Dissertations / Theses on the topic 'Bioactive Peptide Products'

The principle theme of this thesis was the synthesis of bioactive compounds. To this end, this work was focus on two main projects. The first one, which was carried out in the Department of Chemistry of the University of Girona under the supervision of Dr Montserrat Heras, concerned the synthesis of new unnatural amino acids bearing a pyrimidine ring within their side chain for incorporation into the antimicrobial peptide BP100 following a rational design in order to improve its biological profile. On the other hand, the second chapter of this thesis was developed in collaboration with the Laboratoire de Chimie Organique (ESPCI-ParisTech, Paris, France) under the guidance of Pr Janine Cossy and Dr Arseniyadis. This chapter was centered on the total synthesis of three marine natural products with complex structures and interesting biological activities: acremolide B, (–) bitungolide F and lyngbouilloside.
Aquesta tesi s'ha centrat en la preparació de nous compostos bioactius seguint dues estratègies diferents. El primer projecte es va portar a terme sota la supervisió de la Dra. Montserrat Heras del grup LIPPSO del Departament de Química i ha permés el desenvolupament de noves metodologies per la síntesi de nous aminoàcids no naturals. i el seu ús en la preparació d'anàlegs del pèptid antimicrobià BP100 amb l'objectiu de millorar-ne les propietats biològiques. El segon projecte és fruit de la col•laboració amb la Prof. Janine Cossy i el Dr. Stellios Arseniyadis del "Laboratoire de Chimie Organique" de l'Ecole Superieur de Physique et Chimie Industrielles (ESPCI-ParisTech, Paris, França). I ha permés posar a punt tres estratègies sintètiques convergents i versàtils per l’obtenció de tres productes naturals de gran complexitat estructural i interessants activitats biològiques – l'acremolide B, la bitungolide F i la lyngbouilloside – aïllats recentment del fons marí de diferents punts del món.

Malgré l’intérêt et la diversité des propriétés pharmacologiques des 2,5-dicétopipérazines (DKP), les voies de biosynthèse de ces molécules d’origine microbienne sont très peu connues. L’objectif de mes travaux de thèse a été i) de documenter de nouvelles voies de biosynthèse de DKP qui se caractérisent par la présence d’une synthase de cyclodipeptides (CDPS) travaillant souvent de concert avec une ou plusieurs enzymes de modification des cyclodipeptides et ii) d’explorer la diversité chimique codée par ces voies. Dans un premier temps, je me suis intéressée aux CDPS. Après la sélection par bioinformatique de candidats dans les bases de données génomiques, j’ai pu identifier 51 nouvelles CDPS actives et montrer que ces enzymes peuvent incorporer 17 des 20 acides aminés naturels. Par ailleurs, ce travail a permis de mieux caractériser la famille des CDPS, de définir l’existence de plusieurs sous-familles aux signatures fonctionnelles spécifiques et d’établir les premiers éléments d’un code de spécificité pour la synthèse de cyclodipeptides. Dans un second temps, je me suis attachée à caractériser les enzymes de modification associées aux nouvelles CDPS et, en particulier, les dioxygénases dépendant du Fe(II) et du 2-oxoglutarate (OG) qui sont très représentées dans ces voies. J’ai ainsi pu détecter une activité in vivo pour 11 OG et poursuivre la caractérisation in vitro pour l’une de ces OG, ce qui a permis de caractériser les DKP qu’elle synthétise et d’ainsi montrer la complexité des modifications chimiques introduites. L’ensemble de ces travaux a donc permis d’identifier et de caractériser de nouvelles voies de biosynthèse qui donnent accès à une diversité accrue de DKP
Despite the interest and diversity of the pharmacological properties of 2,5-diketopiperazines (DKPs), the biosynthetic pathways of these microbial molecules are poorly documented. The aim of my doctoral work was i) to identify new DKP biosynthetic pathways that are characterized by the presence of a cyclodipeptide synthase (CDPS) often associated with one or more cyclodipeptide-tailoring enzymes and ii) to explore the chemical diversity encoded by these pathways. First of all, my study focused on CDPSs. After the bioinformatics-based selection of candidates, 51 novel CDPS were characterized, revealing the incorporation of 17 of the 20 proteinogenic amino acids. Moreover, this work has allowed a better characterization of the CDPS family, by showing the existence of several subfamilies with specific functional signatures and laying the foundations of a specificity conferring code for the synthesis of cyclodipeptides. Second, I characterized the tailoring enzymes associated with the newly identified CDPSs and, in particular, the Fe(II) and oxoglutarate dependent dioxygenases (OGs) that are highly represented in these pathways. I detected the in vivo activity for 11 OGs and characterized the in vitro activity for one of them, showing the complexity of the chemical modifications introduced into the cyclodipeptide. This work has led to identify and characterize novel biosynthetic pathways that provide access to a greater diversity of DKPs

