Dissertations / Theses on the topic 'Hydrolysis of lipids'

The transport of compounds around the body has been a topic of interest for many years, and the advent of non-invasive biological imaging in living tissue has made huge advances in the characterisation and localisation of cellular receptors for use in drug targeting. However, there remains a significant paucity of knowledge regarding how the majority of drug molecules are transported about the body, when they often exhibit negligible aqueous solubility and the body expresses no trans-membrane pumps or chaperone proteins that recognise them and facilitate their movement. This leads to large attrition rates in drug discovery programmes, as compounds with high binding constants or inhibitive activity in vitro fail to perform in vivo, due to poor bioavailability or non-specific sequestration away from the tissue of interest. In this study, the interactions between a number of drug and lipid molecules were investigated and the effects upon both the lipids’ chemical and bulk membrane structures were analysed. This revealed some of the mechanistic causes of the previously observed hydrolytic activity a number of common drug compounds exhibit toward lipid membranes and identified parameters affecting the observed rates of reaction. The findings also suggest approaches by which this behaviour might be predicted, or even tuned to deliver optimum pharmacological characteristics.

Parmi les œufs de poisson, qui sont une ressource aquatique nutritionnelle intéressante, ceux de la truite arc-en-ciel (Oncorhynchus mykiss) contiennent une quantité élevée de protéines et une huile riche en acides gras polyinsaturés (AGPI), avec une proportion très importante de phospholipides. Cependant, l’œuf de poisson présente une capacité élevée d’auto-protection contre les contraintes extérieures, qui limite la destructuration de son réseau protéique par attaque enzymatique. Ainsi, le degré d’hydrolyse des œufs de la truite l’Alcalase®, la Neutrase® et la Protamex® varie entre 3 et 7 %, ce qui est très faible (20 % dans la majorité des protéines animales). L’extraction des lipides après protéolyse partielle est incomplète, probablement en raison d’interactions fortes avec les protéines faiblement hydrolysées. Ils contiennent une teneur élevée en phospholipides (53 % des lipides totaux) et les acides gras polyinsaturés entrent pour 42 % des acides gras totaux. Les AGPI, notamment le DHA, sont situés préférentiellement en position sn-2 sur la molécule de glycérol ce qui est particulièrement intéressant du point de vue nutritionnel. La stabilité à l’oxydation de l’huile a été étudiée par diverses méthodes, dont la spectrométrie infrarouge à transformée de Fourier. Cette méthode s’est avérée extrêmement intéressante pour une analyse structurale de la dégradation de l’huile en cours d’oxydation. Il peut être conclu que les lipides tirés des œufs de la truite arc-en-ciel ou de poisson en général, ont un réel avenir en matière de complément alimentaire ou nutraceutique, à condition de lever l’obstacle de l’hydrolyse enzymatique des protéines du chorion et du vitellus<br>Fish eggs, especially those of the rainbow trout (Oncorhynchus mykiss) in the present study, are an interesting nutritional aquatic source. They contain proteins of high value, as well as an oil rich in polyunsaturated fatty acids (PUFA) with a large percentage of phospholipids. However, they exhibit a high auto-protection capacity against environmental constraints and thus, the degree of hydrolysis of rainbow trout eggs by Alcalase®, Neutrase® and Protamex® proteases varied solely within 3-7 %. This value was low compared with the 20 % obtained in most animal proteins. The phospholipid content was high (53 % of total lipids) and PUFA accounted for 42 % of total fatty acids. Among PUFA, DHA was found preferably at the sn-2 position of the glycerol backbone, which is of special interest about nutritional properties. The oil release by enzymatic hydrolysis was found limited compared with chemical methods, probably because of the strong interactions engaged with the incomplete destructured protein network. The oxidative stability of the oil was studied through several methods in which the infrared Fourier transform appeared as the best tool for structural analysis along the oxidation process. As a conclusion, lipids from fish eggs, especially from rainbow trout, could be a nutritional breakthrough, as far the enzymatic hydrolysis of the vitellus and of the chorion proteins is achieved

Le cycle biogéochimique des lipides a été étudié en milieu marin côtier anthropisé. Ce travail est basé sur l’analyse chimique des stocks de lipides par CCM/DIF et la mesure de leur hydrolyse par les communautés bactériennes associées à ces stocks. Un développement méthodologique a eu pour objectif d’identifier les bactéries marines possédant l’activité lipase. Le substrat testé (ELF-palmitate), vendu comme marqueur potentiel des lipases, n’est pas hydrolysé spécifiquement par ces dernières. Par conséquent, il ne constitue pas un outil adéquat pour l’identification des bactéries lipases en milieu marin. L’étude temporelle réalisée en baie de Marseille a permis de déterminer les caractéristiques côtières propres à la baie, et de les comparer à des mesures obtenues antérieurement en milieu hauturier (site DYFAMED, Mer Ligure). Alors qu’ils représentent 1 à 7 % du carbone organique dissous (COD) en milieu hauturier, les lipides peuvent représenter jusqu’à 16% du COD en baie de Marseille en période de forte productivité, soulignant une forte accumulation de ces composés dans la matière organique dissoute (MOD). L’influence des apports côtiers dans la distribution des lipides biogéniques n’apparaît pas de manière aussi flagrante qu’on pouvait s’y attendre. Leur distribution est clairement contrôlée par les alternances saisonnières, comme dans le milieu hauturier. Ainsi, leur temps de résidence varie de 0.8-8 jours en fonction de l’activité bactérienne et de la saison. Les apports lipidiques d’origine anthropique (hydrocarbures) sont détectés à des concentrations parfois très élevées en 2007 en baie de Marseille. Entre 2007 et 2008, une forte diminution des concentrations en lipides et hydrocarbures dissous pourrait être liée à la mise en place d’un étage biologique dans le traitement des eaux usées de la ville de Marseille. Ce traitement supplémentaire aurait en effet pour principale conséquence de diminuer les apports en MOD dans les eaux usées rejetées par l’émissaire de Cortiou. Cette hypothèse implique que l’influence du panache de Cortiou soit visible jusqu’à la station Sofcom, située au milieu de la rade sud de la baie de Marseille<br>The biogeochemical cycle of lipids has been studied in a marine coastal anthropized environment. This work is based on chemical analysis of lipids by TLC/FID and the measurement of their hydrolysis rates by the associated bacterial communities. A methodological development aimed to identify lipase marine bacteria. The tested substrate (ELF-palmitate), sold as a potential marker of lipases, was not specifically hydrolyzed by the latter. Therefore, it is not an appropriate tool for the identification of marine lipase bacteria. The time series conducted in the Bay of Marseille (April 2007 – December 2008) allowed the determination of coastal characteristics of the bay and their comparison with previous measurements, obtained in the open Mediterranean Sea (DYFAMED, Ligurian sea). While they represent 1 to 7% of de dissolved organic carbon (DOC) at the offshore station, lipids may represent up to 16% of DOC in the Bay of Marseille, during periods of high productivity, underlining a strong accumulation of these compounds in the dissolved organic matter (DOM). The coastal inputs did not influence the distribution of biogenic lipids as was expected. Their distribution was clearly controlled by season, as in the offshore site. Accordingly, their residence time ranged from 0.8-8 days depending on bacterial enzyme activity and season. Lipidic anthropogenic inputs (hydrocarbons) were occasionally detected at very high concentrations in the Bay of Marseille during the year 2007. Between 2007 and 2008, a significant decrease in dissolved biogenic lipid and hydrocarbon concentrations could be related to the establishment of a biological treatment of wastewaters from Marseille. Indeed, this additional processing was expected to lead to the decrease of DOM inputs in wastewaters. This hypothesis implies that the plume of the wastewaters from Cortiou could influence the quality of OM at the Sofcom station, located in the centre of the Bay of Marseille

Les composés lipidiques et protéiques des têtes de saumon (Salmo salar) ont été extraits par un procédé d'hydrolyse enzymatique. Dans la première partie de cette étude, l'extraction enzymatique des hydrolysats protéiques avec l'Alcalase® 2,4 L a été réalisée et l' optimisation du procédé par l'utilisation de la méthode des surfaces de réponse a permis de quantifier l'influence et les interactions des facteurs (pH, température, rapport enzyme / substrat). Par planification expérimentale, des hydrolysats protéiques avec des degrés d' hydrolyse variant de 10,8 % à 17,3 % et des rendements protéiques compris entre 47 % et 71 % ont été obtenus. Les propriétés fonctionnelles des hydrolysats protéiques ont été améliorées. Dans la deuxième partie, l'huile est extraite à un taux de 19,6 %, omparable à la méthode d'extraction par solvant (21,5 %). Les deux types d'huiles ont des caractéristiques physico-chimiques similaires et les acides docosahexaenoique (DHA) et eicosapentenoique (EPA) sont majoritaires parmi les acides gras polyinsaturés. Les culots protéiques contiennent la majorité des phospholipides (55 %) dont la phosphatidylcholine qui représente environ 55 % des phospholipides. Le potentiel de ce procédé d'extraction peut être étendu à d'autres sources d'acide gras polyinsaturés à longue chaîne sensibles à des conditions d'extraction drastiques<br>A new process for the extraction of lipids and proteins from salmon (Salmo salar) heads was performed by enzymatic treatment. In the first part of this work, proteolysis assisted with Alcalase® 2. 4 L was performed. The use of Response Surface Methodology allowed optimization of temperature, enzyme / substrate ratio and pH leading to various hydrolysates (10. 8 % - 17. 3 % degree of hydrolysis) and protein recovery ranging from 47 % to 71 %. The functional properties of protein hydrolysates were improved. In the second part, the enzymatic extraction of oil yielded 19. 6 % and was comparable to solvent extraction (21. 5 %). The oil fractions resulting from the proteolysis were rich in polyunsaturated fatty acids and their individual fatty acid composition was similar to total lipids extracted by solvent method. Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) constituted predominant fatty acids among total n-3 polyunsaturated fatty acids. The sludge lipids contained most of phospholipids (up to 55 %) with high content of phosphatidylcholine (around 55 % of phospholipids). This process might be extended to any source of long chain polyunsaturated fatty acids, which are sensitive to drastic conditions

