Made available in DSpace on 2014-06-11T19:33:38Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-08-13Bitstream added on 2014-06-13T21:06:45Z : No. of bitstreams: 1 hojo_etd_dr_jabo.pdf: 1539570 bytes, checksum: 9a8c4375124457a8e811faf5f3693cc6 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Visando prolongar a vida útil da lichia, principalmente quanto à manutenção da cor e da qualidade, executaram-se experimentos para avaliar a eficiência dos tratamentos hidrotérmico e com solução de ácido clorídrico (HCl); do armazenamento sob refrigeração, em atmosfera controlada e em diferentes embalagens plásticas e de coberturas com quitosana. No Experimento I, testou-se a imersão em HCl a 0,087M por 6 minutos; o tratamento hidrotérmico por imersão a 52ºC por 1 minuto, seguido de resfriamento em água a 10ºC por 6 minutos; e o tratamento hidrotérmico com resfriamento em HCl a 0,087M a 10ºC por 6 minutos. O tratamento hidrotérmico seguido de resfriamento em HCl conservou a coloração dos frutos até o 3º dia, e a polpa com qualidade adequada até o 12º dia. No Experimento II, utilizou-se o melhor tratamento do experimento anterior (hidrotérmico com resfriamento em HCl) e testaramse diferentes temperaturas de armazenamento: 2ºC (91% UR); 5ºC (98% UR); 10ºC (80% UR); e 20ºC (70% UR). Os frutos foram analisados após 1, 4, 7, 10, 13, 16, 19, 22 e 25 dias. O armazenamento de lichia a 5 ºC manteve a boa aparência por até 13 dias e a qualidade da polpa até o final do período, 25 dias. O armazenamento a 2 ºC levou a maiores prejuízos na aparência. As temperaturas, de 10 ºC e 20 ºC, não foram efetivas para a manutenção da cor vermelha da casca. No Experimento III, foi testado o efeito da atmosfera controlada, associado aos melhores tratamentos dos experimentos anteriores. Os frutos foram armazenados a 5ºC e 94% UR, em atmosfera controlada contendo 5%, 10%, 20% e 80% de O2, com avaliações após 0 (inicial), 3, 7, 14, 21, 28 dias. As lichias de todos os tratamentos mantiveram a boa qualidade da polpa por até 21 dias, com os frutos sob atmosfera com 5% de O2, apresentando menor escurecimento da casca. As lichias apresentaram escurecimento da casca... / Aiming to extend litchi life, especially regarding to color and quality maintenance, experiments were performed to evaluate the treatment efficiency under heat and using hydrochloric acid solution (HCl), refrigerated storage, controlled atmosphere, different plastic containers, and chitosan coatings. In Experiment I, it was tested immersion in 0,087M HCl for 6 minutes; hydrothermal treatment by immersion at 52ºC for 1 minute, followed by water cooling at 10ºC for 6 minutes; and hydrothermal treatment with 0,087M HCl cooling at 10 ºC for 6 minutes. Hydrothermal treatment followed by HCl cooling preserved fruit color until the 3rd day and adequate pulp quality until the 12th day. In Experiment II, it was used the best treatment in the previous experiment (hydrothermal with HCl cooling) and different storage temperatures were tested: 2ºC (91% RH), 5ºC (98% RH), 10ºC (80% RH), and 20ºC (70% RH). Fruits were analyzed after 1, 4, 7, 10, 13, 16, 19, 22, and 25 days. Storage at 5ºC kept the good fruit appearance for up to 13 days, and pulp quality until the 25th day. The 2ºC led to to ligher losses in appearance. The temperatures of 10ºC and 20ºC, were not effective for maintaining the red color of the skin. In Experiment III, the effects of controlled atmosphere combined with improved treatments of previous experiments were tested. Fruits were stored at 5ºC and 94% RH in a controlled atmosphere containing 5%, 10%, 20% and 80% O2, with evaluations after 0 (initial), 3, 7, 14, 21, 28 days. Litchis in all treatments maintained good pulp quality for up to 21 days, with the fruits under a 5% O2 atmosphere showing a lower skin browning. Litchis showed over 50% skin browning after 7 days. In Experiment IV, different concentrations of CO2 (0%, 5%, 10%, 15%, and 20%) combined with the best concentration in the previous experiment, 5% O2, were tested... (Complete abstract click electronic access below)

Orientador: Paulo Mazzafera / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-05T10:18:49Z (GMT). No. of bitstreams: 1 Melo_GeraldoAclecio_D.pdf: 1320706 bytes, checksum: 5f501b7dd60a44ae72341950498d75f7 (MD5) Previous issue date: 2005 / Resumo: Polifenoloxidase - PFO (EC 1.14.18.1 ou EC 1.10.3.2) é uma enzima de ampla distribuição entre as plantas e catalisa a hidroxilação de monofenóis a o-difenóis e a oxidação destes para o-diquinonas. Sua função em plantas tem sido relacionada a mecanismos de defesa contra patógenos e pragas. Em cafeeiro, o ácido 5-cafeoilquínico, também conhecido como ácido clorogênico (CGA) é o principal substrato da PFO e ambos, enzima e substrato, estão presentes em quantidades expressivas nos frutos e nas folhas desta planta. O CGA também está relacionado com mecanismos de defesas das plantas e como tal é considerando importante substrato em reações de oxidação, principalmente aquelas mediadas pela PFO. No presente estudo, com objetivo de conhecer características da PFO de folhas do cafeeiro, de averiguar sua ação em mecanismos de defesa nessa planta e de entender fatores ligados à síntese e ao acúmulo de seu principal substrato foram feitas a purificação e caracterização dessa enzima, estudos da expressão de sua atividade, bem como estudos de expressão de enzimas da via de síntese do CGA em cafeeiro. Com o uso de técnicas de precipitação com sulfato de amônio, cromatografias de troca iônica, interação hidrofóbica e exclusão molecular foi possível obter a PFO com alto grau de pureza. A enzima apresentou massa molecular de 40,5 Kda e preferência pelo ácido 5-cafeoilquínico como substrato. Seqüências de peptídeos obtidas após digestão da proteína e análise por espectrometria de massas mostraram-se homólogas a seqüências de PFO de várias outras plantas. O nível constitutivo de atividade da PFO observado para quinze genótipos de café variou de 3,8 a 88,0 unidades de atividade/mg de proteína, entretanto não teve relação direta com resistência a pragas e doenças nessa planta. A resistência ao bicho mineiro foi significativamente relacionada ao nível de compostos fenólicos, entretanto, ácido 5-cafeoilquínico, o principal substrato da PFO em café, não teve relação com essa resistência, sugerindo a importância de outros compostos fenólicos como substratos da PFO. Dano mecânico, tratamento com ácido metiljasmônico, inoculação com esporos do fungo Hemileia vastatrix e a infestação com ovos do inseto Perileucoptera coffeella levaram a respostas variadas nos níveis de atividade de PFO nos genótipos avaliados. Baseando-se nesses resultados, conclui-se que a ação da PFO na resistência do cafeeiro a pragas e doenças pode estar relacionada ao potencial oxidativo do tecido e não simplesmente uma maior atividade; que o tipo e quantidade de substrato encontrado no tecido podem ser importantes na resistência do cafeeiro e que entre os genótipos pode existir a especialização de mecanismos de resistência envolvendo a ação da PFO. Estudos de expressão por RT-PCR de fenilalanina amônia-liase (PAL), cinamato 4-hiroxilase (C4H), coumarato 3-hidroxilase (C3H), hidroxicinamoil-CoA ligase (4CL) e hidroxicinamoil-CoA:D-quinato hidroxicinamoil transferase (CQT), enzimas da via de síntese do CGA, tiveram sua expressão reduzida à medida que o tecido envelhece. No endosperma foi observado um decréscimo acentuado de expressão no final da maturação dos frutos. Plântulas estioladas obtidas pela germinação de sementes no escuro e transferidas para luz mostraram aumentos significativos no conteúdo de CGA após 24 horas. Esses aumentos foram transientes e coincidiram com a expressão da PAL, C4H, C3H, 4CL e CQT. Os resultados indicam existência de controle da síntese de CGA e a existência de mecanismos de controle da expressão em comum para as cinco enzimas estudadas / Abstract: Polyphenoloxidase - PPO (EC 1.14.18.1 ou EC 1.10.3.2) is an enzyme with broad distribution among plants and catalyzes the hydroxylation of monophenols to o-diphenols and the oxidation of these to o-diquinones. Its function on plants has been related to defense mechanisms against pathogens and plagues. 