Dissertations / Theses on the topic 'L-rhamnose'

Thesis (S.M. in Molecular and Systems Toxicology)--Massachusetts Institute of Technology, Biological Engineering Division, 2003.
Vita.
Includes bibliographical references (leaves 60-62).
The purpose of this work is to probe the dTDP-L-rhamnose pathway in an effort to develop small molecule inhibitors that could act as therapeutics for Mycobacterium tuberculosis. The necessity for newer, more effective treatments for tuberculosis is growing, as the bacteria evolve resistance to traditional treatments. In an effort to develop more effective and perhaps more abbreviated courses of treatment, a plan was developed to investigate a pathway involved in cell wall biosynthesis as a promising target: the dTDP-L-rhamnose pathway. This pathway plays an essential role in linking the peptidoglycan and arabinogalactan portions of the mycolic acid-arabinogalactan-peptidoglycan complex, a significant part of the mycobacterial cell wall. The mounting level of biochemical understanding of this pathway and its importance in bacterial cell wall biosynthesis indicates that it is not only a relevant target but also an accessible one. Of the four enzymes crucial to this biosynthetic pathway, one was chosen as the primary focus: dTDP-D-glucose-4,6- dehydratase (RmlB). There are 3 steps in the reaction mechanism of RmlB: oxidation of the C4 position of dTDP-D-glucose to form a 4-keto structure, dehydration of the C6 position via the elimination of water and a subsequent reduction to result in a 6-deoxy product. Crystal structures of this particular enzyme, dTDP-D-glucose 4,6-dehydratase (RmlB), complexed with single substrates or substrate analogs have provided a foundation for these studies, enabling the rational design of a small library of potential inhibitors. Twelve mechanism-based inhibitors of RmlB are proposed. These compounds reflect the current understanding of the mechanism and mimic the sugar portion of the sugar-nucleotide substrate at various steps throughout the reaction mechanism. Each of the proposed inhibitors is designed to inhibit one of the specific steps of the mechanism. While the intention of this project is to synthesize each compound in this library from commercially available starting materials in 15 steps or less, the primary goal of this particular dissertation is to synthesize 3 of the 12 proposed inhibitors from the commercially available starting material 1,5-anhydro-D-glucitol. The long term goal of this work is to produce these compounds in significant amounts in order to test their efficacy in an animal model of mycobacterial infection.
by Neena Sujata Kadaba.
S.M.in Molecular and Systems Toxicology

La synthèse d’oligosides complexes et glycoconjugués reste difficilement réalisable par voie chimique, freinant ainsi l’exploitation de ces molécules à grande échelle. Le recours aux catalyseurs enzymatiques apparaît comme une alternative prometteuse mais les enzymes naturelles ne présentent pas toujours les propriétés adéquates, en particulier sur le plan de leurs spécificités, pour être intégrées dans des voies de synthèse chimio-enzymatiques. Le défi de cette thèse a consisté à mettre à profit les techniques d’ingénierie enzymatique pour concevoir des glyco-enzymes déployant de nouvelles spécificités, produire à façon des intermédiaires de synthèse chimique et ouvrir la voie à de nouveaux procédés d’obtention d’oligosides d’intérêt thérapeutique. Il s’agissait de développer de nouvelles transglucosidases capables de glucosyler des dérivés de la N-acétyl-D-glucosamine et du L-rhamnose pour produire des disaccharides n’existant pas dans la nature mais pouvant entrer dans une voie de synthèse chimique destinées à synthétiser des haptènes oligosaccharidiques mimant les motifs antigéniques des lipopolysaccharides des sérotypes 1b et 3a de Shigella flexneri. A terme, l’objectif est de développer un vaccin multivalent de troisième génération contre ces pathogènes, responsables du décès de milliers de personnes chaque année dans le monde. Pour relever ce défi, nous avons tout d’abord évalué le potentiel de quatre glucane-saccharases sauvages, spécifiques de la synthèse de liaisons osidiques distinctes, à glucosyler les accepteurs cibles. Les résultats obtenus ont montré qu’aucune des enzymes testées ne catalysait efficacement la réaction attendue. Cette étude préliminaire a cependant permis de sélectionner l’amylosaccharase de Neisseria polysaccharea, comme enzyme candidate au développement de biocatalyseurs à stéréo- et régio-spécificités contrôlées. Le remodelage du catalyseur a été guidé par l’analyse des modèles des complexes enzyme:accepteur. Sept acides aminés situés dans le site de fixation de l’accepteur (sous-site +1) ont été identifiés et individuellement remplacés par les 19 acides aminés possibles par mutagénèse dirigée. Une banque de 133 mutants a été créée qui renfermait 43 mutants d’intérêt pour les réactions de glucosylation considérées. Notamment, le mutant I228Y a révélé une toute nouvelle régio-spécificité vis-à-vis de l’α-methyl L-rhamnopyranose. Il s’agit du premier exemple de création de nouvelle spécificité pour les enzymes de cette famille et plus largement pour des α-transglycosidases. Le mutant F290K, également isolé dans cette première librairie, s’est montré capable de glucosyler l’α-allyl N-acétyl-D-glucoamine avec une efficacité catalytique 130 fois plus élevée que celle observée avec l’enzyme sauvage. Ces deux nouvelles enzymes ont été employées pour la synthèse des disaccharides ciblés et la validation de la voie chimio-enzymatique proposée pour la synthèse des haptènes saccharidiques. Cette stratégie a été poursuivie par la construction de banques de double-mutants permettant de générer une diversité plus importante, focalisée sur ces positions, et rechercher des effets synergiques entre les différentes mutations pour améliorer la réaction de glucosylation des accepteurs cibles. Vingt double-mutants ont pu être ainsi identifiés
The difficult access to complex carbohydrates and glycoconjugates by chemical synthesis impairs their development on a large scale. Therefore, the use of biocatalysts appears as an appealing alternative, yet poorly exploited in spite of the fast-growing development of engineering technologies allowing the search and the construction of better performing enzymes, displaying novel specificities. The objective of this thesis aimed to apply enzyme engineering techniques to conceive novel glyco-enzymes well-adapted for the glucosylation of naturally non- or poorly recognized molecules (N-acetyl-D-glucosamine and L-rhamnose derivatives fulfilling the subsequent chemical step requisites). The glucosylated motives obtained via the enzymatic route, and corresponding to the serotype-specific branched -D-glucopyranosyl linkages of Shigella flexneri 2a, 1b and 3a, are subsequently elongated through a chemical synthetic pathway to synthesize the antigenic oligosaccharides. This chemo-enzymatic road could open the way to oligosaccharide haptens at low cost, that could in the future be used in the development of a third-generation vaccine against S. Flexneri 1b and 3a. In a first study, we have evaluated the potential of four wild-type glucansucrases, specific for the synthesis of distinct osidic linkages, to glucosylate both types of targeted acceptors. Our preliminary results demonstrated that none of the tested glucansucrases allowed an efficient glucosylation of the target acceptors with the desired linkage specificity. On the basis of these results, amylosucrase from Neisseria polysaccharea was chosen as a candidate enzyme for the development of biocatalysts with controlled stereo- and regio-specificities that could enable the glucosylation of both types of acceptors of interest. A semi-rational approach based on the engineering of the acceptor binding site (subsite +1) was undertaken to evolve this enzyme. By individually exploring 7 positions of the active site using site-directed mutagenesis, the strategy led to the isolation of 47 mutants of interest for the considered glucosylation reactions (over the 133 generated mono-mutants) and to the identification of 2 key positions (228 and 290). Noteworthy, I228Y mutant displayed an entirely novel regio-specificity towards the -methyl L-rhamnopyranose and in the presence of allyl N-acetyl-D-glucosamine, F290K mutant permitted a 130 fold enhancement of catalityc efficiency compared to wild-type amylosucrase. This strategy was further pursued with the construction of double-mutant libraries enabling the generation of an enlarged diversity, focused on the identified positions, to investigate the synergistic effects between the different mutations in order to improve the glucosylation reactions of the target acceptors. Twenty double-mutants were thus identified