Le sang brut issu des abattoirs est une source importante de protéines. Il est actuellement peu valorisé, essentiellement par séchage ou par récupération de molécules plasmatiques après séparation centrifuge du plasma et du cruor. Ce co-produit est principalement composé d’hémoglobine, protéine riche en peptides actifs, tels que les peptides antimicrobiens, après hydrolyse par la pepsine porcine. L’objectif est de proposer une nouvelle stratégie de valorisation du sang, sans séparation plasma-cruor, tout en conservant les peptides bioactifs historiquement identifiés lors de l’hydrolyse pepsique de l’hémoglobine purifiée. Cette nouvelle voie, mise au point puis optimisée à l’échelle laboratoire, a été technologiquement transférée à l’échelle pilote. L’hydrolyse pepsique du sang a été premièrement mise au point à la concentration de 1% (p/v) en hémoglobine. Cette hydrolyse a mis en évidence la coexistence des mécanismes enzymatiques de type zipper et one-by-one pour l’apparition de la population peptidique. Les conditions d’hydrolyses (concentration en hémoglobine, choix de la pepsine, rapport enzyme-substrat, acide et temps d’hydrolyse) ont été optimisées en fixant une décoloration complète de l’hydrolysat ainsi que la conservation de la population peptidique. L’hydrolysat bioactif ainsi obtenu présente des propriétés antimicrobiennes et antioxydantes. Son analyse par spectrométrie de masse a permis la caractérisation de cet hydrolysat en terme de peptides issus de l’hémoglobine. Il ne possède aucune masse supérieure à 10 kDa, lui procurant ainsi une bonne digestibilité: son utilisation en alimentation animale en tant que complément alimentaire apparaît prometteuse
Raw blood from slaughterhouses is an important source of proteins. This co-product, currently undervalued, is mainly composed of hemoglobin, a protein rich in active peptides such as antimicrobial peptides, after hydrolysis by porcine pepsin.The aim of this thesis is to propose a new strategy for the valorization of whole blood, without plasma-cruor separation. Preservation of identified bioactive peptides by pepsic hydrolysis of purified hemoglobin is required. This new way of blood valorization, developed and then optimized at laboratory scale, has been technologically transferred on a pilot scale (80 L).The pepsic hydrolysis of 70% bovine 30% porcine blood was first developed at 1% (w/v) of hemoglobin (23°C, 200 mL). This hydrolysis has demonstrated the coexistence of zipper and one by one enzymatic mechanism for the appearance of the peptide population. Hydrolysis parameters (hemoglobin concentration, industrial grade pepsin, enzyme-substrate proportion, acid allowing the sustainability of the hydrolysis pH and hydrolysis time) were optimized by fixing a complete discoloration of the hydrolysate as well as the preservation of the peptide population.The bioactive hydrolysate thus obtained contains antimicrobial and antioxidant properties. Mass spectrometry analysis has shown the hydrolysate composition in terms of peptides derived from hemoglobin. No mass above 10 kDa have been found, providing it with a good digestibility: its use in pet food as a food supplement seems promising

La luzerne est la plante la plus cultivée dans le monde et est une excellente source de protéines. Cependant, la RuBisCO a montré le plus d'intérêt. Cette protéine a été étiquetée comme la protéine la plus abondante sur terre, elle constitue environ 65% (p/p) des protéines solubles de la luzerne. Dans ce travail une nouvelle méthode a été mise en place pour la purification de la RuBisCO à partir de la poudre de luzerne 10% (p/v) en utilisant deux solvants différents et l’effet du pH. Premièrement, des méthodes d’analyse qualitative et quantitative ont été utilisées pour confirmer l’efficacité de la méthode de purification qui pourrait remplacer certains procédés industriels classiques. Puis, une hydrolyse pepsique a été réalisée sur la RuBisCO purifiée qui aboutit à une forte population peptidique bioactive. Les peptides finaux de 24h d’hydrolyse ont montré une meilleure activité antibactérienne ou antioxydante par rapport aux autres hydrolysats. 9 nouveaux peptides antibactériens ont été identifiés et caractérisés par SM et qui ont un CMI entre 2-6mM contre quatre espèces de bactéries: B subtilis, E coli, L innocua et M luteus. En plus, des fractions peptidiques antioxydantes ont également été identifiées dans ce travail. Et leur activité antioxydante a été évaluée par différents tests in vitro et in vivo sur l’huile de Colza. Enfin, l’addition du peptide RDRFL issu de l’hydrolyse pepsique de la RuBisCO a montré un effet positif sur la prolongation de la durée de conservation de la viande hachée et de la purée de tomate