5-Caffeoylquinic acid, also known as chlorogenic acid (CGA), is the main PPO substrate in coffee tissues and both, enzyme and substrate are present on substantial quantities in fruits and leaves. CGA is also referred to having connection with plants defense mechanisms and it is also an important substrate on oxidation reactions, mainly those mediated by PPO. Therefore, in order to increase our knowledge on the coffee PPO characteristics, to verify its role in defense mechanisms and also to understand the factors connected to the synthesis of CGA, coffee leaf PPO was purified and characterized regarding kinetic parameters and its activity in leaves of several coffee species exposed or not to pest (leaf miner) and disease (leaf rust). Also studies on the expression of the enzymes of CGA synthesis were carried out. By using ammonium sulfate precipitation followed by chromatographic steps on ionic exchange, hydrophobic interaction and molecular exclusion resins it was possible to purify PPO to homogeneity. The enzyme presented a molecular mass of 40,5 Kda and used 5-cafeoylquinic acid as the preferred substrate. Peptide sequences obtained after digestion of the purified PPO and analysis through mass spectrometry were homologous to PPO sequences of several other plants. The constitutive level of PPO activity observed for 15 coffee genotypes varied from 3,8 to 88,0 units of activity/mg of protein, but did not have a direct relationship with resistance to plagues in this plant. Resistance to leaf miner was significantly related to the level of phenolic compounds. However, 5-caffeoylquínic acid, the main substrate of PPO on coffee, was not related with resistance, suggesting the importance of other phenolic compounds as PPO substrates. Mechanical damage, treatment with methyljasmonic acid, inoculation with spores from Hemileia vastatrix and the infestation with the insect Perileucoptera coffeella led to varied results of the PPO activity in the evaluated genotypes. Based on these results, we conclude that the PPO role in the coffee resistance to plagues and diseases might be related to the oxidative potential of the tissue and not only on the PPO activity; that the kind and quantity of PPO substrate found in the tissue might be important for the resistance of the coffee tree and that there may be specific mechanisms of resistance involving PPO action among the genotypes. RT-PCR studies of the expression of phenylalanine ammonia-lyase (PAL), cinnamate 4-hyroxylase (C4H), coumarate 3-hydroxylase (C3H), hydroxycinnamoyl-CoA ligase (4CL) and hydroxycinnamoyl-CoA:D-quinate hydroxycinnamoyl transferase (CQT), which code for enzymes of the CGA biosynthetic pathway, showed that the expression of these enzymes decrease with tissue aging. In the endosperm, an evident decrease on the expression was observed in the end of the fruit ripening. Etiolated seedlings obtained by germination of coffee seeds in the dark and transferred into light showed significant increasing on the CGA content after 24 hours. The increase was transient and followed the expression pattern of PAL, C4H, C3H, 4CL and CQT. The results indicate that CGA biosynthesis is coordinately regulated by the expression of the five enzymes / Doutorado / Biologia Vegetal / Doutor em Biologia Vegetal

No description available.

Doctor of Philosophy / Polyphenol oxidase (PPO) catalyses undesirable darkening in wheat products such as Asian noodles. Genetic variation for PPO activity is characterized in bread wheat. Australian wheat breeding programmes recognize that reduced PPO activity is an important quality target. Despite this interest from breeders, no varieties possessing extremely low and null PPO activity exist. The development of null PPO wheat varieties is dependant on an understanding of the genetic control of the null phenotype. Knowledge of these factors will accelerate efforts to develop them. The inheritance of PPO activity was investigated in two populations that were derived from hybrids between a null PPO genotype and Australian wheat varieties Lang and QAlBis. Observed genetic ratios were consistent with two and three gene control, respectively in these populations. QTL mapping was performed in the QALBis x VAW08-A17 population. The Diversity Array Technology (DArT) approach was employed to genotype the QALBis x VAW08-A17 population. Three highly significant QTLs that control PPO activity were identified on chromosomes 2AL, 2BS and 2DL. Close associations between PPO activity and DArT marker loci wPt-7024, wPt-0094 and wPt-2544 were observed, respectively. Collectively, these loci explained 74% of the observed variation in PPO activity across seasons. Significant QTLs on chromosomes 1B and 3B were also identified that together explained an additional 17% of variation in PPO activity. The relationship between PPO activity and yellow alkaline noodles (YAN) colour stability parameters was investigated in a DM5637*B8 x H45 doubled haploid population. PPO activity and changes in YAN brightness (ΔL* 0-24h) and yellowness (Δb* 0-24h) in both seasons were analysed. Quantitative trait analyses of PPO activity, flour yellowness (b*) and YAN colour stability was also conducted in this population. QTL mapping of variation in PPO activity in the DM5637*B8 x H45 DH population identified a highly significant QTL on chromosome 2AL, which explained 52% of the observed variation across seasons. Regression analysis identified that wPt-7024 was highly significantly associated with PPO activity in this population. A highly significant association between this marker and PPO was also identified in the QALBis x VAW08-A17 population. Collectively, the three identified QTLs (on chromosomes 2AL, 7A and 7B) explained 71% of variation in PPO activity across seasons. A highly significant (P<0.001) QTL on chromosome 2B along with significant (P<0.01) QTLs on the chromosomes 1A, 3B, 4B and 5B were found to control flour yellowness. The QTLs on 2B, 4B and 5B were detected in both seasons analysed and accounted for 90% of variation in flour b* across seasons. The study on YAN colour stability located two highly significant (P<0.001) QTLs and two significant (P<0.01) QTLs that controlled the change in brightness of yellow alkaline noodle. The 2A QTL accounted for 64% of observed variation across seasons. It was in the same location as the PPO QTL and shared a common closest marker wPt-7024. Only one significant QTL for YAN a* (0-24h) was identified. It accounted for 12% of variation across seasons and was only detected in one season. One highly significant (P<0.001) QTL and two significant (P<0.01) QTLs were identified that controlled the change in yellowness of yellow alkaline noodle. The 2A QTL accounted for 68% of observed variation across seasons. The location of this QTL corresponded with that of 2A QTLs for PPO activity and L* of YAN in this study. Furthermore, wPt-7024 was also identified as the marker with the most significant association with L*. The identification of a correlation between the characters and a common location of a highly significant QTL for each of these characters indicates that it is likely that PPO activity is directly responsible for a large proportion of the changes in brightness and yellowness of YAN. QTLs for L* and b* of YAN were detected in a common location on chromosome 1A. However, no corresponding QTL was identified that controls PPO activity, highlighting the complexity of the relationship between these traits. Resistance to three rust pathogens (Puccinia graminis, Puccinia striiformis, and Puccinia triticina) was also investigated in the DM5637*B8 x H45 DH population because they are major yield limiting diseases in wheat. Disease response data at the seedling stage were converted to genotypic scores for rust genes Sr24/Lr24, Sr36, Lr13 and Yr7 to construct a genetic linkage map. No recombination was observed between rust resistance genes Sr36, Lr13 and Yr7 in this DH population. Therefore, these genes mapped in the same position on chromosome 2B. The Lr24/Sr24 locus was incorporated into the chromosome 3D map. Interval mapping analysis identified QTLs on chromosomes 2B, 3B, 4B and 5B that control adult plant resistance (APR) to stripe rust. Two QTLs on chromosomes 2B and 3D were identified that controlled APR to leaf rust in this DH population.

