Plants are used to produce many secondary metabolites that are too difficult, expensive or impossible to make by chemical synthesis. Conventional cultivation of plants is of course subject to vagaries of weather, pests and availability of land; hence, the interest in highly controlled culture of plant cells and hairy roots in bioreactors as methods of producing various products. This project focussed on production of blue and red colors of Lavandula augustifolia and Beta vulgaris, respectively. Callus and suspension cell culture were successfully produced from L. augustifolia after extensive trials, but hairy roots could not be generated from this species. In contrast, a successful protocol was developed for consistently producing hairy roots from B. vulgaris, but calli could not be generated from this species. Effects of medium composition on growth of L. augustifolia calli and freely suspended cells and production of the blue pigment by the latter, were investigated. Optimal production of callus occurred in full-strength Murashige and Skoog (MS) medium supplemented with 2 mg/l of indole-3-acetic acid (IAA) and 1 mg/l of kinetin. Stable suspension cultures could be produced and maintained in full-strength MS medium supplemented with 1 mg/l each of IAA and kinetin. In suspension culture in full-strength MS medium, the following hormone combinations were tested: (1) 1 mg/l each of indole-3-acetic acid (IAA) and kinetin; (2) 2 mg/l of IAA and 1 mg/l of kinetin; (3) 2 mg/l of IAA and 1 mg/l of benzyl amino purine (BAP); and (4) 2 mg/l each of IAA and BAP. Combination (3) maximized cell growth, but the highest cell-specific production of the blue pigment was seen in combination (2), although pigment production occurred at all hormone combinations. The medium formulation that gave the best production of the pigment in shake flasks was scaled up to a 2 L aerated stirred tank bioreactor, but both the biomass and pigment productivities were reduced in the bioreactor apparently due to the high shear stress generated by the Rushton turbine impeller. Compared to suspension cultures of L. augustifolia, the hairy root cultures of B. vulgaris grew extremely rapidly. Hairy roots also produced large amounts of the red pigments. Growth of hairy roots was influenced by the composition of the medium. Although the full strength MS medium better promoted biomass growth compared to the half-strength MS medium, the final concentration of the biomass and the pigment were nearly the same in both media. Attempts were made to enhance production by using various hormones (i.e. naphthalene acetic acid, BAP, IAA added individually at a concentration of 0.5 mg/l), but none of the hormones proved useful. BAP adversely affected the growth of hairy roots. In summary, production of pigments by suspension culture of L. augustifolia and hairy root culture of B. vulgaris, is technically possible, but requires substantial further optimization for enhancing productivity than has been possible in this project. iii

Plants are used to produce many secondary metabolites that are too difficult, expensive or impossible to make by chemical synthesis. Conventional cultivation of plants is of course subject to vagaries of weather, pests and availability of land; hence, the interest in highly controlled culture of plant cells and hairy roots in bioreactors as methods of producing various products. This project focussed on production of blue and red colors of Lavandula augustifolia and Beta vulgaris, respectively. Callus and suspension cell culture were successfully produced from L. augustifolia after extensive trials, but hairy roots could not be generated from this species. In contrast, a successful protocol was developed for consistently producing hairy roots from B. vulgaris, but calli could not be generated from this species. Effects of medium composition on growth of L. augustifolia calli and freely suspended cells and production of the blue pigment by the latter, were investigated. Optimal production of callus occurred in full-strength Murashige and Skoog (MS) medium supplemented with 2 mg/l of indole-3-acetic acid (IAA) and 1 mg/l of kinetin. Stable suspension cultures could be produced and maintained in full-strength MS medium supplemented with 1 mg/l each of IAA and kinetin. In suspension culture in full-strength MS medium, the following hormone combinations were tested: (1) 1 mg/l each of indole-3-acetic acid (IAA) and kinetin; (2) 2 mg/l of IAA and 1 mg/l of kinetin; (3) 2 mg/l of IAA and 1 mg/l of benzyl amino purine (BAP); and (4) 2 mg/l each of IAA and BAP. Combination (3) maximized cell growth, but the highest cell-specific production of the blue pigment was seen in combination (2), although pigment production occurred at all hormone combinations. The medium formulation that gave the best production of the pigment in shake flasks was scaled up to a 2 L aerated stirred tank bioreactor, but both the biomass and pigment productivities were reduced in the bioreactor apparently due to the high shear stress generated by the Rushton turbine impeller. Compared to suspension cultures of L. augustifolia, the hairy root cultures of B. vulgaris grew extremely rapidly. Hairy roots also produced large amounts of the red pigments. Growth of hairy roots was influenced by the composition of the medium. Although the full strength MS medium better promoted biomass growth compared to the half-strength MS medium, the final concentration of the biomass and the pigment were nearly the same in both media. Attempts were made to enhance production by using various hormones (i.e. naphthalene acetic acid, BAP, IAA added individually at a concentration of 0.5 mg/l), but none of the hormones proved useful. BAP adversely affected the growth of hairy roots. In summary, production of pigments by suspension culture of L. augustifolia and hairy root culture of B. vulgaris, is technically possible, but requires substantial further optimization for enhancing productivity than has been possible in this project. iii