O objetivo deste estudo foi avaliar o efeito inibitório de Lactobacillus rhamnosus sobre as contagens de Staphylococcus aureus e Listeria monocytogenes, aspergidos isoladamente ou em combinação sobre a superfície de queijo Minas Frescal, durante armazenamento por 21 dias a 7ºC. O delineamento consistiu em esquema fatorial 2x2x2, sendo 8 tratamentos com 4 repetições. As características físico-químicas (pH, atividade de água, umidade, teor de gordura, proteína e perfil de textura) foram determinadas nos queijos dos tratamentos sem adição de L. rhamnosus ou contendo este probiótico (T1 e T2, respectivamente). Verificaramse as contagens de L. rhamnosus, S. aureus e L. monocytogenes nos queijos de todos os tratamentos nos dias 1, 7, 14 e 21 de armazenamento. Foram também analisados os percentuais de sobrevivência dos microrganismos submetidos a condições de simulação do trato gastrointestinal (TGI) utilizando ensaios in vitro. Não houve efeito significativo (P>0,05) entre os parâmetros físico-químicos dos queijos dos tratamentos T1 e T2. As contagens de L. rhamnosus aumentaram (P<0,05) em todos os tratamentos a partir do dia 7 de armazenamento, estabilizando ao redor de 108 UFC/g, sendo que a presença concomitante de L. monocytogenes e/ou S. aureus nos queijos não influenciou a contagem de L. rhamnosus. L. rhamnosus diminuiu em cerca de 1 ciclo log as contagens de L. monocytogenes, e não exerceu efeito inibitório sobre S. aureus após 21 dias. S. aureus não sobreviveu ao teste de simulação ao TGI. No entanto, L. rhamnosus e L. monocytogenes apresentaram percentuais de sobrevivência entre 74,6% a 86,4%, e entre 75,8% a 94,1%, respectivamente. Os resultados demonstraram que a adição de L. rhamnosus não alterou as características físico-químicas dos queijos Minas frescal, porém exerceu efeito inibitório sobre L. monocytogenes, mas nenhum efeito sobre S. aureus. A utilização de L. rhamnosus como probiótico apresenta um potencial para inibição de L. monocytogenes na fabricação de queijos Minas frescal. São necessários estudos sobre os mecanismos envolvidos na competição entre as bactérias por substratos no alimento, bem como sua sobrevivência nas condições do TGI em ensaios in vivo.
The aim of this study was to evaluate the effects of Lactobacillus rhamnosus on growth of Staphylococcus aureus and Listeria monocytogenes in Minas frescal cheese during 21 days of storage at 7ºC. The experimental design was totally randomized, in a 2x2x2 factorial arrangement with 8 treatments and 4 replicates per treatment. Physical chemical parameters such as pH, moisture, water activity, fat, protein and texture profile analysis were carried out in cheeses where no microorganism were inoculated (T1) and in the cheeses inoculated with the probiotic bacteria, L. rhamnosus (T2). The counts of L. rhamnosus, S. aureus and L. monocytogenes were examined on days 1, 7, 14, 21 of storage. Survival percentage of the bacteria after exposure to simulated gastrointestinal conditions was studied in vitro. Statistical analysis indicated that there were no significant differences (P>0,05) among the means of the physical chemical parameters analyzed in treatments 1 and 2. From day 7 on, the counts of L. rhamnosus increased (P<0,05) in all treatments, stabilizing and reaching up to 108 CFU/g. It was noticed that the concurring presence of L. monocytogenes and/or S. aureus in the cheese samples did not show influence in the counts of the probiotic bacteria. The L. rhamnosus caused about 1 log cycle reduction in the counts of L. monocytogenes, but showed no inhibitory effect on S. aureus at the end of the period of storage. S. aureus did not survive the exposure to simulated gastrointestinal conditions. However, L. rhamnosus and L. monocytogenes showed survival percentages varying from 74,6% to 86,4%, and from 75,8% to 94,1%, respectively. The results showed that the addition of L. rhamnosus had no influence on the physical chemical characteristics of the Minas frescal cheese and no inhibitory effect on S. aureus, nevertheless demonstrated inhibitory effect on L. monocytogenes. The addition of probiotic strains of L. rhamnosus in Minas frescal cheese represents potential for L. monocytogenes inhibition. It is essential to carry out studies on the mechanisms involved in the competition for substrate by bacteria, as well as their survival to simulated gastrointestinal conditions in in vivo experiments.

Ce travail de thèse a permis d’étudier l’influence de l’ajout d’ingrédients fonctionnels laitiers sur l’encapsulation de L. rhamnosus GG (LGG). Deux ingrédients laitiers (ß-lactoglobuline et membrane des globules gras du lait - MFGM) ont été identifiés comme étant capables d’adhérer fortement à LGG par l’intermédiaire de ses pili. Le rôle clé de ces adhésions dans la localisation spatiale des bactéries dans la matrice laitière a été mis en évidence, ainsi que le rôle des constituants de la matrice dans sa structuration. Cela a permis de sélectionner, in vitro, une matrice d’encapsulation capable de protéger de manière efficace les bactéries des conditions gastriques et de libérer les bactéries vivantes au niveau de l’intestin. En parallèle, la MFGM dans l’encapsulation des bactéries s’est révélée prometteuse. Ce travail a également démontré l’importance primordiale du choix de la matrice d’encapsulation. En effet, une compétition entre l’adhésion de LGG aux cellules intestinales et l’adhésion de LGG à certains composants de la matrice laitière a été démontrée. Les deux phénomènes impliquent probablement les mêmes mécanismes : adhésion aux pili glycosylés de LGG. Pour terminer, un procédé de séchage par atomisation a été développé pour encapsuler LGG. Il permet une bonne survie des bactéries après séchage et la production de microparticules présentant des propriétés fonctionnelles innovantes liées à la température du milieu de réhydratation
The aim of this work was to understand how functional dairy components influence L. rhamnosus GG (LGG) encapsulation. First, two dairy components (-lactoglobulin and milk fat globule membrane - MFGM) able to strongly adhere to LGG through their pili are identified. The key role of these adhesions on bacteria spatial location in the matrix is highlighted, as well as the role of matrix dairy components in their structuration. This allowed to select, in vitro, a matrix able to protect bacteria in gastric conditions and to release them viable in the intestine. Simultaneously, the use of MFGM in bacteria encapsulation has proven to be promising. This work demonstrated the importance of the matrix choice in the encapsulation procedure. Results demonstrated that adhesion between LGG and dairy matrix may compete with adhesion of LGG to epithelial intestinal cell. The two phenomena likely involve the same mechanisms: adhesion to glycosylated pili of LGG. To finish, a spray drying encapsulation process is developed to encapsulate bacteria. It leads to a high bacteria survival after drying and the production of microparticles with innovative properties depending on rehydration temperature

