Os compostos antimicrobianos naturais são um tema de grande interesse devido ao aumento da demanda por alimentos seguros e de alta qualidade. A utilização de lipossomas é uma alternativa interessante para proteger antimicrobianos nos alimentos, além de fornecer compostos naturais de liberação controlada. Os lipossomas revestidos com polissacarídeos apresentam melhor estabilidade, representando uma alternativa aos lipossomas convencionais. Inicialmente, os nanolipossomas que encapsulam a nisina foram preparados com fosfatidilcolina de soja (PC) e pectina ou ácido poligalacturônico. Os lipossomas desenvolvidos apresentaram alta eficiência de encapsulação, baixo índice de polidispersão e foram estáveis durante 21 dias a 7 °C e 25 °C. A atividade antimicrobiana foi observada contra cinco cepas diferentes de Listeria em placas de ágar de leite, com uma melhor eficiência contra L. innocua 6a. Em um segundo momento, as características estruturais dos lipossomas foram estudadas por dispersão de raios-X de pequeno ângulo (SAXS) e as amostras foram submetidas a ciclos de temperatura (20-60 °C). Para isso, os lipossomas foram desenvolvidos contendo pectina ou ácido poligalacturônico pelos métodos de hidratação de filme e evaporação em fase reversa, para encapsular nisina. A análise de SAXS confirmou a presença de estruturas lamelares em todas as amostras. Além disso, parte da estrutura multilamelar tornou-se cúbica, provavelmente devido à presença de nisina nos lipossomas. A adição de polissacarídeos mostrou diferenças entre as fases cúbicas formadas. Em última análise, a mistura de lisozima e nisina foi encapsulada em lipossomas contendo polissacarídeos. O diâmetro médio dos lipossomas foi de 85,6 e variou para 77,3 e 79,9 nm com a incorporação de pectina ou ácido poligalacturônico, respectivamente. O potencial zeta dos lipossomas com polissacarídeos foi de cerca de -30 mV, mostrando alta eficiência de encapsulação. A atividade antimicrobiana foi avaliada a 37 °C, mostrando que a PC-pectina reduziu a população de L. monocytogenes em 2 log UFC/mL e 5 log UFC/mL em leite integral e desnatado, respectivamente. Em refrigeração, a PC-pectina reduziu a população de L. monocytogenes para quase zero por até 25 dias em leite desnatado. Portanto, pode dizer-se que os lipossomas que contêm polissacarídeos podem ser uma tecnologia promissora para o encapsulamento da lisozima e nisina. Além disso, a existência de estrutura cúbica nos lipossomas pode proporcionar liberação controlada de antimicrobianos. / Natural antimicrobial compounds are a topic of utmost interest due to the increased demand for safe and high-quality foods. The use of liposomes is an interesting alternative to protect antimicrobials in food, also providing controlled release natural compounds. Polysaccharides coated liposomes present better stability, representing an alternative to conventional liposomes. Initially, nanoliposomes encapsulating nisin were prepared with soy phosphatidylcholine (PC) and pectin or polygalacturonic acid. The liposomes developed presented high encapsulation efficiency, low polydispersity index, and were stable for 21 days at 7°C and 25°C. The antimicrobial activity was observed against five different strains of Listeria in milk-agar plates, with a better efficiency against L. innocua 6a. In a second moment, structural characteristics of liposomes were studied by small angle X-ray scattering (SAXS) and the samples were submitted to temperature cycles (20-60°C). For this, liposomes were developed containing pectin or polygalacturonic acid by the thin-film hydration method and reverse phase evaporation method for nisin encapsulation. The analysis of SAXS confirmed the presence of lamellar structures in all the samples. In addition, part of the multilamellar structure became cubic, probably due to the presence of nisin in the liposomes. The addition of polysaccharides showed differences between the cubic phases formed. Ultimately, the mixture of lysozyme and nisin were encapsulated in liposomes containing polysaccharides. The mean diameter of the liposomes was 85.6 and varied to 77.3 and 79.9 nm with the incorporation of pectin or polygalacturonic acid, respectively. The zeta potential of liposomes with polysaccharides were around -30 mV, showing high encapsulation efficiency. The antimicrobial activity was assessed at 37 °C, showing that PC-pectin reduced the population of L. monocytogenes to 2 log CFU/mL and 5 log CFU/mL in whole and skim milk, respectively. At under refrigeration, PC-pectin reduced the population of L. monocytogenes to almost zero for up to 25 days in skim milk. Therefore, it can say that the liposomes containing polysaccharides can be a promising technology for the encapsulation of lysozyme and nisin. In addition, the existence of cubic structure in the liposomes can provide controlled release of antimicrobials.

