastrointestinal digestion and therefore, a significant proportion reaches the infant colon, where they can be substrates for the resident microbiota. Lacto-N-biose (LNB) and galacto-N-biose (GNB) are the core type-1 sugar structures in HMO and mucin glycoproteins, respectively. They are fermented by species of the genus Bifidobacterium, but, there is no data about their utilization by the genus Lactobacillus. There is also no information for this genus about the metabolism of N-acetyllactosamine (LacNAc), which constitutes the type-2 sugar core in HMO, and lacto-N-triose, which forms part of the structure of lacto-N-tetraose, one of the most abundant HMOs. Lactobacillus casei is a lactic acid bacteria isolated from several environmental niches such as milk, meat, and reproductive and gastrointestinal tracts of animals and humans. In addition, some strains are used as starter cultures in the dairy industry and also as probiotics. The capability of L. casei species to survive in the gastrointestinal tract would depend in part of its ability to metabolize the available carbohydrates. In this Thesis we have shown that this strain is able to grow using LNB, GBN, LacNAc, and lacto-N-triose as carbon sources, and we have characterized the corresponding metabolic pathways. L. casei contains a gene cluster, gnbREFGBCDA, involved in the metabolism of GNB, LNB and also N-acetylgalactosamine. Transcriptional analysis showed that the gnb operon is regulated by substrate-specific induction mediated by the transcriptional repressor GnbR. Upstream of the gnb operon, there are two genes, bnaG and manA, encoding a b-N-acetylglucosaminidase precursor and a mannose-6P isomerase. It has been shown that BnaG is an extracellular wall-attached enzyme and that it is involved on lacto-N-triose metabolism. ManA enzyme is involved in the utilization of the mannose moiety of 3'-N-acetylglucosaminyl-mannose, which is a carbon source for L. casei BL23. Finally, in this strain, LacNAc is transported and phosphorylated by the lactose PTS and it is intracellularly hydrolyzed by the phospho-b-galactosidase LacG into galactose-6P and GlcNAc. Transcriptional analysis showed that the lac operon, in addition to lactose, is also induced by LacNAc. In an effort to better understand the metabolism and bioactive potential of HMO, sufficient quantities are required. In order to have enough amounts of LNB and GNB to test their biological activities, both disaccharides have been synthesized in vitro using the transglycosylation activity of the GnbG glycosyl hydrolase isolated from L. casei. Transglycosylation reactions were scaled and the resulting products were purified, and the yields obtained were 10.7 ± 0.2 g/L of LNB and 10.8 ± 0.3 g/l of GNB. Both disaccharides were used in vitro to determine their potential prebiotic properties using 33 Lactobacillus strains corresponding to 13 different species. It was determined that 21 strains, corresponding to the species L. casei, Lactobacillus rhamnosus, Lactobacillus zeae, Lactobacillus gasseri and Lactobacillus johnsonii were able to metabolize both GNB as LNB. Recently, some scientific evidences suggest an immunomodulatory function for HMO. In vitro analyses to assay this function have been performed for LNB and GNB, and for two fucosyloligosaccharides (Fuc-a-1,3-GlcNAc and Fuc-a-1,6-GlcNAc) previously synthesized in our laboratory. The four disaccharides were able to significantly increase IFN-g production in Peripheral Blood Mononuclear Cells (PBMC). Fuc-a-1,6-GlcNAc was also able to significantly reduce the production of IL13. These results suggest a stimulatory effect on the immune system, and in the particular case of Fuc-a-1,6-GlcNAc, a polarizing effect of immune response Th1 / Th2 towards Th1 populations. / [ES] La leche humana contiene una gran cantidad de oligosacáridos, libres o unidos a lípidos y proteínas, y su función fisiológica es mayoritariamente desconocida. Estos oligosacáridos son resistentes a la digestión y una gran proporción llega al intestino del lactante, donde pueden ser substratos para la microbiota. La lacto-N-biosa (LNB) y la galacto-N-biosa (GNB) son las estructuras tipo-1 que forman el núcleo de los oligosacáridos de la leche humana (OLH) y de las glicoproteínas de la mucina, respectivamente. Los dos azúcares son fermentados por especies del género Bifidobacterium, pero no hay datos acerca de su utilización por el género Lactobacillus. Tampoco hay información para este género acerca del metabolismo de la N-acetil-lactosamina (LacNAc), que constituye la cadena de azúcar tipo-2 en los OLH, y de la lacto-N-triosa, que forma parte de la estructura de la lacto-N-tetraosa, uno de los OLH más abundantes. La capacidad de Lactobacillus casei, de prevalecer en el sistema gastrointestinal dependerá en parte de su versatilidad metabólica para utilizar los carbohidratos disponibles. En la presente Tesis Doctoral se ha demostrado que la cepa L. casei BL23 se puede cultivar en presencia de LNB, GBN, LacNAc, y lacto-N-triosa como fuentes de carbono. En el metabolismo de la LNB, GNB y también N-acetilgalactosamina (GalNAc) está implicado el operon gnbREFGBCDA. Análisis transcripcionales demostraron que el operon gnb está regulado por inducción del substrato mediada por el represor transcripcional GnbR. Por encima del operon gnb, hay dos genes bnaG y manA, que codifican para el precursor de una b-N-acetilglucosaminidasa y una manosa-6P isomerasa. Se ha demostrado que BnaG es un enzima extracelular unido a la pared celular y que está implicado en el metabolismo de la lacto-N-triosa. El enzima ManA está implicada en el metabolismo de la manosa presente en el disacárido 3'-N-acetilglucosaminil-manosa, el cual es también una fuente de carbono para L. casei BL23. Por último, en esta cepa se ha demostrado que la LacNAc es transportada y fosforilada por el PTS de la lactosa e hidrolizada intracelularmente por la fosfo-b-galactosidasa