Pivski trop čini približno 85% od ukupnih sporednih proizvoda proizvodnje piva, i dostupan je po veoma niskim cenama tokom čitave godine. Pivski trop ima veliku perspektivu za primenu u biotehnologiji i proizvodnji visoko vrednih proizvoda. Jedna od veoma ekološki i ekonomski isplativih alternativa je upotreba pivskog tropa u proizvodnji mlečne kiseline, jer se poslednjih par decenija uočava intenzivan rast potražnje za mlečnom kiselinom. Mlečna kiselina je najvažnija hidroksikarbonska kiselina široko rasprostranjena u prirodi, sa velikom primenom u prehrambenoj, farmaceutskoj, tekstilnoj i hemijskoj industriji i industriji prerade kože.Cilj istraživanja ove doktorske disertacije je ispitivanje primene pivskog tropa u proizvodnji mlečne kiseline. Prvo je izvršena optimizacija enzimske hidrolize pivskog tropa u cilju dobijanja što je moguće veće koncentacije redukujućih šećera neophodne za mlečno-kiselu fermentaciju. Hidrolizat pivskog tropa je dobijen enzimskom hidrolizom dodatkom komercijalnih enzima za razgradnju skroba i celuloze. Parametri čiji je uticaj na efikasnost enzimske hidrolize ispitanu su: pH vrednost, temperatura hidrolize i količina dodatih enzima. Nakon što su određeni najbolji uslovi razgranje pivskog tropa, dobijeni postupak hidrolize je primenjen u proizvodnji hidrolizata pivskog tropa koji je korišćen u mlečno-kiselim fermentacijama.Nakon toga je ispitana mlečno-kisela fermentacija sa dva proizvodna mikoorganizma. Kao proizvodni mikroorganizmi u mlečno-kiselim fermentacijama primenjena su dva soja bakterija mlečne kiseline: Lactobacillus fermentum PL-1 i Lactobacillus rhamnosus ATCC 7469. Ispitan je uticaj dodatka različitih koncentracija ekstrakta kvasca (0,5-5,0%) uz korekciju pH vrednosti tokom fermentacije sa dodatkom kalcijum-karbonata. U zavisnosti od udela L-(+)- i D-(-)-mlečne kiseline koje nastaju tokom fermentacije izabran je proizvodni mikroorganizam koji proizvodi više L-(+)-mlečne kiseline.U daljim ispitivanjima je ispitan uticaj korekcije pH pomoću natrijum-hidroksida kao i dodatak različitih koncentracija ekstrakta kvasca (0,5-5,0%) i redukujućih šećera (2,7; 5,4 i 8,1%) u hidrolizatu pivskog tropa na mlečno-kiselu fermentaciju pomoću odabranog soja bakterija mlečne kiseline. Na osnovu dobijenih rezultata izabrana je najbolja koncentracija redukujućih šećera i ekstrakta kvasca koji će se koristiti u daljim istraživanjima.Takođe je ispitana i mogućnost zamene skupog ekstrakta kvasca i glukoze sa obnovljivim sirovinama, kao što su pivski kvasac, džibra i bistra džibra.Ispitan je uticaj dodatka različitih koncentracija pivskog kvasca (0,5-5,0%), džibre (5-20%) i bistre džibre (5-50%) pre fermentacije kao i dodatak bistre džibre u dolivnoj fermentaciji, na mlečno-kiselu fermentaciju hidrolizata pivskog tropa.Ispitan je i dolivni postupak fermentacije hidrolizata pivskog tropa dodatkom glukoze, glukoze i ekstrakta kvasca i sladovine. Takođe je ispitana mogućnost izvođenja više uzastopnih fermentacija sa imobilisanim ćelijama odabranog soja bakterija mlečne kiseline u kalcijum-alginatu.Na osnovu eksperimentalnih rezultata zaključujeno je da je dodatak kalcijum-karbonata imao pozitivan uticaj na proizvodnju mlečne kiseline sa L. fermentum i L. rhamnosus. Sa dodatkom kalcijum-karbonata povećali su se utrošak redukujućih šećera, koncentracija i prinos mlečne kiseline i vijabilnost ćelija L. fermentum i L. rhamnosus. Ekstrakt kvasca i kalcijum-karbonat su imali značajan uticaj na proizvodnju mlečne kiseline sa L. fermentum i L. rhamnosus. U fermentacijama sa L. fermentum najveći prinos ukupne mlečne kiseline (44%) je postignut sa dodatkom 5,0% ekstrakta kvasca i 2,0% kalcijum-karbonata. U fermentacijama sa L. rhamnosus najveći prinos ukupne mlečne kiseline (98%) i L-(+)-mlečne kiseline (96%) je ostvaren u fermentaciji sa dodatkom 2,0% ekstrakta kvasca i 2,0% kalcijum-karbonata. Na osnovu rezultata odlučeno je da se u daljim ispitivanjima mlečno-kisele fermentacije hidrolizata pivskog tropa kao proizvodni mikoorganizam koristi L. rhamnosus.Primenom natrijum-hidroksida za korekciju pH je skratila fermentaciju za 48 sati a ostvareno je i značajno povećanje zapreminske produktivnosti L-(+)-mlečne kiseline (za 200%, povećanje sa 0,21 na 0,63 g/l·h-1). Korekcija pH u svim daljim istraživanjima je vršena sa dodatkom natrijum-hidroksida.U mlečno-kiselim fermentacijama sa različitim početnim koncentracijama redukujućih šećera (2,7; 5,4 i 8,1%) i sa dodatkom različitih koncentracija ekstrakta kvasca (0,5-5,0%), najveći prinos L-(+)-mlečne kiseline i zapreminska produktivnost od 91,29% i 1,69 g/l·h-1, kao i vijabilnost ćelija L. rhamnosus od 9,7·109 CFU/ml ostvareni su u fermentaciji sa početniom koncentracijom redukujućih šećera od 5,4% i dodatkom 5,0% ekstrakta kvasca.Na osnovu ostvarenih rezultata u istraživanjima sa dodatkom džibre i dodacima tokom fermentacije kao i u fermentacijama sa imobilisanim ćelijama je korišćen hidrolizat pivskog tropa sa početnom koncentracijom redukujućih šećera od 5,4%.U mlečno kiseloj fermentaciji sa dodatkom pivskog kvasca najveći prinos L-(+)-mlečne kiseline (89,01%) i zapreminska produktivnost (0,89 g/l·h-1) L-(+)-mlečne kiseline su ostvareni u fermentaciji sa dodatkom 5,0% pivskog kvasca i korekcijom početne koncentracije redukujućih šećera na 5,0%. Na osnovu rezultata utvrđeno je da se može izvršiti delimična ili potpuna zamena ekstrakta kvasca pivskim kvascem uz značajno smanjenje cene podloge za mlečno-kiselu fermentaciju, bez značajnog smanjenja efikasnosti mlečno-kisele fermentacije.U mlečno-kiseloj fermentaciji sa dodatkom džibre i bistre džibre najveć koncetracija, prinos i zapreminska produktivnost L-(+)-mlečne kiseline od 31,03 g/l, 86,15% i 0,93 g/l·h-1, ostvareni su u fermentaciji sa dodatkom 50% bistre džibre. Najviša koncentracija, prinos i zapreminska produktivnost L-(+)-mlečne kiseline ostvareni u dolivnoj fermentaciji sa dodatkom glukoze i bistre džibre tokom mlečno-kisele fermentacije su iznosili su 48,02 g/l, 87,82% i 0,96 g/l·h-1.U fermentacijama sa dodatkom nutritijenata tokom mlečno-kisele fermentacije najveća vrednost koncetracije, prinosa i zapreminske produktivnosti L-(+)-mlečne kiseline od 116,08 g/l, 93,32% i 2,04 g/L·h-1, su ostvarene u fermentaciji sa dodatkom glukoze i ekstrakta kvasca tokom fermentacije. Na osnovu rezultata utvrđeno je da se dolivni postupak fermentacije može koristiti u cilju povećanja efikasnosti mlečno-kisele fermentacije.Izvršena je imobilizacija ćelija L. rhamnosus u kalcijum-alginatu uz izuzetno visoku vijabilnost (1010 CFU/ml). Imobilisane ćelije L. rhamnosus su uspešno korišćene u tri mlečno-kisele fermentacije. Prinos L-(+)-mlečne kiseline i zapreminska produktivnost su u sve tri fermentacije bili izuzetno visoki, pri čemu su najveći prinos L-(+)-mlečne kiseline i zapreminska produktivnost od 95,2% i 1,76 g/l·h-1, ostvareni u drugoj fermentaciji. Upotrebom imobilisanih ćelija L. rhamnosus je osim povećanja prinosa i zapreminske produktivnosti L-(+)-mlečne kiseline skraćena fermentacija za 12 sati u poređenju sa šaržnim fermentacijama.
Brewers spent grain represents (BSG) about 85% of the total by-products from brewing process and is available at low price during the whole year. Due to its chemical composition BSG has great potential use in biotechnology and production of high-value products. One of very eco-friendly and economical alternative uses of BSG is in production of lactic acid (LA), since in the last few decades the demand for the LA has significantly risen, mostly because of development of biodegradable lactic polymers, which are eco-friendly and nontoxic.Lactic acid is the most important hydrocarboxylic acid with an asymmetrical carbon atom, widely distributed in nature, and it has shown great potential in fields of food, pharmaceutical, textile, leather and chemical industries.The aim of this doctoral thesis was to investigate the application of BSG in lactic acid production. First, the optimization of enzymatic hydrolysis of BSG was conducted, with the goal to achieve high reducing sugar concentrations, as much as possible, that are necessary on LA fermentation. BSG hydrolysis was conducted by usage of commercial enzymes for degradation of starch and cellulose. Effect of pH value, temperature and enzyme dosage on BSG hydrolysis efficiency was investigated. After the best conditions for BSG hydrolysis were determined, the optimized procedure for BSG hydrolysis was used for the production of BSG hydrolysate that will be used in LA fermentations.After optimization of BSG hydrolysis, LA fermentation by two LA producing microorganisms was investigated. The strains investigated were two LA bacteria strains: Lactobacillus fermentum PL-1 and Lactobacillus rhamnosus ATCC 7469. The effect of yeast extract (0.5; 1.0; 2.0; 3.0; 4.0, and 5.0%) addition in BSG hydrolysate, with the correction of pH value during LA fermentation by the addition of calcium-carbonate, on LA fermentation was investigated. Based on the results achieved for L-(+)- and D-(-)-LA ratio the LAB strains that produced more L-(+)-LA was chosen for further research.In further research the effect of pH correction (with addition of NaOH), yeast extract (0.5, 1.0, 2.0, 3.0, 4.0, and 5.0%) addition and reducing sugar concentration (2.7; 5.4 and 8.1%) in BSG hydrolysate on LA fermentation was investigated. Based on the results achieved the best yeast extract and reducing sugars concentrations was determined and used in further analysis or research. Also the possible replacement of expensive yeast extract and glucose with cheap alternatives, like brewer`s spent grain and stillage was investigated. The effect of brewer`s spent grain (0.5; 1.0; 2.0; 3.0; 4.0, and 5.0%), whole stillage (5, 10, 15 i 20%) and thin stillage (5, 10, 15, 20, 30, 40, 50%) addition before fermentation as well as thin stillage addition in fed-batch fermentation in BSG hydrolysate on LA fermentation were investigated.Also fed-batch fermentation procedure (addition of glucose, glucose and yeast extract and wort during fermentation) was investigated. The possible application of cells immobilized in Ca-alginate for LA fermentation of BSG hydrolysate was also investigated.Based on the results it was concluded that BSG can be successfully utilized as a raw material in production of LA, after optimization of hydrolysis and addition of nitrogen source.According to the results of chemical composition before and after optimized hydrolysis 78.6% of total cellulose was hydrolyzed.Addition of calcium-carbonate had positive effect on LA production by L. fermentum i L. rhamnosus. With the addition of calcium-carbonate reducing sugar utilization, LA yield and concentration and cell viability (both L. fermentum i L. rhamnosus) increased. Addition of calcium-carbonate and yeast extract had a positive effect on LA fermentation by L. fermentum and L. rhamnosus. In LA fermentation by L. fermentum the highest LA yield (44%) was achieved with addition of 5.0% of yeast extract and 2.0% of calcium-carbonate. In L. rhamnosus fermentations the highest total LA yield (98%) and L-(+)-LA yield (96%) was reached when 2.0% of yeast extract and 2.0% of calcium-carbonate were added.Based on the results achieved it was concluded that BSG hydrolysate, with the addition of yeast extract, is a good fermentation media for LA fermentation with L. rhamnosus, and it was decided that L. rhamnosus will be used in further research of LA fermentation on BSG hydrolysate.Addition of NaOH instead of calcium-carbonate for the pH correction shortened the fermentation time by 48 h and increased the L-(+)-LA volumetric productivity (by 200%, from 0.21 to 0.63 g/L·h-1). Based on this results pH correction in further experiments was done by addition of NaOH.In LA fermentation with different reducing sugar (2.7, 5.4 and 8.1%) and yeast extract concentrations (0.5-5.0%), the highest L-(+)-LA yield and volumetric productivity of 91.29%, and 1.69 g/L·h-1, respectively, as well as L. rhamnosus cell viability (9.67 log CFU/mL), were achieved with the reducing sugar content of 5.4% and yeast extract content of 5.0%.Based on this results in further experiment with the addition of stillage, in fed-batch fermentation and fermentation with immobilized cell BSG hydrolysate with 5.4% of reducing sugars and 5.0% yeast extract was used.In fermentation with the addition of brewer’s spent yeast the highest L-(+)-LA yield (89.01%) and volumetric productivity (0.89 g/L·h-1) were achieved in the fermentation of BSG hydrolysate with 5.0% of reducing sugar and 5.0% of brewer’s yeast. Based on the results achieved it was concluded that yeast extract can be partial or complete replaced by brewer’s spent yeast with significant decrease of media cost, without the decrease in LA fermentation efficiency.In fermentation with the addition of thin stillage the highest L-(+)-LA concentration, yield, and volumetric productivity of 31.03 g/L, 86.15%, and 0.93 g/L·h-1, respectively, was obtained in fermentation with the addition of 50% of thin stillage. The highest L-(+)-LA concentration, yield, and volumetric productivity achieved in fed-batch fermentation with the addition of glucose and thin stillage during fermentation, were 48,02 g/L, 87,82% i 0,96 g/L·h-1.In fed-batch fermentation the highest L-(+)-LA concentration, yield, and volumetric productivity of 116.08 g/L, 93.32%, and, 2.04 g/L h-1, respectively, were achieved in fermentation with glucose and yeast extract addition during fermentation. The results showerd that fed-batch fermentation could be used to increase L-(+)-LA fermentation efficiencyImmobilization of L. rhamnosus cells with high viability (1010 CFU/mL) in Ca-alginate was conducted. Immobilized cells we successfully utilized in three repeated batch fermentation. L-(+)-LA yield and volumetric productivity were very high in all three batch fermentation, with the highest results achieved (95.20% and 1.76 g/L·h-1, respectively) in second fermentation. Application of immobilized L. rhamnosus cells increased L-(+)-LA yield and volumetric productivity and shortened the fermentation time for 12 h in comparison with batch fermentation.