Plants are used to produce many secondary metabolites that are too difficult, expensive or impossible to make by chemical synthesis. Conventional cultivation of plants is of course subject to vagaries of weather, pests and availability of land; hence, the interest in highly controlled culture of plant cells and hairy roots in bioreactors as methods of producing various products. This project focussed on production of blue and red colors of Lavandula augustifolia and Beta vulgaris, respectively. Callus and suspension cell culture were successfully produced from L. augustifolia after extensive trials, but hairy roots could not be generated from this species. In contrast, a successful protocol was developed for consistently producing hairy roots from B. vulgaris, but calli could not be generated from this species. Effects of medium composition on growth of L. augustifolia calli and freely suspended cells and production of the blue pigment by the latter, were investigated. Optimal production of callus occurred in full-strength Murashige and Skoog (MS) medium supplemented with 2 mg/l of indole-3-acetic acid (IAA) and 1 mg/l of kinetin. Stable suspension cultures could be produced and maintained in full-strength MS medium supplemented with 1 mg/l each of IAA and kinetin. In suspension culture in full-strength MS medium, the following hormone combinations were tested: (1) 1 mg/l each of indole-3-acetic acid (IAA) and kinetin; (2) 2 mg/l of IAA and 1 mg/l of kinetin; (3) 2 mg/l of IAA and 1 mg/l of benzyl amino purine (BAP); and (4) 2 mg/l each of IAA and BAP. Combination (3) maximized cell growth, but the highest cell-specific production of the blue pigment was seen in combination (2), although pigment production occurred at all hormone combinations. The medium formulation that gave the best production of the pigment in shake flasks was scaled up to a 2 L aerated stirred tank bioreactor, but both the biomass and pigment productivities were reduced in the bioreactor apparently due to the high shear stress generated by the Rushton turbine impeller. Compared to suspension cultures of L. augustifolia, the hairy root cultures of B. vulgaris grew extremely rapidly. Hairy roots also produced large amounts of the red pigments. Growth of hairy roots was influenced by the composition of the medium. Although the full strength MS medium better promoted biomass growth compared to the half-strength MS medium, the final concentration of the biomass and the pigment were nearly the same in both media. Attempts were made to enhance production by using various hormones (i.e. naphthalene acetic acid, BAP, IAA added individually at a concentration of 0.5 mg/l), but none of the hormones proved useful. BAP adversely affected the growth of hairy roots. In summary, production of pigments by suspension culture of L. augustifolia and hairy root culture of B. vulgaris, is technically possible, but requires substantial further optimization for enhancing productivity than has been possible in this project. iii

Dans cette thèse, nous avons démontré la faisabilité de la préparation de polymères à empreinte moléculaires (MIP) hautement sélectifs pour la reconnaissance dans des matrices aqueuses complexes avec des applications dans les cosmétiques et en technologie alimentaire. Les MIP (de l'anglais molecularly imprinted polymers) sont des récepteurs synthétiques comparables aux anticorps, qui sont synthétisés par co-polymérisation de monomères fonctionnels et réticulants en présence d'un gabarit moléculaire. Leurs propriétés de reconnaissance moléculaire, associées à leur grande stabilité, robustesse mécanique, faible coût et leur synthèse facile les rendent extrêmement intéressants comme matériaux de capture sélective, avec des applications dans les séparations analytiques, la détection et la vectorisation des médicaments. Cependant, leur reconnaissance sélective dans des milieux aqueux reste toujours problématique et c'est l'une des raisons de leur expansion commerciale restreinte. Dans une première partie, nous avons développé un MIP fonctionnant en milieu aqueux pour son application comme ingrédient actif dans un déodorant. Les odeurs corporelles sont principalement dues à des acides gras volatils générés à partir de leurs précurseurs, des conjugués de glutamine par des enzymes hydrolytiques produites à partir de bactéries présentes sur la peau. La plupart des anti-transpirants et des déodorants actuellement commercialisés contiennent des sels d'aluminium et des agents antibactériens non spécifiques, respectivement. Cependant, l'utilisation extrêmement étendue de ces produits nécessite des solutions alternatives en ce qui concerne divers problèmes (environnement, respect de