Alfalfa is an excellent source of protein. However, RuBisCO proteins showed most interest. Indeed, this protein has been labelled the most abundant on earth; it constitutes about 65% (w/w) of soluble leaf protein of Alfalfa. In this work, a new method was introduced for the purification of RuBisCO from alfalfa powder 10% (w /v), using two different solvents and pH effect. In a first step, the performance of the proposed RuBisCO recovery method was evaluated through qualitative and quantitative analysis and the results obtained showed that this new method could replace some conventional industrial processes. In a second step, enzymatic hydrolysis was carried out on the purified RuBisCO, which resulted in a large bioactive peptide population. The final peptides after 24h of hydrolysis showed better antibacterial or antioxidant activity compared to the other peptide hydrolysates. Nine new antibacterial peptides have been identified and characterized by MS and have a MIC of 2-6 mM against four species of bacteria: B subtilis, E coli, L innocua and M luteus. In addition, antioxidants peptide fractions were identified in this work, their antioxidant activity was evaluated by various in vitro and in vivo tests on oil of Colza. Finally, the addition of peptide RDRFL derived from the peptic hydrolysis of RuBisCO has a positive effect on the prolongation of the shelf life of minced meat and of tomato puree

Le diabète de type 2 (DT2) est un trouble multifactoriel complexe de l’homéostasie du glucose. Cette maladie, bien qu’elle possède une composante génétique, est principalement induite par des causes socio-environnementales comme de mauvaises habitudes alimentaires. En dépit des mesures hygiéno-diététiques et des traitements médicaux utilisés pour prévenir et traiter la maladie, le DT2 ne cesse de progresser. L’identification et la production de peptides bioactifs à partir de sources naturelles offrent une alternative intéressante aux médicaments de synthèse, dont les préoccupations concernant les effets secondaires ne cessent d’augmenter. Ainsi, en raison de leur abondance et leur richesse en molécule bioactive, les coproduits de la transformation du poisson offrent une source presque inépuisable de peptides bioactifs. En effet, lors d’études précédentes, il a été démontré que des protéines de morue et de saumon permettaient d’améliorer la santé cardio-métabolique in vivo, et d’améliorer la captation du glucose musculaire, de diminuer la production du glucose hépatique et l’inflammation. De plus, avec un nombre de plus en plus important d’individus à nourrir, l’industrie de la transformation doit augmenter sa production, et les déchets ne cessent de s’accumuler. Néanmoins, afin d’exercer leur effet bioactif, il est nécessaire de libérer ces peptides bioactifs des structures protéiques au sein desquelles ils sont imbriqués et sous forme inactive générant ainsi des hydrolysats complexes. Par la suite, une ou plusieurs étapes de séparation sont utilisées afin de concentrer ces peptides dans des fractions plus actives, par exemple en fractionnant ces hydrolysats complexes par électrodialyse avec membranes d’ultrafiltration. En effet, Il a été démontré l’efficacité de l’électrodialyse avec membranes d’ultrafiltration pour la génération de fractions améliorant, in vitro, la captation du glucose à partir d’hydrolysats de protéines de soya et de saumon. Ainsi, l’objectif principal de cette thèse était de concentrer et d’identifier des peptides bioactifs par le fractionnement d’un hydrolysat de protéines issu d’un coproduit de saumon iv par l’électrodialyse avec membranes d’ultrafiltration, et d’étudier l’impact de ces fractions et peptides sur le DT2. Lors de la première étude, il a été démontré qu’un empilement judicieux de membranes permettait de fractionner les peptides issus d’un hydrolysat de coproduit de saumon, en générant des fractions possédant des peptides avec des propriétés physico-chimiques (charge et masse) différentes. De plus, cet empilement de trois membranes d’ultrafiltration de seuils de coupure différents a permis de moduler la réponse in vitro de la captation du glucose. En effet, des peptides cationiques de plus haut poids moléculaire ont amélioré la captation du glucose dans une étude in vitro, alors que les peptides cationiques de plus faible poids moléculaire ont démontré un effet inhibiteur de la bioactivité. Finalement, l’analyse par spectrométrie de masse des fractions a permis de caractériser (temps de rétention et charge) 17 peptides cationiques et 21 peptides anioniques, potentiellement responsables de l’effet bioactif des fractions. Lors de la deuxième étude, le fractionnement par EDUF des fractions finales récupérées lors de la précédente séparation et l’étude in vitro de la bioactivité de ces fractions (captation du glucose, production du glucose hépatique et inflammation) ont permis d’identifier deux fractions très prometteuses, démontrant un effet sur ces trois bioactivités. De plus, l’analyse par spectrométrie de masse en tandem de ces fractions a permis d’identifier, à l’aide de base de données, la séquence peptidique de 24 peptides anioniques, potentiellement responsables de ces effets bioactifs. Finalement, lors de la troisième étude, basées sur l’analyse des spectres obtenus par spectrométrie de masse en tandem, 13 peptides ont été sélectionnés et synthétisés, puis testés individuellement pour leurs capacités à augmenter l’absorption du glucose au niveau de cellules musculaires, à diminuer la production de glucose hépatique et finalement à diminuer la réponse inflammatoire de macrophages. Ainsi, pour la première fois, quatre nouveaux peptides ont été identifiés à partir de coproduits de saumon, et leurs propriétés glucorégulatrices in vitro ont été démontrées.