Bacterial cell division requires formation of the cytokinetic cell division septum at the mid-cell position, a process that is determined by three Min proteins; MinC, MinD and MinE. Regulation of cell division by Min proteins occurs via a multi-step process involving interactions between various Min proteins, as well as the membrane. In this cycle, ATP-bound MinD binds to the membrane surface where it can recruit MinC to inhibit formation of the cell division septum. MinE binding to this complex displaces MinC and stimulates ATP hydrolysis, leading to the dissociation of MinD from the membrane. These interactions give rise to a dynamic pattern of Min protein localization that appears to involve a polymeric state that is designed to create a zone that is permissive to cell division at the mid-point of the cell. The interaction between MinD and the membrane is a critical aspect of this cycle, yet the role of the lipid bilayer in MinD activation, localization and polymerization is not well understood. To probe the role of membrane charge and fluidity on MinD activation and polymerization, we developed a kinetic assay of MinE-stimulated MinD ATPase activity. We found that membrane charge is essential for MinD activation and that differences in membrane fluidity give rise to changes in its activity. Moreover, a burst phase was also observed during the first few minutes of reaction, but only on the most fluid anionic lipid tested. To help determine if the observed membrane-dependent changes in MinD activity are linked to any changes in MinD polymer structure, we have begun to develop a method to identify surface exposed regions of MinD through a combination of covalent labeling and mass spectrometry. Optimization of various steps for the assay has been done, and the assay can be applied to the future characterization of MinD polymer structure. Results from this assay, in combination with those from the kinetic measurements described here, will help to improve understanding about how membrane properties modulate MinD ATPase activity, and how this can influence the Min protein oscillation that is required to ensure normal bacterial cell division.

Submitted by Raquel Vergara Gondran (raquelvergara38@yahoo.com.br) on 2016-04-27T01:06:48Z No. of bitstreams: 1 renata aline dos santos da fonseca - atividade antioxidante de peptdeos provenientes de hidrolisado proteico de bijupir rachycentron canadum.pdf: 1691827 bytes, checksum: e9a7f8b7b6d0b7f659f997831b433f42 (MD5)<br>Approved for entry into archive by Gilmar Barros (gilmargomesdebarros@gmail.com) on 2016-05-03T18:51:23Z (GMT) No. of bitstreams: 1 renata aline dos santos da fonseca - atividade antioxidante de peptdeos provenientes de hidrolisado proteico de bijupir rachycentron canadum.pdf: 1691827 bytes, checksum: e9a7f8b7b6d0b7f659f997831b433f42 (MD5)<br>Made available in DSpace on 2016-05-03T18:51:23Z (GMT). No. of bitstreams: 1 renata aline dos santos da fonseca - atividade antioxidante de peptdeos provenientes de hidrolisado proteico de bijupir rachycentron canadum.pdf: 1691827 bytes, checksum: e9a7f8b7b6d0b7f659f997831b433f42 (MD5) Previous issue date: 2014<br>Algumas proteínas além das propriedades tecnológicas, funcionais e nutricionais apresentam atividade biológica, entre esta a antioxidante, associada a peptídeos bioativos liberados após hidrólise. Antioxidantes são utilizados em alimentos para retardar a peroxidação lipídica que gera compostos indesejáveis afetando a qualidade. Espécies reativas de oxigênio (ERO) presentes em organismos vivos podem danificar proteínas, lipídios e ácidos nucléicos, levando ao desenvolvimento de várias doenças. Uma grande fonte de proteínas são os pescados, principalmente seus rejeitos do beneficiamento industrial, assim as proteínas do bijupirá (Rachycentron canadum), pescado de grande porte e facilmente adaptável ao cultivo em aquicultura, desponta como uma alternativa na obtenção de peptídeos com atividade antioxidante. Neste contexto, o objetivo deste trabalho foi obter peptídeos provenientes de hidrolisados proteicos do músculo e do resíduo do bijupirá com diferentes enzimas comerciais, ultrafiltrar em frações peptídicas, comparar o efeito antioxidante dos hidrolisados integrais e suas frações por métodos in vitro, avaliar a toxicidade e a inibição de ERO em células in vivo e aplicar os hidrolisados e frações peptídicas em sistemas alimentícios. Foram obtidos seis hidrolisados através da hidrólise do músculo e resíduo do bijupirá com Alcalase, Flavourzyme e Protamex, em que a última apresentou maior capacidade hidrolítica alcançando grau de hidrólise de 27,94% em 760 min para o músculo e 33,14% em 580 min para o resíduo. O teor de proteínas solúveis (tirosina) variou de acordo com substrato e enzima. A atividade antioxidante foi determinada através dos métodos do sequestro do DPPH, em que o músculo hidrolisado por Protamex apresentou maior efetividade (50 mg/mL) alcançando efeito sequestrante de 81,35%, seguido pelos hidrolisados de músculo e resíduo com Flavourzyme – 60,77 e 60,25%. O resíduo hidrolisado por Protamex apresentou-se como o mais promissor para o poder redutor e todos os hidrolisados apresentaram inibição da peroxidação lipídica do ácido linoleico por seis dias semelhante ou superior aos controles comerciais. A ultrafiltração forneceu frações de peptídeos maiores e menores que 3 kDa. As frações maiores que 3 kDa apresentaram maior quantidade de grupos sulfidrila, mas não foram capazes de sequestrar o DPPH, demonstraram maior potencial no poder redutor e aumentaram a inibição da peroxidação lipídica, chegando a mais de 80% frente ao branco. As frações menores que 3 kDa apresentaram capacidades menores ou semelhantes de sequestro do DPPH, poder redutor e na inibição da peroxidação do ácido linoleico quando comparados aos hidrolisados integrais. Foram selecionados os hidrolisados com Protamex (músculo e resíduo) menores que 3 kDa para avaliar seu efeito sobre células de hepatócitos de zebrafish, demonstrando não haver citotoxicidade nas concentrações estudadas (0,1 – 100 μg/mL), contudo não foram capazes de diminuir as ERO. A incorporação dos hidrolisados e suas frações em toucinho e carne bovina moída evidenciou que a maioria dos compostos reduziu o índice de substâncias reativas ao ácido tiobarbitúrico alcançando mais de 80% frente ao branco. Portanto, através da hidrólise enzimática das proteínas do músculo e resíduo do bijupirá sob a atuação das enzimas Alcalase, Flavourzyme e Protamex foi possível obter hidrolisados e frações peptídicas antioxidantes com potencial para serem utilizados em alimentos.<br>Some proteins, in addition to their technological, functional and nutritional properties, present biological activity, being one of them, the antioxidant activity, associated to bioactive peptides released after the hydrolysis. The antioxidants are used in foods to slow the lipid peroxidation that generates undesirable compounds, affecting the quality. Reactive oxygen species (ROS) present in organisms can damage proteins, lipids and nucleic acids, leading to the development of various diseases. A great source of proteins is the fish, specially their industrial processing wastes. Therefore, the proteins from the cobia (Rachycentron canadum), a large fish that is easily adaptable to cultivation in aquaculture, emerges as an option to obtain peptides with antioxidant activity. Thus, the aim of this study was to obtain peptides from protein hydrolysates from the muscles and residues of cobia with different enzymes, ultrafiltrate in peptide fractions, compare the antioxidant effect between the whole hydrolysates and their peptides fractions using in vitro methods, measure the toxicity and the ROS inhibition in vivo cells and apply both hydrolysates and peptide fractions in food matrix. Six hydrolysates were obtained through the hydrolysis of the muscles and residues of the cobia with Alcalase, Flavourzyme and Protamex, wherein the last one presented greater hydrolytic capacity for the subtrates, reaching a degree of hydrolysis of 27.94% in 760 min for the muscle and 33.14% in 580 min for the residue. The content of soluble protein (tyrosine) varied depending on the substrate and the enzyme. The antioxidant activity was determined through the DPPH free radical scavenging assay, in which the muscle with Protamex showed itself more effective (50 mg/ml) reaching 81.35%, followed by the muscle and the residue with Flavourzyme – 60.77 and 60.25%. The residue, also hydrolyzed by Protamex, showed the best effect in the reducing power and, in the 6 days lipid peroxidation of the linoleic acid, in which all the hydrolysates presented the same or higher inhibition compared to the commercial controls. The ultrafiltration provided larger and smaller than 3 kDa fractions. The larger than 3 kDa fractions showed a higher amount of sulfhydryl groups, but were not capable to sequestrate the DPPH, also showed higher potential in the reducing power and increased the inhibition of the lipid peroxidation, achieving more than 80% comparing to the blank. The smaller than 3 kDa fractions results were similar or smaller in the DPPH sequestering, in the reducing power and in the lipid peroxidation of the linoleic acid when compared to the whole hydrolysates. Smaller than 3 kDa hydrolysates (muscle and residue) with Protamex were selected to measure its effect over zebrafish hepatocyte cells, demonstrating no cytotoxicity with the studied concentrations (0.1 - 100 μg/ml), however they were not capable to reduce the ROS. The incorporation of the hydrolysates and its fractions in bacon and ground beef showed that most of the composites reduced the tiobarbituric acid reactive substances index, achieving more than 80% in comparison to the blank. Therefore, through the enzymatic hydrolysis of proteins from muscles and residues of cobia, under the action of Alcalase, Flavourzyme and Protamex, it was possible to obtain antioxidant hydrolysates and peptide fractions with potential to be used in foods.