Doctor of Philosophy / Polyphenol oxidase (PPO) catalyses undesirable darkening in wheat products such as Asian noodles. Genetic variation for PPO activity is characterized in bread wheat. Australian wheat breeding programmes recognize that reduced PPO activity is an important quality target. Despite this interest from breeders, no varieties possessing extremely low and null PPO activity exist. The development of null PPO wheat varieties is dependant on an understanding of the genetic control of the null phenotype. Knowledge of these factors will accelerate efforts to develop them. The inheritance of PPO activity was investigated in two populations that were derived from hybrids between a null PPO genotype and Australian wheat varieties Lang and QAlBis. Observed genetic ratios were consistent with two and three gene control, respectively in these populations. QTL mapping was performed in the QALBis x VAW08-A17 population. The Diversity Array Technology (DArT) approach was employed to genotype the QALBis x VAW08-A17 population. Three highly significant QTLs that control PPO activity were identified on chromosomes 2AL, 2BS and 2DL. Close associations between PPO activity and DArT marker loci wPt-7024, wPt-0094 and wPt-2544 were observed, respectively. Collectively, these loci explained 74% of the observed variation in PPO activity across seasons. Significant QTLs on chromosomes 1B and 3B were also identified that together explained an additional 17% of variation in PPO activity. The relationship between PPO activity and yellow alkaline noodles (YAN) colour stability parameters was investigated in a DM5637*B8 x H45 doubled haploid population. PPO activity and changes in YAN brightness (ΔL* 0-24h) and yellowness (Δb* 0-24h) in both seasons were analysed. Quantitative trait analyses of PPO activity, flour yellowness (b*) and YAN colour stability was also conducted in this population. QTL mapping of variation in PPO activity in the DM5637*B8 x H45 DH population identified a highly significant QTL on chromosome 2AL, which explained 52% of the observed variation across seasons. Regression analysis identified that wPt-7024 was highly significantly associated with PPO activity in this population. A highly significant association between this marker and PPO was also identified in the QALBis x VAW08-A17 population. Collectively, the three identified QTLs (on chromosomes 2AL, 7A and 7B) explained 71% of variation in PPO activity across seasons. A highly significant (P<0.001) QTL on chromosome 2B along with significant (P<0.01) QTLs on the chromosomes 1A, 3B, 4B and 5B were found to control flour yellowness. The QTLs on 2B, 4B and 5B were detected in both seasons analysed and accounted for 90% of variation in flour b* across seasons. The study on YAN colour stability located two highly significant (P<0.001) QTLs and two significant (P<0.01) QTLs that controlled the change in brightness of yellow alkaline noodle. The 2A QTL accounted for 64% of observed variation across seasons. It was in the same location as the PPO QTL and shared a common closest marker wPt-7024. Only one significant QTL for YAN a* (0-24h) was identified. It accounted for 12% of variation across seasons and was only detected in one season. One highly significant (P<0.001) QTL and two significant (P<0.01) QTLs were identified that controlled the change in yellowness of yellow alkaline noodle. The 2A QTL accounted for 68% of observed variation across seasons. The location of this QTL corresponded with that of 2A QTLs for PPO activity and L* of YAN in this study. Furthermore, wPt-7024 was also identified as the marker with the most significant association with L*. The identification of a correlation between the characters and a common location of a highly significant QTL for each of these characters indicates that it is likely that PPO activity is directly responsible for a large proportion of the changes in brightness and yellowness of YAN. QTLs for L* and b* of YAN were detected in a common location on chromosome 1A. However, no corresponding QTL was identified that controls PPO activity, highlighting the complexity of the relationship between these traits. Resistance to three rust pathogens (Puccinia graminis, Puccinia striiformis, and Puccinia triticina) was also investigated in the DM5637*B8 x H45 DH population because they are major yield limiting diseases in wheat. Disease response data at the seedling stage were converted to genotypic scores for rust genes Sr24/Lr24, Sr36, Lr13 and Yr7 to construct a genetic linkage map. No recombination was observed between rust resistance genes Sr36, Lr13 and Yr7 in this DH population. Therefore, these genes mapped in the same position on chromosome 2B. The Lr24/Sr24 locus was incorporated into the chromosome 3D map. Interval mapping analysis identified QTLs on chromosomes 2B, 3B, 4B and 5B that control adult plant resistance (APR) to stripe rust. Two QTLs on chromosomes 2B and 3D were identified that controlled APR to leaf rust in this DH population.

