Dissertations / Theses on the topic 'High pressure homogenization (HPH)'

The aim of this PhD thesis was to evaluate the effect of a sub-lethal HPH treatment on some probiotic properties and on cell response mechanisms of already-known functional strains, isolated from Argentinean dairy products. The results achieved showed that HPH treatments, performed at a sub-lethal level of 50 MPa, increased some important functional and technological characteristics of the considered non intestinal probiotic strains. In particular, HPH could modify cell hydrophobicity, autoaggregation and resistance to acid gastric conditions (tested in in vitro model), cell viability and cell production of positive aroma compounds, during a refrigerate storage in a simulated dairy product. In addition, HPH process was able to increase also some probiotic properties exerted in vivo and tested for two of the considered strains. In fact, HPH-treated cells were able to enhance the number of IgA+ cells more than other not treated cells, although this capacity was time dependent. On the other hand, HPH treatment was able to modify some important characteristics that are linked to the cell wall and, consequently, could alter the adhesion capacity in vivo and the interaction with the intestinal cells. These modifications, involving cell outermost structures, were highlighted also by Trasmission Electron Microscopy (TEM) analysis. In fact, the micrographs obtained showed a significant effect of the pressure treatment on the cell morphology and particularly on the cell wall. Moreover, the results achieved showed that composition of plasma membranes and their level of unsaturation are involved in response mechanisms adopted by cells exposed to the sub-lethal HPH treatment. Although the response to the treatment varied according to the characteristics of individual strains, time of storage and suspension media employed, the results of present study, could be exploited to enhance the quality of functional products and to improve their organoleptic properties.

Ce travail de recherche se concentre sur l'extraction et le fractionnement des biomolécules à partir de microalgues par des traitements physiques: les champs électriques pulsés (CEP), les décharges électriques de hautes tensions (DEHT) et les ultrasons (US). Dans cette étude, trois espèces de microalgues Nannochloropsis sp., Phaeodactylum tricornutum (P. tricornutum) et Parachlorella kessleri (P. kessleri) ont été étudiées. Les espèces ont différentes formes cellulaires, structure et contenu intracellulaire. L'effet des techniques testées sur l'extraction des biomolécules a été mis en évidence à travers une analyse quantitative et qualitative: suivi du rendement des composés ioniques, des glucides, des protéines, des pigments et des lipides. Une étude comparative des traitements physiques (CEP, DEHT et US), à la même énergie, pour la libération des biomolécules intracellulaires à partir des trois espèces de microalgues, a permis de mieux comprendre les différents mécanismes de désintégration. Pour chaque microalgue, à la même énergie consommée, le traitement par DEHT s'est révélé le plus efficace en terme d'extraction des glucides, tandis que les US sont plus efficaces pour l'extraction des protéines et des pigments. Le traitement par CEP a été moins efficace en terme du rendement d’extraction. Cependant, la meilleure sélectivité (extraction des glucides) a été obtenue en utilisant les CEP ou les DEHT. Les prétraitements physiques (CEP, DEHT ou US) des suspensions plus concentrées suivis d'une homogénéisation haute pression (HHP) de suspensions diluées ont permis d'améliorer l'efficacité de l'extraction et de diminuer la consommation énergétique totale et le nombre de passages. Le prétraitement physique permet de réduire la pression mécanique de l’HHP, pour atteindre le même rendement d’extraction. Pour la valorisation maximale de la biomasse de microalgues, une procédure d'extraction assistée par DEHT (40 kV/cm, 1-8 ms) suivie de plusieurs étapes d'extraction aqueuses et non aqueuses semble être utile pour l'extraction sélective et le fractionnement de différentes biomolécules à partir de microalgues. Des effets significatifs du prétraitement HVED sur l'extraction par solvant organique des pigments (chlorophylles, caroténoïdes) et des lipides ont été observés
This research work focuses on extraction and fractionation of bio-molecules from microalgae using physical treatments: pulsed electric fields (PEF), high voltage electrical discharges (HVED) and ultrasonication (US) techniques. In this study, three microalgae species Nannochloropsis sp., Phaeodactylum tricornutum (P. tricornutum) and Parachlorella kessleri (P. kessleri) were investigated. These species have different cell shapes, structure and intracellular contents. The effects of tested techniques on extraction of bio-molecules have been highlighted in a quantitative and qualitative analysis by evaluating the ionic components, carbohydrates, proteins, pigments and lipids. A comparative study of physical treatments (PEF, HVED and US) at the equivalent energy input for release of intracellular bio-molecules from three microalgal species allowed us to better understand the different disintegration mechanisms. For each microalga at the same energy consumption, the HVED treatment proved to be the most efficient for extraction of carbohydrates, while the US treatment for extraction of proteins and pigments. In general, the smallest efficiency was observed for the PEF treatment. However, the highest selectivity towards carbohydrates can be obtained using the mild PEF or HVED technique. The preliminary physical treatments (PEF, HVED or US) of more concentrated suspensions followed by high pressure homogenization (HPH) of diluted suspensions allowed improving the extraction efficiency and decreasing the total energy consumption. The physical pretreatments permit to reduce the mechanical pressure of the HPH and number of passes, to reach the same extraction yield. For the maximum valorisation of microalgal biomass, extraction procedure assisted by HVED treatment (40 kV/cm, 1-8 ms) followed by aqueous and non-aqueous extraction steps seems to be useful for selective extraction and fractionation of different bio-molecules from microalgae. The significant effects of HVED pre-treatment on organic solvent extraction of pigments (chlorophylls, carotenoids) and lipids were also observed

El consumo de licuado de soja está experimentado un notable incremento debido a su consideración de producto saludable. El licuado de soja, además de ser una alternativa a la leche de vaca, sobre todo para las personas que poseen alguna intolerancia a los productos derivado de la leche, tiene componentes bioactivos (flavonoides, vitamina E y poliaminas) que pueden contribuir a prevenir algunas dolencias crónicas prevalentes en la sociedad actual. En este estudio se planteó la utilización de una tecnología emergente, la ultra alta presión de homogenización (UHPH) para la obtención de licuado de soja. Esta tecnología no térmica consiste en la aplicación de presiones de hasta 400 MPa utilizando un sistema de homogenización, especialmente diseñado para producir un efecto conservador, al mismo tiempo que se mejora la estabilidad coloidal y se mantiene la calidad nutricional y sensorial. Con esta hipótesis de partida, la UHPH podría ser una tecnología alternativa a las comúnmente aplicadas a nivel industrial. Por ello, en el planteamiento de este trabajo se incluyó el estudio comparativo de la UHPH con los tratamientos térmicos de pasteurización y UHT. En la primera parte de esta tesis se llevaron a cabo diferentes tratamientos UHPH (200 y 300 MPa con temperaturas de entrada de 55, 65 y 75ºC) con la finalidad de seleccionar las condiciones óptimas para obtener productos de buena calidad, tanto de almacenamiento en refrigeración, como de larga duración de almacenamiento a temperatura ambiente. El estudio se realizó a dos niveles independientes. Por una parte se evaluaron parámetros característicos de la calidad química, coloidal, enzimática y microbiológica de licuados de consumo habitual, y por la otra, se realizó un estudio con licuados inoculados con diversas cepas microbianas para conocer su cinética de destrucción frente a tratamientos UHPH. De estos estudios se concluyó que los tratamientos a 300 MPa produjeron licuado de soja con muy buena estabilidad coloidal y química y que, aplicando una temperatura de entrada de 85ºC en combinación con dicha presión, se alcanzó una excelente reducción de las esporas bacterianas. La segunda parte del trabajo consistió en el estudio de la evolución durante el almacenamiento de los licuados UHPH tratados en las condiciones óptimas seleccionadas del estudio previo. De este modo, se obtuvieron tanto licuados frescos similares a los pasteurizados, como licuados de larga duración, similares a los tratados por UHT. Para lograr este propósito, se evaluaron una serie de aspectos, tales como microbiológicos, estabilidad coloidal, cambios de color, parámetros químicos y sensoriales que permitieron evaluar la calidad global de los productos de soja, así como su aceptación por los consumidores. Los licuados de soja fresco y de larga duración alcanzaron respectivamente 1 y 6 meses de caducidad con mejor calidad que aquellos tratados térmicamente.
Soymilk consumption is experiencing a noticeable increase due to it being considered as a healthy product. Soymilk has often been used as an alternative to dairy milk for people who have intolerance to dairy products. Nowadays, it is known for its important health benefits that can contribute to the reduction of chronic illness commonly prevalent in the modern style life. This is due, primarily, to characteristics of protein fraction and minor components rich in antioxidant activity (flavonoids, tocopherols and poliamines) taking into account the excellent nutritional profile of soymilk. This thesis project was focused on the application of an emerging technology, Ultra High Pressure Homogenization (UHPH), in the production of soy vegetable milk. This non-thermal technology consists of a high pressure machine capable of applying pressures of up to 400 MPa using a special homogenizing system designed to produce a conserving effect, improving the colloidal stability while maintaining good nutritional and sensory qualities. Considering this hypothesis, UHPH could be an alternative technology to those commonly applied in the food industries. For that, a comparative study of UHPH with thermal treatments (pasteurization and UHT) was carried out in this work. In the first part of this thesis, different UHPH conditions (200 and 300 MPa at 55, 65 and 75ºC of inlet temperature) were performed on soymilk in order to select optimal treatment conditions for producing a good quality product whether intended for refrigeration or long-term storage at room temperature. In this first step, two independent evaluations were performed. On one hand, quality parameters related to chemical, enzymatic, microbiological and colloidal characteristics were evaluated and on the other hand, an inoculation study with different strain spores was carried out in order to determine the inactivation kinetic of the UHPH treatment. Results indicated that treatments at 300 MPa were able to produce soymilk with high chemical and colloidal stability. It is also worth noting that an excellent reduction of bacterial spores was reached applying inlet temperature of 85ºC at the same pressure. The second part consisted in the shelf-life evaluation of soymilk treated by UHPH using the selected optimal conditions determined in the previous step. As a result, soymilk was obtained with similar characteristics to those produced by pasteurization and with extended shelf-life similar to those obtained by UHT treatments. To achieve this purpose, microbiological aspects, colloidal stability, color changes, chemical parameters and sensory quality were applied to evaluate the overall quality of soymilk and its acceptance by the consumers. Refrigerated soymilk and that produced for an extended shelf-life respectively reached 1 and 6 months of storage in good conditions for consumption and with better quality than those obtained by thermal treatments.