Les enzymes lipolytiques sont solubles en phase aqueuse mais hydrolysent des substrats insolubles. Leurs activités lipolytiques dépendent donc fortement de l’organisation des substrats lipidiques présents sous forme de structures interfaciales telles que des émulsions, des micelles, des liposomes, ou des bicouches lipidiques. Les propriétés cinétiques et la spécificité de substrat de ces enzymes résultent de l’étape initiale d’adsorption à l’interface lipide-eau et des interactions entre le substrat et le site actif. Dans le cadre de ce travail de thèse, la technique des films monomoléculaires a été utilisée pour étudier en détail les étapes séquentielles d’adsorption, de catalyse et d’inhibition de l’enzyme à l’interface lipide-eau. Dans une première partie, nous avons réalisé la caractérisation physico-chimique de la lipase gastrique de chien (DGL), avec l’étude :  de son adsorption sur un film non substrat de dilauroylphosphatidylcholine ; ‚ de l’hydrolyse interfaciale de la 1,2-dicaprine dans des films mixtes en présence d’Orlistat. Concernant l’étape de catalyse, nous avons étudié l’effet du propeptide sur la spécificité de substrat et l’activité interfaciale de la phospholipase A2 sécrétée de groupe X de souris. Enfin, dans une troisième partie, nous avons comparé les propriétés interfaciales de la lipase YLLIP2 de la levure Yarrowia lipolytica qui serait un bon candidat pour l’enzymothérapie de substitution chez les patients atteints d’insuffisance pancréatique exocrine (IPE), la lipase pancréatique humaine et la DGL. Nos résultats ont confirmé le rôle d’YLLIP2 en tant qu’excellent « substitut » non seulement de la HPL en cas d’IPE, mais aussi de la DGL<br>Lipolytic enzymes are water-soluble whereas their substrates are insoluble in water. Their lipolytic activities depend strongly on the organization of the lipid substrates present in interfacial structures such as oil-in-water emulsions, micelles, liposomes, and membrane bilayers. The kinetic properties and substrate specificity of these enzymes result from both their adsorption at the lipid-water interface, and the interactions occurring between the substrate and the active site. In this thesis work, the monomolecular film technique was used to study in details the sequential steps of adsorption, catalysis and inhibition of model enzymes at the lipid-water interface. In a first part, we performed the physico-chemical characterization of the dog gastric lipase (DGL), by studying:  its adsorption onto a dilauroylphosphatidylcholine non-substrate film; ‚ its interfacial hydrolysis of 1,2-dicaprin in mixed films with various amounts of Orlistat. Regarding the catalysis step, we studied the effect of the propeptide on the substrate specificity and interfacial activity of the murine group X secreted phospholipase A2. A model of this enzyme with its propeptide was built from the available 3D structure of the corresponding mature human enzyme. Finally, in the third part, we compared the interfacial kinetic properties of YLLIP2 lipase of the yeast Yarrowia lipolytica which has been identified as a good candidate for enzyme replacement therapy for patients with exocrine pancreatic insufficiency (EPI), human pancreatic lipase and DGL. Our results confirmed the role of YLLIP2 as an excellent "substitute" not only for HPL in case of PEI, but also for the DGL at acidic pH values

L’OBJECTIF DE CE TRAVAIL EST DE VALORISER LES CO-PRODUITS DE SARDINE (SARDINA PILCHARDUS) PAR LA MISE EN ŒUVRE DE TECHNIQUES DOUCES AVEC UN INVESTISSEMENT ET UNE DEPENSE ENERGETIQUE MODERES AFIN D’OBTENIR DES COMPOSES D’INTERET MAJEUR. LES TECHNIQUES D’HYDROLYSE ENZYMATIQUE ET DE SEPARATION MEMBRANAIRE ONT ETE CHOISIES POUR CETTE ETUDE. LES ENZYMES RETENUES POUR CETTE ETUDE ONT TOUT D’ABORD ETE ETALONNEES DE FAÇON A DETERMINER LEURS CONDITIONS OPERATOIRES OPTIMALES. LES HYDROLYSES ONT ENSUITE ETE MENEES EN DEUX ETAPES, UNE PREMIERE POUR DETERMINER L’EFFICACITE DES ENZYMES SUR LES MATRICES VISEES ET CIBLER LE DOMAINE D’ETUDE, LA SECONDE ETAPE A QUANT A ELLE PORTE SUR L’OPTIMISATION DE L’HYDROLYSE PAR PLAN D’EXPERIENCES AVEC POUR OBJECTIF D’OBTENIR LA LIBERATION DE LIPIDES LA PLUS IMPORTANTE POSSIBLE EN JOUANT SUR LES PARAMETRES INFLUENTS DE L’HYDROLYSE. UNE FOIS CES CONDITIONS DETERMINEES, LA PHASE SOLUBLE RESULTANT DE L’HYDROLYSE OPTIMISEE A ETE TRAITEE PAR ULTRAFILTRATION AFIN DE SEPARER LES LIPIDES DES PEPTIDES. LES LIPIDES DES CO-PRODUITS DE SARDINE ONT MAJORITAIREMENT ETE RECUPERES DANS LES PHASES LIQUIDES RESULTANTES DE L’HYDROLYSE. LA FRACTION AQUEUSE A DE PLUS MONTRE UNE FORTE TENEUR EN PHOSPHOLIPIDES. LES ACIDES GRAS DE LA FAMILLE DES w3 SONT REPRESENTE A HAUTEUR DE 20% DANS CES FRACTIONS. CONCERNANT LES MATRICES DIFFICILES A BROYER, L’EXTRACTION DES LIPIDES A ETE AMELIOREE EN REALISANT UNE HYDROLYSE ENZYMATIQUE PAR RAPPORT A UNE EXTRACTION CLASSIQUE. LE TRAITEMENT PAR ULTRAFILTRATION DE LA PHASE SOLUBLE A PERMIS, APRES DETERMINATION DES PARAMETRES OPERATOIRES, DE SEPARER LES FRACTIONS LIPIDIQUES ET PEPTIDIQUES EN CONCENTRANT LES PHOSPHOLIPIDES DANS LE RETENTAT<br>THE MAIN GOAL OF THIS WORK WAS TO UP-GRADE SARDINE (SARDINA PILCHARDUS) BY-PRODUCTS USING MILD PROCEDURE, ENVIRONMENTALLY SOUND, IN ORDER TO OBTAIN VALUABLE COMPOUNDS INVOLVING MODERATE INVESTMENT AND LOW ENERGY CONSUMPTION. HYDROLYSIS AND ULTRAFILTRATION TECHNIQUES HAVE BEEN INVESTIGATED. FIRSTLY, SELECTED ENZYMES HAVE BEEN CALIBRATED IN ORDER TO DETERMINE THEIR OPTIMAL CONDITIONS WITH A MODEL SUBSTRATE AND TO PERMIT THE COMPARISON BETWEEN THEM. THEN, HYDROLYSIS ON SARDINE HEAD AND VISCERA HAVE BEEN CARRIED OUT IN TWO STEPS. THE FIRST ONE HAD THE OBJECTIVE TO DETERMINE THE ENZYME EFFICIENCY AND THE STUDY AREA. THE SECOND STEP WAS TO OPTIMISE ENZYMATIC HYDROLYSIS USING EXPERIMENTAL DESIGNS. THE AIM OF THIS SECOND STEP WAS TO OBTAIN THE HIGHEST LIPID RECOVERY IN THE LIQUID FRACTIONS USING THE VARIATION OF THE INFLUENTS HYDROLYSIS PARAMETERS SUCH AS TEMPERATURE, HYDROLYSIS TIME AND ENZYME CONCENTRATION. THE SOLUBLE PHASE OBTAINED AFTER THIS OPTIMISED STEP HAVE BEEN FILTERED USING ULTRAFILTRATION TECHNIQUE IN ORDER TO SEPARATE LIPIDS FROM PEPTIDES. LIPIDS FROM SARDINE BY-PRODUCTS HAVE BEEN MAINLY RECOVERED IN THE LIQUID FRACTIONS OF THE HYDROLYSATES. MOREOVER, AQUEOUS FRACTION HAS SHOWN A HIGH CONTENT OF PHOSPHOLIPIDS. W3 FATTY ACIDS REPRESENT AROUND 20% OF THE TOTAL FATTY ACIDS INTO THOSE FRACTIONS. REGARDING VISCERA, WHICH IS A HARDLY CRUSHING MATRIX, THE LIPID EXTRACTION YIELDS HAVE BEEN IMPROVED USING ENZYMATIC TREATMENT COMPARED TO TRADITIONAL EXTRACTION. THE ULTRAFILTRATION TREATMENT OF THE SOLUBLE PHASE HAS ALLOWED, AFTER THE DETERMINATION OF OPERATING CONDITIONS, TO SEPARATE LIPIDS FROM PEPTIDES AND TO CONCENTRATE THE PHOSPHOLIPIDS IN THE RETENTATE