Orientador: José Fernando Durigan / Banca: Ben-Hur Mattiuz / Banca: Ricardo Alfredo Kluge / Banca: Jairo Osvaldo Cazetta / Banca: Marcos David Ferreira / Resumo: Visando prolongar a vida útil da lichia, principalmente quanto à manutenção da cor e da qualidade, executaram-se experimentos para avaliar a eficiência dos tratamentos hidrotérmico e com solução de ácido clorídrico (HCl); do armazenamento sob refrigeração, em atmosfera controlada e em diferentes embalagens plásticas e de coberturas com quitosana. No Experimento I, testou-se a imersão em HCl a 0,087M por 6 minutos; o tratamento hidrotérmico por imersão a 52ºC por 1 minuto, seguido de resfriamento em água a 10ºC por 6 minutos; e o tratamento hidrotérmico com resfriamento em HCl a 0,087M a 10ºC por 6 minutos. O tratamento hidrotérmico seguido de resfriamento em HCl conservou a coloração dos frutos até o 3º dia, e a polpa com qualidade adequada até o 12º dia. No Experimento II, utilizou-se o melhor tratamento do experimento anterior (hidrotérmico com resfriamento em HCl) e testaramse diferentes temperaturas de armazenamento: 2ºC (91% UR); 5ºC (98% UR); 10ºC (80% UR); e 20ºC (70% UR). Os frutos foram analisados após 1, 4, 7, 10, 13, 16, 19, 22 e 25 dias. O armazenamento de lichia a 5 ºC manteve a boa aparência por até 13 dias e a qualidade da polpa até o final do período, 25 dias. O armazenamento a 2 ºC levou a maiores prejuízos na aparência. As temperaturas, de 10 ºC e 20 ºC, não foram efetivas para a manutenção da cor vermelha da casca. No Experimento III, foi testado o efeito da atmosfera controlada, associado aos melhores tratamentos dos experimentos anteriores. Os frutos foram armazenados a 5ºC e 94% UR, em atmosfera controlada contendo 5%, 10%, 20% e 80% de O2, com avaliações após 0 (inicial), 3, 7, 14, 21, 28 dias. As lichias de todos os tratamentos mantiveram a boa qualidade da polpa por até 21 dias, com os frutos sob atmosfera com 5% de O2, apresentando menor escurecimento da casca. As lichias apresentaram escurecimento da casca... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Aiming to extend litchi life, especially regarding to color and quality maintenance, experiments were performed to evaluate the treatment efficiency under heat and using hydrochloric acid solution (HCl), refrigerated storage, controlled atmosphere, different plastic containers, and chitosan coatings. In Experiment I, it was tested immersion in 0,087M HCl for 6 minutes; hydrothermal treatment by immersion at 52ºC for 1 minute, followed by water cooling at 10ºC for 6 minutes; and hydrothermal treatment with 0,087M HCl cooling at 10 ºC for 6 minutes. Hydrothermal treatment followed by HCl cooling preserved fruit color until the 3rd day and adequate pulp quality until the 12th day. In Experiment II, it was used the best treatment in the previous experiment (hydrothermal with HCl cooling) and different storage temperatures were tested: 2ºC (91% RH), 5ºC (98% RH), 10ºC (80% RH), and 20ºC (70% RH). Fruits were analyzed after 1, 4, 7, 10, 13, 16, 19, 22, and 25 days. Storage at 5ºC kept the good fruit appearance for up to 13 days, and pulp quality until the 25th day. The 2ºC led to to ligher losses in appearance. The temperatures of 10ºC and 20ºC, were not effective for maintaining the red color of the skin. In Experiment III, the effects of controlled atmosphere combined with improved treatments of previous experiments were tested. Fruits were stored at 5ºC and 94% RH in a controlled atmosphere containing 5%, 10%, 20% and 80% O2, with evaluations after 0 (initial), 3, 7, 14, 21, 28 days. Litchis in all treatments maintained good pulp quality for up to 21 days, with the fruits under a 5% O2 atmosphere showing a lower skin browning. Litchis showed over 50% skin browning after 7 days. In Experiment IV, different concentrations of CO2 (0%, 5%, 10%, 15%, and 20%) combined with the best concentration in the previous experiment, 5% O2, were tested... (Complete abstract click electronic access below) / Doutor

Les légumineuses permettent de réduire les intrants en élevage (engrais, concentrés) en raison notamment de leurs niveaux élevés en protéines. Cependant, à la fois pendant le processus d'ensilage et celui de fermentation dans le rumen, les protéines peuvent subir une importante dégradation, ce qui affecte la valeur nutritive des fourrages et induit des rejets d'azote (N) importants, notamment dans l'urine. Certaines légumineuses peuvent alors être d'un intérêt particulier car elles produisent des composés secondaires qui peuvent modifier positivement les processus fermentaires et digestifs. Ainsi, les tannins condensés (CT) présents dans le sainfoin (SF, Onobrychis viciifolia) sont capables de se lier aux protéines, réduisant leur dégradation dans le silo et le rumen et se traduisant par un transfert de l'excrétion d'azote de l'urine vers les fèces. Le trèfle violet (RC, Trifolium pratense) contient la polyphénoloxydase (PPO), une enzyme qui catalyse l'oxydation de différents composés phénoliques en quinones. Comme les CTs, les quinones sont capables de former des complexes avec les protéines permettant de réduire leur dégradation dans le silo et le rumen. L'objectif de cette thèse était alors d'étudier et de quantifier les bénéfices potentiels de l'utilisation de ces deux espèces de légumineuses bioactives sur i) la qualité et la conservation des ensilages, ii) la fermentation ruminale, l'efficacité digestive et les performances des ovins, et iii) l'empreinte environnementale (excrétion d'N et de CH4). Nous avons effectué deux essais in vitro et deux essais in vivo, basés sur des ensilages composés de ces deux légumineuses, seules ou en mélange avec une graminée (la fléole- T, Phleum pratense L.) qui nous a servie de contrôle. Les essais in vitro nous ont permis de nous focaliser sur la qualité et la conservation des ensilages ainsi que sur la fermentation ruminale, tandis que les essais in vivo se sont concentrés sur la performance et l'efficacité digestive des agneaux, ainsi que sur leur bilan azoté et leurs émissions de CH4. L'inclusion de légumineuses bioactives dans les ensilages d'herbe a amélioré la qualité du fourrage, la fermentation pendant le processus d'ensilage ainsi que la protection des protéines contre une dégradation au sein du silo et du rumen. Globalement, l'alimentation des agneaux avec des mélanges comportant ces légumineuses s'est traduite par une augmentation de l'ingestion de matière sèche, en comparaison des agneaux alimentés avec la graminée pure. Néanmoins, en raison de la digestibilité nettement plus faible de T-SF, probablement due à une composition et une nature des fibres différentes ainsi qu'à la présence de CT, les agneaux ayant reçu T-SF ont montré une ingestion et des performances plus faibles que ceux ayant reçu les ensilages contenant RC. Dans le rumen, il semble que les protéines de RC aient été plus protégées de la dégradation que celles de SF, tandis que dans la suite du tractus digestif, les complexes formés entre protéines et CT (avec SF) se seraient moins dissociés