Dans un contexte de fort développement des biotechnologies, de plus en plus de composés sont extraits, généralement par l'utilisation d'un solvant, à partir de bio-ressources renouvelables. L'éthanol (parfois en mélange avec de l'eau) est un des solvants utilisés car il permet de solubiliser des molécules polaires moyennement hydrophobes et peut être, lui-même, produit par fermentation à partir de bio-ressources renouvelables. Après l'étape d'extraction, les procédés baro-membranaires, et en particulier l'ultrafiltration (UF), peuvent être des solutions pertinentes en vue d'un fractionnement plus fin des constituants de l'extrait. Cependant, à ce jour, si ce procédé est largement développé en milieu aqueux, peu d'études portent sur l'UF en milieu hydro-alcoolique à des teneurs supérieures à 15% en éthanol. Le but de ces travaux est d'étudier l'impact du changement de solvant sur les mécanismes de transfert en UF. Pour cela, une étude systématique de l'UF a été réalisée dans des mélanges eau/éthanol de composition variable allant jusqu'à 30% d'éthanol en volume. L'UF a été conduite en utilisant une protéine modèle, le lysozyme, choisie en raison simultanément de sa bonne stabilité dans les milieux filtrés et de la variation de sa charge en fonction de son environnement physico-chimique (pH, force ionique). L'ajout de sels (NaCl, KH2PO4) aux milieux hydro-alcooliques a permis de montrer l'impact de la modulation des interactions électrostatiques, connues pour jouer un rôle important sur les performances de rétention en UF en milieu aqueux. Les rétentions expérimentales, obtenues au moyen d'une membrane en oxyde de zirconium, ont été interprétés à l'aide du modèle CDE (convection – diffusion – migration électrophorétique) précédemment développé à l'ISCR pour les milieux aqueux et contribuent ainsi à l'extension du domaine d'application de ce modèle. / In the context of the rapid growth of the biotechnological sector, more and more molecules are extracted from renewable resources using solvent extraction. Ethanol (alone or in mixture with water) is one of the most used solvent as it allows solubilizing polar molecules exhibiting moderately hydrophobic nature. Furthermore, it can be itself produced by fermentation using renewable bioresources as a substrate. Following the extraction step, pressure-driven membrane processes, and ultrafiltration (UF) in particular, can be used for further fractionation of the alcoholic extract. However, to date, UF is widely studied in aqueous solution but only few studies deal with its application to the filtration of hydro-alcoholic medium for ethanol concentration above 15%. The objective of this work is thus to study the consequences of the solvent modification on UF transfer mechanisms. To this end, a systematic study of UF in water/ethanol mixture of variable composition up to 30 % (v/v) has been carried out. Lysozyme has been chosen as the model solute due to its high stability in the tested conditions and its surface charge modification according to the physicochemical environment (pH, ionic strength). Thereby, electrostatic interactions known to play a key role in aqueous UF, where modulated by salt addition (NaCl, KH2PO4) to the hydro-alcoholic mixtures. Experimental results obtained using a zirconium oxide membrane were analyzed using the CDE (convection, diffusion and electrophoretic migration) model previously developed in our group for aqueous filtration. The range of application of the CDE model is thus enlarged to the case of water/ethanol mixtures.