Les enzymes lipolytiques sont solubles en phase aqueuse mais hydrolysent des substrats insolubles. Leurs activités lipolytiques dépendent donc fortement de l’organisation des substrats lipidiques présents sous forme de structures interfaciales telles que des émulsions, des micelles, des liposomes, ou des bicouches lipidiques. Les propriétés cinétiques et la spécificité de substrat de ces enzymes résultent de l’étape initiale d’adsorption à l’interface lipide-eau et des interactions entre le substrat et le site actif. Dans le cadre de ce travail de thèse, la technique des films monomoléculaires a été utilisée pour étudier en détail les étapes séquentielles d’adsorption, de catalyse et d’inhibition de l’enzyme à l’interface lipide-eau. Dans une première partie, nous avons réalisé la caractérisation physico-chimique de la lipase gastrique de chien (DGL), avec l’étude :  de son adsorption sur un film non substrat de dilauroylphosphatidylcholine ; ‚ de l’hydrolyse interfaciale de la 1,2-dicaprine dans des films mixtes en présence d’Orlistat. Concernant l’étape de catalyse, nous avons étudié l’effet du propeptide sur la spécificité de substrat et l’activité interfaciale de la phospholipase A2 sécrétée de groupe X de souris. Enfin, dans une troisième partie, nous avons comparé les propriétés interfaciales de la lipase YLLIP2 de la levure Yarrowia lipolytica qui serait un bon candidat pour l’enzymothérapie de substitution chez les patients atteints d’insuffisance pancréatique exocrine (IPE), la lipase pancréatique humaine et la DGL. Nos résultats ont confirmé le rôle d’YLLIP2 en tant qu’excellent « substitut » non seulement de la HPL en cas d’IPE, mais aussi de la DGL<br>Lipolytic enzymes are water-soluble whereas their substrates are insoluble in water. Their lipolytic activities depend strongly on the organization of the lipid substrates present in interfacial structures such as oil-in-water emulsions, micelles, liposomes, and membrane bilayers. The kinetic properties and substrate specificity of these enzymes result from both their adsorption at the lipid-water interface, and the interactions occurring between the substrate and the active site. In this thesis work, the monomolecular film technique was used to study in details the sequential steps of adsorption, catalysis and inhibition of model enzymes at the lipid-water interface. In a first part, we performed the physico-chemical characterization of the dog gastric lipase (DGL), by studying:  its adsorption onto a dilauroylphosphatidylcholine non-substrate film; ‚ its interfacial hydrolysis of 1,2-dicaprin in mixed films with various amounts of Orlistat. Regarding the catalysis step, we studied the effect of the propeptide on the substrate specificity and interfacial activity of the murine group X secreted phospholipase A2. A model of this enzyme with its propeptide was built from the available 3D structure of the corresponding mature human enzyme. Finally, in the third part, we compared the interfacial kinetic properties of YLLIP2 lipase of the yeast Yarrowia lipolytica which has been identified as a good candidate for enzyme replacement therapy for patients with exocrine pancreatic insufficiency (EPI), human pancreatic lipase and DGL. Our results confirmed the role of YLLIP2 as an excellent "substitute" not only for HPL in case of PEI, but also for the DGL at acidic pH values

Les lipides cationiques sont parmi les agents chimiques les plus efficaces pour le transfert de gênes in vitro : ils forment avec l'ADN des particules appelées lipoplexes capables de transporter le gêne thérapeutique jusqu'au noyau des céllules. Malheureusement, ces systèmes interagissent non spécifiquement avec les milieux biologiques ce qui résulte en une faible sélectivite et une mauvaise efficacité in vivo. Cette thèse a pour objectif de concevoir des vecteurs chimiques induisant un transfert de gênes sélectif vers les tumeurs après injection intraveineuse. Les voies d'activation du polyethylene glycol (peg) mises au point au début de ce travail ont d'abord permis la synthèse de dérives lipidiques du peg. L'enrobage des lipoplexes par ces polymères modifie la bio-distribution et diminue les interactions non spécifiques de ces particules mais entraine une forte inhibition de leur pouvoir transfectant. Deux stratégies ont été développées pour augmenter sélectivement le transfert de gênes dans les tumeurs : la fixation de l'acide folique à l'extremité des lipides pegyles pour induire une reconnaissance spécifique par le recepteur au folate sur-exprime en surface des cellules cancéreuses, et la synthese de lipides pegyles comportant une fonction orthoester dégradable à faible ph pour exploiter l'acidite naturelle des tissus tumoraux. Les premiers résultats obtenus in vitro et in vivo avec ces deux strategies sont prometteurs : certains dérivés de l'acide folique sont reconnus de manière sélective et compétitive par le récepteur au folate et l'hydrolyse selective de la fonction orthoester dans les tumeurs permet d'y accroitre selectivement le transfert de gênes. La dernière partie de cette thèse est dediée à l'optimisation du trafic intracellulaire du gêne thérapeutique : l'incorporation d'une fonction orthoester dans la structure des lipides cationiques devrait favoriser la liberation de l'ADN dans la cellule par hydrolyse dans les endosomes.

Estudos recentes indicam que os peptídeos obtidos pela hidrolise enzimática da caseína podem apresentar atividades antioxidantes. Neste trabalho, previamente obteve-se os peptídeos através de hidrolise da caseína utilizando as enzimas Alcalase e Flavourzyme (4h, a 50ºC e pH 8), selecionando os que apresentaram as melhores características, in vitro, relativas à atividade antioxidante. A hidrolise enzimática utilizando a enzima Flavourzyme mostrou melhores resultados, com alto valor de proteína solúvel e conteúdo de aminoácidos livres, além de peptídeos de menor peso molecular do que com a Alcalase, como observado nas análises de cromatografia de permeação em gel e eletroforese em gel de poliacrilamida. Os peptídeos de caseína obtidos com a Flavourzyme também apresentaram melhores resultados utilizando o método ABTS na determinação da capacidade antioxidante. O hidrolisado obtido a partir da enzima Flavourzyme foi aplicado em produtos cárneos e em chocolate branco. Em produtos cárneos, os peptídeos de caseína (2.0%) inibiram, efetivamente, a peroxidação lipídica em carne moída (100%) e em carne mecanicamente separada de ave (CMS) (cerca de 20%) indicando que estes peptídeos podem ser utilizados nestes produtos, auxiliando na prevenção da formação de flavor desagradável e aumentando sua vida útil. Relativamente a sua aplicação em chocolate branco, esta adição teve o intuito de inibir escurecimento deste produto, fator considerado como limitante na sua vida-útil sendo conseqüência tanto de reações de escurecimento não enzimático quanto da oxidação de lipídeos. Os parâmetros que indicaram alteração lipidica e reações não enzimáticas foram mensurados em três diferentes amostras de chocolate branco: uma amostra com 0,2%, de manteiga de cacau, de antioxidante sintético Grindox 562, outra com 0,2%, de manteiga de cacau, dos peptídeos de caseína e a terceira amostra sem qualquer tipo de antioxidante. As amostras foram expostas a duas temperaturas diferentes: 20 ± 2 e 28 ± 2ºC. Os resultados das análises realizadas indicaram que as amostras armazenadas à temperatura de 20ºC apresentaram resultados significativamente melhores àqueles das amostras armazenadas à temperatura de 28ºC, relativos ao índice de acidez, à atividade de água, ao índice de peróxido, à cor e às substâncias reativas ao ácido tiobarbitúrico (TBARS), indicando melhor conservação deste produto. Também foi observado que a adição de quaisquer dos antioxidantes empregados não influenciou de forma significativa os resultados obtidos, evidenciandose assim, que o principal parâmetro responsável pelas alterações do chocolate branco em sua vida útil refere-se à temperatura de armazenamento a qual as amostras foram submetidas.<br>Recent studies indicate that peptides obtained by casein hydrolysis may have antioxidant activity. In this work, previous casein peptides were obtained by enzymatic hydrolysis using Alcalase and Flavourzyme (4h, at 50ºC and pH 8), selecting the ones that showed the best characteristics in vitro, related to the antioxidant activity. The enzymatic hydrolysis using Flavourzyme showed the best results, with higher soluble protein and free amino acid content and producing lower molecular weight peptides than Alcalase, as observed by gel permeation chromatography and polyacrylamide gel electrophoresis. Related to its application in meat products, casein peptides obtained with Flavourzyme also exhibited greater antioxidant capacity using the ABTS method. The casein hydrolyzed from Flavourzyme enzyme was applicated in ground beef homogenates, mechanically deboned meat (MDM) of poultry and white chocolate. In meat products, casein peptides (2.0%) effectively inhibited lipid peroxidation in ground beef homogenates (100%) and mechanically deboned meat (about 20%) of poultry. Casein peptides may be useful in meat processing as another naturally occurring antioxidant, helping to prevent off-flavor formation in meat products and increasing shelf life. In the use for white chocolate, the goal was to inhibit its browning, the main problems that limit the white chocolate’s shelf-life. Non-enzymatic browning reaction and lipid oxidation were involved directly in the browning of white chocolate. Thus, parameters which indicated fat alteration and non-enzymatic reactions were measured in three different samples of white chocolate. One sample with 0,2% of cocoa butter, with the synthetic antioxidant Grindox 562, other with 0,2% of cocoa butter, with the natural antioxidant and the third sample without any kind of antioxidant. The samples were exposed to two different temperatures: 20 ± 2 and 28 ± 2ºC. The results of the analysis made indicated that the samples stored at the temperature of 20ºC showed results significantly better to those samples stored at the temperature of 28ºC, related to the conservation of the white chocolate. Besides, the results indicated that the addition of any antioxidants employees has not influenced in a significant way the results obtained. Thus, it was evidenced that the main responsible parameter for the alterations of the white chocolate’s shelf-life is related to the storage temperature to which the samples were submitted.