que ceux formés entre protéines et quinones (avec RC), ce qui pourrait en partie expliquer le transfert d'excrétion de l'N de l'urine vers les fèces, observé chez les agneaux alimentés avec T-SF et bénéfique pour l'environnement. SF a également permis de réduire légèrement les émissions de CH4. Ainsi, utiliser des légumineuses bioactives dans les pratiques d'alimentation des ruminants apparaît une stratégie prometteuse pour fournir des produits animaux de façon plus durable. Nos résultats montrent que chaque espèce apporte des avantages différents, plutôt orientés vers la qualité de l'aliment et les performances animales pour RC mais plutôt orientés vers la réduction des rejets pour SF. Des recherches complémentaires sont donc nécessaires pour mieux caractériser ces avantages et élargir les investigations à d'autres espèces, d'autres mélanges et d'autres bénéfices potentiels. (...) / Fodder legume species allow to reduce inputs in livestock breeding systems (fertilizer, concentrates) notably because they contain high levels of crude proteins which are of primary importance in ruminant nutrition. However, during both silage and rumen fermentation processes, proteins are submitted to degradation which affects forage nutritive value and leads to nitrogen (N) losses notably via urine. Some specific legumes can then be of particular interest as they produce plant secondary compounds that can positively affect silage and digestive processes. Condensed tannins (CTs) present in sainfoin (SF, Onobrychis viciifolia) are able to bind with proteins thereby reducing their degradation in the silo and the rumen, resulting in a shift in N excretion from urine to faeces. Red clover (RC, Trifolium pratense) contains polyphenol oxidase (PPO), an enzyme that catalyses the oxidation of different phenolics into quinones. As CTs, quinones are able to form complexes with proteins that will similarly reduce their degradation in the silo and the rumen. The aim of this thesis was to investigate and quantify the potential benefits of using these two bioactive legume species on i) quality and conservation of silages, ii) rumen fermentation, digestive efficiency and sheep performance, and iii) environmental footprint (N excretion and CH4 emissions). We conducted two in vitro and two in vivo trials which were based on silages of pure legumes or of different mixtures with the grass species (timothy T, Phleum pratense L.), which served as control. In the in vitro trials, we focussed on silage quality, silage conservation and rumen fermentation, while in the in vivo trials, we focussed on lambs' performance, digestion efficiency, N balance and CH4 emissions. Including bioactive legumes in mixtures with grass improved, compared to pure grass, forage quality and fermentation during the silage making process, as well as proteins' protection from degradation within both the silos and the rumen. Lambs fed with the mixtures involving legumes responded with an increase in DM intake compared to their counterparts fed with T. Nevertheless, due to a possibly different fibre composition and to the presence of CT which impaired SF digestibility, lambs that consumed T-SF showed lower intake and performance than those that received RC-containing silages. In the rumen, RC proteins appeared more protected from degradation than SF ones, while in the subsequent parts of the digestive tract, the proteins-CT complexes (from SF) might less dissociate than the proteins-quinones ones (from RC). This could partly explain the environment-friendly shift in N excretion from urine to faeces when animals are fed with T-SF. SF also allowed to slightly reduce CH4 emissions. Thus, utilizing bioactive legumes in livestock feeding practices is a promising strategy to produce animal products more sustainably. Our results show different benefits relative to the bioactive legume species involved, directed towards boosted forage quality and animals' performance for RC but towards lowered wastes for SF. Further research is thus needed to better characterize these benefits and enlarging investigations to other plant species, mixtures and potential benefits (e.g. health). This will help to determine the appropriate choice of plant species according to the objectives.

Submitted by Renata Lopes (renatasil82@gmail.com) on 2016-07-20T19:32:21Z No. of bitstreams: 1 virginiafernandesbraga.pdf: 2075207 bytes, checksum: 720e5dba0516d172aec1843372dcecc2 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2016-07-22T15:25:58Z (GMT) No. of bitstreams: 1 virginiafernandesbraga.pdf: 2075207 bytes, checksum: 720e5dba0516d172aec1843372dcecc2 (MD5) / Made available in DSpace on 2016-07-22T15:25:58Z (GMT). No. of bitstreams: 1 virginiafernandesbraga.pdf: 2075207 bytes, checksum: 720e5dba0516d172aec1843372dcecc2 (MD5) Previous issue date: 2011-04-20 / Pitcairnia albiflos Herb. (Bromeliaceae) atualmente se encontra na lista de espécies ameaçadas de extinção. Essa espécie é endêmica dos afloramentos rochosos do município do Rio de Janeiro, RJ, e vem sofrendo com o pisoteio de alpinistas, queimadas, invasão de gramíneas exóticas e extrativismo vegetal. A micropropagação pode ser utilizada como alternativa às condições de risco em que as populações dessa espécie se encontram submetidas, visando à recomposição de populações ameaçadas em ambiente natural, assim como o abastecimento do mercado consumidor. A etapa final da micropropagação é a aclimatização, período em que as plantas ficam mais susceptíveis e sofrem com o estresse oxidativo devido às mudanças nas condições ambientais. No presente trabalho foram avaliadas as atividades enzimáticas antioxidativas da CAT, SOD, POD, PPO e o conteúdo de prolina, além dos teores de pigmentos fotossintéticos em plantas de Pitcairnia albiflos cultivados in vitro, em meios de cultura contendo duas concentrações de sacarose (15 ou 30 g L-1), tampas não vedadas que permitiam trocas gasosas e frascos vedados com tampas e filme plástico de PVC, que impediam a ventilação. Sob essas condições, as plantas foram cultivadas em meios contendo GA3 ou ANA. Após o período de crescimento in vitro, as plantas foram transferidas para condições ex vitro em casa de vegetação. As análises supra-citadas e a determinação dos teores de carboidratos solúveis totais, sacarose, amido, açúcares redutores, conteúdo relativo de água e suculência também foram realizadas nas plantas previamente cultivadas in vitro com 15 ou 30 g L-1 de sacarose e GA3 em tubos com tampas vedadas, após submissão das mesmas a estresse hídrico durante 24, 38 ou 52 dias. Após o período de estresse hídrico, as plantas foram reidratadas durante 34 dias sob irrigação periódica em casa de vegetação. Nos tecidos cultivados in vitro percebeu-se o surgimento de características de hiperidricidade nas plantas cultivadas com 15 g L-1 de sacarose, GA3 e tubos com tampas vedadas, o que foi evidenciado pelo menor acúmulo de prolina, aumento das atividades das enzimas antioxidantes e menor acúmulo de pigmentos fotossintetizantes. Na condição ex vitro, as plantas