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CNPQ
Os alimentos com probióticos à base de vegetais vêm ganhando destaque no mercado por serem uma ótima fonte de vitaminas e minerais, como é o caso da goiaba (Psidium guajava L.), uma fruta largamente produzida no Brasil. Probióticos são microorganismos vivos que tem a função de modular a microbiota intestinal, oferecendo benefícios a saúde. Para que o produto se torne mais nutritivo, outras substâncias podem ser adicionadas, como é o caso da inulina, um conhecido prebiótico que melhora a adesão intestinal dos probióticos, e a Stevia (Stevia rebaudiana Bertoni), um adoçante natural fonte de antioxidantes. O objetivo desse estudo foi avaliar a estabilidade de sucos probióticos de goiaba fermentado e não-fermentado, contendo Lactobacillus rhamnosus ATCC 7469, durante 28 dias. Foram realizados ensaios de sobrevivência gastrointestinal simulada com 3 condições de sucos-controle, para determinar se a inulina e Stevia influenciariam na sobrevivência do micro-organismo, sendo as seguintes condições: não-fermentado, sem inulina e sem Stevia (SCNF); não-fermentado, com 5 g/L de inulina e sem Stevia (SCNFI); e fermentado, com 5 g/L de inulina e sem Stevia (SCFI). Os sucos probióticos não-fermentado (SNF) e fermentado (SF), ambos adicionados de inulina 5g/L e Stevia 10% v/v, foram analisados quanto a composição química, viabilidade, sobrevivência após ensaio de simulação gastrointestinal, pH, ácidos orgânicos cítrico e lático e concentração de glicose e frutose. A aceitabilidade dos sucos também foi avaliada quanto a intensidade de doçura, acidez, aceitabilidade global, preferência e intenção de compra. Os testes com as 3 condições de sucos demonstraram que a inulina não apresentou efeito e a Stevia foi capaz de aumentar a sobrevivência do micro-organismo no SNF e SF. Em relação à composição, os valores de umidade, cinzas, proteínas, carboidratos, lipídios e grau Brix foram similares em ambos os sucos, diferindo significativamente apenas na acidez titulável (5,5 - 10,3g/100g), carotenóides totais (543 - 833 mg/g) e compostos fenólicos (0,11 - 0,15 AGE mg/100 mL). O SF manteve a viabilidade inicial de 8 Log UFC/mL ao fim do estoque, enquanto o SNF reduziu de 9 para 8 Log UFC/mL. A sobrevivência após simulação gastrointestinal no dia inicial foi de 55% e 35% para o SNF e SF, respectivamente; porém, no último dia, essa sobrevivência reduziu para 25% no SNF e 28% no SF. Outros paramêtros como pH, ácido lático e concentração de glicose e frutose tiveram menores alterações no SF durante o estudo. A análise sensorial demonstrou que o SNF foi melhor aceito e preferido pelos provadores, entretanto, o SF obteve escores que possibilitam a compra e o consumo. Na análise de componentes principais (ACP), não houve diferença entre os sucos quando os atributos de aceitabilidade foram analisados ao mesmo tempo. Portanto, como o suco probiótico de goiaba fermentado apresentou maior estabilidade, contém o ácido lático e não apresentou diferença significativa na análise multivariada, foi considerado um produto mais adequado para elaboração e consumo.
Foods with vegetable-based probiotics have been gaining prominence in the market because they are a great source of vitamins and minerals, as is the case of guava (Psidium guajava L.), a fruit widely produced in Brazil. Probiotics are living microorganisms that have the function of modulating the intestinal microbiota, offering health benefits. To make the product more nutritious, other substances can be added, such as inulin, a known prebiotic that improves bowel adhesion of probiotics, and Stevia (Stevia rebaudiana Bertoni), a natural sweetener source of antioxidants. The objective of this study was to evaluate for 28 days the stability of fermented and nonfermented probiotic guava juices containing Lactobacillus rhamnosus ATCC 7469. Simulated gastrointestinal survival trials with 3 control juice conditions were performed to determine if inulin and Stevia would influence the survival of the microorganism, with the following conditions: unfermented, without inulin and without Stevia (NFCJ); Unfermented, 5 g / L inulin and without Stevia (NFICJ); And fermented with 5 g / L inulin and without Stevia (FICJ). Non-fermented juice (NFJ) and fermented juice (FJ), both added with 5 g/L inulin and 10% v/v Stevia were analyzed for chemical composition, viability, survival after gastrointestinal simulation test, pH, organic acids citric and lactic and glucose and fructose concentrations. The acceptability of juices was also evaluated as to the intensity of sweetness, acidity, overall acceptability, preference and buying intention. Tests with the 3 juice conditions showed that inulin had no positive or negative effect and Stevia increased the survival of the microorganism in the NFJ and FJ. In relation to the composition, the values of moisture, ashes, proteins, carbohydrates, lipids and total soluble solids were similar in both juices, differing only in titratable acidity (5,5 – 10,3g / 100g), total carotenoids (543 - 833 mg / g) and phenolic compounds (0.11 - 0.15 GAE mg / 100 mL). FJ maintained the initial viability of 8 log CFU / mL at the end of storage, while the NFJ reduced from 9 to 8 log CFU / mL. Survival after gastrointestinal simulation with 0 days was 55% and 35% for SNF and SF, respectively; however, the last day, that survival decreased to 25% in the NFJ and 28% in FJ. Other parameters such as pH, lactic acid and glucose and fructose concentration varied less in FJ during the study. The sensory analysis showed that the NFJ was better accepted and preferred by the tasters, however, the FJ obtained scores that make it possible to buy and consume. In the principal component analysis (PCA), there was no difference between juices when acceptability attributes were analyzed at the same time. Therefore, as the guava fermented probiotic juice presented greater stability, it contains lactic acid and did not present significant difference in the multivariate analysis, it was considered a more suitable product for elaboration and consumption.