The overall objective of the doctoral thesis was the development of starch-based (S) biodegradable active films for food packaging applications, by applying both casting method and thermoprocessing. Different blends of S with protein material have been studied in order to improve the functional properties of the films or confer antimicrobial/antioxidant activity. The following protein materials were used: powder buttermilk (BM); lactoferrin (LF) and/or lysozyme (LZ), and bovine gelatin (BG). Ethyl lauroyl arginate (LAE, E243) was also incorporated as antimicrobial compound. Likewise, S:BG blend films, either with or without LAE, with previously oxidized S, have been studied to enhance the crosslinking of polymer chains and to improve the film properties. The films have been characterized as to their functional properties as packaging material, their antioxidant and/or antimicrobial properties, as well as their capacity for preserving different food systems, in terms of lipid oxidation and microbial spoilage. Blends of S with BM gave rise to films with a heterogeneous structure, in which the formation of a protein gel was observed when BM dispersion was heated with S at 90 ºC for 30 min. The heat treatment promoted an increase in the resistance to break and stretchability of films, together with a decrease in water vapour permeability. Only those films subjected to heat treatment exhibited antioxidant activity, probably due to the release of active peptides as a result of high temperatures. However, no antilisterial activity was observed for any film containing BM. The incorporation of LF and/or LZ into S films, obtained by the casting method, led to a partial compatibility between polymers, thus affecting the microstructure of S films, as well as leading to an rise in the glass transition temperature. Films with proteins were less extensible, especially when LF was incorporated. All of the films tested were effective at controlling the progress of lipid oxidation in pork lard, whereas only films with LF/LZ blend reduced the growth of coliforms in minced pork meat, as a result of their synergistic action. Films based on S and BG blends (1:1) were obtained by both casting method and thermo-processing. Phase separation of both polymers (stratified structure or separated domains of both polymers, respectively) was observed in both cases. The incorporation of LZ, but mainly LAE, into films, enhanced the compatibility between polymers. Thermo-processed films were more permeable to water vapour and oxygen, less rigid and resistant and more stretchable, in comparison with those films obtained by casting. While LAE incorporation improved the water vapour barrier capacity, it worsened the oxygen barrier properties, contrary to the effect produced by LZ. All films with LAE exhibited high antilisterial activity. Films based on oxidized S and BG (1:1), obtained by casting, showed a high polymer compatibility, and crosslinking between the polymer chains occurred due to the carbonyl-amino condensation reaction. As a result, the water uptake ability of the films decreased and the mechanical and barrier properties improved, although film browning was induced due to the formation of Maillard compounds. LAE incorporation implied its involvement in condensation reactions, due to its bi-functional character (carbonyl-amino), thus affecting crosslinking and the film properties. These reactive processes progressed throughout storage time, leading to an increase in the mechanical resistance and browning of the films. The obtained Maillard compounds conferred antimicrobial capacity on the films, which increased as the storage time progressed. The application of blend films of native or oxidized S and BG with LAE, for the purposes of preserving vacuum packaged chicken breast fillets, extended the shelf-life through the inhibition of bacterial growth (total viable counts; psicrotrophic, anaerobic,lactic acid bacteria and coliforms). Samples packaged i / El objetivo general de la presente tesis doctoral se basa en el desarrollo de films activos biodegradables a base de almidón (S) para su aplicación en sistemas de envasado de alimentos, por medio de dos métodos diferentes de obtención, método en húmedo por extensión y secado (casting) y método en seco (termoprocesado). Se estudiaron mezclas de S con diferentes materiales proteicos, con fin de disminuir la alta higroscopicidad de los films de S y su retrogradación a lo largo del tiempo de almacenamiento y mejorar sus propiedades funcionales, así como conferirles actividad antimicrobiana/antioxidante. Los materiales proteicos utilizados fueron los siguientes: suero de mantequilla en polvo (BM); lactoferrina (LF) y/o lisozima (LZ), y gelatina bovina (BG). El etil lauroil arginato (LAE, E243) fue también incorporado como compuesto