cultivadas anteriormente em meio de cultura contendo 15 g L-1 de sacarose apresentaram maior atividade das enzimas antioxidantes não havendo, em alguns casos, diferenças significativas em comparação com a concentração mais elevada de sacarose. Nessa condição o acúmulo de prolina foi menor, o que é indicativo de maior estresse oxidativo nessas plantas durante a aclimatização. Durante o estresse hídrico houve queda na atividade de todas as enzimas estudadas, embora essa queda tenha sido mais acentuada para as plantas que inicialmente foram cultivadas com 15 g L-1 de vi sacarose. O acúmulo de prolina aumentou com o prolongamento do estresse hídrico, sendo maior nas plantas que foram cultivadas in vitro com 30 g L-1 de sacarose. Não houve diferenças significativas no conteúdo de pigmentos fotossintetizantes e nas suas relações para nenhuma das concentrações de sacarose, exceto para os carotenóides totais, que apresentaram aumento significativo ao longo do período de estresse hídrico para as plantas previamente cultivadas com a menor concentração de sacarose. Os conteúdos de carboidratos solúveis totais e sacarose aumentaram com o prolongamento do estresse, sendo mais acentuados na concentração de 30 g L-1 de sacarose. Os conteúdos de amido, o conteúdo relativo de água e a suculência apresentaram redução com o aumento do estresse hídrico. Após a reidratação, todas as plantas mostraram capacidade de recuperação, apresentando valores próximos aos dos controles para todas as variáveis analisadas. Ressalta-se, todavia, que as plantas tratadas com 30 g L-1 de sacarose tiveram melhor recuperação quando comparadas com aquelas que foram tratadas com 15 g L-1 de sacarose. Em função dos resultados obtidos, é possível concluir que a concentração de sacarose utilizada in vitro apresenta influência no processo de aclimatização ex vitro e também, posteriormente, no campo, na capacidade de recuperação das plantas à seca quando elas são submetidas a estresse hídrico. As plantas cultivadas in vitro com 15 g L-1 de sacarose se mostraram mais sensíveis à seca e, possivelmente, não sobreviveriam caso fossem transferidas dos tubos de ensaio diretamente para o campo. As plantas cultivadas in vitro com 30 g L-1 de sacarose aparentemente eram mais resistentes ao processo de aclimatização ex vitro, apresentando maiores chances de sobrevivência em campo, maior tolerância à seca e maior capacidade de recuperação após períodos prolongados de estresse hídrico. / Pitcairnia albiflos Herb. (Bromeliaceae) is currently in the list of endangered species. This species is endemic of the inselbergs of the city of Rio de Janeiro, RJ, and has been suffering with the mountaineer’s trampling, wildfires, invasion of exotic grasses and plant extraction. The micropropagation can be used as an alternative to the risk conditions under which populations of this species are submitted, aiming at the recomposition of endangered populations in the natural environment, as well as the supply of the consumer market. The final stage of the micropropagation is the acclimatization, period in which the plants become more susceptible and suffer from oxidative stress due to the changes in the environmental conditions. In the present study it were evaluated the antioxidative enzymatic activities of CAT, SOD, POD, PPO and proline content, besides the levels of photosynthetic pigments in plants of Pitcairnia albiflos grown in vitro in culture mediums containing two sucrose concentrations (15 or 30 g L-1). Part was covered with unsealed lids that allowed gas exchanges and part was kept in sealed flasks with lids and PVC plastic film, that didn’t allow the ventilation. Under these conditions, the plants were cultivated in culture mediums containing GA3 or NAA. After the in vitro growth period, the plants were transferred to ex vitro conditions at a greenhouse. The above mentioned analyses and the determination of total soluble carbohydrate levels, sucrose, starch, reducer sugars, relative water content and succulence were also performed on the plants previously grown in vitro with 15 or 30 g L-1 of sucrose and GA3 in tubes with sealed lids, after the submission of these to water stress during 24, 38 or 52 days. After the water stress period, the plants were rehydrated for 34 days under regular irrigation at the greenhouse. In the in vitro cultivated tissues it was noted the emergence of hyperhydricity characteristics in the plants grown with 15 g L-1 of sucrose, GA3 and tubes with sealed lids, which was evidenced by the lowest proline accumulation, the increased in the antioxidative enzymatic activities and the lowest accumulation of photosynthetic pigments. In the ex vitro condition, the plants previously grown in culture mediums containing 15 g L-1 of sucrose presented larger antioxidative enzymatic activities, which did not show, in some cases, significant differences compared with the largest concentration of sucrose. In this condition, the proline accumulation was lower, which is an indicative of larger oxidative stress in these plants during acclimatization. During the water stress, there was a fall in the activity of all studied enzymes, although that fall had been more evident in the plants that were initially cultivated with 15 g L-1 of sucrose. The proline accumulation increased with the extension of the water stress, being larger in the plants grown in vitro with 30 g L-1 of sucrose. There were no viii significant differences in the content of photosynthetic pigments and in their relation with any sucrose concentrations, except for total carotenoids, which significantly increased over the period of water stress for the plants previously grown with the lowest concentration of sucrose. The contents of total soluble carbohydrates and sucrose increased with the extension of the stress, being more accentuated in the 30 g L-1 of sucrose concentration. The contents of starch, the relative content of water and succulence presented a reduction with the increase of water stress. After the rehydration, all plants showed recovery capacity, presenting values close to those from the control groups for all the analyzed variables. It should be noted, however, that the plants treated with 30 g L-1 of sucrose had better recovery compared with those that were treated with 15 g L-1 of sucrose. According to the obtained results, it is possible to conclude that the concentration of sucrose used in vitro presents influence on the process of ex vitro acclimatization and also, later in the field, in the recovery capacity of the plants to drought when they are submitted to water stress. The plants grown in vitro with 15 g L-1 of sucrose were more sensitive to drought and, possibly, would not survive if they were transferred from the test tubes directly to the field. The plants grown in vitro with 30 g L-1 of sucrose were apparently more resistant to the ex vitro acclimatization process, presenting greater survival chances in the field, larger drought tolerance and larger recovery capacity after extended periods of water stress.