Les capacités de survie des lactobacilles en milieu acide en font le genre prédominant dans la carie dentinaire profonde. Le suivi des espèces associées à la carie au sein de l'écosystème buccal est indispensable à une meilleure compréhension du déroulement du processus carieux et à sa prévention. Il nécessite le développement d'outils permettant la caractérisation précise de ces bactéries, indépendamment de leur phénotype qui peut être instable. Des méthodes génotypiques ont donc été appliquées aux principales espèces retrouvées dans le milieu salivaire (souches de référence de l'ATCC) ainsi qu'à des souches sauvages d'origine buccale pour permettre leur identification. Le travail a été plus particulièrement orienté vers l'espèce Lactobacillus rhamnosus qui prédomine dans les prélèvements issus de dentine cariée. Un profil d'amplification par réaction de polymérisation en chaine (PCR) permettant une première discrimination de cette espèce par rapport aux autres espèces de lactobacilles salivaires a été obtenu. Une sonde spécifique a également été développée et testée en hybridation sur taches. Elle permet d'identifier cette espèce et de suivre son implantation dans la lésion carieuse, mais aussi de surveiller son évolution dans la salive et dans la plaque dentaire. A partir d'un prélèvement de salive, de plaque dentaire ou de dentine cariée, la détection de l'espèce L. Rhamnosus peut se faire par amplification sur cellules entières après incubation du prélèvement sur milieu MRS gélosé. Les profils PCR obtenus en RAPD comme en REP permettent également le typage des souches. Ils pourront autoriser le suivi de leur évolution dans un écosystème particulier, leur transmission à l'intérieur d'un groupe familial, ou aider à mieux comprendre la résistance de certains individus à la carie malgré des charges microbiennes élevées, certains biotypes pouvant être spécifiquement associées à la carie. La détermination du biotype d'une souche est indissociable de celle de son milieu d'origine ou de ses capacités métaboliques. La taxonomie modernes (taxonomie polyphasique) à côté des informations génotypiques ou phylogéniques, doit intégrer des informations phénotypiques sur les micro-organismes
The Lactobacillus genus is involved in the progression of dental decay. The high acid tolerance of these bacteria make them predominant in dentinal caries. Conventional methods often lead to ambigous results or even to misidentifications. However, very few taxonomic tools have been developed to allow accurate identification of oral lactobacilli. This work develops reliable genotypic methods for identification and detection of the species relative to caries, and particularly of the Lactobacillus rhamnosus species, which dominate in samples from carious dentine, with the aim to monitor its development in this particular ecosystem. Methods based on hybridization with DNA probes and DNA amplification by PCR were used. The dominant salivary Lactobacillus species (reference strains from the ATCC) were selected for this purpose as well as some oral wild strains. DNA profiling using random polymorphic DNA amplification (RAPD) generated specific patterns for L. Rhamnosus ATCC 7469. A species-specific probe was developed and used on dot blots ; it may help to locate this species within its ecological niche and elucidate the progression of the carious process. The detection of L. Rhamnosus from oral samples was obtained after growth in nutritive medium and direct PCR on cells. Moreover, DNA profiles obtained by rep- or RAPD-PCR allow strain typing. This may help to elucidate the progression of the carious process, the transmission within familygroups, or some unexplained resistance to caries that could be related to a particular biotype. Typing do not exclude the description of the metabolic specifities of a strain. Modern taxonomy (polyphasic taxonomy) must include phenotypic characteristics besides genotypic and phylogenetic informations