Huit lipases industrielles, d'origine bactérienne, fongique et de levure, sont étudiées en vue de leur immobilisation sur support et leur application à l'hydrolyse des matières grasses végétales et animales. Deux d'entre elles présentent un rapport coût/efficacité attrayant. Il s'agit de la lipase SP 398 de Humicola lanuginosa et la lipase OF de Candida rugosa. Leur poids moléculaire est de l'ordre de 50 kDa et leur activité lipasique de 100 000 UL/g de produit brut et 360 000 UL/g de produit brut, respectivement (d'après les données des fournisseurs). La lipase SP 398 de Humicola lanuginosa présente une activité stable sur un domaine de pH allant de 4 à 11 et une résistance à l'inactivation thermique qui la classe dans la catégorie des enzymes thermostables. L'activité de la lipase OF de Candida rugosa est stable sur un domaine de pH plus restreint et elle est davantage sensible à la température. Toutefois, son importante activité catalytique permet d'étendre son domaine de tolérance (pH et température) jusqu'à une activité équivalente à celle de la lipase SP 398 de Humicola lanuginosa. Une nouvelle technique d'analyse, rapide et non destructive, est employée pour mettre en évidence les modifications structurales responsables du processus d'inactivation des lipases lors de la montée de température : la spectroscopie micro-ondes. En effet, les données thermodynamiques de l'inactivation thermique des lipases ont pu être corrélées aux variations structurales observées par la spectroscopie micro-ondes pour expliquer la thermostabilité de la lipase SP 398 de Humicola lanuginosa et la sensiblité de la lipase OF de Candida rugosa. La lipase SP 398 de Humicola lanuginosa est choisie pour l'immobilisation de lipases sur différents supports en raison de son coût modique qui permet de palier le prix onéreux des supports synthétiques. Son adsorption sur une matrice hydrophobe donne des résultats bien plus performants que son immobilisation par liaisons covalentes sur une matrice activée par le glutaraldéhyde, ou son adsorption sur une silice hydrophile. L'hydrolyse de l'huile d'olive en réacteur à lit fluidisé, par le système de lipases immobilisées, est prometteur et mérite une optimisation sur la stabilité du système et son utilisation en continu. Sous sa forme libre, la lipase OF de Candida rugosa, se révèle extrêmement efficace dans l'hydrolyse des graisses animales. Elle permet l'hydrolyse complète de la graisse brute présente à plus de 50% dans l'effluent en moins de 4 heures. Le traitement enzymatique mis en place respecte les exigences, économiques et pratiques, des industriels et permet, ainsi, d'envisager l'application du procédé à l'échelle pilote avec sérénité.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior<br>The objective of this study was to develop a liquid protein hydrolysate from Mechanically Deboned Chicken Meat (MDCM) by enzymatic hydrolysis and to evaluate the addition of different concentrations (10, 20 and 30%) in a reduced-fat mortadella-type sausage. First, it was evaluated the addition of BHT antioxidant (0.01%) and curing salts (0.25%) in the production of protein hydrolysate at different hydrolysis times (5, 15, 30 and 60 minutes) in order to reduce lipid oxidation and brownish color formation. There were evaluated the degree of hydrolysis, pH, proximate composition, lipid oxidation and instrumental color. It was observed that the addition of BHT antioxidant and curing salts did not affect the degree of hydrolysis at 60 minutes. The pH was similar to the MDCM and the proximate composition was not affected by the hydrolysis process or by the addition of BHT antioxidant and curing salts. However, there was a synergistic action between the BHT antioxidant and the curing salts in lipid oxidation prevention of the hydrolysates during the whole period of hydrolysis. Also, there was observed a reduction in the formation of a brownish color mainly by the use of curing salts, evidenced by high levels of values of a* (redness). Subsequently, there was evaluated the addition of different concentrations protein hydrolysate containing BHT antioxidant and curing salts in a reduced-fat mortadella-type sausage. Thus, four treatments were made containing 0, 10, 20 and 30% of MDCM protein hydrolysate. There were evaluated the proximate composition, pH, lipid oxidation, colorimetric parameters, texture profile and microbiological and sensory characteristics during 60 days of storage at 4 ºC. The proximate composition, pH and microbiological characteristics were considered normal for this kind of product. The values of lipid oxidation of products containing MDCM protein hydrolysate increased up to 30th day of storage with subsequent decrease until the end of the storage period (60 days). The products containing MDCM protein hydrolysate had low values of L* (lightness) and a* (redness), higher lipid oxidation and soft textured, evidenced both by the panelists and by instrumental texture profile. The addition of up to 10% protein hydrolysate proved to be viable in the production of reduced-fat mortadella-type sausage, showing quality characteristics closer to the control treatment.<br>O objetivo deste trabalho foi desenvolver um hidrolisado proteico líquido de carne mecanicamente separada (CMS) de frango, através de hidrólise enzimática e avaliar a adição de diferentes concentrações (10, 20 e 30%) em mortadela com teor de gordura reduzido. Primeiramente, foi avaliada a adição de antioxidante BHT (0,01%) e sal de cura (0,25%) na produção do hidrolisado proteico em diferentes tempos de hidrólise (5, 15, 30 e 60 minutos) com o objetivo de reduzir a oxidação lipídica e a formação da coloração amarronzada. Avaliou-se o grau de hidrólise, pH, composição centesimal, oxidação lipídica e cor instrumental. Foi possível observar que a adição de antioxidante BHT e sal de cura não afetou o grau de hidrólise ao final de 60 minutos. O pH foi similar ao encontrado na CMS original e a composição centesimal não foi afetada pelo processo de hidrólise nem pela adição de antioxidante BHT e sal de cura. Entretanto, houve uma ação sinergística entre o antioxidante BHT e o sal de cura na prevenção da oxidação lipídica dos hidrolisados durante todo o período de hidrólise. Também, foi observada uma redução na formação da coloração amarronzada pelo uso principalmente do sal de cura, evidenciada pelos maiores teores de vermelho (a*). Posteriormente, foi avaliada a adição do hidrolisado proteico com antioxidante BHT e sais de cura, em mortadela com teor de gordura reduzido, contendo 0, 10, 20 e 30% de hidrolisado proteico de CMS. Foram avaliadas a composição centesimal, pH, oxidação lipídica, parâmetros de cor e as características microbiológicas, sensoriais e perfil de textura durante 60 dias em armazenamento a 4 ºC. A composição centesimal, pH e as características microbiológicas foram consideradas normais para o produto em questão. Os valores de oxidação lipídica dos produtos contendo hidrolisado proteico de CMS aumentaram até o 30º dia de armazenamento com posterior decréscimo até o final do período de armazenamento (60 dias). Os produtos contendo hidrolisado proteico de CMS apresentaram valores de luminosidade (L*) e valores de a* menores e textura mais amolecida, evidenciada tanto pelos provadores quanto pelo perfil de textura instrumental. A adição de até 10% do hidrolisado proteico se mostrou viável na fabricação de mortadela com teor de gordura reduzido, pois apresentou características de qualidade mais próximas ao tratamento controle.