Type 2 diabetes (T2DM) is a complex multifactorial disorder of glucose homeostasis. This disease has a genetic basis but is mainly caused by socio-environmental behaviours, such as overeating and a lack of physical activity. Despite dietary measures and medical treatments used to prevent and treat the disease, T2D continues to progress. The identification and production of bioactive peptides from natural sources offer an interesting alternative to synthetic drugs, whose concerns about side effects are constantly increasing. Thus, because of their abundance and richness in bioactive molecules, fish processing co-products offer an almost inexhaustible source of bioactive peptides. Indeed, in previous studies, cod and salmon proteins have been shown to improve cardio-metabolic health in in vivo studies, and to improve muscle glucose uptake, decrease hepatic glucose production, and inflammation. In addition, with a growing number of people to feed, the processing industry is at its height, and waste continues to accumulate. Nevertheless, in order to exert their bioactive effect, it is necessary to release these bioactive peptides from native proteins. Subsequently, one or more separation, using for example electrodialysis with ultrafiltration membranes, are needed to concentrate these peptides and generate bioactive fractions. Indeed, it was previously demonstrated the effectiveness of electrodialysis with ultrafiltration membranes to generate bioactive fractions, from complex matrices, able to improve the glucose uptake in vitro, from soy and salmon protein hydrolysates. In this context, the main objective of this thesis was to concentrate and identify bioactive peptides, by fractionating a protein hydrolysate from a salmon co-product, by electrodialysis with ultrafiltration membranes, and to study the impact of these fractions and peptides on T2D. In the first study, results demonstrated that a triple size selective separation by EDUF allowed to generate peptide fractions with different physicochemical properties (charge and mass). Moreover, it was demonstrated that such a separation allowed to modulate the in vitro response of the fractions for glucose metabolism. Indeed, from a single EDUF separation, cationic peptides with higher molecular weights were concentrated and demonstrated to enhance their glucose uptake capacity. Whereas, cationic peptides with lower molecular weights have decreased the glucose uptake capacity. In addition, analyses by mass spectrometry of the vi fractions allowed to characterize (retention time and charge) 17 cationic peptides and 21 anionic peptides, potentially responsible for the bioactive effect of the fractions. In a second study, a second EDUF fractionation, using as feed solution the final fractions recovered during the previous separation was performed. The selectivity of the process was confirmed by liquid chromatography-mass spectrometry analyses. Moreover, in vitro study of the bioactivities (glucose uptake, hepatic glucose production and inflammation) effect of these fractions, led to the identification of two very promising fractions, demonstrating a simultaneous effect on all three bioactivities tested. In addition, the tandem mass spectrometry analysis of these fractions allowed the sequence identification of 24 anionic peptides, potentially responsible for these bioactive effects. Finally, in a third study, based on the analysis of the spectra obtained by tandem mass spectrometry, 13 peptides were selected and synthesized, then individually tested for their ability to increase glucose uptake in muscle cells, to reduce glucose production by hepatic cells, and to decrease the inflammatory response of macrophages. Thus, for the first time, four new peptides identified from salmon by-products, demonstrated in vitro glucoregulatory properties.