l'écosystème de la peau, toxicité, etc.). Pour cette raison, un MIP a été synthétisé pour capturer les précurseurs conjugués de glutamine afin qu'ils ne soient plus disponibles aux bactéries, empêchant ainsi leur transformation en composés malodorants. Afin de générer des liaisons sélectifs dans des environnements aqueux, un monomère à base d'amidinium qui peut former une interaction électrostatique stoechiométrique forte avec les groupes carboxyle sur le gabarit moléculaire a été synthétisé. Le MIP, mélangé dans une formulation dermo-cosmétique, pourrait capter sélectivement les précurseurs conjugués de glutamine, au milieu d'une multitude d'autres molécules présentes dans la sueur humaine. En outre, le MIP n’affecte pas les bactéries de la peau, ouvrant la voie à des déodorants innovateurs de nouvelle génération, moins problématiques pour la santé. Dans une deuxième partie, nous avons développé une procédure rapide et efficace basée sur l'extraction en phase solide à empreinte moléculaire (MISPE) pour la purification sélective de la bétanine et de son stéréoisomère l’isobétanine à partir d'extraits de betterave. La bétanine est un pigment naturel ayant un fort pouvoir antioxydant et dont les propriétés pharmacologiques sont de plus en plus étudiées. Ce pigment est actuellement utilisé comme simple colorant alimentaire. Dans notre étude, l'acide dipicolinique a été utilisé comme gabarit moléculaire pour la synthèse de MIP, en raison de sa similarité structurelle avec le groupe chromophore de la bétanine. Les procédures MISPE ont été optimisées permettant l'élimination presque complète des glucides issus de la matrice végétale ainsi que la majorité des protéines, ce qui permet d'obtenir un rendement élevé d'extraction de la bétanine / isobétanine en une seule étape. De plus, toute la procédure d'extraction a été réalisée dans des solvants respectueux de l'environnement, tels que l'éthanol ou l'eau. Pour conclure, nous sommes convaincus que ce travail pave le chemin au développement d'une nouvelle génération des MIP fonctionnant en milieu aqueux avec des propriétés de reconnaissance améliorées dans des environnements complexes, qui pourra s'appliquer également à d'autres domaines biotechnologiques et biomédicaux. / In this thesis, we have demonstrated the feasibility of preparing highly selective molecularly imprinted polymers (MIPs) for recognition in complex aqueous matrices with applications in cosmetics and food technology. MIPs are synthetic tailor-made receptors, with binding affinities and specificities comparable to those of natural antibodies. Their molecular recognition properties, combined with their high stability, mechanical robustness, low cost and easy synthesis make them extremely attractive as selective capture materials with applications in analytical and preparative separations, sensing and drug delivery, among others. However, their selective recognition in aqueous samples still remains problematic and is one of the reasons for their so far lilited commercial expansion. In the first part, we developed a water compatible MIP for its application as an active ingredient in a deodorant. Body odors are mainly due to volatile fatty acids generated from their glutamine conjugate precursors by hydrolytic enzymes from bacteria present on the skin. Most currently marketed anti-perspirants and deodorants contain, respectively aluminum salts and unspecific antibacterials. However, the extremely wide use of these products requires alternative solutions with regard to various problems (environmental, respect of skin ecosystem, toxicity, etc.). For this reason, a MIP was developed to capture the glutamine conjugate precursors so that they are no longer available to the bacteria, thus preventing their transformation to malodorous compounds. In order to generate binding selectivity in aqueous environments, an amidinium-based monomer which can form a strong stoichiometric electrostatic interaction with the carboxyl groups on the template, was synthesized. The MIP, blended in a dermo-cosmetic formulation, could capture selectively the glutamine precursors, amidst a multitude of other molecules present in human sweat. Furthermore, the MIP did not affect the skin bacteria, paving the way to an innovative and 'safer ' future-generation deodorant. In the second part, we developed a fast and efficient procedure based on molecularly imprinted solid­ phase extraction (MISPE) for the selective clean-up of betanin and its stereoisomer isobetanin from red beetroot extracts. Betanin is a natural pigment with significant antioxidant and biological activities currently used as food colorant. Dipicolinic acid was used as template for the MIP synthesis, because of its structural similarity to the chromophore group of betanin The MISPE procedures were optimized allowing the almost complete removal of carbohydrates and the majority of proteins, resulting in high extraction recovery of betanin / isobetanin in a single step. Moreover, the whole extraction procedure was performed in environmentally friendly solvents with either ethanol or water. To conclude, we believe that this study paves the way towards the development of a new generation of water compatible MIPs with improved recognition properties in highly complex aqueous environments, and should be applicable to other biotechnological and biomedical areas as well.