Les peptides chélateurs de métaux (PCMs), issus d'hydrolysats de protéines, présentent diverses applications dans les domaines de la nutrition, de la pharmacie, des cosmétiques, etc. Cependant, l'approche empirique généralement utilisée pour découvrir des peptides bioactifs à partir d'hydrolysats est longue et coûteuse en raison de nombreuses étapes de fractionnement, de séparation et d'évaluation des activités biologiques. Cette thèse a donc pour but de développer une nouvelle approche pour la prédiction de la séparation des PCMs en utilisant la modélisation et la simulation chromatographiques basées sur l'analogie entre la chromatographie d'affinité sur ions métalliques immobilisés (IMAC) et la résonance plasmonique de surface (SPR). Pour la première fois, l'analogie SPR-IMAC a été étudiée expérimentalement sur 22 peptides et 70% d'entre eux ont validé cette analogie, puisque les peptides bien retenus en IMAC étaient également dotés d'une bonne affinité pour Ni2+ en SPR. Dans un deuxième temps, des peptides ayant une forte affinité pour Ni2+ (c'est-à-dire une faible constante de dissociation KD en SPR et un temps de rétention élevé en IMAC) ont été utilisés pour étudier la simulation des profils de concentration de peptides à la sortie de la colonne en IMAC. La connaissance des isothermes d'adsorption étant nécessaire pour effectuer la simulation, il a fallu développer une méthodologie pour prédire les paramètres de l'isotherme de Langmuir en IMAC à partir des données SPR. La simulation a été évaluée en comparant les temps de rétention expérimentaux et simulés qui devraient être proches pour une prédiction fiable. Par conséquent, plusieurs approches ont été étudiées pour déterminer les paramètres de sorption de Langmuir. L‘approche la plus intéressante a introduit un facteur correctif sur la capacité d'adsorption maximale qmax seulement. En effet, l'affinité des peptides pour le Ni2+ immobilisé étant supposée constante quelle que soit technologie utilisée (SPR vs. IMAC), la constante d'affinité KA n'a pas été modifiée. Parallèlement, les applications industrielles des PCMs et des hydrolysats peptidiques ont été étudiées. Tout d'abord, des hydrolysats de protéines de pois ont été produits par protéolyse enzymatique soit avec l'Alcalase® suivi de la Flavourzyme® (Alc+Flav≤1kDa), soit par la Protamex® suivi de la Flavourzyme® (Prot+Flav≤1kDa). La technologie SwitchSENSE® a mis en évidence la présence de peptides chélateurs de Ni2+ et les tests antioxydants ont montré que l'hydrolysat Prot+Flav≤1kDa avait une activité antiradicalaire et un pouvoir réducteur plus élevés, liés à son degré d'hydrolyse plus élevé et à la quantité de peptides de petite taille. Les hydrolysats de protéines de pois et les PCMs ont ensuite été étudiés pour leur capacité à inhiber l'oxydation des lipides dans les émulsions. Ils ont ralenti l'oxydation des lipides par chélation des métaux pro-oxydants (tels que Fe2+) en réduisant les produits d'oxydation primaires et secondaires, responsables de la détérioration des produits contenant des lipides. Ainsi, les hydrolysats de pois et les PCMs pourraient être utilisés comme antioxydants dans les produits alimentaires et cosmétiques, comme alternatives aux produits chimiques tels que l'EDTA, le BHT et le TBHQ<br>Metal-Chelating Peptides (MCPs), from protein hydrolysates, present various applications in nutrition, pharmacy, cosmetic etc. Yet, the empirical approach generally used to discover bioactive peptides from hydrolysates is time consuming and expensive due to many steps of fractionation, separation and biological activities evaluation. Thus, this PhD aimed to develop a novel approach for MCPs separation prediction using chromatography modelling and simulation based on the analogy between Immobilized Metal ion Affinity Chromatography (IMAC) and Surface Plasmon Resonance (SPR). For the first time, the SPR-IMAC analogy was experimentally investigated on 22 peptides and 70% of them validated this analogy, since peptides well retained in IMAC were also endowed with a good affinity for Ni2+ in SPR. In the second time, peptides with high affinity for Ni2+ (i.e low dissociation constant KD in SPR and a high retention time in IMAC) were used to study the modelling and simulation of peptide concentration profiles at the column outlet in IMAC. Since knowledge of adsorption isotherms was required to perform simulation, it was necessary to develop a methodology for predicting Langmuir isotherm parameters in IMAC from SPR data. The validity of simulation was evaluated by comparing experimental and simulated retention times that should be close for reliable prediction. Therefore, several approaches were evaluated to determine Langmuir sorption parameters, the most interesting one introduces a correction factor on the maximum adsorption capacity qmax alone, assuming that the affinity of peptides for immobilized Ni2+ did not change depending on the technology used (SPR vs. IMAC), thus affinity constant KA was not modified. Meanwhile, industrial application of MCPs and hydrolysates were studied. First, pea protein hydrolysates were produced by either Alcalase® followed by Flavourzyme® (Alc+Flav≤1kDa) or Protamex® followed by Flavourzyme® (Prot+Flav≤1kDa). SwitchSENSE® technology evidences the presence of Ni2+ chelating peptides and antioxidants tests showed that Prot+Flav≤1kDa has higher radical scavenging and reducing power, related to its higher degree of hydrolysis and small-size peptides quantity. Secondly, pea hydrolysates and MCPs were investigated for their ability to inhibit the lipid oxidation in emulsions. They slowed down lipid oxidation through chelation of prooxidant (metals such as Fe2+) reducing primary and secondary oxidation products responsible of deterioration of lipid containing products. Thus, pea hydrolysates and MCPs could be used as antioxidants in food and cosmetic products, as alternative to chemicals such as EDTA, BHT and TBHQ

Thesis (M.S.)--University of Wisconsin--Madison, 1989.<br>Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 104-110).

Metal ions and complexes utilized as cleavage agents have influenced many synthetic approaches of scientists to assist in the cleavage and transformation of biomolecules. These metal-based synthetic cleavage agents have potential applications in biotechnology or as molecular therapeutic agents. Herein, we have examined Ce(IV) metal ion and complexes as acidic hydrolytic agents in lipid hydrolysis reactions (Chapter 2 and 3), and a copper(II) complex that photo-oxidizes DNA upon exposure to ultraviolet light (Chapter 4). In Chapter 2 we examined the hydrolysis of sphingomyelin vesicles by Ce(NH4)2(NO3)6 (Ce(IV)) and compared the results to twelve d- and f-block metal salts, hydrolysis of mixed lipid vesicles and mixed micelles of sphingomyelin by Ce(IV), and hydrolysis of phosphatidylcholine vesicles by Ce(IV), using either MALDI-TOF mass spectrometry or colorimetric assays. In Chapter 3, we described the study of a Ce(IV) complex based on 1,3-bis[tris(hydroxymethyl)methylamino]propane as a potential acidic hydrolytic agent of phospholipids using colorimetric assays and NMR spectroscopy. The hydrolytic agent provided markedly enhance hydrolysis at lysosomal pH (~ 4.8), but suppress hydrolysis when pH was raised to near-neutral pH (~ 7.2). This was due to the pKa values of the donor atoms of the ligand, in which the metal’s electrophilicity was reduced to a greater extent at ~ pH 7.2 compared to ~ pH 4.8. Chapter 4 describes the synthesis and study of a Cu(II) complex based on a hexaazatriphenylene derivative for photo-assisted cleavage of double-helical DNA. Scavenger and chemical assays suggested the formation of DNA damaging reactive oxygen species, hydroxyl and superoxide radicals, and hydrogen peroxide, in the photocleavage reactions. Thermal denaturation and UV-vis absorption studies suggested that the Cu(II) complex binds in a non-intercalative fashion to duplex DNA.