LacG en galactosa-6P y GlcNAc. Análisis transcripcionales demostraron que el operon lac, además de por lactosa, está también inducido por LacNAc. Para intentar comprender mejor el metabolismo y potencial bioactivo de los OLH se necesitan cantidades adecuadas de éstos. Con el objeto de disponer de LNB y GNB en cantidad suficiente para ensayar su actividad biológica, se han sintetizado in vitro ambos disacáridos utilizando para ello la capacidad de transglicosidación de la glicosil hidrolasa GnbG aislada de L. casei. Las reacciones de transglicosidación se escalaron, los productos resultantes se purificaron, y se obtuvieron rendimientos de 10.7 ± 0.2 g/l de LNB y 10.8 ± 0.3 g/l de GNB. Ambos disacáridos fueron utilizados in vitro para determinar sus propiedades prebióticas potenciales con 33 cepas de Lactobacillus correspondientes a 13 especies diferentes. Se determinó que 21 de las cepas, correspondientes a las especies L. casei, Lactobacillus rhamnosus, Lactobacillus zeae, Lactobacillus gasseri y Lactobacillus johnsonii fueron capaces de metabolizar tanto GNB como LNB. Existen evidencias científicas recientes que les atribuyen a los OLH propiedades inmunomoduladoras, así que se han determinado éstas in vitro para LNB y GNB, y para dos fucosiloligosacáridos (Fuc-a-1,3-GlcNAc y Fuc-a-1,6-GlcNAc) sintetizados anteriormente en nuestro laboratorio. Se ha demostrado que los cuatro disacáridos son capaces de incrementar significativamente la producción de IFN-g en Células Mononucleares de Sangre Periférica. El Fuc-a-1,6-GlcNAc además fue capaz de reducir significativamente la producción de IL13. Estos resultados sugieren un efecto estimulante del sistema inmune y en el caso particular del Fuc-a-1,6-GlcNAc un efecto polarizador de la respuesta inmune Th1/Th2 / [CAT] La llet humana conté una gran quantitat d'oligosacàrids, lliures o conjugats amb lípids o proteïnes i la seua funció fisiològica és majoritàriament desconeguda. Aquests oligosacàrids són resistents a la digestió i una gran proporció arriba a l'intestí dels lactants, on poden ser substrat per a la microbiota. La lacto-N-biosa (LNB) i la galacto-N-biosa (GNB) són les estructures tipus-1 que conformen el nucli dels oligosacàrids de la llet humana (OLH) i de les glicoproteïnes de la mucina, respectivament. Els dos sucres són fermentats per espècies del gènere Bifidobacterium, però no existeixen dades sobre l'ús pel gènere Lactobacillus. Tampoc no hi ha informació per a aquest gènere sobre el metabolisme de la N-acetil-lactosamina (LacNAc), que cosntitueix la cadena de sucre tipus-2 als OLH i de la lacto-N-triosa, que forma part de l'estructura de als OLH tipus-1 i 2. La capacitat de L. casei, una bactèria làctica aïllada de múltiples nínxols ambientals, de prevaler al sistema gastrointestinal dependrà en part de la seua versatilitat metabòlica per a utilitzar els carbohidrats disponibles. A la present tesi doctoral s'ha demostrat que la soca L. casei BL23 es pot cultivar en presència de LNB, GBN, LacNAc i lacto-N-triosa com a fonts de carboni. Al metabolisme de la LNB, GNB i també de la N-acetilgalactosamina (GalNAc) està implicat l'operó gnbREFGBCDA. Anàlisi transcripcionals demostraren que l'operó gnb està regulat per inducció del substrat mitjançant el repressor transcripcional GnbR. A sobre de l'operó gnb, hi ha dos gens bnaG i manA, que codifiquen per al precursor d'una b-N-acetilglucosaminidasa i una manosa-6P isomerasa. S'ha demostrat que BnaG és un enzim extracel·lular unit a la paret cel·lular i que està implicat en el metabolisme de la lacto-N-triosa. Aquesta és hidrolitzada per BnaG en lactosa i N-acetilglucosamina (GlcNAc). L'enzim ManA està implicada en el metabolisme de la manosa present al disacàrid 3-N-acetilglucosaminil-manosa, el qual també representa una font de carboni per a L. casei BL23. Per concloure, en aquesta soca s'ha demostrat que la LacNAc és transportada i fosforilada pel PTS de la lactosa i hidrolitzada intracel·lularment per la fosfo-b-galactosidasa LacG en galactosa-6P i GlcNAc. Anàlisi transcripcionals demostraren que l'operó lac, a més de per lactosa està induït per LacNAc. Per intentar comprendre millor el metabolisme i potencial bioactiu dels OLH es necessiten quantitats adequades d'aquests. A fi de disposar de LNB i GNB en quantitats suficients per a assajar la seua activitat biològica, s'han sintetitzat in vitro ambdós disacàrids utilitzant per a tal la capacitat de trasglicosidació de la glicosil hidrolasa GnbG aïllada de L. casei. Les reaccions de transglicosidació s'escalaren, els productes resultants es purificaren i s'obtingueren rendiments de 10.7 ± 0.2 g/l de LNB y 10.8 ± 0.3 g/l de GNB. Ambdós disacàrids foren utilitzats in vitro per a determinar les seues propietats prebiòtiques potencials amb 33 soques de Lactobacillus corresponents a 13 espècies diferents. Es determinà que 21 de les soques, corresponents a les espècies L. casei, Lactobacillus rhamnosus, Lactobacillus zeae, Lactobacillus gasseri i Lactobacillus johnsonii foren capaces de metabolitzar tant GNB com LNB. Existeixen evidències científiques recents que atribueixen als OLH propietats immunomoduladores, així que s'han determinat aquestes in vitro per a LNB i GNB i per als dos fucosiloligosacàrids (Fuc-a-1,3-GlcNAc y Fuc-a-1,6-GlcNAc) sintetitzats anteriorment al nostre laboratori. S'ha demostrat que els quatre disacàrids són capaços d'incrementar significativament la producció de IFN-g en Cèllules Mononuclears de Sang Perifèrica. El Fuc-a-1,6-GlcNAc a més, fou capaç de reduir significativament la producció de IL13. Aquests resultats suggereixen un efecte estimulant del sistema immune i en el cas particular del Fuc-a-1,6-GlcNAc un efecte pol / Bidart Costoya, G. (2016). Metabolismo y síntesis de oligosacáridos de la leche humana mediante la utilización de enzimas glicosil hidrolasas de Lactobacillus casei [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/70898 / TESIS