<p>Etanolni ekstrakti tropa odabranih sorti cvekle (Detroit, Cardeal-F1, Egipatska, Bikor i Kestrel) preči&scaron;ćeni su primenom ekstrakcije na čvrstoj fazi (SPE). Sadržaj ukupnih<br />fenolnih jedinjenja, flavonoida i betalaina u preči&scaron;ćenim ekstraktima određeni su spektrofotometrijskim metodama. HPLC analizom utvrđen je kvalitativni i kvantitativni sastav fenolnih jedinjenja i betalaina ekstrakata tropa odabranih sorti cvekle. ESR spektroskopijom ispitana je antiradikalska aktivnost ekstrakata topa cvekle na stabilne DPPH i reaktivne superoksid anjon i hidroksil radikale.<br />Spektrofotometrijski je određena antioksidativna aktivnost na DPPH radikale i redukciona sposobnost po Oyaizu u ekstraktima odabranih sorti cvekle. Ispitana je in vitro<br />antiproliferativna aktivnost frakcija ekstrakata, njihovim delovanjem na rast tri histolo&scaron;ki različite humane ćelijske linije: MCF-7 (adenokarcinom dojke), HeLa (epitelni karcinom cerviksa)i MRC-5 (fetalni fibroblastni karcinom pluća). U zavr&scaron;noj fazi rada određena je antimikrobna aktivnost ekstrakata tropa odabranih sorti cvekle.</p> / <p> Beetroot (Detroit, Cardeal-F1, Egipatska, Bikor i Kestrel)<br /> pomace ethanol extracts were purified using solid phase<br /> extraction (SPE). Contents of total phenols, flavonoids and<br /> betalains in purified extracts were determined by spectrophotometric<br /> methods. HPLC analysis were used for quantitative<br /> and qualitative characterization of phenolic compounds<br /> and betalains in investigated extracts. ESR spectroscopy<br /> was used for investigation of antiradical activity of<br /> beetroot pomace extracts on stable DPPH and reactive<br /> superoxide anion and hydroxyl radicals. Antioxidant activity<br /> was determined spectrophotometrically on DPPH radicals<br /> and reducing power according to Oyaizu in the beetroot pomace<br /> extracts. Antiproliferative activity of investigated extracts<br /> was determined in vitro, testing their influence on the<br /> growth of three histologically different human cell lines:<br /> MCF-7 (breast adenocarcinoma), HeLa (cervix epithelioid<br /> carcinoma) and MRC-5 (fetal lung). Also, antimicrobial activity<br /> of beetroot pomace extracts was determined.</p>

There is a global rise in the use of herbal products in combination with allopathic medicines, while most patients do not inform their health care providers of the use of these natural products. Both pharmacodynamic and pharmacokinetic interactions between herbal products and conventional drugs must be avoided for the wellbeing of the patient. Increasing evidence from in vitro and in vivo studies indicate that changed drug pharmacokinetics by co-administered herbs may be attributed to modulation of efflux drug transporters such as P-glycoprotein (P-gp). Garlic (Allium sativum), lemon (Citrus limonum) and beetroot (Beta vulgaris) are widely used by human immunodeficiency virus (HIV) patients, especially following the pronouncement by a former President of South Africa and the Ministers of Health at that time who promoted the use of these botanicals in HIV patients. The aim of this study was to measure the bi-directional in vitro transport of indinavir, a protease inhibitor, in the presence of crude extracts and pure phytoconstituents of A. sativum (L-alliin and diallyl disulphide), C. limonum (hesperidin and eriocitrin) and B. vulgaris (betaine monohydrate and ß-carotene) across excised porcine intestinal tissue in Sweetana-Grass diffusion chambers. In the negative control group, the transport of indinavir alone (200 M) was determined with no modulator added. In the positive control group, the transport of indinavir was determined in the presence of verapamil (100 M), a known P-gp related efflux inhibitor. The control experiments were used to indicate that the effects of the test compounds were caused by their action and not by chance interferences or external factors. Samples collected at pre-determined time intervals were analysed by means of a validated high performance liquid chromatography (HPLC) method and the transport was expressed as the apparent permeability coefficient (Papp) and the transepithelial flux (J) from which the efflux ratio (ER) and the net flux (Jnet) values were calculated. Statistical analysis was used to compare the results of the test compounds with the control groups in order