El consumo de las bebidas de soja y de almendra está aumentando en Europa porque son útiles como alternativa a la leche de vaca para los individuos intolerantes a la lactosa, con alergia a las proteínas de la leche, o para aquellos que evitan la leche por otras razones. También su popularidad aumenta por los efectos beneficiosos para la salud que se le atribuyen. Las bebidas de soja y de almendra comerciales están normalmente procesadas por tratamientos térmicos, especialmente UHT, no sólo para estabilizarlas sino también para mejorar su palatabilidad y digestibilidad ya que, entre otros efectos, destruyen componentes indeseables, tales como los inhibidores de tripsina. No obstante, los tratamientos térmicos pueden afectar a sus componentes nutritivos y compuestos bioactivos. La ultra alta presión de homogeneización (UHPH) es una tecnología no térmica emergente que consiste en la aplicación de presiones de hasta 400 MPa utilizando un sistema de homogeneización, especialmente diseñado para producir un efecto conservador, al mismo tiempo que mejora la estabilidad coloidal. En el marco de un proyecto del Plan Nacional (AGL 2008-05430-C02-02) realizado en coordinación con el grupo del Centre Especial Planta de Tecnologia dels Aliments (CERPTA) de la Universidad Autónoma de Barcelona, se pretende aplicar la UHPH para la obtención de bebidas de soja y almendra y comparar los efectos de esta tecnología con los de los convencionalmente aplicados a este tipo de productos (pasteurización y UHT). En el primer apartado de esta memoria se valora el efecto que esta tecnología provoca sobre una serie de parámetros nutritivos y biofuncionales (perfil de aminoácidos, perfil de ácidos grasos, digestibilidad de las proteínas, disponibidad de la lisina, tocoferoles, fitoesteroles, aminas biógenas, poliaminas e isoflavonas), extendiendo el estudio a no solo al efecto inmediato del tratamiento si no también durante la vida útil de los productos. En términos generales, se concluye que los tratamientos por UHPH mantienen o mejoran, en comparación con los térmicos, el valor nutritivo y biofuncional de las bebidas de soja y de almendra. La soja aporta compuestos bioactivos, como las isoflavonas, cuyo consumo puede presentar efectos beneficiosos para la salud pero hay controversia sobre la seguridad de su consumo por parte de lactantes, ya que se he señalado que pueden ejercer tiene efectos endocrinos indeseables sobre el desarrollo y el sistema reproductor. En la segunda parte de esta tesis, se realiza una aproximación a una estimación de la exposición y del riesgo de estos compuestos bioactivos en productos derivados de la soja de mayor consumo. Aún no existen datos de consumo de productos derivados de soja en Europa, pero la estimación muestra que una dieta que incluya habitualmente productos derivados de la soja aporta contenidos de isoflavonas suficientes para lograr sus potenciales efectos beneficiosos. Por otro lado, el consumo de fórmulas infantiles a base de soja por lactantes supone un cierto riesgo de sufrir los efectos adversos de las isoflavonas, ya que su consumo sobrepasa los niveles de seguridad establecidos por el Panel de Expertos (NTP-CERHR). Además, los productos derivados de la soja, especialmente los fermentados, contienen niveles elevados de algunas aminas biógenas. La ingesta de algunos de estos productos de soja puede suponer un riesgo de aparición de los efectos adversos de estas aminas, especialmente cuando hay un tratamiento con fármacos específicos. En la tercera parte, se estudia algunos efectos biológicos de las poliaminas e isoflavonas. Las poliaminas son compuestos naturales con un papel reconocido como antioxidante endógeno en frente al estrés oxidativo celular. Se estudia su capacidad antioxidante in vitro y el mecanismo de acción y se concluye que su capacidad antioxidante es superior al de otros antioxidantes reconocidos (galato de octilo, palmitato de ascorbilo y α-tocoferol) y que su potencia antioxidante se debe a su capacidad para secuestrar metales y radicales libres. Por otro último, se estudia el metabolismo intracelular de las isoflavonas en células endoteliales por su importancia potencial en los mecanismos de acción cardioprotectores del sistema vascular. Se concluye que las isoflavonas entran en las células endoteliales y son metabolizadas mediante glucuronidación, sulfatación y metilación.
Soymilk and almond milk are particularly useful as an alternative of cow’s milk for people who are lactose-intolerant, allergic to milk proteins, or for those who avoid milk for other reasons, and their consumption also has potential health benefits. Ultra high pressure homogenization (UHPH) is a useful non-thermal technology to obtain safe and high-quality liquid foods. UHPH is based on the same principle as conventional homogenization, but it works at significantly higher pressures (up to 400 MPa). In the first part of this thesis, in the frame of the project “The application of Ultra high pressure homogenization (UHPH) to produce high quality vegetable milks (soy and almond)” (AGL 2008- 05430- C02-02), the aim was to study the effects of the UHPH treatment on the nutritional and biofunctional compounds of soymilk and almond milk compared to conventional thermal pasteurization and UHT treatments. As a result, it was concluded that UHPH treatments maintained or improved the nutritional and biofunctional value of soymilk and almond milk, compared to conventional thermal pasteurization and UHT treatments. Soybean provides bioactive compounds, such as isoflavones, whose consumption has potential beneficial health effects for adults and also could have significant effects on the endocrine and reproductive system development in infants. In the second part of this thesis, an estimation of exposure and risk of these bioactive compounds in soybean products was performed. In general, the consumption of these products provides similar or higher isoflavone contents to achieve the beneficial effects. On the other hand, the consumption of soybean based infant formulas could involve a potential risk to infants. Additionally, the fermented soybean products contain high levels of certain biogenic amines. Adverse effects of some biogenic amines as a result of the consumption of the soybean products in general would be unlikely, except for individuals under specific drug therapies. In the third part, some biological effects of polyamines and isoflavones were studied. The mechanism by which polyamines act as antioxidants was evaluated by in vitro methods, concluding that it seems to be related to its ability to chelate metals. Furthermore, the intracellular metabolism of isoflavones in endothelial cells was studied because it can be important in the elucidation of the mechanism of action of isoflavones in the cardiovascular system.

An industrial Fourier transform infrared (FTIR) milk analyser has been adapted for the proximate analysis of fresh or cooked meat and meat products. Stable freeze-dried samples of ground beef and bologna were prepared for the calibration of an FTIR spectrometer equipped with a 37-mum transmission cell maintained at a constant temperature of 65°C and were analysed for fat, protein, moisture, and ash by the official methods of analysis of the Association of Official Analytical Chemists (AOAC) prior to instrumental measurement. The requirement to prepare a "milk-like" emulsion of meat for FTIR analysis led to the development of two prototype high-pressure homogenizers specifically designed to produce analytical volumes of emulsions in which the largest residual colloids present in suspension would have dimensions smaller than 1 mum. Emulsified samples were examined by transmission electron microscopy and laser light scattering spectroscopy to determine the size distribution of fat globules and the dimensions of the residual insoluble fragments of protein.
"Milk-like" emulsions of meat passed three times through a high-pressure homogenizer operating at 20,000 psi (138 MPa) had an average fat globule diameter of less than 320 nm. Also, the use of high-pressure homogenization eliminated the need to filter out insoluble proteins from connective tissues prior to the infrared analysis, resulting in a more accurate determination of the protein content in the meat samples. The results of validation studies conducted with both fresh and freeze-dried samples demonstrated that it is possible to analyse meat samples simultaneously for fat, protein, carbohydrates and moisture with good accuracy in approximately 7½; minutes per sample employing existing FTIR instrumentation used for the routine analysis of milk and dairy products.