The main objective of this thesis was to use grape pomace (GP) as a source of carbohydrates for yeast growth and subsequent production of lipids, or carotenoids, thereby achieving the valorization of a very important agro-industrial residue. To that effect, subcritical water (SBW) technology was used as a treatment to overcome the resilience of the GP lignocellulosic matrix, and depolymerize it into more elementary structures. Experiments were conducted on the growth of yeast strains on the GP extract resulting from the carbohydrate-rich liquor generated during SBW treatment, under conditions conducive to lipid accumulation and carotenoid production. SBW extraction / hydrolysis experiments were carried out at a constant pressure of 70 bar, a water flow rate of 10 mL/min, and maximum temperatures between 190°C and 240 °C. Two temperature programs were used, namely one in which temperature was increased continuously to the target temperature, and another consisting of a series of steps, each step consisting of a ramp followed by a plateau, aiming at the fractionation of GP into extractives (plateau at 130 ºC), hemicellulose constituents (plateau at 190 ºC), and glucose or gluco-oligosaccharides from cellulose (plateau at 240 ºC). The best results were obtained in a plateau-type assay reaching 240 ºC. At these conditions, the extraction / hydrolysis efficiency of the SBW treatment of GP was 71 wt.%, leading to a yield of GP extract of 31 wt.%, and a yield of carbohydrates of 27 wt.%. The latter value corresponds to a recovery of approximately 84% of the total amount of carbohydrates (both soluble and structural) of GP, which indicates that the hydrolysis of cellulose occurred to some extent. Of the total amount of carbohydrates measured in the GP extract, approximately 80% were in the form of oligosaccharides. The most abundant monosaccharides in GP extract were glucose and fructose, which exist in GP as soluble carbohydrates, followed by arabinose, xylose and galactose, from hemicellulose. With a view to breaking down the oligosaccharides in the liquors produced in the SBW treatment, Viscozyme, an enzyme complex exhibiting cellulase and hemicellulase activity, was immobilized on glutaraldehyde-activated chitosan microparticles. The yield of enzyme immobilization was approximately 79%, and the enzyme kept about 75% of its specific activity, as determined in the hydrolysis of a model substrate (arabinogalactan). The enzymatic hydrolysis of GP and GP residue left in the reactor after SBW treatment was found to be very slow. That of GP extract was carried out but HPLC analysis was inconclusive. GP extract was used as carbon source for the growth of two oleaginous yeasts: Rhodotorula babjevae, and Lipomyces starkeyi. A comparative analysis of biomass and lipid production was made, in media with different pH and composition. Similar results were found with media at pH 4 and pH 6. Lipomyces starkeyi exhibited both highest cell dry weight and lipids dry weight in both the control medium (9.6 g/L cells, of which 5.3 g/L were lipids) containing glucose as carbon source, and medium with GP extract (0.9 g/L cells, of which 0.2 g/L are lipids) containing carbohydrates in an amount identical to that of glucose in the control medium. R. babjevae produced the highest carotenoid content, reaching 6.3 mg carotenoids / g cell dry weight in control assays with glucose.

碩士<br>國立清華大學<br>通訊工程研究所<br>94<br>The phospholipase A2 (PLA2) catalyze specially the hydrolysis of the center(sn-2) ester bond of substrate phospholipids. The hydrolysis products of the PLA2 reaction are free fatty acid and lysophospholipid. The fatty acids released by PLA2, such as arachidonic acid (AA) and oleic acid (OA), can be important as stores of energy, but more importantly AA can also function as a second messenger and as the precursor of eicosanoids, which are potent mediators of inflammation and signal transduction. In this thesis, Fourier transform infrared and Langmuir monolayer are utilized to explore the processes of surface pressure and area decrease of lipid monolayer during PLA2 hydrolysis. Our study showed that PLA2 not only hydrolyzed phospholipid but also carried transported free fatty acid away from membrane interface. The free fatty acid transport phenomenum require calcium ion but is fatty acid chain length independent. Naja nigricolius PLA2 decreased surface area of DMPG monolayer but it cannot transport free fatty acid to the subphase. It implied that Naja nigricolius PLA2 transported Lyso PG instead of free fatty acid chain in negative charge lipid membrane. The aforementioned difference renders new potential research of PLA2 in toxic, physical and pharmacology.

碩士<br>元培科學技術學院<br>生物技術研究所<br>96<br>Phospholipase A2 (PLA2、E.C. 3.1.1.4) converts phospholipids into lysophospholipids and free fatty acids by hydrolyzing the ester bond of phospholipids at the sn-2 position. Some released fatty acids are precursors of eicosanoids or work as secondary messengers、which might be potentially involved in inflammation、immune response and signal transduction. In this thesis、we would like to determine whether enzyme activities of PLA2 from different species could be influenced as they reacted with different molecular species of phospholipids. By making use of techniques of genetic engineering and site-directed mutagenesis、we constructed Taiwanese cobra (Naja atra) PLA2 (aPLA2) gene and four mutated genes with a point mutation at D23A、D23F、D23K and D23N positions、respectively. Then、these recombinant aPLA2 proteins in inclusion bodies with the correct structure were over-expressed、collected and refolded by using E. coli expression system and protein engineering techniques. To purify aPLA2 and identify its secondary structures、we utilized combined techniques of reverse phase-high performance liquid chromatography (RP-HPLC) and Circular Dichroism spectra (CD spectra). Enzyme activities of these proteins were detected by colorimetric assay. Results in this study agreed with previous reports that PLA2 from cobra venom exerted the most powerful activity to hydrolyze phosphocholine (PC)、and also indicated that recombinant aPLA2 preferably hydrolyzed phospholipids with three methyl groups on the polar head、as compared to those containing no、one or two methyl groups. When enzyme activities of mutated aPLA2 were examined、hydrolytic activity on phosphotidylcholine (PC) was significantly reduced as phospholipids appeared、and the degree of hydrolysis was as same as that of phosphatidylethanolamine (PE) and phosphatidylglycerol (PG). These findings suggested that the position D23 of aPLA2 DNA sequence might be responsible for enzyme substrate specificity. Beside、we determined the other type of phospholipase A2、human Group IIa PLA2、and found the enzyme hydrolyzed 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (POPG) powerfully、however、no such effect was observed when worked with 1,2-Dimyristoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (DMPG). In conclusion、results from this thesis help us have a better understanding of the substrate specificity of phospholipase A2、and provide an approach to explore the possible mechanism of phospholipase-mediated hydrolysis occurs in the cell membrane.

<p>The outer-leaflet of the outer membrane of Gram-negative bacteria is composed of lipopolysaccharide (LPS), which is attached to the membrane via a hexa-acylated saccharolipid called lipid A. The fourth step of lipid A biosynthesis involves the cleavage of the pyrophosphate group of UDP-2,3-diacyl-GlcN to form lipid X; this step is carried out by LpxH in E. coli and the majority of Gamma- and Beta-Proteobacteria. LpxH has been previously characterized, however sample impurity and non-optimized assay conditions hindered meaningful conclusions. The enzyme was suggested to contain signature motifs found in the calcineurin-like phosphoesterase (CLP) family of metalloenzymes, however the extent of biochemical data fails to demonstrate a significant level of metal activation in LpxH assays. We report cloning, purification, and detailed enzymatic characterization with a highly purified sample of H. influenzae LpxH (HiLpxH). HiLpxH shows over 600-fold stimulation of activity in the presence of Mn2+. Furthermore, EPR studies reveal the presence of a Mn2+ cluster in LpxH. Finally, point mutants of residues in the conserved metal-binding motifs of the CLP family greatly inhibit HiLpxH activity, highlighting their importance in enzyme function. Overall, through optimized purification and assay methods, our work unambiguously establishes LpxH as a membrane-associating CLP containing a Mn2+ cluster coordinated by conserved residues. These results set the scene for further structural investigation of the enzyme and for design of novel antibiotics targeting lipid A biosynthesis.</p><p>Several species of Gram-negative bacteria lack LpxH orthologs, yet retain other lipid A biosynthetic enzymes and still produce lipid A. An unrelated protein, LpxI, is responsible for UDP-DAGn hydrolysis is several such organisms. Interestingly, some bacteria, such as the human pathogen Chlamydia trachomatis, have neither LpxH nor LpxI orthologs, suggesting the presence of a third UDP-DAGn hydrolase. Through implantation of a novel complementation screen that used a C. trachmatis genomic library and a conditional-lethal lpxH mutant E. coli strain, we were able to identify an open reading frame encoding an new enzyme capable of lipid X production. Due to its ability to complement UDP-DAGn hydrolase function in vivo and catalyze the formation of lipid X in vitro, we have designated the enzyme LpxG. Further biochemical analysis with purified LpxG revealed it facilitates hydrolysis through attack on the alpha phosphate of its substrate and is activated by Mn2+ in vitro. LpxG is in the same CLP superfamily as LpxH, however it shows very little homology to LpxH or LpxI. Identification of LpxG improves our understanding of the lipid A biosynthetic pathway in C. trachomatis. More broadly, as limited genetic tools are available for the study of the prevalent pathogen, it provides an advantageous method for the functional screening of other C. trachomatis genes.</p><br>Dissertation

碩士<br>國立成功大學<br>化學工程學系碩博士班<br>100<br>In recent years, microalgae were considered as potential alternative source for biofuel production. Due to the high cost of cultivation and harvesting, the microalgae biofuel became uncompetitive with fossil fuel. Thus enhancing the interest in offsetting the cost of lipids by co-production of other valuable products (such as: chlorophyll) from microalgae may make microalgae biomass economically competitive for biofuels production. In this study, lipid and chlorophyll were extracted simultaneously from microalgae by solvent extraction. Enzymatic hydrolysis was applied as a pretreatment to break the cell walls of microalgae in order to facilitate the penetration of the solvent through the microalgae cell wall. The cellulase was immobilized onto PAN coated magnetic nanoparticles by activating the nitrile group in the PAN layer with the amidination reaction. The immobilized cellulase could be removed easily from the reaction system due to the induced moderate magnetic field of magnetic nanoparticles. Under the optimal immobilization conditions, the immobilization yielded a protein loading of 88.6 mg/g particle. The optimal conditions for hydrolysis were also determined by measuring the reducing sugar released at temperature of 50 C and pH 7. The immobilized cellulase retained 70.2% residual activity after ten hydrolysis cycles and 58.2% residual activity after 7 days storage. The extraction of lipid and chlorophyll by using a solvent mixture of ethanol and hexane was examined in this study. The influences of the operating parameters including the hydrolysis time, the ratio between ethanol to hexane, the ratio of mixed solvents to algal biomass (dry weight), and the extraction time were investigated. After hydrolysis, the recovery of lipid and chlorophyll yields of 33.93% (30.2 mg of lipid) and 30% (10.05 mg of chlorophyll) could be extracted from one gram of microalgae. These results represent the feasibility of the proposed applications of immobilized cellulase for extraction of lipid and chlorophyll from microalgae.