to indicate significant differences. The mean ER value for indinavir in the negative control group was 1.41 ± 0.170 and in the positive control group it was 0.56 ± 0.0426. Statistically significant (p < 0.05) inhibition of indinavir efflux as indicated by reduced ER values was obtained for L-alliin (ER = 0.280 ± 0.030), diallyl disulphide (ER = 0.505 ± 0.034) and ß-carotene (ER = 0.664 ± 0.075). Inhibition of indinavir efflux will lead to increased transport and therefore a potentially higher bioavailability. Statistically significant (p < 0.05) promotion of indinavir efflux as indicated by increased ER values was obtained for C. limonum crude extract (ER = 5.551 ± 0.575) and hesperidin (ER = 3.385 ± 0.477), which potentially may lead to lower bioavalability. B. vulgaris crude extract (p = 0.8452), betaine monohydrate (p = 0.9982), A. sativum crude extract (p = 0.7161) and eriocitrin (p = 0.4431) displayed no statistically significant effect compared to the negative control group on indinavir transport across excised porcine intestinal tissue. The results from this study demonstrate that L-alliin, diallyl disulphide and ß-carotene have an inhibitory effect on indinavir efflux, which may significantly increase indinavir plasma levels after oral administration. C. limonum crude extract and hesperidin promote indinavir efflux, which may significantly reduce indinavir plasma levels. These pharmacokinetic interactions between certain drugs and plant extracts may negatively affect the anti-retroviral treatment of HIV patients, but deliberate and controlled inclusion of L-alliin, diallyl disulphide and ß-carotene in dosage forms may possibly cause more effective delivery of protease inhibitors after oral administration resulting in less frequent dosing intervals. / Thesis (MSc (Pharmaceutics))--North-West University, Potchefstroom Campus, 2013.

There is a global rise in the use of herbal products in combination with allopathic medicines, while most patients do not inform their health care providers of the use of these natural products. Both pharmacodynamic and pharmacokinetic interactions between herbal products and conventional drugs must be avoided for the wellbeing of the patient. Increasing evidence from in vitro and in vivo studies indicate that changed drug pharmacokinetics by co-administered herbs may be attributed to modulation of efflux drug transporters such as P-glycoprotein (P-gp). Garlic (Allium sativum), lemon (Citrus limonum) and beetroot (Beta vulgaris) are widely used by human immunodeficiency virus (HIV) patients, especially following the pronouncement by a former President of South Africa and the Ministers of Health at that time who promoted the use of these botanicals in HIV patients. The aim of this study was to measure the bi-directional in vitro transport of indinavir, a protease inhibitor, in the presence of crude extracts and pure phytoconstituents of A. sativum (L-alliin and diallyl disulphide), C. limonum (hesperidin and eriocitrin) and B. vulgaris (betaine monohydrate and ß-carotene) across excised porcine intestinal tissue in Sweetana-Grass diffusion chambers. In the negative control group, the transport of indinavir alone (200 M) was determined with no modulator added. In the positive control group, the transport of indinavir was determined in the presence of verapamil (100 M), a known P-gp related efflux inhibitor. The control experiments were used to indicate that the effects of the test compounds were caused by their action and not by chance interferences or external factors. Samples collected at pre-determined time intervals were analysed by means of a validated high performance liquid chromatography (HPLC) method and the transport was expressed as the apparent permeability coefficient (Papp) and the transepithelial flux (J) from which the efflux ratio (ER) and the net flux (Jnet) values were calculated. Statistical analysis was used to compare the results of the test compounds with the control groups in order to indicate significant differences. The mean ER value for indinavir in the negative control group was 1.41 ± 0.170 and in the positive control group it was 0.56 ± 0.0426. Statistically significant (p < 0.05) inhibition of indinavir efflux as indicated by reduced ER values was obtained for L-alliin (ER = 0.280 ± 0.030), diallyl disulphide (ER = 0.505 ± 0.034) and ß-carotene (ER = 0.664 ± 