El principal objetivo de esta tesis ha sido evaluar la capacidad de la tecnología de ultra alta presión de homogeneización (UHPH) como alternativa a la pasteurización convencional para garantizar la seguridad y calidad de los alimentos líquidos. Para probar la eficacia de la UHPH y garantizar la seguridad microbiana de los alimentos se inoculó Listeria monocytogenes y/o S. enterica serovar Senftenberg 775W en diferentes alimentos líquidos como son huevo entero líquido, leche y zumo de frutas (naranja y uva). y se trataron a 150, 200 y 250 MPa en el caso del huevo liquido y a 200, 300 y 400 MPa en el de leche y zumos de frutas, realizándose la evaluación de su supervivencia durante el almacenamiento en refrigeración a 4ºC durante 20 días en las muestras de huevo y 12 días en las muestras de leche y zumo de frutas. Los recuentos de células viables y dañadas se realizaron utilizando agar triptona soya enriquecida con extracto de levadura y el mismo medio suplementado con sal. Para evaluar los factores que pudieran afectar a la inactivación bacteriana en la eficacia del tratamiento UHPH, se estudió la influencia de la concentración bacteriana baja (3 log CFU/ml) y alta (7 log CFU/ml) en huevo y leche, así como la influencia del contenido de grasa en leche (0.3, 3.6, 10, y 15% de grasa). Adicionalmente, con la finalidad de explicar algunos de los resultados de supervivencia de los patógenos inoculados en los zumos de frutas se estudió la eficacia a la respuesta de ácido tolerancia (ATR) en la protección de Listeria monocytogenes y S. enterica en fase exponencial o estacionaria en zumo de naranja y uva conservados a 4 y 25ºC. En huevo, el tratamiento a 250 MPa disminuyo los recuentos de Salmonella enteritidis serovar Senftenberg 775W a niveles similares a los obtenidos en la pasteurización térmica, produciéndose una disminución posterior por debajo de los límites de detección durante el almacenamiento 4ºC, aunque se detecto su presencia. En las muestras de leche (0.3 and 3.6%), se observó que 300 y 400 MPa dañaban considerablemente a L. monocytogenes, pero durante el almacenamiento fue capaz de recuperarse y desarrollarse. No obstante, los mayores valores de letalidad se consiguieron en las muestras de leche con un contenido de 15 y 10% de grasa. En las zumos, el tratamiento a 400 MPa inactivó completamente Salmonella enteritidis serovar Senftenberg 775W, este cepa se mostró mas sensible que L. monocytogenes a los tratamientos UHPH aplicados. Sin embargo, al finalizar el periodo de almacenamiento del zumo de uva control y homogenizado no se detectaron recuentos de L. monocytogenes, lo que podría ser atribuido a la presencia de compuestos naturales de la uva con efecto antilisteria. La respuesta de ácido tolerancia se indujo a L. monocytogenes y Salmonella enteritidis serovar Senftenberg 775W para evaluar si se presentaba un efecto protector ante estrés por acidez. Se observó que las células en fase estacionaria mostraban una resistencia natural a pH bajos. Tomando como modelo al zumo de naranja, se evaluó el efecto de la UHPH en la actividad enzimática (pectin metilesterasa) y microbiológica (bacterias aerobias mesófilas, psicotrófas, ácido lácticas y levaduras), en los atributos físicos (turbidez y distribución del tamaño de partícula), en las propiedades bioactivas (contenido de Lacido ascórbico, carotenoides y polifenoles), en la actividad antioxidante y en otros los parámetros generales de calidad (color, pH, °Brix, acidez titulable, azúcares reductores y índice de oscurecimiento no enzimático). Los tratamientos UHPH aplicados consistieron de dos temperaturas de entrada (10 o 20ºC), tres niveles de presión (100, 200 y 300 MPa) y dos tiempos de retención (≤ 0.7 o 30 segundos). Los resultados se compararon con los resultados obtenidos por pasteurización térmica (1 o 2 min a 90 ºC). Los tratamientos de UHPH iguales o superiores a 200 MPa fueron igualmente efectivos que la pasteurización para el control de la actividad enzimática y de las bacterias alterantes del zumo no apreciándose actividad enzimática ni incremento de los recuentos microbianos tras 50 días de almacenamiento a 4ºC. Los zumos tratados por UHPH tuvieron la mejor distribución de tamaño de partícula y los mejores valores de turbidez en comparación con los zumos pasteurizados, obteniendo una mayor reducción de del tamaño de partícula en las muestras tratadas a 300 MPa. La retención de L-acido ascórbico y carotenoides dependió de la presión aplicada y específicamente de la temperatura alcanzada durante el tratamiento. El contenido de flavonoides se incrementó en el zumo con los tratamientos por UHPH, obteniendo el mayor contenido a 200 y 300 MPa. Además, los zumos tratados por UHPH mostraron niveles de capacidad equivalente antioxidante trolox (TEAC) mayores que los de los zumos frescos o pasteurizados. Además, se realizó un estudio sensorial de preferencia y aceptabilidad en zumo homogeneizado en las condiciones consideras de elección (20ºC de temperatura de entrada y 300 MPa) y se evaluó la vida útil a 6ºC y 20ºC por 90 días. En el estudio de aceptabilidad, la muestras tratada por UHPH obtuvieron la menor puntuación en términos de valoración de color con respecto a la muestra pasteurizada. Durante los 90 días de almacenamiento a 6ºC los recuentos de las muestras tratadas por UHPH así como las pasteurizadas se mantuvieron por debajo del límite de detección (1 Log CFU/ml).
The main objective of this thesis has been to evaluate the capacity of the ultra-high pressure homogenization (UHPH) technology as alternative to the conventional pasteurization in ensuring the safety and quality of liquid foods. To test the effectiveness of the technology to guaranty the microbial safety of foods we inoculated strains of Listeria monocytogenes and/or S. enterica serovar Senftenberg 775W into different liquid foods such as whole egg, milk and fruit juices (orange and grape). They were submitted to a single cycle of UHPH treatment at 150, 200 and 250 MPa for liquid whole egg and at 200, 300, and 400 MPa for milk and fruit juices. The effectiveness of the UHPH treatments over low (3 log CFU/ml) and high (7 log CFU/ml) bacteria loads was evaluated in both liquid whole egg and milk. Moreover, the influence of milk fat content (0.3, 3.6, 10, and 15% of fat) in the Listeria monocytogenes inactivation by UHPH treatments was also studied. Samples counts were followed during of storage at 4ºC over 20 days for liquid whole egg and 15 days for milk and fruit juices. Viable and injured bacterial counts were evaluated by means of a differential plating method using tryptone soy agar enriched with yeast extract and the same medium supplemented with salt. Additionally, with the purpose to explain some of the results of the survival of pathogens inoculated in fruit juices, the effectiveness of the response acid tolerance (ATR) on the protection in the exponential or stationary phase of Salmonella Seftenberg and L. monocytogenes inoculated in orange and grapefruit juice during their conservation at 4 °C and 25ºC was studied. In liquid whole egg, UHPH treatments at 250 MPa effectively reduced Salmonella enteritidis serovar Senftenberg 775W to similar levels than reported for thermal pasteurization. Surviving Salmonella counts decreased below the detection limit during the storage of the liquid egg at 4ºC, although Salmonella was immunologically detected during all the storage period. In milk samples which a low fat concentration (0.3 and 3.6 %), pressures of 300 and 400 MPa damaged considerably L. monocytogenes cells but they were able to recover and grow up during the subsequent cold storage. Nevertheless, higher lethality values were achieved in milk with the highest fat content (15 and 10%). In fruit juices, UHPH treatments at 400 MPa inactivated completely Salmonella enteritidis serovar Senftenberg 775W, being this strain more sensitive than L. monocytogenes to the UHPH treatments applied. However, in grape juice L. monocytogenes viable counts were undetectable at the end of storage in both control and pressurized samples, which could be attributed to the presence of natural compounds with antilisterial effect. Acid tolerance response (ATR) was induced in L. monocytogenes and Salmonella enteritidis serovar Senftenberg 775W to assess if it was able to protect cells from a most severe acid stress. Cells in stationary phase, used on the UHPH experiment with juices, showed a natural resistance to low pH values. Taking orange juice as food model, we also evaluated the effect of UHPH treatments on enzymatic activity (pectin methylesterase PME) and microbiological (mesophilic aerobic bacteria, psychrotrophic aerobic bacteria, lactic-acid bacteria and yeast) spoiling activity, as well as on physical attributes (cloud stability and particle size distribution), bioactive properties (L-ascorbic, carotenoid and polyphenol content), antioxidant activity and other general quality parameters (color, pH, °Brix, titratable acidity, reducing sugars and non-enzymatic browning index). In this case the UHPH treatments used consisted in combinations of two inlet temperatures (10 or 20ºC), three pressures (100, 200 or 300 MPa) and two holding times (≤ 0.7 or 30 seconds). Results were compared with two thermal pasteurization treatments (1 or 2 min at 90 ºC). UHPH treatments above 200 MPa were as effective as pasteurization to control both PME activity and spoilage bacteria in orange juice. Neither pectin methyl esterase activity nor microbial counts increased significantly after 50 days of storage at 4ºC. UHPH treated juices showed a better particle size distribution and cloudiness values than the pasteurized juices. In particular, the smallest particles were observed in samples treated at 300 MPa. L-ascorbic acid and carotenoid retention of orange juice depended on the high pressure used and more specifically on the maximum temperature achieved during the UHPH treatment. Content of flavonoids in orange juice increased after the UHPH treatments, achieving the maximum concentration in the samples treated at 200 and 300 MPa. Moreover, UHPH treated juices showed higher levels of trolox equivalent antioxidant capacity (TEAC) values than did fresh and heat-treated samples. Additionally, a preliminary sensorial test of preference and acceptability was made with the orange juice treated at 20ºC of inlet temperature and 300 MPa. Moreover with these samples a shelf-life test was carried out by 90 days at 6ºC and 20ºC. In the consumer acceptability study, the UHPH samples in term of color obtained the lowest score. Microbial counts of both UHPH treated and pasteurized samples kept below of the detection limit (1 Log CFU/ml) during the 90 days of storage at 6ºC.