This diploma thesis deals with the biodegradation of whey on selected microbial products (carbohydrates, lactic acid, acetic acid and ethanol) thermophilic bacteria of genus Thermus aquaticus and mesophilic bacteria of genus Lactobacillus casei and Bacillus coagulans. For cultivation was used as medium whey, from which the proteins were removed and which was enriched with nutrients. On the basis of culture in the fermentor were determined growth curve and the HPLC method were determined individual bioremediation products.

The master’s thesis discusses the comparison of microbial metabolic production from waste and powder whey. Whey is obtained as a by-product of cheese production. Lactose (preferably whey) using lactic acid bacteria (eg. Lactobacillus) under suitable temperature conditions is converted to lactic acid. Effect of temperature, effect of salts and effect of yeast extract on lactic acid production by L. casei were investigated. HPLC metod was determined lactid acid. In the practical part were used bacteria of the genus Lactobacillus. It was found that for dried and waste whey is the optimal temperature of 35 °C. At this temperature is the greatest gain of lactic acid. The highest concentration of lactic acid was obtained by using 20 g of yeast extract for dried whey and for waste whey were used 24 g of yeast extract. When monitoring the effect of salt concentration on the production of lactic acid, it was found that using only MnSO4·H2O gain most of lactic acid.

Nanotechnology is currently one of the fastest growing scientific disciplines. An interesting area of research is the biosynthesis of metal nanoparticles using microorganisms including lactic acid bacteria. In the first part this diploma thesis focuses on verification of identity of bacterial species Lactobacillus casei and Lactobacillus paracasei by genus- and species-specific PCR. In the next part of experimental work the capability of six Lactobacillus strains to produce silver nanoparticles is tested. Synthesis of nanoparticles was confirmed for all the strains depending on the amount of added AgNO3 and on time of cultivation. Differences between the strains were detected.