0.075). Inhibition of indinavir efflux will lead to increased transport and therefore a potentially higher bioavailability. Statistically significant (p < 0.05) promotion of indinavir efflux as indicated by increased ER values was obtained for C. limonum crude extract (ER = 5.551 ± 0.575) and hesperidin (ER = 3.385 ± 0.477), which potentially may lead to lower bioavalability. B. vulgaris crude extract (p = 0.8452), betaine monohydrate (p = 0.9982), A. sativum crude extract (p = 0.7161) and eriocitrin (p = 0.4431) displayed no statistically significant effect compared to the negative control group on indinavir transport across excised porcine intestinal tissue. The results from this study demonstrate that L-alliin, diallyl disulphide and ß-carotene have an inhibitory effect on indinavir efflux, which may significantly increase indinavir plasma levels after oral administration. C. limonum crude extract and hesperidin promote indinavir efflux, which may significantly reduce indinavir plasma levels. These pharmacokinetic interactions between certain drugs and plant extracts may negatively affect the anti-retroviral treatment of HIV patients, but deliberate and controlled inclusion of L-alliin, diallyl disulphide and ß-carotene in dosage forms may possibly cause more effective delivery of protease inhibitors after oral administration resulting in less frequent dosing intervals. / Thesis (MSc (Pharmaceutics))--North-West University, Potchefstroom Campus, 2013.

Tesis por compendio / [ES] Se ha estimado que obtenemos más de las tres cuartas partes del nitrato que ingerimos de la fruta y la verdura. Los vegetales ricos en nitratos incluyen verduras de hoja verde y ciertos tubérculos (p. ej., remolachas y rábanos). Las glándulas salivales concentran activamente el nitrato plasmático, lo que da lugar a concentraciones elevadas de nitrato en la saliva (5 a 8 mM) después de una comida rica en nitratos. El nitrato es un factor ecológico que puede inducir cambios rápidos en la estructura y función de las comunidades polimicrobianas. Sin embargo, los efectos sobre la microbiota oral no se han estudiado en detalle, mientras que un número limitado de estudios previos a esta tesis indican que es probable que el nitrato sea beneficioso para la salud bucal. El objetivo de esta tesis es, por tanto, estudiar los cambios microbiológicos inducidos por nitratos e identificar posibles mecanismos de homeostasis generados por este compuesto, con el fin de determinar si el nitrato puede considerarse un prebiótico para la salud bucal. Un segundo objetivo fue aislar cepas reductoras de nitrato y probar su potencial probiótico in vitro. En el capítulo 1, se realizó un estudio in vitro para testar el efecto del nitrato 6,5 mM en comunidades orales cultivadas a partir de la saliva de 12 individuos sanos. En el capítulo 2, se obtuvieron 53 aislados de bacterias reductoras de nitrato y se probó el efecto de seis candidatos a probióticos en comunidades orales sanas cultivadas a partir de saliva de diferentes donantes con o sin nitrato 6,5 mM. En el capítulo 3, se estudió el efecto de un extracto de remolacha rico en nitrato sobre la acidificación oral después de un enjuague con azúcar en 24 individuos sin caries activas. Se tomaron sobrenadantes (capítulos 1 y 2) o muestras de saliva (capítulo 3) para mediciones de nitrato, nitrito, amonio, lactato y pH. Además, la composición bacteriana de la biopelícula in vitro y del pellet salivar se determinó usando secuenciación Illumina del rRNA 16S y/o qPCR del género nitratorreductor Rothia. Los datos demuestran que el nitrato estimula el crecimiento de los géneros beneficiosos Rothia y Neisseria en nuestro modelo in vitro, mientras que potencialmente disminuye las bacterias asociadas a la caries, la halitosis y la enfermedad periodontal. Además, los datos in vitro e in vivo presentados en esta tesis indican que el nitrato puede limitar o prevenir caídas de pH cuando los azúcares son fermentados por la microbiota oral, un mecanismo de resiliencia que podría ser estimulado por el consumo de extractos vegetales ricos en nitratos. Los principales mecanismos de amortiguación del pH por parte del nitrato son el uso de acido láctico durante la desnitrificación (observado tanto in vivo como in vitro) y durante la reducción de nitrito a amonio, así como la producción potencial de amoníaco (observado in vitro). En esta tesis, los efectos