Alguns decênios atrás, o advento da nanotecnologia abriu perspectivas inovadoras permitindo alcançar novos alvos. Na área farmacêutica, as nanopartículas abriram alternativas ineditas de acesso para tratar órgãos e tecidos. Dentre as diversas nanopartículas existentes, este trabalho assentou sobre as nanopartículas lípidicas sólidas porque elas permitem a utilização de substâncias naturais, que representam um interesse cada vez maior devido às suas potencialidades diversificadas e comprovadas. Assim, foram escolhidos o óleo de copaíba e a alantoína devido às suas propriedades farmacológicas. Da mesma maneira que existem vários tipos de nanopartículas, existem várias técnicas para produzi-las. Neste estudo, o método por homogeneização à alta pressão foi selecionado devido às diversas vantagens. Para validar essas opções tecnológicas e caracterizar as nanopartículas, análises morfológicas, físico-químicas e térmicas foram realizadas. O segundo propósito desta pesquisa foi a avaliação do potencial antifúngico das nanopartículas contra fungos leveduriformes e filamentosos multirresistentes, devido às diversas propriedades microbiológicas do óleo de copaíba. Consequentemente, diversos ensaios micológicos foram feitos a fim de determinar onde e como essas nanopartículas agiram sobre esses fungos. Os resultados mostraram que a produção de nanopartículas lípidicas sólidas homogêneas e estáveis físico-quimicamente foi possível. Além disso, essas nanopartículas, compostas por substâncias naturais, demonstraram atividade antifúngica contra fungos multiresistentes, fato que não ocorreu com as matérias-primas isoladas. A nanotecnologia foi fundamental e levou a desenvolver suspensões antifúngicas.
Some decades ago, the advent of nanotechnology has opened new perspectives allowing reaching new targets. In the pharmaceutical area, nanoparticles have opened new ways for treating organs and tissues. Among the various existing nanoparticles, this work was based on solid lipid nanoparticles because they permitted the use of natural substances which represent a growing interest due to their diverse and proven strengths. So, copaiba oil and allantoin were chosen. Just as there are several types of nanoparticles, there are several techniques to produce them. In this study, the method of high pressure homogenization was selected because of several advantages. To validate these technological options and characterize nanoparticles, morphological, physico-chemical and thermal analysis were performed. The second purpose of this research was to evaluate the antifungal potential of nanoparticles against multiresistant yeasts and filamentous fungi due to various microbiological properties of copaiba oil. Consequently, many mycological tests were performed to determine where and how these nanoparticles acted on these fungi. The results showed the production of homogeneous and physico-chemically stable solid lipid nanoparticles is possible and, moreover, these nanoparticles produced with natural substances demonstrated their antifungal activity against multiresistant fungi, which did not happen with isolated raw materials. The nanotechnology was fundamental and led to develop antifungal suspensions.

The scope of these studies was two-fold: to evaluate high pressure homogenized slurries as a rapid means of screening wood pulps for their Cu, Mn and Fe content and to evaluate the high pressure homogenization in combination with chelating agents or enzymatic digestion for the liberation/extraction of metal ions from this matrix.
Cu, Mn and Fe were determined successfully in pulp samples using high-pressure homogenization prior to slurry introduction-GF-AAS. The analysis time of the method from sample acquisition to determination was of the order of a few minutes per sample.
Different cellulose swelling/dissolution agents were evaluated for the generation of quasi-stable pulp suspensions, rich in cellulose. High-pressure homogenization alone or in combination with chelating agents or enzymatic digestion was also investigated as a means of quantitatively releasing these metal ions into the liquid phases.
A new model of homogenizer equipped with ceramic homogenizing valve with a few modifications was evaluated in terms of metal contamination levels within the final sample dispersion. (Abstract shortened by UMI.)

The overall goal of the present PhD thesis was to study some factors related to the choice of emulsifier (whey protein isolate or sodium caseinate) concentration, oil-phase volume fractions (10-50%) and homogenization conditions (100-300 MPa) that could influence physical stability and lipid oxidation in nano/submicron oil-in-water emulsions by using a rotor-startor system (colloidal mill, CM, at 5000 rpm for 5 min) for obtaining the coarse emulsions and stabilized by ultra high-pressure homogenization (UHPH), in comparison to conventional homogenization (CH, 15 MPa). Emulsions were characterized for their physical properties (droplet size distribution, microstructure, surface protein concentration, emulsifying stability against creaming and coalescence, and viscosity) and oxidative stability (hydroperoxide content and TBARs) under light (2000 lux/m2 for 10 days). The first study focused on using whey protein isolate (WPI) as emulsifier in different concentrations (1, 2 and 4%) with a fixed oil concentration (20%) of sunflower and olive oils (3:1). UHPH produced emulsions with lipid particles of small size in the sub-micron range (100-200 nm) and low surface protein with unimodal distribution in emulsions treated at 200 MPa using whey proteins at 4%. Long term physical stability against creaming and coalescence was observed in UHPH emulsions, compared to those obtained by CM and CH. Oxidative stability of emulsions was also improved by UHPH compared to CM and CH, especially when 100 MPa was applied. All emulsions exhibited Newtonian behavior (n ≈ 1). These results led us to use the best conditions obtained in the previous work (4% of protein concentration and pressure treatments of 100 and 200 MPa) to study the physical and oxidative stability of emulsions containing different oil-phase volume fractions (10, 30 and 50%). Increasing the oil concentration from 10 to 50%, in general, increased the particle size, decreased the surface protein concentration and resulted in a high degree of flocculation and coalescence, especially in emulsions treated at 200 MPa. All UHPH emulsions, except those treated at 100 MPa containing 10% oil, and CH emulsions with 50% oil displayed an excellent stability vs. creaming during storage at ambient temperature. The lowest oxidation rate was observed in UHPH emulsions, especially those containing 30% oil. The third study was conducted on using sodium caseinate (SC) as emulsifier in different concentrations (1, 3 and 5%) with a fixed oil concentration (20%) of sunflower and olive oils (3:1). UHPH emulsions containing 1% protein presented a high particle size (especially in emulsions treated at 100 MPa) but increasing the protein content to 3 and 5% in UHPH emulsions reduced the particle size, and tended to change the rheological behaviour from Newtonian to shear thinning, improving the creaming and oxidative stabilities of emulsions. From the previous study, the best droplet breakdown, physical and oxidative stability were obtained with pressures in the range of 200 and 300 MPa and sodium caseinate (5%). Therefore, the objective of the last study was to evaluating the emulsions containing different oil-phase volume fractions (10, 20, 30 and 50%) treated by UHPH in the conditions above mentioned, in comparison to CH emulsions containing 1 and 5% SC. Increasing the oil content to 50% tended to produce emulsions with a gel structure such as a mayonnaise type product so, the results of this study focused only on emulsions containing 10, 20 and 30% oil. CM and CH emulsions containing 1% SC and different oil contents (10, 20 and 30%), exhibited a Newtonian flow behavior with a slow creaming rate, whereas the oxidation rate was faster in these emulsions. On the other hand, high degree of flocculation with a shear thinning behavior, higher creaming rates, but low oxidation rates were observed in CH emulsions containing 5%. UHPH-treated emulsions containing high oil contents (20 and 30%) exhibited excellent creaming stability, and with a shear thinning rheological behavior only in emulsions containing 30% oil. UHPH produced stable emulsions against oxidation, especially when high oil contents (20-30%) were used. Increasing the oil concentration from 10 to 30%, in general, resulted in an increase in the oxidative stability in all emulsions, except in CH emulsions containing 1% of SC. Emulsions produced by both whey protein (4%) and caseinate (5%), and treated by UHPH have a good physical stability to flocculation, coalescence and creaming and also high stability to lipid oxidation, opening a wide range of opportunities in the formulation of emulsions containing bioactive components with lipid nature.