Les probiotiques font partie du microbiote commensal. Ils ont le potentiel de stimuler la réponse immunitaire intestinale et systémique. Le virus de la grippe est à l’origine de morbidité importante. De plus, il favorise les infections bactériennes secondaires telles que Neisseria meningitidis. Nous nous sommes intéressés à l’étude des mécanismes de protections conférées par la souche Lactobacillus paracasei CNCM I-1518 sur une infection par le virus de la grippe dans un modèle de souris. Nos résultats ont montré que le gavage des souris avec la souche L. paracasei pendant 7 jours avant l’infection grippale a permis une activation des cytokines pro-inflammatoires et un recrutement massif des cellules immunitaires dans les poumons. Cette activation du système immunitaire est responsable après infection grippale d’une meilleure clairance du virus de la grippe et d’une réponse inflammatoire moins importante comparée aux souris témoins. L’administration orale à des souris du peptidoglycane de la souche L. paracasei a permis de retrouver partiellement le phénotype protecteur observé avec la bactérie entière. Les effets protecteurs induits par L. paracasei CNCM I-1518 lui sont spécifiques car l’utilisation de 2 souches L. rhamnosus CNCM I-3690 et L. paracasei CNCM I-3689 n’ont pas conférées aux souris une protection contre la grippe. L’étude de l’impact de la souche L. paracasei CNCM I-1518 sur une infection secondaire par N. meningitidis après infection grippale a montré que le gavage des souris par la souche L. paracasei permet un meilleur état clinique associé à une modulation de la réponse immunitaire et une clairance de l’infection bactérienne secondaire plus efficace
Probiotics are part of the commensal microbiota. They play a potential role in stimulating the intestinal and systemic immune response. Several clinical studies addressed beneficial effect of probiotics against respiratory infections in particular on influenza infections. These infections are responsible for significant morbidity. The burden of flu is also worsened by secondary bacterial infections such as Neisseria meningitidis. In this work, we investigated the mechanisms of protection against influenza infection conferred by Lactobacillus paracasei CNCM I-1518 strain in mice. Our results showed that, L. paracasei consumption allow an early activation of pro-inflammatory cytokines and a massive recruitment of immune cells in the lungs prior to influenza infection. This activation of immune system was associated with a faster clearance of influenza virus after infection. We able to show that feeding mice with purified peptidoglycan from L. paracasei reproduced partially the above mentioned effects observed with L. paracasei bacteria feeding.The protective effects induced by L. paracasei CNCM I-1518 against the flu infection are strain specific as L. rhamnosus CNCM I-3690 and L. paracasei CNCM I-3689, tested under the same conditions did not confer to mice protection against influenza infection. Subsequently, we investigated the impact of L. paracasei CNCM I-1518 on secondary bacterial infection with N. meningitidis following influenza infection. Our results showed that consumption of L. paracasei CNCM I-1518 strain was associated with a better clinical status and a modulation of the immune response with a better clearance of secondary bacterial infection

Conselho Nacional de Desenvolvimento Científico e Tecnológico
Lignocellulosic agroindustrial wastes, such as rice husks (RH), are abundant, low cost, renewable sources, available to biotransformation in value-added products. Thus, the main objective of this study was to investigate the use of RH with the purpose of its biotransformation in lactic acid (LacA) starting from hydrolysates of this residual biomass. At the same time, aiming to contribute to the mitigation of the growing environmental concern about the illegal disposal of this waste in the State of Rio Grande do Sul the biggest Brazilian rice producer. For this, a central composite rotational design (CCRD) of the hydrolysis experiments of RH in pressurized conversion system with diluted HCl and H2SO4, revealed that experiments conducted at 175 °C, for 46 min, with acid concentrations of 2.2% v v-1, generate the highest glucose contents. For hydrolysis involving HCl, 17.8 g glucose L-1, with an yield of 106.6 mg glucose g-1 RH; for hydrolysis with H2SO4, 14.1 g glucose L-1, with an yield of 84.5 mg glucose g-1 RH. Previous fermentative experiments were carried out with the aim to select resilient and efficient micro-organisms for the LacA production and, also, to evaluate the need of supplementation of the RH hydrolyzate with specific nutrients. L. rhamnosus, in this study, was the most suitable bacteria for the lactic fermentation, among all the Lactobacillus tested, being able to to produce 5.6 g LacA L-1, with a yield of 33.8 mg LacA g-1 RH (hydrolysed with HCl, without nutrient addition, 96 h fermentation). The acid hydrolysis, in this particular case, was carried out at 160 °C, for 70 min, using 4.0% HCl v v-1, generating 14.7 g glucose L-1. The lactic fermentation via CCRD was batch conducted in 4 mL vials, at 37 °C, pH 6.0, using L. casei subsp. rhamnosus ATCC 10863, specialized in catabolization of both hexoses and pentoses (except arabinose). Experiments with RH hydrolysates containing nutrients added produced up to 18.8 g LacA L-1 (maximum yield of 997,1 mg LacA g-1 sugars) in substrate hydrolyzed with HCl, after 52 h fermentation; and 14.6 g LacA L-1 (maximum yield of 999.8 mg LacA g-1 sugars) in substrate hydrolyzed with H2SO4, after 24 h fermentation. Therefore, in this study, the choices of most influence in the LacA production were fermentation with sulfuric acid and employment of L. rhamnosus.
Resíduos lignocelulósicos agroindustriais, como a casca de arroz (CA), são fontes renováveis, abundantes e de baixo custo, disponíveis para a biotransformação em produtos com valor agregado. O principal objetivo deste trabalho foi investigar o aproveitamento da CA com vistas à sua biotransformação em ácido láctico (ALac), partindo-se de hidrolisados desta biomassa residual. Ao mesmo tempo, objetivou-se contribuir para a mitigação do crescente problema ambiental decorrente da disposição irregular deste resíduo no estado do Rio Grande do Sul, maior produtor brasileiro. Para tanto, o delineamento composto central rotacional (DCCR) do planejamento experimental da hidrólise ácida da CA em sistema de conversão à pressão, com HCl e H2SO4 diluídos, revelou que, experimentos conduzidos a 175 °C, por 46 min, com concentração de 2,2% (v v-1) de ácido, produziram os teores máximos de glicose. Para hidrólises envolvendo HCl, teor de 17,8 g de glicose L-1, com rendimento de 106,6 mg de glicose g-1 CA; para hidrólises com H2SO4, teor de 14,1 g de glicose L-1, com rendimento de 84,5 mg de glicose g-1 CA. Ensaios fermentativos prévios foram feitos com o intuito de selecionar micro-organismos resilientes e eficientes para a produção de ALac e, também, para avaliar a necessidade de suplementação do hidrolisado da CA com nutrientes específicos. L. rhamnosus, neste trabalho, foi a bactéria mais apropriada à fermentação láctica, dentre os Lactobacillus testados, sendo capaz de produzir 5,6 g ALac L-1, com rendimento de 33,8 mg ALac g-1 CA (hidrolisado com HCl, sem adição de nutrientes, 96 h de fermentação). A hidrólise ácida, neste caso específico, foi feita a 160 °C, por 70 min, com 4,0% HCl v v-1, gerando 14,7 g L-1 de glicose. A fermentação láctica via DCCR foi conduzida em vials de 4 mL, em batelada, a 37 °C, pH 6,0, empregando-se L. casei subsp. rhamnosus ATCC 10863, cepa especializada em catabolização de hexoses e pentoses (exceto arabinose). Experimentos com hidrolisados da CA adicionados de nutrientes produziram até 18,8 g ALac L-1 (rendimento máximo de 997,1 mg ALac g-1 açúcares) em substrato hidrolisado com HCl, após 52 h de fermentação; e 14,6 g ALac L-1 (rendimento máximo de 999,8 mg ALac g-1 açúcares) em substrato hidrolisado com H2SO4, após 24 h de fermentação. Portanto, neste trabalho, as escolhas de maior influência na produção de ALac foram a fermentação com hidrolisado sulfúrico e emprego de L. rhamnosus.