antimicrobiano. Asimismo, se estudiaron los films mezcla de S con BG, con y sin LAE incorporado, habiendo oxidado previamente el S, para así potenciar el entrecruzado de las cadenas poliméricas y mejorar las propiedades de los films. Estos fueron caracterizados en sus propiedades funcionales como material de envase, sus propiedades antioxidantes y/o antimicrobianas, así como por su capacidad de conservación de diferentes sistemas alimentarios, en términos de su oxidación lipídica y deterioro microbiológico. Las mezclas de S con BM dieron lugar a películas con una estructura heterogénea, en las que se observó la formación de un gel proteico como resultado del calentamiento de la dispersión BM con S a 90 ºC durante 30 min. El tratamiento térmico promovió un aumento de la resistencia a la rotura y extensibilidad de los films, junto con una disminución en la permeabilidad al vapor de agua. Sólo las películas sometidas a tratamiento térmico y homogeneización con cizalla mostraron actividad antioxidante, probablemente debido a la liberación de péptidos activos en consecuencia de la alta temperatura y fuerza de cizalla aplicada Sin embargo, no se observó actividad antilisteria para ninguno de los films con BM. La incorporación de LF y/o LZ en films de S condujo a una compatibilidad parcial entre polímeros, afectando así a la microestructura de los films de S, y produciendo un aumento de la temperatura de transición vítrea y disminución de la capacidad de alargamiento de los films, especialmente cuando se incorporó LF. Todos los films resultaron eficaces en el control del progreso de la oxidación lipídica de la manteca de cerdo, mientras que sólo las películas con mezcla LF/LZ redujeron el crecimiento de coliformes en carne picada de cerdo, como resultado de su acción sinérgica. Los films basados en la mezcla S y BG (1: 1) fueron obtenidos por casting y termo-moldeado y compresión, llevando a la separación de fases entre ambos polímeros (estructura esratificada o separación de dominios de ambos polímeros, respectivamente). La incorporación de LZ, y principalmente de LAE, en los films, aumentó la compatibilidad entre ambos polímeros. Los films termoprensados fueron más permeables al vapor de agua y al oxígeno, menos rígidos y resistentes y más extensibles, en comparación con aquellos obtenidos por casting. La incorporación de LAE mejoró la capacidad de barrera contra el vapor de agua, mientras que incurrió en un empeoramiento de la barrera frente al oxígeno, contrariamente al efecto producido por la LZ. Los films con LAE, moldeados o termoprensados, mostraron una alta eficacia antilisteria. Los films basados en S oxidado y BG (1: 1), fueron obtenidos por casting y mostraron una alta compatibilidad polimérica, lo cual condujo al entrecruzado de las cadenas como resultado de la reacción de condensación carbonilo-amino producida entre ambos polímeros. En consecuencia, la capacidad de absorción de agua de los films disminuyó y se mejoraron las propiedades mecánicas y de barrera, aunque también se indujo a un pardeamiento de los films, indicando / L'objectiu general de la tesi doctoral és el desenvolupament de films actius biodegradables a base de midó (S) per a la seua aplicació en sistemes d'envasat d'aliments, amb l'utitització del mètode d'extensió i assecat (casting) i termoprocessat (barrejat en fos i termo-compressió). Es van estudiar barreges de S amb diferents materials proteics, per millorar les propietats funcionals dels films o conferir activitat antimicrobiana. Els materials protèics utilitzats van ser: sèrum de mantega en pols (BM); lactoferrina (LF) i/o lisozima (LZ), i gelatina bovina (BG). L'ètil lauroil arginat (LAE, E243) va ser també incorporat com a compost antimicrobià. Així mateix, es van estudiar els films barreja de S amb BG, amb i sense LAE, havent oxidat prèviament el S, per potenciar l'entrecreuat de les cadenes polimèriques i millorar les propietats dels films. Aquests van ser caracteritzats en les seues propietats funcionals com a material d'envàs, les seues propietats antioxidants i/o antimicrobianes, així com en la seua capacitat de conservació en diferents sistemes alimentaris, en termes de la seua oxidació lipídica i deteriorament microbià. Les barreges de S amb BM van donar lloc a films amb una estructura heterogènia, en què es va observar la formació d'un gel protèic com a resultat del calfament de la dispersió BM amb S a 90 ºC durant 30 min. El tractament tèrmic va promoure un augment de la resistència al trencament i extensibilitat dels films, juntament amb una disminució en la permeabilitat al vapor d'aigua. Només el films sotmesos a tractament tèrmic van mostrar activitat antioxidant, probablement a causa de l'alliberament de pèptids actius com a conseqüència de l'alta temperatura. No obstant això, no es va observar activitat antilisteria per cap