En els últims 10 anys s'ha incrementat la recerca en emulsions per a l'alliberament de compostos bioactius. Les emulsions convencionals d'oli-en-aigua (> 1 µm) presenten una estabilitat fisicoquímica i oxidativa insuficient; per tant, i per superar aquest problema, s'han dut a terme molts esforços en el desenvolupament de noves tecnologies. La tecnologia d'Homogeneïtzació a Ultra-Alta Pressió (UHPH) s'ha aplicat en diferents emulsions alimentàries per tal de millorar la seva estabilitat física. Per això, és necessari el desenvolupament d'emulsions UHPH d'oli-en-aigua que continguin bioactius lipòfils, com l'àcid linoleic conjugat (CLA), per tal de millorar les característiques nutricionals i fisicoquímiques de les emulsions, així com la seva vida útil. En aquesta tesi doctoral, es va avaluar la influència de diferents tecnologies (UHPH i homogeneïtzació convencional; CH) i ingredients (aïllat proteic de soja, SPI; aïllat proteic de pèsol, PPI; oli de soja i CLA) en emulsions d'oli-en-aigua. A més, es va desenvolupar un producte final de base làctia UHT incorporant emulsions amb CLA. La present recerca es va dividir en quatre parts. En un primer estudi, es va avaluar l'efecte de la UHPH (100-300 MPa, Ti=20 °C) en comparació de la CH (15 MPa, Ti=20 °C) sobre l'estabilitat física i oxidativa d'emulsions d'oli de soja (10 i 20%) i estabilitzades amb SPI per tal de determinar les formulacions més estables. A més, la desnaturalització completa de l'SPI (95 °C, 15 min) es va realitzar abans del tractament CH amb l'objectiu d'analitzar la possibilitat d'obtenir característiques millorades de les emulsions. En concret, les emulsions UHPH tractades a 100 i 200 MPa amb un 20% d'oli van ser les més estables a nivell físic i oxidatiu a causa de la disminució de la mida de partícula, la major quantitat d'oli, i una major viscositat i quantitat de proteïna a la interfase oli-en aigua. El tractament tèrmic de l'SPI no va millorar l'estabilitat física de les emulsions, però sí l'estabilitat oxidativa quan es va utilitzar un 10% d'oli. El segon estudi consistí en la producció de diferents emulsions (20% d'oli) estabilitzades amb SPI i PPI i emprant 1 i 5 etapes de CH (55 MPa, Ti=20 °C) per avaluar l'eficiència d'encapsulació de CLA (6%), així com el seu alliberament i bioeficàcia utilitzant la digestió in vitro i un model cel·lular Caco-2 d'absorció intestinal. Totes les emulsions van protegir millor el CLA encapsulat a diferència del control (CLA en forma lliure), el qual es va oxidar durant l'emmagatzematge, així com després de la digestió in vitro i consegüent alliberament en les cèl·lules Caco-2. Es van trobar percentatges similars de biodisponibilitat del CLA en totes les emulsions. Un tercer estudi va avaluar l'efecte de la UHPH (200 MPa, Ti=20 °C) en les emulsions amb CLA en comparació a la CH (15 MPa, Ti=20 °C). Per a un altre lot de emulsions CH, també es va dur a terme un tractament tèrmic de pasteurització. La UHPH va produir una emulsió estèril i durant l'emmagatzematge, va mostrar millor estabilitat física en comparació amb els altres tractaments, i a més, va mantenir una òptima estabilitat oxidativa. Un cop més, es varen trobar percentatges similars de biodisponibilitat del CLA en totes les emulsions. En un l'últim estudi, es va avaluar la incorporació d'emulsions amb CLA tractades per UHPH (200 MPa, Ti=20 °C) i CH (15 MPa, Ti=20 °C) en un producte de base làctia UHT. El producte final que contenia les emulsions UHPH va mostrar una estabilitat fisicoquímica i propietats sensorials millorades durant 4 mesos d'emmagatzematge.
En los últimos 10 años se ha incrementado la investigación en emulsiones para la liberación de compuestos bioactivos. Las emulsiones convencionales de aceite-en-agua (> 1 µm) presentan una estabilidad fisicoquímica y oxidativa insuficiente; por lo tanto, y para superar este problema, se han llevado a cabo muchos esfuerzos en el desarrollo de nuevas tecnologías. La tecnología de Homogeneización a Ultra-Alta Presión (UHPH) se ha aplicado en diferentes emulsiones alimentarias con el fin de mejorar su estabilidad física. Por estas razones, es necesario el desarrollo de emulsiones UHPH de aceite-en-agua que contengan bioactivos lipófilos, como el ácido linoleico conjugado (CLA), con el fin de mejorar las características nutricionales y físico-químicas de las emulsiones, así como su vida útil. En esta tesis doctoral, se estudió la influencia de diferentes tecnologías (UHPH y homogeneización convencional; CH) e ingredientes (aislado proteico de soja, SPI; aislado proteico de guisante, PPI; aceite de soja y CLA) en emulsiones de aceite-en-agua. Además, se desarrolló un producto final de base láctea UHT conteniendo emulsiones con CLA. La presente investigación se dividió en cuatro partes. En un primer estudio, se evaluó el efecto de la UHPH (100-300 MPa, Ti=20 °C) en comparación a la CH (15 MPa, Ti=20 °C) sobre la estabilidad física y oxidativa de emulsiones de aceite de soja (10 y 20%) y estabilizadas con SPI, con el fin de determinar las formulaciones más estables. Además, se desnaturalizó el SPI (95 ° C, 15 min) antes del tratamiento CH con el objetivo de analizar la posibilidad de obtener características de las emulsiones mejoradas. En concreto, las emulsiones UHPH tratadas a 100 y 200 MPa con un 20% de aceite fueron las más estables a nivel físico y oxidativo debido a la disminución del tamaño de partícula, la mayor cantidad de aceite, y una mayor viscosidad y cantidad de proteína en la interfase aceite-en-agua. El tratamiento térmico del SPI no mejoró la estabilidad física de las emulsiones, pero sí la estabilidad oxidativa cuando se utilizó un 10% de aceite. El segundo estudio consistió en la producción de diferentes emulsiones (20% de aceite) estabilizadas con SPI y PPI, y se emplearon 1 y 5 pases de CH (55 MPa, Ti=20 °C) para determinar la eficiencia de encapsulación de CLA (6%), su liberación y bioeficacia utilizando la digestión in vitro y un modelo celular Caco-2 de absorción intestinal. Todas las emulsiones protegieron mejor el CLA encapsulado a diferencia del control (CLA en forma libre), el cual se oxidó durante el almacenamiento, así como después de la digestión in vitro y consiguiente liberación en las células Caco-2. Se encontraron porcentajes similares de biodisponibilidad del CLA en todas las emulsiones. Para evaluar el efecto de la UHPH (200 MPa, Ti=20 °C) en las emulsiones con CLA en comparación a la CH (15 MPa, Ti=20 °C), se realizó un tercer estudio. Para otro lote de emulsiones CH, también se llevó a cabo un tratamiento térmico de pasteurización. La UHPH produjo una emulsión estéril y durante el almacenamiento, mostró mejor estabilidad física en comparación a los otros tratamientos, y además, mantuvo una óptima estabilidad oxidativa. Una vez más, se encontraron porcentajes similares de biodisponibilidad del CLA en todas las emulsiones. En un el último estudio, se evaluó la incorporación de emulsiones con CLA tratadas por UHPH (200 MPa, Ti=20 °C) y CH (15 MPa, Ti=20 °C) en un producto de base láctea UHT. El producto final que contenía las emulsiones UHPH mostró una mayor estabilidad físico-química y propiedades sensoriales mejoradas durante 4 meses de almacenamiento.
Research on delivery of bioactive compounds by emulsion-based systems has increased over the last 10 years. Conventional oil-in-water emulsions (>1 μm) have poor physicochemical and oxidative stability; therefore, many efforts on novel technologies development have been employed to overcome this problem. Ultra-High Pressure Homogenization (UHPH) technology has been applied in different food emulsions in order to improve their physical stability. For these reasons, development of UHPH oil-in-water emulsions containing lipophilic bioactives, such as conjugated linoleic acid (CLA), to improve their physicochemical and nutritional characteristics and shelf-life is needed. In this thesis dissertation, the influence of different technologies (UHPH and Conventional Homogenization; CH) and ingredients (soy protein isolate, SPI; pea protein isolate, PPI; soybean oil and CLA) in oil-in-water emulsions was evaluated. In addition, a final UHT milkbased product containing CLA-emulsions was developed. This research has been divided into four parts. The first part studied the effect of UHPH (100- 300 MPa, Ti=20 °C) compared to CH (15 MPa, Ti=20 °C) on physical and oxidative stability of soybean oil (10 and 20%) emulsions stabilized by SPI was assessed in order to determine the most stable formulations. Also, complete denaturation of SPI (95 °C, 15 min) before CH treatment was performed with the aim of analyzing possible improvement of emulsion characteristics. Particularly, UHPH emulsions treated at 100 and 200 MPa with 20% of oil were the most physically and oxidatively stable due to low particle size values, higher amount of oil, and greater viscosity and protein surface load at the interface. The heating of SPI did not improve physical stability of emulsions but enhanced oxidative stability when 10% oil was used. The second study consisted in the assessment of different emulsions (20% oil) stabilized by SPI and PPI and homogenized by CH (55 MPa, Ti=20 °C) for 1 and 5 passes on the efficiency of 6% CLA encapsulation, its release and bioefficacy using in vitro digestion and a Caco-2 intestinal cell absorption model. All emulsions protected the encapsulated CLA better than the nonemulsified control in which CLA was oxidized during storage, as well as after in vitro digestion and delivery in Caco-2 cells. Similar percentages of CLA bioavailability were found for all emulsion treatments. To evaluate the effect of UHPH (200 MPa, Ti=20 °C) in CLA-emulsions in comparison to CH (15 MPa, Ti=20 °C), a third study was performed. For another batch of CH emulsions, a heat treatment was also conducted applying a pasteurization treatment. UHPH produced a sterile CLA-emulsion and showed better physical stability during storage compared to other treatments, maintaining an optimal oxidative stability. Again, similar percentages of CLA bioavailability in Caco-2 cells were found for all emulsion treatments. In the last study, the incorporation of CLA-emulsions obtained by UHPH (200 MPa, Ti=20 °C) and CH (15 MPa, Ti=20 °C) in a UHT milk-based product was evaluated. The final product containing the UHPH emulsions showed enhanced physicochemical stability and sensory properties during 4 months of storage.