碩士
國立臺灣海洋大學
食品科學系
100
The research work can be concluded into two parts: optimization of conditions for immobilization of L-rhamnose isomerase (L-RhI) on Amberlite and characterization of immobilized L-RhI. The recombinant L-RhI from Thermoanaerobacterium saccharolyticum NTOU1 was immobilized on two types of Amberlites, Amberlite® XAD7HP (XAD7) and Amberlite® XAD4 (XAD4), respectively. The studies on the optimal conditions of immobilization included time, pH and glutaraldehyde concentration. From the studies for immobilization time and pH, the results showed that XAD7 carrier was better than XAD4 carrier on the absorption of enzyme and relative activity of immobilized enzyme. But the result from the immobilization with glutaraldehyde concentration showed that XAD4 carriers were better than XAD7 carriers on the absorption of enzyme and relative activity of the immobilized enzyme. Under all the tested immobilization conditions, the activity on XAD7 carrier immobilized in pH 8.5 for 2 h was higher than that on XAD4 carrier immobilized in pH 7 and 4% glutaraldehyde for 2 h, and XAD7 carrier immobilized in pH 8.5 for 2 h has the highest immobilization efficiency. The optimal pH and temperature for immobilized L-RhI were 7.5 and 80℃, respectively. The optimal pH and temperature were higher than those of free enzyme. Afer being used sixteen times, the activity of the immobilized L-RhI was 80 % retained.

碩士
國立中正大學
化學所
94
Xestovanin A have been isolated from extracts of the marine sponge Xestospongia Vanillia, and it shows antifungal activity. How to synthetic xestovanin glycoside, a part of Xestovanin A, was a point of this thesis. Xestovanin glycoside composed of ?Rhamanaose and ?Fucose is a very rare combination in nature. Modified sugars within different regents and conditions could yield four different kinds of disaccharides by ? β selectivity

碩士
國立臺灣海洋大學
食品科學系
101
In this study, we immobilized C-terminally lysine-tagged L-rhamnose isomerase (L-RhI-7K) by carboxylated magnetic particles, and optimised the immobilization conditions and characterized of the immobilized L-RhI-7K. L-rhamnose isomerase (L-RhI) not only catalyzes the aldose-ketose isomerization between L-rhamnose and L-rhammulose, but also between many aldose and ketoses. The activity of L-RhI from T. saccharolyticum is three times higher than L-rhamnose isomerases from other sources, so it has the potential to be immobilized. In this study, the carboxylated magnetic particles were prepared by the co-precipitation of Fe2O3 and FeO in aqueous medium and then modified with adipic acid. The gene of the recombinant L-RhI is obtained from T. saccharolyticum, and the recombinant L-RhI is tagged C-terminally with hepta-lysines (lysine tag). The positive charge of lysine residues allows the emzyme to bind to the negatively charged magnetic particles. The pET-21b-rhi-7k was constructed, and transformed into E. coli BL21-CodonPlus(DE3)-RIL (BL21C), and expressed under the presence of IPTG. The molecular weights of monomer of L-RhI-7K, which is close to that of wild-type L-RhI is 48kDa. For raising the purifity of L-RhI-7K, the crude enzyme is heated at 80 ℃ in 15 min to denature other proteins, and then purified by ion-exchange chromatography. L-RhI-7K shows the optimal temperature at 75 ℃ and the optimal pH at pH 7, and presents wide pH stability (pH 5-9) and good thermostability. However the residual activity is decreased to about 50% at 65 ℃ and pH 5-9 for 24 h. When L-RhI-7K is incubated in the condition of pH 8 and 80 mM Tris-HCl and immobilized 10 min, the best immobilized conditionc was obtained and allowed the adsorption efficiency to reach maximum, so we use this condition to immobilize L-RhI-7K. The optimal temperature and pH of immobilized L-RhI-7K estimated by using response surface methodology are 73.55 ℃ and pH 7.6. The thermostability and pH stability at 25 ℃ are not changed obviously, but the residual activity is decreased about 90% at 65 ℃ under various pH values. After the storage of 30 days, the immobilized L-RhI-7K has decreased 49.5% activity. After 7 times of usage, the immobilized L-RhI-7K remains only 20% relative activity of the first reaction.

碩士
國立臺灣海洋大學
食品科學系
102
Abstract D-Allose is a C-3 epimer of D-glucose, which have only slightly amount existed in nature. It has many physiological activities including inhibiting cancer cell proliferations, anti-tumor, decreasing oxidative pressures and immunosuppression. Moreover, it possesses nearly no calories and toxicity, and therefore has a potential to be used as a food additive. L-Rhamnose isomerase (L-RhI) can catalyze sugar isomerization between aldoses and ketoses. Some L-RhIs have a broad range of substrate selectivity, they can be used in the presence of AsDPE to produce D-allose from D-fructose which is a cheap material, and the intermediate product D-psicose is also an expensive and functional sugar. This study aims to alter the substrate specificity of Thermoanaerobacterium saccharolyticus NTOU1 L-RhI against D-allose by protein engineering. First, we performed the computer simulation to obtain modeling structures, and docked target substrates to the modeling structures. After analyzing relative sites of the substrate binding, residue Ile102 was mutated to other nine polar or charged amino acid residues, and residues Phe142 and His100 were mutated to six other polar or charged amino acid residues with similar side-chain lengths. Quick-screening result shows that mutant I102N Ts L-RhI has higher relative specific activity against L-rhamnose, mutant I102N and I102R Ts L-RhIs have higher relative specific activities against D-allose. Comparing to wild-type Ts L-RhI, I102N and I102R Ts L-RhIs have more preference against D-allose, while I102Y Ts L-RhI has more preference against L-rhamnose. Mutations on both Phe142 and His100 residues have resulted poor relative specific activities and only mutant H100D Ts L-RhI has more preference against D-allose than wild-type. We also have performed several partial sequence replacements around Leu59 which is located in a lid-like loop region, and the quick-screening result shows that double mutant L59M/S60G Ts L-RhI has more preference against D-allose. In a consequence of their lower KM values, mutant I102Y, I102Q and L59M/S60G Ts L-RhIs have higher catalytic efficiencies against L-rhamnose, and I102N, I102R, I102Q, L59M/S60G Ts L-RhIs have higher catalytic efficiencies against D-allose comparing to wild-type Ts L-RhI. The wild-type and mutant Ts L-RhIs were used in the presence of AsDPE to produce D-allose from 10% (w/v) D-fructose, there are no significantly differences in final predicted yields between wild-type and mutant Ts L-RhIs, and the final predicted yields can reach 22% D-psicose and 15% D-allose. Mutant I102Q Ts L-RhI consumed less time to reached the final predicted yields suggesting that this enzyme has the potential to be used in D-allose production. Keywords : Protein engineering, L-rhamnose isomerase, Substrate specificity.