dels films amb BM. La incorporació de LF i/o LZ en films de S obtinguts per casting va donar lloc a una compatibilitat parcial entre polímers, afectant a la microestructura dels films de S, i produint un augment de la temperatura de transició vítria. Els films amb les proteïnes van ser menys extensibles, especialment quan es va incorporar LF. Tots els films van resultar eficaços en el control de l'oxidació lipídica de la mantega de porc, mentre que només el films amb barreja LF/LZ van reduir el creixement de coliforms en carn picada de porc, com a resultat de la seua acció sinèrgica. Els films amb barreges S i BG (1: 1) van ser obtinguts per casting i termo-processat. En tots dos casos es va observar separació de fases entre els dos polímers (estructura estratificada o separació de dominis d'ambdós polímers, respectivament). La incorporació de LZ, i principalment de LAE, en els films, va augmentar la compatibilitat entre tots dos polímers. Els films termo-processats van ser més permeables al vapor d'aigua i l'oxigen, menys rígids i resistents i més extensibles, en comparació amb els obtinguts per càsting. La incorporació de LAE va millorar la capacitat de barrera al vapor d'aigua, a l'hora que va disminuir la capacitat de barrera davant l'oxigen, contràriament a l'efecte produït per la LZ. Tots els films amb LAE, van mostrar una alta capacitat antilisteria. Els films amb S oxidat i BG (1: 1), van ser obtinguts per casting i van mostrar una alta compatibilitat dels polímers, tot produint entrecreuat de les cadenes com a resultat de la reacció de condensació carbonil-amino. En conseqüència, va disminuir la capacitat d'absorció d'aigua dels films i es van millorar les propietats mecàniques i de barrera, encara que es va promoure l'enfosquiment dels films, cosa que indica la formació de compostos de Maillard. La incorporació de LAE va implicar la seua participació en les reaccions de condensació, a causa del seu caràcter bi-funcional (carbonil-amino), el que va afectar a l'entrecreuat i les propietats dels films. Aquests processos reactius van progressar al llarg del temps d'emmagatzematge, donant lloc a un augment de l / Moreno Marro, O. (2017). Starch-protein active films for food preservation [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/80616 / TESIS

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Simplex algorithms have been used to optimize for size, number and morphology of lysozyme and apoferritin crystals. This approach requires fewer experiments than the single-factor-at-a-time method or factorial designs and will be useful in conserving materials on the International Space Station. The simplex method has the possible advantage that it conserves on materials by reducing the number of experiments required to optimize a crystallization system. The process is iterative and exploratory and should allow optimum microgravity conditions to be determined which might very well be different from the optimum conditions on Earth. Because the simplex method uses simple mathematical operations to calculate the next set of crystallization conditions it will be easier for crystal growers to implement than factorial designs. Factorial experiments are based on varying all factors simultaneously at a limited number of factor levels. This results in a model that is used to determine the influence of each factor and their interactions. Factorial design experiments are especially useful at the beginning of an experimental study and as a screening tool to investigate a large number of factors. The simplex method is an optimization method which is model-independent and requires no fitting of models to data. Also, when applied to protein crystal growth the simplex method does not rely on an absolute quality score. Instead, with each iteration a comparison is made to the last experiment and the results are assigned as being "better or worse". In this study, commercially obtained apoferritin was purified from 65% monomeric apoferritin to 92% monomeric apoferritin by size exclusion chromatography. Simplex optimization found the best apoferritin crystals were obtained at 15 mg/ml apoferritin, 2.0% CdSO4, 25°C using the hanging drop vapor diffusion method of crystallization and at 24 mg/ml apoferritin, 1.5% CdSO4, 25°C using the containerless crystallization method. For lysozyme, the simplex method found the best crystals at 19 mg/ml lysozyme, 7.0% (w/v) NaCl, pH 4.0, 25°C using the hanging drop vapor diffusion method of crystallization. For both proteins, the optimum conditions were found with less than ten experiments using very little protein. Finally, we report that the factors to be considered in the successful application of this method to crystallization are the number of variables to be studied, the initial conditions, step size and analysis of crystal quality.