Orientador: Marcelo Cristianini
Texto em português e inglês
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: A homogeneização à alta pressão (HAP) é uma operação unitária capaz de alterar a conformação e, consequentemente a funcionalidade de polissacarídeos,proteínas e enzimas. O objetivo deste trabalho foi avaliar o efeito da HAP na atividade e estabilidade de cinco enzimas comerciais com aplicação na indústria de alimentos (x-amilase de Aspergillus niger, protease neutra de Bacillus subtilis, b-galactosidase de Kluyveromyces lactis, amiloglicosidase de A. niger e glicose oxidase de A. niger). Para cada enzima, a atividade foi avaliada antes e após a HAP (até 200 MPa) em diferentes temperaturas e pH. Além disso, a reversibilidade dos efeitos do processo foi determinada indiretamente através da medida de atividade da enzima após um período de repouso. Os resultados de x-amilase demonstraram que a enzima é altamente estável ao processo de HAP (em pressões de até 150 MPa), independentemente do pH e temperatura de processo e da ausência de cálcio no tampão de diluição da enzima. Os resultados da b-galactosidase, por outro lado, mostraram que a enzima é pouco estável, apresentando redução da atividade (~30%) após HAP a 150 MPa quando processadas em pH não ótimo para atividade da enzima. Os resultados obtidos para a protease neutra, amiloglicosidase e glicose oxidase indicaram que o efeito da HAP foi dependente dos parâmetros utilizados no processo (pH, temperatura e pressão de homogeneização) e das condições utilizadas na medida de atividade (pH, temperatura e tempo de estocagem). Para estas três enzimas, significativos ganhos de atividade e/ou estabilidade foram observados para pelo menos uma das condições avaliadas, sendo que os mais importantes foram: (i) redução da temperatura ótima de atividade da protease neutra de 55 para 20ºC após HAP a 200 MPa, (ii) aumento da atividade da glicose-oxidase à 75ºC após HAP a 150 MPa, (iii) aumento da atividade residual entre 100 e 400% após armazenamento refrigerado de glicose-oxidase homogeneizada em diferentes pressões, (iv) aumento da atividade de amiloglicosidase à 80ºC após a HAP a 100 MPa. A reversibilidade das alterações observadas foi inferida pela avaliação da atividade da enzima após um período de repouso, sendo as alterações determinadas como reversíveis (protease neutra, glicose-oxidase, amiloglicosidase) ou irreversíveis (protease neutra, glicose-oxidase, b-galactosidase) em função dos parâmetros de processo. O efeito de processamentos sequenciais sobre a glicose oxidase, a protease e a amiloglicosidase também foi avaliado e os resultados demonstraram que, para a maioria das condições estudadas, a atividade da enzima se manteve igual à obtida após o primeiro ciclo de homogeneização ou apresentou uma redução. Uma exceção foram os resultados da glicose oxidase homogeneizada a 150 MPa por 3 vezes, que apresentou aumento de atividade de aproximadamente 150% em relação à enzima nativa. A partir dos resultados, conclui-se que o efeito da HAP é diferente para cada enzima e que as maiores alterações ocorrem em condições de atividade não ótima e para enzimas de estruturas mais complexas, como é o caso da glicose oxidase. Os resultados obtidos apresentam aplicação direta, para modificação e melhoria do desempenho de enzimas comerciais, e preenchem uma lacuna científica importante sobre o conhecimento dos efeitos do processo de HAP em enzimas
Abstract: High pressure homogenization (HPH) is a unitary operation capable to alter the conformation and, consequently, the functionality of polyssacharides, proteins and enzymes. This work aimed to study the HPH effects on activity and stability of five commercial enzymes intensively applied in food industry (x-amylase from Aspergillus niger, b-galactosidase from Kluyveromyces lactis, neutral protease from Bacillus subtilis, amyloglucosidase from A. niger and glucose-oxidase from A. niger). The activity of each enzyme was studied before and after HPH process (up to 200 MPa) at different temperatures and pH. Moreover, the process reversibility was indirectly determined by the activity measured after a rest period under refrigeration (8ºC).The results revealed that x-amylase was highly stable to HPH up to 150 MPa,independent on the pH or temperature used in the HPH process or the presence of calcium in buffer. On the other hand, the results of b-galactosidase indicated that enzyme was partially inactivated (~ 30%) after homogenization at 150 MPa when processed at non optimum pH. The HPH effects on neutral protease, amyloglucosidase (AMG) and glucose oxidase (GO) were dependent on the process parameters (pH, temperature and homogenization conditions) and the activity measurement conditions (pH, temperature and storage time). For these enzymes, it was observed activity and/or stability improvement after some process. The main improvements were: (i) change of the optimum temperature of neutral protease from 55 to 20ºC after HPH at 200 MPa, (ii) improvement of GO activity at 75ºC after HPH at 150 MPa, (iii) enzyme activity improvement between 100 and 400% after GO refrigerated storage, (iv) improvement on amyloglucosidase activity at 80ºC after HPH at 100 MPa. The reversibility of the HPH effects was evaluated after a rest period.The reversibility was dependent on the process parameters; but, in general, neutral protease, GO and AMG were reversible, while the results of neutral protease, GO and b-galactosidase were irreversible. The effects of sequential homogenization processes (sequential passes) on GO, AMG and neutral protease were evaluated and the results showed that the enzyme activity remained equal or reduced after 2 or 3 cycles of homogenization. An exception was the result obtained for GO homogenized at 150 MPa, which showed an activity improvement of 150% after three passes. The results evaluation of this research showed that HPH effects on enzymes were different for each enzyme. The main alterations occured at non optimum condition of enzyme activity and for enzymes with complex structure as the GO. The obtained results can be directly applied for improvement of enzyme industrial production. Also, the results enriched the scientific knowledge about the HPH effects on enzymes
Doutorado
Tecnologia de Alimentos
Doutora em Tecnologia de Alimentos