Tesis por compendio / [ES] El oscurecimiento de los tejidos de frutas y verduras dañados puede provocar cambios indeseables y el rechazo por parte del consumidor. Este deterioro, conocido como pardeamiento enzimático, es causado principalmente por la enzima polifenol oxidasa (PPO), que oxida los compuestos fenólicos en pigmentos rojizos llamados melanoides. De esta forma, la prevención de la actividad de PPO en el procesamiento postcosecha tanto en frutas y verduras como en sus licuados, ha recibido desde siempre mucha atención por parte de la industria alimentaria. Sin embargo, los tratamientos actuales presentan diversos inconvenientes, entre los que podemos destacar los efectos negativos en la calidad nutricional de los productos y su elevado coste. El cometido de esta tesis doctoral se centra en el desarrollo y evaluación de nuevas estrategias no térmicas para la inhibición de la PPO con el objetivo final de detener el pardeamiento enzimático. Para esto se abordaron dos estrategias diferentes. Por un lado, se seleccionaron y evaluaron varias poliaminas macrocíclicas. Se determinó que la estructura química influye fuertemente en el poder inhibidor, existiendo dos compuestos altamente eficaces contra el pardeamiento enzimático, los cuales presentan IC50 de 10 µM y 0.30 mM. Su eficacia se validó en zumo de manzana recién licuado retrasando el pardeamiento enzimático y la pérdida de compuestos fenólicos totales. Por otro lado, se estudió el desarrollo y aplicación de partículas de sílice mesoporosas funcionalizadas con diversos grupos químicos. Los resultados mostraron que tanto la estructura del material como la funcionalización son determinantes. El soporte UVM-7 ofreció la inhibición más fuerte sobre la enzima PPO, y una vez funcionalizado con grupos tiol este aumentó notablemente su poder inhibidor, deteniendo el pardeamiento enzimático en zumo de manzana. Por el contrario, los grupos amino, aunque mostraron menor poder inhibidor, fueron capaces de inmovilizar a la enzima y eliminarla del medio. Finalmente, el soporte UVM-7 fue magnetizado para su fácil eliminación del medio, evitando así la etapa de filtración. El soporte UVM-7 magnetizado y funcionalizado con tioles logró mantener la concentración inicial de vitamina C y flavonoides en el zumo de manzana. Además, la capacidad antioxidante y el contenido de fenoles totales se mantuvieron casi sin cambios. / [CA] L'enfosquiment de fruïtes i verdures provoca un alt rebuig per part del consumidor. Aquest fet genera grans pèrdues econòmiques i un gran desaprofitament d'aliments. Aquest deteriorament, conegut com enfosquiment enzimàtic, és causat principalment per l'enzim polifenol oxidasa (PPO), que oxida els compostos fenòlics en pigments vermellosos anomenats melanoïdines. D'aquesta manera, la prevenció de l'activitat de la PPO en el processament postcollita tant en fruites i verdures com en els seus liquats, ha rebut des de sempre molta atenció per part de la indústria alimentària. No obstant això, els tractaments actuals presenten diversos inconvenients, entre els quals podem destacar els efectes negatius en la qualitat nutricional dels productes o el seu elevat cost. L'objectiu d'aquesta tesi doctoral es centra en el desenvolupament i avaluació de noves estratègies no tèrmiques per a la inhibició de la PPO amb la finalitat de detindre el enfosquiment enzimàtic. Per això s'aborden dues estratègies diferents. D'una banda, es va seleccionar i avaluar diverses poliamines macrocícliques. Determinat que l'estructura química influeix fortament en el poder inhibidor, existint dos compostos altament eficaços contra el enfosquiment enzimàtic, els quals presenten IC50 de 10 µM i 0.30 mM. La seua eficàcia es va validar en el suc de poma recentment liquat retardant el enfosquiment enzimàtic i la pèrdua de compostos fenòlics totals. D'altra banda, es va estudiat el desenvolupament i aplicació de partícules de sílice mesoporoses funcionalitzades amb diversos grups químics. Els resultats van mostrar que tant l'estructura del material com la funcionalització foren determinants. El suport UVM-7 va oferir la inhibició més forta sobre l'enzim PPO, i una vegada funcionalitzat amb grups tiol aquest va augmentar notablement el seu poder inhibidor, detenint el enfosquiment enzimàtic en el suc de poma. Tot i això, els grups amina, encara que mostren menys poder inhibidor, foren capaços d'immobilitzar l'enzim i eliminar-la del medi. Finalment, el suport UVM-7 va ser magnetitzat per la seva fàcil eliminació del mig, evitant així l'etapa de filtració. El suport UVM-7 magnetitzat i funcionalitzat amb grups tiol va aconseguir mantindre la concentració inicial de vitamina C i flavonoides en el suc de poma. A més, la capacitat antioxidant i el contingut de fenols totals es van mantindre quasi sense canvis. / [EN] The browning of injured fruit and vegetable tissues can cause undesirable changes and consumers' rejection resulting in high economic losses and food waste. This damage, known as enzymatic browning, is mainly caused by the polyphenol oxidase enzyme (PPO) which oxidize the phenolic compounds found in these tissues into reddish pigments named melanonids. Thus, preventing PPO activity in post-harvest processing fruits and vegetables including their juices has received a lot of attention from the food industry. Nonetheless, the current alternatives have some drawbacks such as negative effects on the nutritional quality of products or high cost. Hence, the purpose of this doctoral thesis is focused on the development and evaluation of new non-thermal strategies for PPO inhibition and prevention of the enzymatic browning. For this purpose, two different strategies were addressed; On one hand, several macrocyclic polyamine compounds were selected and evaluated. The chemical structure was found to strongly influence the inhibitor power, and two different compounds were found to be efficient against the enzymatic browning with IC50 of 10 µM and 0.30 mM. Their effectiveness was proved in cloudy apple juice resulting in a delay of the enzymatic browning and the loss of total phenolic compounds. On the other hand, the development and application of mesoporous silica particles functionalized with diverse chemical groups were studied. The results showed that both the structure of the material and the type of functionalization are decisive. The UVM-7 support offered the strongest inhibition of the PPO. The functionalisation with thiol groups enhanced the inhibitor power stopping the enzymatic browning in cloudy apple juice. Alternatively, amine groups, although showing less inhibitory power, were able to immobilise the enzyme. Finally, the UVM-7 support was magnetized for easily elimination of the medium, thus preventing the juice filtration need. Cloudy apple juice treated with magnetized UVM-7 functionalized with tiol groups maintained the initial concentration of both vitamin C and flavonoids. Moreover, the antioxidant capacity and the total phenolic content remained almost unchanged. / Financial support by the Spanish Ministerio de Ciencia, Innovación y Universidades (project RTI2018-100910-B-C44 and MAT2015-64139-C4-1), Ministerio de Economía y Competitividad (projects CTQ2016-78499-C6-1-R, Unidad de Excelencia MDM 2015- 0038 and CTQ2017-90852-REDC) and Generalitat Valenciana (projects PROMETEOII2015-002 PROMETEO/2018/024 and GVA/2014/13) is gratefully acknowledged. / Muñoz Pina, S. (2021). Mesoporous silica particles and macrocyclic ligands as modulators of polyphenol oxidase activity in food systems [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/174934 / TESIS / Compendio