Les maladies inflammatoires chroniques de l'intestin sont caractérisées par une inflammation anormale et récurrente du tractus digestif. De nombreuses études ont démontré des effets bénéfiques de souches probiotiques anti-inflammatoires recombinantes ou non. La première partie de cette thèse décrit différentes stratégies d'optimisation de souches de bactéries lactiques en tant que vecteurs de protéines d'intérêt santé. Nous avons ainsi démontré qu'une modification du peptidoglycane de la paroie de Lactococcus lactis influençant la lyse bactérienne ne permettait pas de moduler l'immunogénicité de l'antigène E7 délivré par L. lactis. Nous avons également démontré que la nature du vecteur bactérien était un paramètre essentiel dans la vectorisation de la protéine délivrée : ainsi l'espèce Bifidobacterium infantis induit une réponse immunitaire spécifique à l'antigène E7 supérieure à celle obtenue avec les vecteurs L. lactis et Lactobacillus plantarum. La deuxième partie de cette thèse porte sur l'étude des effets anti-inflammatoires de bactéries recombinantes ou non. Nous avons ainsi démontré que la souche Lb. casei BL23 produisant une superoxyde dismutase à manganèse permettait de diminuer significativement des colites murines induites par administration de dextran sodium sulfate. Enfin, nous avons mis en évidence des propriétés anti-inflammatoires sur divers modèles d'inflammation in vitro / in vivo de Faecalibacterium prausnitzii, première bactérie commensale anti-inflammatoire identifiée sur la base de données cliniques humaines.

This Diploma thesis deals with preparation of probiotic cultures Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium breve enriched with prebiotics meant for application in baby food products. Natural extracts from matcha, moringa, young beat, young barley, chlorella and spirulina were selected as prebiotics. The theoretical part is focused on probiotic bacteria, their biological effects and their effects on the child´s body. The experimental part deals with the cultivation of probiotic bacteria with plant extracts, monitoring their viability and stabilization in an encapsulated form. Mixtures of probiotic cells with prebiotics were encapsulated into alginate particles to increase stability. Some of the alginate particles were processed by freeze drying. Mixtures of probiotic cultures with plant extracts were subjected to model human digestion by the action of model digestive juices in unencapsulated, encapsulated and lyophilized form. Selected extracts of plant materials were characterized in terms of amount of total and reducing sugars, total phenolic substances, individual phenolic substances and antioxidant activity. Further, two baby commercial dietary supplements containing probiotics were selected, which were characterized in terms of cell number and viability. Probiotic products were also subjected to model digestion.

Although strong protective immune response is essential for preventing invasion by pathogens, equivalent responses against antigens originating from commensal bacteria can lead to chronic inflammatory diseases, such as inflammatory bowel disease (IBD). Manipulating the mucosal immune responses with microbial antigens might be an excellent tool to IBD therapy or prevention. Our aim was to gain some insight into the regulation of the intestinal inflammation and to isolate bacterial immunomodulatory components that could be used in intestinal inflammation therapy and prevention. One particular mechanism of how healthy colon tissue regulates the inflammation during acute experimental colitis is through modulation of bioavailability of glucocorticoids (GCs) in gut mucosa. Here, we show that intestinal inflammation changes the local GC metabolism, which ultimately leads to decrease in inflammatory readiness of cells in the gut mucosa and in mesenteric lymph nodes. This pre-receptor regulation of GC function could represent an important homeostatic function of the gut mucosa. The actual triggers of intestinal inflammation in IBD seem to be either microbial dysbiosis or microbes with special "pathogenic" abilities, which both could be rectified by feeding with probiotics. Here, we report that oral feeding with live...

Although strong protective immune response is essential for preventing invasion by pathogens, equivalent responses against antigens originating from commensal bacteria can lead to chronic inflammatory diseases, such as inflammatory bowel disease (IBD). Manipulating the mucosal immune responses with microbial antigens might be an excellent tool to IBD therapy or prevention. Our aim was to gain some insight into the regulation of the intestinal inflammation and to isolate bacterial immunomodulatory components that could be used in intestinal inflammation therapy and prevention. One particular mechanism of how healthy colon tissue regulates the inflammation during acute experimental colitis is through modulation of bioavailability of glucocorticoids (GCs) in gut mucosa. Here, we show that intestinal inflammation changes the local GC metabolism, which ultimately leads to decrease in inflammatory readiness of cells in the gut mucosa and in mesenteric lymph nodes. This pre-receptor regulation of GC function could represent an important homeostatic function of the gut mucosa. The actual triggers of intestinal inflammation in IBD seem to be either microbial dysbiosis or microbes with special "pathogenic" abilities, which both could be rectified by feeding with probiotics. Here, we report that oral feeding with live...