Orientadores: Marcelo Cristianini, Alline Artigiani Lima Tribst
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: A homogeneização à alta pressão (HAP) é um processo capaz de alterar a conformação e funcionalidade de enzimas. Os objetivos deste trabalho foram: (i) avaliar a influência da HAP até 190 MPa nas atividades proteolítica e de coagulação do leite bem como na estabilidade de quatro enzimas coagulantes do leite, (ii) acompanhar o processo de coagulação por ensaios reológicos e (iii) avaliar o desenvolvimento dos géis por 24 horas por meio das análises de proteólise, sinérese, reologia e microscopia. As avaliações foram feitas comparando-se os resultados obtidos com as enzimas processadas e não processadas. O coalho de vitelo processado a 190 MPa apresentou redução de 52% na atividade proteolítica, aumento da taxa de coagulação do leite e gel formado mais consistente. A avaliação deste gel por 24h indicou a formação de uma rede proteica com menor proteólise, maior sinérese, maior consistência e menor porosidade. Após processamento a 150 MPa, o coalho de bovino adulto apresentou redução da atividade proteolítica, aumento da atividade e estabilidade de coagulação do leite, maior taxa de coagulação do leite e formação de gel com maior consistência. O gel se mostrou mais compacto, firme e com maior expulsão do soro da matriz proteica nas 24h em que foi avaliado. A protease fúngica do Rhizomucor miehei foi a enzima mais resistente ao processo de HAP, sofrendo mínima ou nenhuma alteração na atividade proteolítica e de coagulação do leite quando processada até 190 MPa em diferentes concentrações e em múltiplos processos consecutivos. Entretanto, na avaliação reológica da coagulação do leite utilizando-se a protease fúngica homogeneizada a 190 MPa por até 3 ciclos ou quando homogeneizada a 190 MPa em soluções com concetração de 20 % foi observado aumento da consistência do gel. Para pepsina suína, as alterações na atividade proteolítica e de coagulação do leite só foram observadas durante a estocagem, com redução na atividade proteolítica e um aumento na atividade de coagulação do leite para enzima processada a 150 MPa. No entanto, esta enzima processada promoveu uma coagulação do leite mais rápida formando um gel mais consistente, mesmo imediatamente após o processamento por HAP. Durante a observação deste gel por 24h, este se mostrou mais compacto, firme, menos poroso e com maior liberação de soro da matriz proteica. De uma forma geral foi possível concluir que as maiores pressões aplicadas (150 MPa e 190 MPa) afetaram positivamente as enzimas avaliadas, com redução da atividade proteolítica inespecífica e aumento da atividade de coagulação de leite, com consequente formação de géis com menores níveis de proteólise, o que favorece a manutenção da rede proteica rígida, firme e coesa. Desta forma, conclui-se que a HAP é um processo promissor que pode ser aplicado como uma tecnologia para melhorar as características hidrolíticas das enzimas coagulantes do leite, especialmente quando se deseja diminuir atividade proteolítica e aumentar sua atividade de coagulação do leite. Além disso, a menor proteólise no gel pode resultar numa extensão da vida de prateleira de queijos frescos, por, possivelmente, reduzir a formação de compostos de sabor indesejável
Abstract: High pressure homogenization (HPH) is a process that can alter the conformation and functionality of enzymes. The objectives of this study were: (i) evaluate the influence of HPH up to 190 MPa on the proteolytic and milk-clotting activities and stability of four milk-clotting enzymes, (ii) monitor the coagulation process by rheological assays and (iii) evaluate the gel development for 24 hours analyzing proteolysis, syneresis, rheological and microstructural behavior. The evaluations were performed by comparing the results between the processed and non-processed enzymes. The calf rennet processed at 190 MPa decreased 52 % its proteolytic activity, increased the rate of milk-clotting and a more consistent gel was formed. The evaluation of the gel for 24 hours indicated the formation of a protein network with lower proteolysis, higher syneresis, higher consistency and lower porosity. After processing at 150 MPa adult bovine rennet showed a reduction proteolytic activity, increase activity and stability of milk-clotting, higher milk-clotting rate and formed more consistent gels. This gel was more compact, firm and higher whey separation of protein matrix during the 24 hours of evaluation. The fungal protease from Rhizomucor miehei was the most resistant enzyme to the HPH process, showing minimal or no change in proteolytic activity and milk coagulation when processed up to 190 MPa at different concentrations and multiple consecutive processes. However, in the rheological evaluation of milk coagulation using fungal protease homogenized to 190 MPa for up 3 cycles or when homogenized in a solution with a concentration of 20% observed increase in the consistency of the gel. For porcine pepsin, changes on proteolytic activity and milk coagulation were only observed during storage, with reduction of proteolytic activity and an increase on the milk-clotting activity for the enzyme processed at 150 MPa. However, this enzyme promoted a faster coagulation of milk forming more consistent gel immediately after the processing by HPH. During the observation of this gel for 24 hours, this was more compact, firm, less porous and more release of whey of the protein matrix. Overall it was concluded that the highest applied pressures (150 MPa and 190 MPa) positively affected the enzymes with reduced nonspecific proteolytic activity and increased milk-clotting activity, with consequent formation of gels with lower levels of proteolysis, which favors the maintenance of a network of protein rigid, firm and cohesive. Thus, it is concluded that HPH is a promising process that can be applied as a technology to improve the hydrolytic characteristics of milk coagulating enzymes, especially to reduce proteolytic activity and increase the milk-clotting activity. Furthermore, the lower proteolysis in the gel may result in an extension of the shelf life of fresh cheese, by possibly reducing the formation of bitterness flavor
Mestrado
Tecnologia de Alimentos
Mestre em Tecnologia de Alimentos

O uso de princípios ativos a base de plantas em alimentos pode ser interessante devido à sua atividade (ex.: antioxidante, antimicrobiana, dentre outros), entretanto, alguns desses aditivos são hidrofóbicos, sendo, portanto, de difícil dispersão na formulação do alimento, que é geralmente rico em água. Do mesmo modo, a diminuição da concentração utilizada de antisépticos é um desafio atual. Uma alternativa é a utilização desses princípios ativos na forma de nanoemulsões com baixas concentrações de princípios ativos. O objetivo geral desta tese foi o desenvolvimento de nanoemulsões a base de eugenol e extrato de pimenta dedo-demoça para uso em alimentos, e a base de triclosan, para uso tópico, como alternativa para higienização de mãos de manipuladores de alimentos. O desenvolvimento das nanoemulsões foi realizado com o auxílio de um homogeneizador de alta velocidade seguido de um homogeneizador de alta pressão. Foram estudados os seguintes parâmetros, em vários experimentos: tipo e concentração de tensoativos, valor ideal de HLB, relação fase lipídica/fase aquosa, relação tensoativo/óleo, e composição e processo. As nanoemulsões foram otimizadas por desenho experimental, avaliadas quanto à estabilidade por estresse térmico e força centrífuga e estabilidade ao armazenamento por 90 dias, caracterizadas por diferentes análises, incluindo a determinação do potencial antioxidante e antibacteriano. Foram desenvolvidas nanoemulsões com tamanho médio de gota menor que 200 nm, sendo que, a estabilidade frente ao estresse térmico e força centrífuga variaram para cada sistema, mas, todas nanoemulsões foram estáveis pelo período de 90 dias em temperatura ambiente. A nanoemulsão de eugenol demonstrou bom potencial antioxidante e a nanoemulsão de triclosan demonstrou alta atividade bacteriostática na concentração de 3% e atividade bactericida em concentração menor que 40µg/mL pelo período de 90 dias. As nanoemulsões desenvolvidas têm potencial de aplicação nas indústrias de alimentos e sanitária.
The utilization of plant bioactives in food can be interesting because of its activity (eg.: antioxidant, antimicrobial, among others) and to minimize the use of their high concentration of antiseptics. However, some of these additives are hydrophobic, and, therefore, are more difficult to be dispersed in food, which are generally water-based. A good alternative is the use of low concentration of these actives as nanoemulsions. The aim of this thesis was the development of nanoemulsions with eugenol, \"dedo-de-moça\" pepper extract for food applications, and of triclosan for topical application as hand\'s cleaner. The studies for development of these nanoemulsions were carried out with a high speed homogenizer followed by a high pressure homogenizer. The following parameters were tested: type and concentration of surfactants, ideal HLB value, lipid phase/aqueous phase relation, surfactant/oil relation, and composition and processing conditions. Nanoemulsions were optimized by experimental design, evaluated by preliminary and storage stability, characterized by different analysis, including antioxidant and antimicrobial activities. Nanoemulsions with mean droplet size lower than 200 nm were developed; the thermal stress and centrifuge stabilities were different for each system, but, all nanoemulsions were stable over 90 days on ambient temperature. The eugenol nanoemulsion had good antioxidant activity and triclosan nanoemulsion had high antimicrobial activity in the concentration of 3% and a bactericidal activity at concentration lower than 40µg/mL over 90 days period. The nanoemulsions developed have potential for applications in food and sanitary industries.