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No. of bitstreams: 1 cristianoferraraderesende.pdf: 2182147 bytes, checksum: a1f62e02cb7c97296c57ea6461357835 (MD5) Previous issue date: 2012-03-05 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Bromeliaceae é uma família essencialmente neotropical, cujos representantes são muito utilizados para fins paisagísticos. Uma das principais fontes de abastecimento do mercado consumidor de bromélias é o extrativismo o que, associado à destruição do ambiente natural, tem levado a perdas irreparáveis na biodiversidade da família, especialmente na Mata Atlântica, onde cerca de 40% das espécies encontram-se sob alguma categoria de ameaça. Pitcairnia encholirioides L. B. Sm. é uma espécie “criticamente em perigo” de extinção, sendo conhecida somente uma população dessa espécie, com cerca de 900 indivíduos, encontrada em 2004 em um afloramento rochoso muito degradado e sujeito ao fogo e ao pisoteio de animais, no município de Santa Maria Madalena, RJ. A utilização das técnicas de micropropagação pode reverter os riscos de extinção garantindo taxas elevadas de multiplicação, fornecendo material necessário ao mercado consumidor, evitando, dessa forma, o extrativismo das plantas nos seus locais de origem. A instalação e a conservação in vitro de bancos de germoplasma possuem especial importância, garantindo a sobrevivência de espécies raras e/ou endêmicas e fornecendo plantas para iniciativas de reintrodução. A etapa final da micropropagação é a aclimatização ex vitro, realizada após os procedimentos de multiplicação e enraizamento in vitro. A aclimatização é um período crítico para as plantas devido à perda de água, o que causa estresse hídrico e, como consequência, estresse oxidativo, podendo provocar problemas metabólicos e perdas elevadas. No presente trabalho, Pitcairnia encholirioides foi cultivada in vitro por 150 dias em meio de cultura adicionado de dois reguladores de crescimento (GA3 ou ANA), além de duas concentrações de sacarose (15 ou 30 g L-1) e sob dois tipos de vedação dos tubos de ensaio (vedação hermética com tampas e filme de PVC ou vedação com tampas que permitiam trocas gasosas), totalizando 8 tratamentos, período após o qual as plantas foram transferidas para condições ex vitro, em casa de vegetação, permanecendo por mais 180 dias. Dois desses tratamentos foram selecionados para as análises de desidratação, quando suas plantas foram submetidas a 30, 42 ou 54 dias sem irrigação, além do controle irrigado periodicamente, sendo posteriormente reidratadas durante 90 dias. Após os períodos de cultivo in vitro e permanência das plantas em casa de vegetação, foram realizadas análises dos conteúdos de prolina, proteínas totais, atividades das enzimas do metabolismo antioxidativo SOD, CAT, POD e PPO, além 2 dos teores de pigmentos fotossintetizantes. Além desses parâmetros, após desidratação e reidratação foram também avaliados os teores de carboidratos. De maneira geral, o cultivo in vitro em meio adicionado da menor concentração de sacarose e em tubos vedados hermeticamente se mostrou prejudicial, o que foi evidenciado pelo menor acúmulo de prolina, aumentos nas atividades das enzimas antioxidativas e menor acúmulo de pigmentos fotossintéticos. Após o período de aclimatização, não foram encontradas diferenças entre os tratamentos para a maioria dos parâmetros analisados, ou os dados oscilaram de maneira a não permitir uma generalização quanto aos efeitos residuais da sacarose, do tipo de tampa e dos reguladores de crescimento utilizados durante o cultivo in vitro. No período em que as plantas foram submetidas à desidratação progressiva, houve maior acúmulo de prolina e proteínas solúveis totais, além de aumento nos teores de pigmentos fotossintéticos nos tecidos provenientes de plantas cultivadas in vitro em meio contendo 30 g L-1 de sacarose e GA3. Houve também, em geral, queda mais acentuada nas atividades das enzimas em meio suplementado com a menor concentração de sacarose e ANA, além de oscilações nos teores de pigmentos. As relações entre os pigmentos sofreram leve redução em ambos os tratamentos, e os teores de carboidratos solúveis totais, sacarose, açúcares redutores e amido aumentaram com o prolongamento do estresse. Após a reidratação, todas as plantas demonstraram elevada capacidade de recuperação, apresentando em todas as análises realizadas valores muito similares aos das plantas do controle, não submetidas à desidratação. Os resultados obtidos no trabalho permitem concluir que o cultivo in vitro em meio adicionado de 30 g L-1 de sacarose e em tubos de ensaio que permitem trocas gasosas é o mais apropriado para plantas de P. encholirioides. Ademais, a concentração de sacarose adicionada ao meio de cultura também influencia o desenvolvimento das plantas após aclimatização, sendo encontrados melhores resultados em plantas cultivadas nos meios de cultura suplementados com a maior concentração desse carboidrato. Acredita-se que as plantas cultivadas nessas condições apresentariam maior capacidade de sobrevivência ao transplantio e maior resistência a períodos prolongados de estresse hídrico, aos quais, frequentemente, estão submetidas no ambiente natural. / Bromeliaceae is essentially a neotropical family, whose representatives are widely used for landscaping. One major source of supply for the consumer market is the extraction, which coupled with the destruction of the natural environment, has led to irreparable losses in biodiversity of the family, especially in the Atlantic Forest, where about 40% of bromeliad species are under some category of threat. Pitcairnia encholirioides L. B. Sm. is a critically endangered species. In 2004, a unique population of this species was found, with about 900 individuals in a very degraded rocky outcrop, subject to fire and trampling by animals, located in Santa Maria Madalena, RJ. The use of micropropagation can guarantee high multiplication rates, providing necessary material for the consumer market, effectively obviating the extraction of plants in their places of origin. In addition, installation and in vitro conservation of germplasm banks have special importance, ensuring the survival of rare species and/or endemic and providing micropropagated plants for reintroduction initiatives. Following the procedures of in vitro multiplication and rooting, the final stage of micropropagation is the ex vitro acclimatization, a critical period for the plants due to water loss, which causes water stress and, consequently, oxidative stress, which can cause metabolic problems and high losses. In this study, Pitcairnia encholirioides was grown for 150 days in vitro in a culture medium supplemented with two growth regulators (GA3 or NAA), and two concentrations of sucrose (15 or 30 g L-1) in two types of sealing of test tubes (hermetic seal with lids and PVC film and seal with lids that allowed gas exchange), totaling eight treatments, after which the plants were transferred to ex vitro conditions in a greenhouse, staying for more than 180 days. Two of these treatments were selected for analysis of dehydration when their plants were subjected to 30, 42 or 54 days without irrigation, apart from control irrigated periodically, being subsequently rehydrated for 90 days. After periods of in vitro cultivation and maintenance of the plants in the greenhouse, they were analyzed for proline content, total protein, activities of antioxidant metabolism enzymes SOD, CAT, POD and PPO, besides the content of photosynthetic pigments. Apart from these parameters, after dehydration and rehydration, levels of carbohydrates were also assessed. In general, the in vitro culture in medium containing the lowest concentration of sucrose and hermetically sealed tubes proved to be harmful, which was evidenced by lower levels of proline, increased 4 activity of antioxidant enzymes and lower accumulation of photosynthetic pigments. After the acclimatization period, no differences were found between treatments for most parameters, or data varied so as to not allow a generalization about the residual effects of sucrose, the type of cover and of the growth regulators used in the in vitro cultivation. During the period in which the plants were subjected to water stress, higher accumulation of proline and total soluble proteins occurred, and also increased levels of photosynthetic pigments in tissues from plants grown in vitro in medium containing 30 g L-1 sucrose and GA3. There was also, in general, sharper decrease in enzyme activity in medium with the lowest concentration of sucrose and NAA, as well as fluctuations in pigment. Relations between pigments suffered slight reduction in both treatments, and total soluble carbohydrates, sucrose, reducing sugars and starch increased with increasing stress. After rehydration, all plants showed high resilience, presenting for all analyzes values very similar to those of control plants not subjected to dehydration. The results indicate that the in vitro culture in medium supplemented with 30 g L-1 sucrose and tubes that allow gas exchange are the most appropriate. Moreover, the concentration of sucrose added to the culture medium also influences the development of plants after acclimatization, with best results found in plants grown in media supplemented with higher amount of this carbohydrate. It is believed that plants grown under these conditions would have higher capacity of resistance to transplanting and could survive for prolonged periods of water stress, which are often subjected in the natural environment.