L'hydrolyse pepsique de l’hémoglobine bovine purifiée ou à partir d'un coproduit des abattoirs : le cruor, peut être considérée comme une voie importante d'obtention de peptides antibactériens. Une étude cinétique nous a permis de maîtriser cette hydrolyse et de déterminer un modèle mathématique capable de prédire la concentration de chaque peptides antibactériens des deux familles de peptides α 1-32 et α 107-141 et ceux pour un intervalle de température de 15-37°C, de pH de 3,5-5,5 et de rapport enzyme/substrat de 1/5-1/20. Le calcul des énergies d'activation pour les différentes réactions impliquées dans le mécanisme était effectué grâce à l’équation d’Arrhenius qui a permis d’étudier l’effet de la température sur les différents coefficients cinétiques. L’effet du pH et du rapport E/S était également étudié et le modèle trouvé a démontré une augmentation linéaire de la vitesse d’hydrolyse en diminuant le pH (entre 3,5 et 5,5) et une vitesse invariable avec le rapport E/S (1/5-1/20). L'étude de la relation structure-fonction des peptides antibactériens α 1-32 et α 137-141 a été effectuée grâce à un suivie de la cinétique de K+ extracellulaire en présence de Lisrea innocua et le déterminant antibactérien minimal a été déterminé pour le peptide α 137-141. La possibilité de valoriser les peptides antibactérien α 1-32 et α 137-141 dans un emballage bioactif pour la conservation des aliments contre le développement de bactéries pathogènes a été étudiée par l'adsorption de ces peptides en surface d'un film de polyéthylène basse densité, traité avec le plasma froid
The pepsin hydrolysis of purified bovine hemoglobin or from a co-product of slaughterhouses: cruor, can be considered as an important route for obtaining antibacterial peptides. A kinetic study has allowed us to control the hydrolysis and to determine a mathematical model able to predict the concentration of each antibacterial peptides of two families of peptides α 1-32 and α 107-141 and those of an interval of temperature 15-37°C, of pH 3,5-5,5 and ratio enzyme/substrate of 1/5-1/20 . The calculation of activation energies for the different reactions involved in the mechanism was made by the Arrhenius equation, which was used to study the effect of temperature on the various kinetic coefficients. The effect of pH and ratio E / S was also studied and the model found showed a linear increase in the rate of hydrolysis decreasing the pH ( between 3,5 and 5,5 ) and an invariable speed with the ratio E / S ( 1/5-1/20 ). The study of the structure-function relationship of antibacterial peptides α 1-32 and α 137-141 was carried out thanks to a followed the kinetics of extracellular K + in the presence of Lisrea innocua and the minimal determinant antibacterial was determined for the peptide α 137-141. The possibility to recovery the antibacterial peptides α 1-32 and α 137-141 in to a bioactive food packaging against the growth of pathogenic bacteria has been studied by the adsorption of these peptides on the surface of a low density polyethylene film treated with cold plasma

La valorisation des co-produits de poisson est un enjeu économique et environnemental. Depuis plusieurs années, des recherches ont montré que les co-produits de poisson contenaient des molécules actives pour la santé humaine comme des acides gras polyinsaturés et des peptides bioactifs. L’objectif principal de cette thèse était d’évaluer l’utilisation potentielle d’hydrolysats de laitance de hareng pour l’amélioration de la santé humaine, spécifiquement en agissant positivement sur certaines fonctions physiologiques associées au syndrome métabolique, et l’effet de leur séparation par électrodialyse avec membranes d’ultrafiltration (EDUF) sur la production de fractions bioactives. Dans un premier temps, il a été démontré qu’une supplémentation avec différents hydrolysats de laitance de hareng d’une diète riche en gras et en sucre chez des souris permettait de moduler certains paramètres physiologiques impliqués dans le développement du syndrome métaboliques (MetS) : amélioration de la tolérance au glucose, augmentation de l’apport énergétique total et protection de la population de Lactobacillus dans le microbiote intestinal. De plus, les hydrolysats ont aussi montré des activités antiinflammatoires in vitro à des concentrations de 1ng/ml et 100pg/ml. Dans un second temps, la faisabilité d’une séparation par EDUF de deux hydrolysats de laitance de hareng différents a été évaluée : le premier plus complexe était un mélange de molécules (lipides, acides nucléiques, peptides, acides aminés libres), alors que le second était principalement composé de peptides et d’acides aminés libres. Une nouvelle configuration utilisant quatre membranes d’ultrafiltrations (deux de 50kDa et deux de 20kDa) a permis un double fractionnement simultané des composés anioniques et cationiques en seulement une étape. Il a d’abord été montré que seuls les peptides chargés ainsi que les acides aminés libres pouvaient être séparés