碩士
國立臺灣海洋大學
食品科學系
103
In this study, D-psicose 3-epimerase (DPE) and L-rhamnose isomerase (L-RhI) were used to produce D-psicose and D-allose. D-Psicose was firstly converted from D-fructose by D-psicose 3-epimerase (DPE); then the isomerization of D-allose from D-psicose by L-rhamnose isomerase (L-RhI) was carried out, and finally response surface methodology (RSM) was used to optimize the productions of D-psicose and D-allose. To investigate the effect of single factor on the production of D-allose, the experiments were carried out at different temperatures in the presence of different concentrations of D-fructose and Co2+ by using DPE and L-RhI to produce D-allose. The results had shown that the higher D-allose yield were obtained at 50℃ in the presence of 0.1 mM Co2+, and the D-allose yield would be reduced by the increasing concentrations of D-fructose. The highest D-allose yield was obtained from 5% D-fructose, and the D-allose yields have no significant differences using 10-30% D-fructose as substrate. To optimize D-psicose production, 50℃, 30% D-fructose and 0.2 mM Co2+ were used as the center point of response surface methodology (RSM). The results had shown that temperature and D-fructose concentration significantly affected D-psicose yield, while the concentration of Co2+ made no significant difference to the D-psicose production. When the temperature and D-fructose concentration increased, the D-psicose yield was also increased. By using different concentrations of DPE (7, 14, and 21 U/mL), the D-psicose production reached equilibrium after 6 h and 26.81, 28.91, and 28.56% of D-psicose were produced after 24 h under the optimized conditions, which were 65℃, 46.8% D-fructose, and 0.3 mM Co2+. The optimize D-allose production, 50℃, L-RhI: DPE = 15: 1 and 0.2 mM Co2+ were used as the center point of response surface methodology (RSM). The results had shown that temperature and Co2+ concentration significantly affected D-allose yield, while the L-RhI: DPE ratio made no significant difference to D-allose production. When the temperature and Co2+ concentration increased, the D-allose yield also increased. The optimized conditions were 64.8℃, L-RhI: DPE = 7.1: 1, and 0.25 mM Co2+. Keywords: D-allose, D-psicose, D-psicose 3-epimerase, L-rhamnose isomerase

碩士
國立臺灣海洋大學
食品科學系
106
D-allose is a rare sugar, aldohexose and the C-3 epimer of D-glucose. It has many physiological activities including inhibiting cancer cell proliferation, anti-tumor, decreasing oxidative pressures and immunosuppression. Besides, it prossesses nearly zero calories and non-toxicity, and it can be used as food additives to enhance product functionality. Previous studies have indicated that D-fructose can be firstly converted to D-psicose by D-psicose-3-epimerase (DPE) and further it can be isomerized by L-rhamnose isomerase (L-RhI) to produce D-allose. The intermediate D-psicose is also a functional rare sugar. The study aims to improve the catalytic efficiency of Thermoanaerobacterium saccharolyticum NTOU1 L-RhI against D-allose by protein engineering. The amino acid sequence aligement of different sources of L-RhIs and TsRhI was performed and the residues at the +2 and -2 positions of the important residues in the substrate recognition region and and enzyme catalytic region were considered as the potential mutation residues. The PC software was used for preliminary evaluation, and the suitable positions selected for mutations were A66V, F236Y, M266T and L290G. After obtaining the mutation, fast screening was applied to analyze their relative specific activities. The results of the fast screening showed that the relative activity of A66V was better than the wild-type, therefore, A66V was further studied and compared with wild-type enzyme. The enzymes expressed by ClearColi BL21(DE3)-CodonPlus -RIL have the specific activities of 140 and 167 U/mg for wild-type and A66V, respectively, after purified from ion-exchange chromatography. In the enzyme kinetic, A66V increases the catalytic efficiency (kcat/KM) when D-allose was used as the substrate. The optimal conditions of immobilization are 1.2% alginate, 143 g/L cell mass and 0.3 M CaCO3, and the highest immobilization efficiency was 72.5%. The immobilized cells containing TsRhI has its optimal pH and temperature at 7.0 and 75⁰C, respectively. The immobilized cells have been stored in room temperature, and it still have 70% activity after 8-day storage, while only 40% activity left after 14-day storage.

碩士
國立成功大學
化學工程學系
88
Soluble particles initiate heterogeneous nucleation at very low supersaturation, particle formation and growth on soluble nuclei lead to depletion of supersaturated vapor , preventing other mechanisms of vapor to happen. So the most likely mechanism of aerosol formation is condensation of vapor on soluble nuclei . In the study, a flow cloud chamber(FCC) is used to examine the heterogeneous nucleation of supersaturated n-butanol vapor on D-Mannose and L-Rhamnose submicrometer particles. The dependence of the heterogeneous nucleation is elucidated on the particle size, material, solubility, supersaturation, etc,furthermore, we also examine the charge effect on the heterogeneous nucleation process caused by singly positive or negative charged particles and the dependence of the critical supersaturation temperature. Both classical nucleation theory and Kohler''s theory are used to calculate the critical supersaturation required to induce heterogeneous nucleation on soluble particles, and the results are compared with experimental values. The experimental result show that critical supersaturation will decrease when diameter or solubility of particle increases. For the same size particles, charged particles need lower critical supersaturation than neutral particles, but the difference is very small and within the uncertainty interval further research is necessary. Particles behave as if they have the ability to induce heterogeneous nucleation better than that of theory predicted. Experimental value is smaller than theoretic value in quantitative but is consistent with theoretic value in qualitatively.

碩士
國立中興大學
食品暨應用生物科技學系所
102
Many health food claimed containingspecific lactic acid bacteria(LAB)with attractive probiotic effects.However, it is difficult for consumers to confirm the microorganisms in the commercial or home made products. The identification of LAB strains become an important issue on their utilization. It is time-consuming and labor intensive work for traditional identification of LAB which rely on the specific biochemical metabolic profiles and plate counts on the selective media.Several commercial rapid detection kitsare developed for this purpose.LABidentification by the unique nucleic acid sequence are also well established. In this study we developed a microfluidic platform for detecting LAB base on the principles of their unique metabolic characteristics. Media with various carbon sources are selectively preloaded on the microfluidic chip. 100μL liquid sample with concentration between 107 CFU/mL-105CFU/mL are injected and flow through the reaction wells of the microchip. Six strains of LAB can be distinguished within 24 hours. Commercial API 50 CHL kit and polymerase chain reaction method are adopted to confirm the LAB identification. Two commercial fermented milk were analyzed by the microfluidic lactic acid bacteria detecting chip.The detecting results are consistent with the product labels. The microfluidic system demonstrated in this study provides the advantages of miniaturization, sample reduction and rapid response thus reveal the application potential on the portable detection system.

Preterm birth remains a major challenge in obstetrics. It complicates up to 13% of all pregnancies and accounts for approximately 80% of neonatal mortality and morbidity. Bacterial Vaginosis (BV) is associated with a 1.4-fold increased risk of preterm birth. Due to ineffectiveness of antibiotics in preventing preterm labour, probiotics have been proposed to serve as an alternative for treatment of BV and prevention of preterm birth. The objectives of this thesis were to determine 1) the effect of Lactobacillus rhamnosus GR-1 (L. rhamnosus GR-1) supernatant on cytokine profile and prostaglandin (PG)-regulating enzyme expression in lipopolysaccharide (LPS)-stimulated human chorion and placental trophoblast cells from human placentae, 2) the potential signaling pathways through which lactobacilli act and 3) the potential role of immune and placental trophoblast cells in initiating a response to LPS and L. rhamnosus GR-1 treatments. Primary cultures of human placental trophoblast cells were pre-treated with lactobacilli supernatant and then with LPS. In addition, immune cells were removed from cell suspensions using a magnetic purification technique to determine their role in modulating cytokine levels. The expression of pro- and anti-inflammatory cytokines and prostaglandin-regulating enzymes was then determined. We found sex-specific differences in the ability of LPS to increase the output of TNF-α, IL-10, and PTGS2. We also showed that L. rhamnosus GR-1 is able to act through the JAK/STAT and MAPK pathways to increase IL-10 and G-CSF, and independently down-regulates PTGS2 and TNF-α and up-regulates PGDH. The increase in G-CSF and PGDH were only observed in women carrying a female fetus. L. rhamnosus GR-1 may serve as an alternative to antibiotics in preventing some infection/inflammation-mediated cases of preterm birth.