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Les variations dans la qualité des fromages affinés sont expliquées par plusieurs facteurs. Parmi ceux-ci, certains influencent la nature du microbiote des fromages, par exemple la présence de protéines antimicrobiennes natives du lait. Ce projet vise à déterminer la concentration et l'activité de la lactoferrine (Lf), de la lactopéroxydase (Lpo) et du lysozyme (Lz) au cours de la fabrication fromagère et dans différents types de fromages (Cheddar frais, Cheddar doux, Camembert et Suisse). Chaque protéine antimicrobienne fut analysée par deux méthodes. La technique de dosage d'immunoabsorption par enzyme liée (ELISA) fut employée pour chaque protéine. De plus, la Lpo fut mesurée par spectrométrie, la Lz par fluorescence et la Lf par HPLC. La mesure de Lf, Lpo et Lz dans le lait cru, le lait thermisé et le fromage frais a permis de déterminer qu'il n'y avait pas de différences significatives entre les teneurs dans le lait cru et le lait thermisé. Les résultats ont aussi démontré qu'il y a une quantité plus importante des trois antimicrobiens dans le fromage que dans le lait, et ce par un facteur de 2,78. Lors de l'étude des différents types de fromage commerciaux, diverses méthodes ont été comparées à l'ELISA. Il y a un manque de corrélation entre les valeurs obtenues par ELISA et celles obtenues par les autres méthodes. Les résultats révèlent que le fromage Camembert a une concentration moindre d'antimicrobiens que les trois autres fromages. Ceci semble être associé à une protéolyse plus avancée dans le Camembert. Les résultats ont aussi été rapportés par « gramme de protéines » et il y a tout de même des différences significatives entre les différents types de fromage. Ces résultats permettre une meilleure compréhension du passage des antimicrobiens présents dans le lait vers le fromage pour pouvoir, éventuellement, contrôler leur concentration. / Variations in cheese quality are explained by several factors. Among them, some influence the microbiota of cheeses such as native milk antimicrobial proteins. This project aims to determine the concentration and activity of lactoferrin (Lf), lactoperoxidase (Lpo) and lysozyme (Lz) during cheese making and in different cheese types (fresh Cheddar, mild Cheddar, Camembert and Suisse). Each antimicrobial protein was analyzed by two methods. An enzyme-linked immunosorbent assay (ELISA) technique was employed the analysis of each protein. In addition, the Lpo was measured by spectrometry, the Lz by fluorescence and the Lf by HPLC. The measurement of Lf, Lpo and Lz in raw milk, thermized milk and fresh cheese revealed that there were no significant differences between level in raw milk and thermized milk. Results also showed that there is a higher quantity of the three antimicrobials in cheese than in milk by a factor of 2.78. When studying different types of commercial cheese, the various methods were compared to ELISA. There is a lack of correlation between the results obtained by ELISA and those of the other methods. Results show that Camembert cheese has lower levels of antimicrobials than the other 3 cheeses and is different from Swiss cheese for all antimicrobials. This seems linked to a higher degree of proteolysis in Camembert. The results were also reported by "grams of protein" and there are still significant differences between the different types of cheese. These results allow a better understanding of the transition of the antimicrobials present in the milk towards the cheese to be able, possibly, to control their concentration.