par EDUF alors que les lipides et les acides nucléiques ne migraient pas dans les fractions de récupérations. De plus, l’utilisation de membranes présentant deux tailles de pores distinctes a permis le fractionnement des hydrolysats en différentes classes de poids moléculaires. En effet, l’utilisation de membranes de 20kDa a permis la concentration de peptides de faibles poids moléculaires (< 600Da) et IV d’acides aminés libres, alors que les populations peptidiques des fractions obtenues avec les membranes de 50kDa présentaient des poids moléculaires supérieurs et plus variés. Dans un troisième temps, les bioactivités des fractions de récupérations et des hydrolysats de laitance de hareng ont été évaluées in vitro. Ainsi, la séparation du premier hydrolysat a permis l’obtention d’une fraction finale stimulant la captation du glucose in vitro et d’une fraction anionique antioxydante. Le fractionnement du second hydrolysat a permis quant à lui la production de deux fractions cationiques anti-inflammatoires ainsi que l’identification subséquente de deux séquences peptidiques bioactives. L’ensemble de cette thèse a donc démontré que les hydrolysats de laitance de hareng contenaient des composés actifs, comme des acides gras polyinsaturés et des peptides, modulant positivement certains paramètres physiologiques impliqués dans le MetS et pouvant ainsi permettre la diminution de son occurrence. De plus, la séparation des hydrolysats par EDUF a permis la production de fractions bioactives ainsi que l’identification de deux nouvelles séquences peptidiques anti-inflammatoires (IVPAS et FDKPVSPLL). Ces travaux ont démontré l’effet bénéfique pour la santé humaine des hydrolysats de laitance de hareng et de ses fractions, permettant d’envisager une valorisation plus efficace de ces coproduits de poisson par les industries de transformation dans les secteurs liés à la santé humaine.
Fish by-product valorization is an economic and environmental issue. For several years, scientific researches have shown that fish by-products contained active molecules for human health, as polyunsaturated fatty acids and peptides. The aim of this thesis was to evaluate the potential use of herring milt hydrolysates for human health, especially by evaluating their potential actions in physiological parameters involved in the metabolic syndrome and the effect of their separation by electrodialysis with ultrafiltration membrane (EDUF) for the production of bioactive fractions. First, we have demonstrated that the supplementation of three different herring milt hydrolysates in a high fat high sucrose diet in mice was able to modulate some physiological functions involved in the metabolic syndrome: improvement of glucose tolerance, increase of the total energy intake and protection against the Lactobacillus disappearance in the gut microbiota. Moreover, the hydrolysates decreased the inflammation induction in macrophages stimulated with LPS at 1ng/ml and 100pg/ml. Secondly, we have evaluated the separation of two herring milt hydrolysates by EDUF: the first one was more complex with a mix of different molecules (lipids, nucleic acids and peptides) while the second one was mainly composed of peptides. A new configuration using four ultrafiltration membranes (two of 50kDa and two of 20kDa) allowed a simultaneous double separation of anionic and cationic compounds. It has been shown that only charged peptides and free amino acids were fractionated in EDUF, while the lipids and nucleic acids didn’t migrate to the recovery fractions. Moreover, the use of membranes with different cut-off allowed a separation of the hydrolysates in different molecular weight ranges. Indeed, the use of 20kDa membranes allowed the concentration of peptides with small molecular weights (<800Da) and free amino acids, while the recovery fractions obtained with the 50kDa membranes were composed oh peptide with higher molecular weights.Thirdly, the potential bioactivities of the recovery fractions and the herring milthydrolysates were evaluated in vitro. Hence, the separation of the first hydrolysate allowed the production of a final fraction increasing the glucose uptake and an antioxidant anionicfraction. While the separation of the second hydrolysate allowed the production of two antiinflammatory cationic fractions as well as the identification of two bioactive peptides sequences. All these results showed that milt herring hydrolysate contained bioactive compounds such as polyunsaturated fatty acids and peptides, improving some physiological functions involved in the MetS and may decrease its occurrence. More over, the separation of the hydrolysates by EDUF allowed the production of bioactive fractions and the identification of two new anti-inflammatory peptide sequences. This work demonstrated the existence of a beneficial effect of herring milt hydrolysate and its fractions for the human health, allowing a better valorization of this by-product of the food industry for the health sector.