del nitrato se observaron después de períodos cortos, es decir, después de 5-9 h de incubación in vitro y 1-4 horas después de la ingesta del suplemento de nitrato in vivo. Los estudios futuros deberían centrarse en los efectos longitudinales de la ingesta diaria de nitratos. En el capítulo 2, se aislaron bacterias reductoras de nitrato pertenecientes a los géneros Rothia y Actinomyces. Una selección de aislados de Rothia aumentó el uso de lactato y la capacidad de reducción de nitratos de las comunidades bucales, lo que potencialmente beneficiaría la salud dental y la salud sistémica, respectivamente. Los datos in vitro e in vivo presentados en esta tesis sugieren que el nitrato puede modular la microbiota oral en aspectos que son beneficiosas para el huésped y, por lo tanto, podría considerarse una sustancia prebiótica para la microbiota oral. Además, los aislados reductores de nitratos pueden estimular los efectos beneficiosos del metabolismo del nitrato, sobre todo en personas con bajos niveles de estas bacterias. / [CA] S'ha estimat que obtenim més de les tres quartes parts del nitrat que ingerim de la fruita i la verdura. Els vegetals rics en nitrats inclouen verdures de fulla verda i uns certs tubercles (p. ex., remolatxes i raves). Les glàndules salivals concentren activament el nitrat plasmàtic, la qual cosa dona lloc a concentracions elevades de nitrat a la saliva (5 a 8 mm) després d'un menjar ric en nitrats. El nitrat és un factor ecològic que pot induir canvis ràpids en l'estructura i funció de les comunitats polimicrobianes. No obstant això, els efectes sobre la microbiota oral no s'han estudiat detalladament, mentre que un nombre limitat d'estudis previs a aquesta tesi indiquen que és probable que el nitrat siga beneficiós per a la salut bucal. L'objectiu d'aquesta tesi és, per tant, estudiar els canvis microbiològics induïts per nitrats i identificar possibles mecanismes d'homeòstasi generats per aquest compost, amb la finalitat de determinar si el nitrat pot considerar-se un prebiòtic per a la salut bucal. Un segon objectiu va ser aïllar soques reductores de nitrat i provar el seu potencial probiòtic in vitro. En el capítol 1, es va realitzar un estudi in vitro per a testar l'efecte del nitrat 6,5 mm en comunitats orals cultivades a partir de la saliva de 12 individus sans. En el capítol 2, es van obtindre 53 aïllats de bacteris reductors de nitrat i es va provar l'efecte de sis candidats a probiòtics en comunitats orals sanes cultivades a partir de saliva de diferents donants amb o sense nitrat 6,5 mm. En el capítol 3, es va estudiar l'efecte d'un extracte de remolatxa ric en nitrat sobre l'acidificació oral després d'un glopeig amb sucre en 24 individus sense càries actives. Es van prendre sobrenadants (capítols 1 i 2) o mostres de saliva (capítol 3) per a mesuraments de nitrat, nitrit, amoni, lactat i pH. A més, la composició bacteriana de la biopel·lícula in vitro i del pèl·let salivar es va determinar usant seqüenciació Illumina del RNAr 16S i/o qPCR del gènere nitratorreductor Rothia. Les dades demostren que el nitrat estimula el creixement dels gèneres beneficiosos Rothia i Neisseria en el nostre model in vitro, mentre que potencialment disminueix els bacteris associats a la càries, l'halitosi i la malaltia periodontal. A més a més, les dades in vitro i in vivo presentades en aquesta tesi indiquen que el nitrat pot limitar o previndre caigudes de pH quan els sucres són fermentats per la microbiota oral, un mecanisme de resiliència que podria ser estimulat pel consum d'extractes vegetals rics en nitrats. Els principals mecanismes d'amortiment del pH per part del nitrat són l'ús de àcid làctic durant la desnitrificació (observat tant in vivo com in vitro) i durant la reducció de nitrit a amoni, així com la producció potencial d'amoníac (observat in vitro). En aquesta tesi, els efectes del nitrat es van observar després de períodes curts, és a dir, després de 5-9 h d'incubació in vitro i 1-4 hores després de la ingesta del suplement de nitrat in vivo. Els estudis futurs haurien de centrar-se en els efectes longitudinals de la ingesta diària de nitrats. En aquesta tesi es van aïllar bacteris reductors de nitrat pertanyents als gèneres Rothia i Actinomyces. Una selecció d'aïllats de Rothia va augmentar l'ús de lactat i la capacitat de reducció de nitrats de les comunitats bucals, la qual cosa potencialment