Nowadays, nearly 80% of world energy demand is satisfied by the use of fossil fuels, which provoke the increasing accumulation of CO2 in atmosphere. Thus, the capture of this gas and the prevention of its release will play a key role to prevent climate changes, which are causing adverse effects. Great efforts have been focused on the production of biodiesel, bioethanol, or biogas. The use of biofuels does not contribute to an increase in the atmospheric CO2 concentration, since the carbon released has previously been taken from atmosphere by photosynthesis. In this scenario, the utilization of microalgae cultures can contribute to CO2 capture and storage, converting it into a biomass rich in valuable products, such as carotenoids, aminoacids and lipids for biodiesel production. Another advantage is that the conversion efficiency of solar energy into biomass for algal cultures and the productivity per hectare are much greater than those obtainable with traditional crops. Moreover, algal cultures do not compete for fertile land, do not require pesticides and can be grown in seawaters or wastewater from where microalgae take nutrients that they transform in biomass. One of the factors hindering the large-scale use of microalgae as a lipid source is the high energy consumption associated with the recovery of lipids. In fact, in most microalgal species, lipids are located inside the cell, which must be disrupted to allow their extraction. The hardness of algal walls and their organization into a complex multi-layered structure make disruption an energy-intensive process. Common methods of cell disruption involve the use of mechanical (e.g., ultrasonication, high-pressure homogenization, bead beating) or chemical (e.g., alkali, acid, detergent) means. In addition to their request for a large consumption of energy or chemicals, these treatments may cause damage to the most easily degradable algal components, such as proteins and carotenoids, which could be coextracted with lipids in a biorefinery perspective. In this context, the primary objective of this doctoral thesis was the development of an enzyme-assisted lipid extraction method using unpurified low cost industrial enzyme preparations. Enzymatic treatment of microalgae is an attractive, but still little explored method of cell wall disruption. It is based on the selective degradation of cell wall components by specific enzymes. This method has the potential to facilitate the recovery of lipids or the post-extraction use of the algal biomass and to preserve the most labile compounds. However, the choice of suitable enzymes is strictly related to the characteristics of the algal wall which, in turn, depend on the microalgae species, the growth conditions and the harvesting and dewatering steps. Unfortunately, microalgal cell walls are still poorly characterized. The first part of the work focused on the selection and the screening of enzyme preparations on the basis of the characteristics of the cell wall of Nannochloropsis sp., the microalga that was used as model organism. This microalga is of great industrial interest because of its ability to accumulate large amounts of lipids and other valuable components, such as the carotenoids astaxanthin and zeaxanthin and the omega-3 polyunsaturated fatty acid EPA. However, it shows unusual resistance towards mechanical and chemical treatments, which seems to be at least partly due to the presence in the outer cell layer of the aliphatic biopolymer algaenan. This makes the extraction of intracellular algal components a challenging and energy-consuming process. Subsequently, an enzyme-assisted extraction processes based on the development of optimized cell wall-degrading enzyme cocktail was developed by the use of the statistical methodology of mixture design. The influence of the most important process parameters (pretreatment time, enzymes to biomass ratio, pH and temperature of pre-treatment) on the yield of lipid extraction was then analyzed using the pretreatment mixture of optimized composition. A factorial design has been identified with the purpose to analyze their effect on the yield of lipid extraction. Numerical optimization of the obtained model allowed identifying the optimal pretreatment conditions, in terms of extraction yield and cost of the pretreatment. The recovery of the enzyme pre-treatment solution for the re-use in subsequent cycles was also evaluated to lower the operating cost. Moreover, a characterization of the untreated and enzymatically treated biomass was performed through the use of instrumental methods, such as Fourier transform infrared spectroscopy (FTIR), X-ray diffraction spectrometry (XRD), thermogravimetry (TGA/DTG) and scanning and transmission electron microscopy (SEM and TEM) in order to understand the action of each enzyme and, consequently, to properly design the process. Finally, the exhausted algal biomass after lipid extraction process has been studied to assess its implementation as a low cost absorbent material for textile azo-dyes, in order to find new application that could allow a further decrease of the cost of the entire biodiesel production chain from microalgae.

碩士<br>國立臺灣大學<br>生化科技學系<br>105<br>Obesity and its related chronic diseases, such as type 2 diabetes, hypertension, and non-alcoholic fatty liver disease, become a key issue in public health. Previous studies indicated that bitter gourd can ameliorate hyperglycemia and dyslipidemia. The bioactive compound in bitter gourd have been identified as cucurbitane-type triterpenoids, 9c, 11t, 13t-conjugated linolenic acid (CLN) and peptide, etc. Particularly, triterpenoids can improve hyperglycemia via AMPK to enhance glucose uptake, and CLN can regulate lipid metabolism by PPARα activation. Multiple cultivars of wild bitter gourd (WBG) have been crossbred by Hualien District Agricultural Research and Extension Station. The first part of this study aims to examine triterpenoids and CLN content in various cultivars of bitter gourd, as well as PPARα and LXRα transactivation activities of bitter gourd ethyl acetate (EA) extract. The results showed that cultivars 55M, H4 and N81 have the highest triterpenoids content. 55M and N81 have the highest CLN content. In the cell-based transactivation assay, EA extracts of CKP55, 1758 and V81 showed the highest transactivation activities of PPARα. Interestingly, WBG EA extracts dose-dependently inhibited the transactivation of T0901317, a synthetic LXRα ligand, and 55M showed the highest inhibition. Based on results from CLN content and LXRα transactivation assay, we speculate that CLN in WBG might modulate transcriptional activities of LXRα. The first part of this study also examined effects of various cultivars of WBG in an animal study. WBG was supplemented to a high-fat diet and fed C57BL/6J mice for 4 weeks. Among the cultivars, H4 performs outstandingly in ameliorating hyperglycemia and dyslipidemia, as well as fat accumulation in WAT. 55M, 1758 and P81 display advantages in reducing cholesterol accumulation in mice liver. Intriguingly, cultivar 55M up-regulated LXRα-related genes in the liver, including Srebf1, Fasn, Abcg1 and Cyp7a1. Previous studies reported that triterpenoid aglycones had higher activities than glycoside froms in enhancing glucose uptake of adipocytes. Previous studies in our lab have developed a hydrolysis procedure using the endogenous β-glucosidase of WBG. Previous animal studies in our lab also showed that 37℃ hydrolyzed WBG powder ameliorates the obesity-induced fatty liver more effectively than the non-hydrolyzed WBG. The second part of this study further examined effects of hydrolysis on composition and biological activities in various cultivars of WBG. Among various cultivars tested, H4 has the highest β-glucosidase activity. Triterpenoids content in EA extract of H4 WBG increases sharply under 37℃ and 60℃ hydrolysis as expected. In contrast, CLN content and PPARα transactivation activities of EA extracts decrease after hydrolysis. Surprisingly, antagonistic effect on LXRα significantly elevated after hydrolysis of WBG. The antagonistic effect is further examined in HepG2 cells. The T0901317-induced triglyceride accumulation in these cells was suppressed by lower dose of H4 EA extract. In addition, the mRNA expression levels of genes related to fatty acid synthesis were also down-regulated. In conclusion, WBG and hydrolyzed WBG both can improve hepatic lipid metabolism. Cultivar selected by the cell-based LXRα transactivation assay, i.e. 55M, also modulate LXRα target genes in animal model. Noticeably, this is the first study that demonstrates WBG extracts can modulate the transactivation activities of LXRα. Results of this study implies that WBG/CLN might act as a “selective modulator” of LXRα.

碩士<br>朝陽科技大學<br>應用化學系<br>102<br>ABSTRACT Enzyme has been found highly specific, highly reactive and highly stereo-selective. Because Lipase is able to change the taste of red wine, the purpose of the study is to select the red wine which has the best taste after being added with different potency of enzyme and evaluated with sensory evaluation. It is hoped that the result of the study can be used to enhance the quality of red wine. The experiment adopted 520 SUPER RED WINE as the sample which was added with four different potency of Yeast and Rhizopus. Being storaged with temperature control for a week, the sample wine was taken out and placed for one hour at room temperature for sensory evaluation. From then on, the sensory evaluation was carried out every week and lasted for twenty weeks. The experiment data was analyzed statistically by nine-point hedonic scale. The result shows that the red wine added with Yeast Lipase has the most significant catalytic reaction on the taste of the red wine, especially the potency of 0.8g which is the favorite of the evaluators. The red wine added with Rhizopus Lipase has no significant catalytic reaction on the taste of the red wine. As the potency of Rhizopus Lipase is higher, the taste of the wine is worse.