During my research career in Prof. V.J.Hruby's laboratory I worked on two different projects. The first project, which was initiated by the author, was planned to serve the need of our laboratory for a novel method of peptide cyclization. This method was planned to use recent advances in Pd0-catalyzed asymmetric synthesis combined with the structural richness offered by the Baylis-Hillman chemistry which could open new ways to diverse areas of drug design, molecular immunology and chemotherapy. This approach would provide cyclic peptides featuring N-alkylated amino acids that would confer high resistance to degradation by proteases. Because of numerous synthetic problems imposed, this strategy was not of considerable current use in peptide synthesis, especially on solid supports. However, despite a substantial amount of effort invested, this method faced serious drawbacks such as multistep synthesis and side reactions when applied to solid supports. Moreover, recent introduction of microwave technology which has helped to solve a great number of problems has led to a renaissance in the classical lactam and thioester bond cyclizations which overshadowed our quest for a novel methodology. The second project was focused on application of 4-anilidopiperidines for the synthesis of chimeric bioactive peptides. It was an effort towards the development of novel analgesics with reduced toxicity and enhanced potency. This project linked small molecule and multimeric ligand designs that were ongoing in our laboratory at the time. Major accomplishments in this project were made possible by successful resolution of several research challenges. I was able to find a straightforward, convenient and economical approach for the synthesis of novel analogues on a solid support. These developments led to novel compounds which showed substantial increases in their binding affinity relative to corresponding opioid analogues. To illustrate, compounds PET25, 26, 27, 29, 30, 31, and 32 showed high bioactivity and sub-nanomolar binding affinity to opioid receptors. Most of the peptides generated in the second project are still being investigated for their biological activities by our colleagues at the Department of Pharmacology, but the results to date indicate that some highly potent novel compounds have been made.

This thesis is an investigation of the natural products deriving from marine algae and cyanobacteria and has resulted in the discovery of eleven new secondary metabolites. The structure elucidations of these new molecules were performed using a variety of spectroscopic techniques. Four new macrolides were isolated and characterized from the Madagascar marine cyanobacterium Geitlerinema sp. These ankaraholides are structurally similar to the potently cytotoxic swinholides and were found to have cytotoxicities ranging from 178 nM to 354 nM against human lung cancer (NCI-H460) and mouse neuro-2a cell lines. Since swinholide-type compounds were previously localized to the heterotrophic bacteria of sponges, these findings raise intriguing questions about their true metabolic source. Geitlerinema sp. was found to be particularly rich in chemistry, and also produced the new linear lipopeptide mitsoamide with unusual structural features including an aminal moiety, a homolysine residue and a polyketide unit (3,7- dimethoxy-5-methyl- nonanedioic acid) (DMNA). A collection of the red marine alga Portieria hornemannii from the south of Madagascar (Tolagniaro, Fort Dauphin), led to the isolation of the previously reported halogenated monoterpene, halomon, and the discovery of three new related metabolites. These molecules were found to inhibit DNA methyltransferase 1 (DNMT-1). As a result of efforts to identify bioactive agents from the marine cyanobacterium Lyngbya majuscula, tanikolide dimer, a novel SIRT2 inhibitor (IC50 = 176 nM), and tanikolide seco-acid were isolated. The depside molecular structure of tanikolide dimer, which is likely a meso compound, was established by NMR, MS and chiral HPLC analyses. The structure of tanikolide dimer raises a number of intriguing configurational and biosynthetic questions for further study. The bioassay guided fractionation of a collection of the brown marine alga Dictyota sp. from Netherland Antilles Playa Fort, led to the identification of a novel HDAC inhibitor with a dolastane carbon skeleton. The novel molecule was also found to possess antimalarial activity. Other known HDAC inhibitors with interesting antimalarial activity have been reported previously, and based on this efficacy against malaria, HDAC appears to be a viable target for the development of antiparasitic agents.
Graduation date: 2006

Cancer is the most widely recognized reason for human deaths globally. Conventional anticancer therapies, including chemotherapy and radiation, are very costly and induce severe side effects on the individual. The discovery of anticancer compounds including dairy-derived peptides may thus be a better alternative for cancer prevention and management. Anticancer peptides exist in the amino acid chain of milk proteins and can be generated during proteolytic activities such as gastrointestinal digestion or food processing including fermentation by lactic acid bacteria (LAB) and probiotics. However, proteolytic capacity of these bacteria is strain specific. The study was conducted to establish proteolytic activity of Lactobacillus (L.) acidophilus (ATCC® 4356™), L. casei (ATCC® 393™) and L. paracasei subsp. paracasei (ATCC® BAA52™) in yogurt. Crude peptides were separated by ultra-high centrifugation and tested for antioxidant and antimutagenic activities. The degree of proteolysis highly correlated with these bioactivities, and its value (11.91 %) for samples containing all the cultures was double that of the control. Liberated peptides showed high radical scavenging activities with 1,1- diphenyl-2-picrylhydrazyl and 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), IC50 1.51 and 1.63 mg/ml respectively and strong antimutagenicity (26.35 %). These probiotics enhanced the generation of bioactive peptides in yogurt.