beneficiaria la salut dental i la salut sistèmica, respectivament. Les dades in vitro i in vivo presentats en aquesta tesi suggereixen que el nitrat pot modular la microbiota oral en aspectes que són beneficiosos per a l'hoste i, per tant, podria considerar-se una substància prebiòtica per a la microbiota oral. A més, els aïllats reductors de nitrats poden estimular els efectes beneficiosos del metabolisme del nitrat, sobretot en persones amb baixos nivells d'aquests bacteris. El nitrat i els bacteris reductors de nitrat són, per tant, components prometedors per a futurs productes de salut oral. / [EN] It has been estimated that we obtain over three quarters of dietary nitrate from vegetables and fruits. Nitrate-rich vegetable types include leafy greens and certain root vegetables (e.g., beetroots and radishes). The salivary glands actively concentrate plasma nitrate, leading to high salivary nitrate concentrations (5-8 mM) after a nitrate-rich meal. Nitrate is an ecological factor that can induce rapid changes in structure and function of polymicrobial communities. However, the effects on the oral microbiota have not been clarified, whilst a limited number of previous studies did indicate that nitrate is likely to be beneficial for oral health. The aim of this thesis was therefore to study nitrate-induced microbiome changes and identify potential mechanisms for nitrate-induced homeostasis, in order to determine if nitrate can be considered a prebiotic compound for oral health. A second aim was to isolate nitrate-reducing isolates and test their probiotic potential in vitro. In chapter 1, an in vitro study was set up testing the effect of 6.5 mM nitrate on oral communities grown from saliva of 12 healthy individuals. In chapter 2, fifty-three nitrate-reducing isolates were obtained and the effect of six probiotic candidates was tested on healthy oral communities grown from saliva of different donors with or without 6.5 mM nitrate. In chapter 3, the effects of nitrate-rich beetroot extracts on oral acidification after sugar rinsing was tested in 24 individuals without active caries. Supernatants (chapters 1 and 2) or saliva samples (chapter 3) were taken for nitrate, nitrite, ammonium, lactate and pH measurements. Additionally, the bacterial composition of in vitro biofilms and salivary pellets were determined using 16S rRNA gene Illumina sequencing and/or qPCR of the nitrate-reducing genus Rothia. We showed that nitrate stimulates the growth of the beneficial genera Rothia and Neisseria in our in vitro model, while potentially decreasing caries-, halitosis- and periodontal disease-associated bacteria. Additionally, the in vitro and in vivo data presented in this thesis indicate that nitrate can limit or prevent pH drops when sugars are fermented by the oral microbiota - a mechanism of resilience that could be stimulated by the consumption of nitrate-rich vegetable extracts. The main pH buffering mechanisms of nitrate were lactic acid usage during denitrification (observed both in vivo and in vitro) and during the reduction of nitrite to ammonium, as well as the potential production of ammonia (observed in vitro). In this thesis, the effects of nitrate were observed after short periods, i.e., after 5-9 h incubation in vitro and/or after 1-4 hours after nitrate supplement intake in vivo. Future studies should focus on the longitudinal effects of daily nitrate intake. In chapter 2, nitrate-reducing species belonging to the genera Rothia and Actinomyces were isolated. A selection of Rothia isolates increased lactate usage and nitrate reduction capacities of oral communities, potentially benefitting dental health and systemic health, respectively. The in vitro and in vivo data presented in the current thesis suggest that nitrate can modulate the oral microbiota in ways that are beneficial for the host and could thus be considered a prebiotic substance for the oral microbiota. Additionally, nitrate-reducing isolates can stimulate certain beneficial effects of nitrate metabolism. Nitrate and nitrate-reducing bacteria are thus promising components for future oral care products to prevent or treat oral diseases and this should be further investigated. / Rosier, BT. (2022). Nitrate as a Prebiotic and Nitrate-Reducing Bacteria as Probiotics for Oral Health [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/181578 / TESIS / Compendio