Proteolytic activity is very important characteristic of Lactic Acid Bacteria (LAB) They produce therapeutic benefits and also increase physiological activity of cultured dairy products by liberating a number of biologically active peptides. The main aim of this project was to determine the release of bioactive peptides from milk proteins by selected Lactobacillus species.

碩士
國立臺灣海洋大學
食品科學系
104
Processing co-products from tilapia are abundant in proteins and can be great sources to produce bioactive peptides. The major purpose in this study was to investigate potential bioactive peptides in tilapia proteins and measure ACE inhibitory activity of tilapia protein hydrolysates. The proteins from tilapia frame and skin were extracted by acid and alkaline pretreatment-isoelectric point precipitation method, then those proteins were identified by SDS-PAGE, in gel digestion, mass spectrometry techniques of proteomic. A total of 7 proteins were identified as alpha actin, collagen alpha-1(I) chain-like isoformX2, collagen alpha-2(I) chain-like isoformX1, creatine kinase (M-type-like), fructose- bisphosphate aldolase A, myosin heavy chain (fast skeletal muscle-like), and troponin T. Sequences of identified proteins were further analyzed using BIOPEP database. Frequency of ACE inhibitory peptides embedded in identified tilapia proteins are 0.350-0.756, and antiamnesic (frequency: 0.187-0.188) and antithrombotic (0.203-0.217) peptides were also discovered from sequences of tilapia collagen proteins. Results from BIOPEP recommended that tilapia proteins can be great precursor to produce ACE inhibitory, antiamnesic and antithrombotic peptides. Frame and skin protein isolates were hydrolysed by 3 proteases (bromelain、papain、pepsin) and ACE inhibitory effect of hydrolysates was evaluated. Among different hydrolysates, frame pepsin hydrolysate showed highest ACE inhibitory activity with 79.7 ± 2.39% value at 1 mg/mL concentration. Frame pepsin hydrolysate was further separated by hollow fiber and gel filtration, it showed highest ACE-inhibitory activity in ＜5 kDa fraction (88.27 ± 0.18%). According to BIOPEP analysis, predicted numbers of ACE inhibitory peptides have positive relation with in vitro ACE inhibitory activity, but such relation can not be observed in papain hydrolysate. Preliminary results in this study indicated that mass spectrometry techniques of proteomic coupled with BIOPEP can be potent tool to assist production of functional hydrolysates or bioactive peptides.

Fish by-products can account up to 75% of total weight of fish catch, harvested or processed. Despite their high economic value, by-products have been used mainly for the production of low-value products resulting in a low profit for the fish industry. Thus, it is imperative to develop efficient and effective technologies for the recovery of valuable ingredients from fish by-products. The main focus of this thesis was to develop a simple hydrolysis process to extract valuable compounds and liberate bioactive peptides from fish by-products of fish species endemic to Australia as well as exploring the essential physiological properties of these bioactive peptides, especially their potent use for colon cancer treatment.

碩士
國立臺灣大學
食品科技研究所
91
In this study, two commercial enzymes were used to hydrolyze the proteins of chicken head and black soybean to produce a health-promoting product containing bioactive peptides. It was found that the chicken head hydrolysate possessed the highest angiotensin I converting enzyme (ACE) inhibitory and antioxidative activities when the substrate was pre-heated and then hydrolyzed at 50℃ for15 hours at E/S ratio 0.5%. The antioxidative properties of the chicken head hydrolysate could be further enhanced by addition of black soybean in the substrate during enzymatic hydrolysis. However, addition of black soybean in the chicken head did not affect the ACEI activity of the hydrolysate. Cell line models were used to investigate the bioactivities of chicken head / black soybean hydrolysates. It was found that the hydrolysate had proliferation effect on the skin fibroblast, Detroit 551 and neuronal cell, PC12. For the immunomodulatory effect, the chicken head / black soybean hydrolysates could not stimulate RAW 264.7 cell to secret tumor necrosis factorα(TNF-α). On the other hand, the commercial products of essence of chicken were able to stimulate the RAW 264.7 cell line to secret TNF-α. For the tests of anti-inflammatory effect using NO synthesis as indicator, we found that neither chicken/black soybean hydrolysates nor commercial essence of chicken had anti-inflammatory effects on LPS / IFNγ- activated murine macrophages, RAW264.7.