Orientador: Maria Helena Andrade Santana
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química
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Resumo: O mitotano, fármaco utilizado para a terapia medicamentosa do carcinoma adrenocortical, apresenta baixa solubilidade aquosa e baixa permeabilidade intestinal. Nanopartículas lipídicas sólidas (SLNs) e carreadores lipídicos nanoestruturados (NLCs) são úteis para a incorporação de fármacos com essas características. O objetivo deste trabalho foi preparar e caracterizar SLNs e NLCs encapsulando mitotano racêmico, estáveis durante a estocagem, como uma estratégia nanotecnológica para melhorar a solubilidade aquosa e permeação intestinal do mitotano. As SLNs foram compostas do lipídeo cetil palmitato e as NLCs de estearato de polioxietileno (40) e triacilgliceróis de ácido cáprico/caprílico e dos tensoativos polissorbato 80 e trioleato de sorbitano. A proporção mássica entre os tensoativos que conferiu estabilidade à fase oleosa foi determinada. Para avaliar estabilidade, as SLNs e NLCs foram caracterizadas através do diâmetro médio, polidispersidade, potencial zeta. A encapsulação do mitotano foi evidenciada por calorimetria diferencial de varredura e termogravimetria, e quantificada através da eficiência encapsulação e carregamento do fármaco pela matriz lipídica. Adicionalmente, as SLNs e NLCs foram avaliadas pela retenção do mitotano em diferentes pHs in vitro e permeação intestinal ex vivo através do modelo de saco intestinal invertido. Os resultados experimentais mostraram que as composições mássicas que asseguraram a estabilidade durante 28 dias de armazenamento a 4 °C nas nanopartículas foram: cetil palmitato (10%), polissorbato 80 (2,35%) e trioleato de sorbitano (2,75%) nas SLNs. Em NLCs foi: estearato de polioxietileno (7%), triacilgliceróis (3%), polissorbato 80 (3,23%) e trioleato de sorbitano (1,76%). O diâmetro médio das SLNs isentas de fármaco foi 185,20±1,39 nm, polidispersidade 0,14 e potencial zeta -24,80±0,27 mV. Nas mesmas condições, as NLCs tiveram diâmetro médio 91,79±0,69 nm, polidispersidade de 0,27 e potencial zeta e -11,50±0,92 mV. A inclusão do mitotano aumentou o diâmetro médio das SLNs e NLCs para 187,40±0,17 nm e 94,50±0,26 nm, reduziu a polidispersidade para 0,12 e 0,22 bem como o potencial zeta -21,10±0,55 mV e -8,05±0,20 mV, respectivamente. Ambas as partículas apresentaram eficiências de encapsulação do mitotano superiores a 90%, porém o carregamento das NLCs (0,25 g mitotano /g nanopartículas) foi cinco vezes maior que as SLNs (0,05 g mitotano /g nanopartículas). O comportamento térmico evidenciou a internalização do mitotano nas partículas. Em condições de pH e tempo de residência que mimetizam o trato gastrointestinal (pHs1,2 e 6,8, 3h), foi liberado, aproximadamente, 90% e 100% do mitotano das SLNs, 30% do mitotano das NLCs, em ambos os pHs. A pH 7,4, houve total liberação do mitotano em 15 minutos das SLNs, enquanto a liberação das NLCs foi aproximadamente 50% em 24 horas. Os ensaios de permeação intestinal ex vivo mostraram que a absorção do mitotano das partículas foi maior quando comparados ao veiculado em óleo de oliva ou a partir do pó micronizado do fármaco. Estes resultados sugerem que as NLCs sejam carreadores promissores para melhorar a biodisponibilidade oral do mitotano e melhorando o tratamento do carcinoma adrenocortical
Abstract: Mitotane is used for adrenocortical carcinoma drug therapy and has low aqueous solubility and low intestinal permeability. Solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs) are useful for incorporation of drugs with these characteristics. The aim of this work was to prepare and to characterize SLNs and NLCs encapsulating racemic mitotane, stable during storage as a nanotechnology strategy to improve aqueous solubility and intestinal permeability of mitotane. The SLNs were composed by cetyl palmitate and the NLCs of polyoxyethylene (40) stearate and capric/caprylic triglycerides as lipids and the surfactants polysorbate 80 and sorbitan trioleate. The weight ratio between the surfactants that conferred storage stability to the oil phase was determined. To evaluate storage stability, the SLNs and NLCs were characterized by mean diameter, polydispersity index and zeta potential. Encapsulation of mitotane was evidenced by differential scanning calorimetry and thermogravimetry, and quantified using the encapsulation efficiency and drug loading by the lipid matrix. Additionally, the SLNs and NLCs were evaluated by the retention of mitotane in vitro and ex vivo intestinal permeation by everted gut sac model. The experimental results showed that the mass compositions of the nanoparticles which ensured storage stability over 28 days of storage at 4 °C were: cetyl palmitate (10%), polysorbate 80 (2.35%) and sorbitan trioleate (2.75%) in SLNs. In NLCs was: polyoxyethylene (7%), triglycerides (3%), polysorbate 80 (3.23%) and sorbitan trioleate (1.76%). The mean diameter of the unloaded SLNs was 185.20±1.39 nm, polydispersity index 0.14 and zeta potential -24.80±0.27 mV. At the same conditions, the NLCs had mean diameter 91.79±0.69 nm, polydispersity index 0.27 and zeta potential -11.50±0.92 mV. The inclusion of mitotane increased the mean diameter of SLNs and NLCs to 187.40±0.17 nm and 94.50±0.26 nm, decreased the polydispersity index to 0.12 and 0.22 and so well the zeta potential to -21.10±0.55 mV and -8.05±0.20 mV, respectively. Both particles showed mitotane entrapment efficiencies greater than 90%, however drug loading of NLCs (0.25 g mitotane /g nanoparticles) were 5-fold higher than the SLNs (0.05 g mitotane /g nanoparticles). The thermal behavior showed internalization of mitotane in the particles. Under conditions of pH and residence time that mimic the gastrointestinal tract (pHs 1.2 to 6.8, 3h) mitotane was released approximately 90% and 100% from SLNs, 30% from NLCs at both pHs. At pH 7.4, there was a complete release of mitotane from SLNs over 15 minutes, while the release from NLCs was approximadely 50% in 12 hours. The ex vivo intestinal permeation assay showed that the absorption of mitotane by the particles were greater than dissolved in olive oil or from micronized powder. These results suggest that NLCs might be promising carriers for improving the oral bioavailability of mitotane and treatment of adrenocortical carcinoma
Mestrado
Desenvolvimento de Processos Biotecnologicos
Mestra em Engenharia Química

Orientador: Marcelo Cristianini
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: A utilização de métodos inovadores de estabilização microbiológica em alimentos como alternativa ao método térmico tem se mostrado promissora para a obtenção do melhor compromisso entre segurança e qualidade. Alta pressão dinâmica e bioproteção são tecnologias inovadoras: alta pressão dinâmica (APD) ou homogeneização por ultra alta pressão (HUAP) utiliza-se de sistemas de compressão de fluido a pressões superiores a 100MPa, para então forçar o fluido através de uma estreita fenda causando brusca aceleração e forças de cisalhamento resultando na ruptura de células de microrganismos; bioproteção, por sua vez, consiste no emprego de substancias naturais ou produzidas a partir de fontes alimentícias com atividade antimicrobiana para a estabilização microbiológica de alimentos e processos. Concentrações inibitórias mínimas (MIC) de nisina, lisozima, extrato de lúpulo e sakacina foram medidas contra Lactobacillus brevis e delbruecki, Acetobacter aceti, Pediococcus sp. e duas cepas de leveduras selvagens. Nisina foi capaz de inibir o crescimento de Pediococcus sp (2,0 mg.L-1), L. brevis (3,0 mg.L-1) e L. delbruecki (0,8 mg.L-1), no entanto, o A. aceti não foi inibido até concentrações de 0,2 g.L-1. Lisozima inibiu L. delbruecki (1,0 mg.L-1) e exibiu uma inibição transitória sobre L. brevis (50 mg.L-1). Alta pressão dinâmica foi empregada sobre os mesmos microrganismos a pressões de 60, 100, 150, 250 MPa em cerveja (pH 4,5; 2,5oP; 4,7oGL). O valor de Dp, taxa de redução logarítmica do microrganismo mais resistente foi calculada. Um experimento de múltiplas passagens foi realizado a pressões subletais até 3 passagens pelo sistema. Todas as linhagens foram inativadas a pressões de até 250 MPa, mesmo em contagens iniciais altas da ordem de (106UFC/mL). L. delbruecki foi a linhagem mais resistente com um Dp de 38,9 MPa. No ensaio de múltiplas passagens pelo sistema foi observado que o efeito de tratamentos consecutivos resultou em um efeito aditivo, não tendo sido observado sinergismo ou antagonismo por seleção de bactérias resistentes. Foi avaliado o efeito da temperatura de entrada (6, 10, 20, 23, 30, 44, 50oC) e concentração de CO2 na cerveja (0; 0,5; 1,0 e 2,0 volumes a 25oC) na destruição de L. delbruecki, a 150 e 170 MPa. A temperatura de entrada causou um efeito positivo sobre a destruição e 3 ciclos logarítmicos de diferença no número de reduções decimais foi observado entre 6 e 50°C. Por sua vez, a concentração de CO2 causou um efeito negativo sobre a destruição. Lactobacillus brevis, um dos microrganismos mais resistentes ao tratamento de cerveja por alta pressão, foi utilizado nos estudos de efeito combinado entre alta pressão dinâmica e bioproteção, uma vez que L. delbruecki apresentou alta sensibilidade aos antimicrobianos. A inibição pela lisozima foi transitória e a adição de 50 mg.L-1 foi capaz de reduzir um ciclo logarítmico da contagem após duas horas de contato. O tratamento por alta pressão dinâmica da suspensão de L.brevis resultou em uma redução de sete ciclos logarítmicos a 200 MPa. Lisozima mostrou-se resistente ao processo APD, não perdendo atividade enzimática ou antimicrobiana até a pressão de 200 MPa. Um tratamento combinado utilizando 50 mg.L-1 e APD (150 a 170 MPa) resultou em uma redução de 6 ciclos logarítmicos estabilizando a contagem após o tratamento por uma semana em tampão fosfato, a temperatura ambiente (25°C). L. brevis foi inoculado a seguir em cerveja a uma concentração de 106 UFC/mL, em presença de 100mg.L-1 de lisozima e reservado por uma hora. A suspensão foi dividida em alíquotas e cada uma tratada por alta pressão dinâmica a 100 e 140 MPa. A contagem de viáveis em cada uma das etapas mostrou que a lisozima promoveu uma destruição de um ciclo seguida de um e quatro ciclos logarítmicos devido ao tratamento por alta pressão a 100 e 140 MPa, respectivamente. Uma completa redução da carga inicial foi observada ao final de 10 dias a temperatura ambiente em cerveja nas amostras tratadas por pressão. Nisina por si só provou ser um bioprotetor eficaz no controle de Lactobacillus em cerveja, e mostrou-se resistente ao processo de alta pressão. O efeito do processo de alta pressão dinâmica sobre a qualidade da cerveja foi avaliado. Cerveja tipo pilsen, não filtrada e não estabilizada físico-quimicamente foi submetida ao tratamento por alta pressão entre 100 e 300 MPa. Foi medida a cor e turbidez segundo os padrões Cie L*a*b* e ASBC/Hunterlab. Foi realizado um teste de vida útil por 100 dias após tratamento de cerveja a 250 MPa, medindo-se o potencial óxido redutor, a turbidez e cor (ASBC), periodicamente (aproximadamente mês a mês). Os resultados mostraram que o processo foi efetivo na retenção da cor mas a turbidez foi aumentada em 5 vezes em tratamentos a pressão de 300 MPa. Também o teste de vida útil resultou em boa retenção da cor durante o período e do potencial de óxido redução, mas a turbidez aumentou a 140 FTU (Formazin Turbidity Units) em um mês e um precipitado foi formado. Alta pressão dinâmica provou ser um método eficiente na estabilização microbiológica de cervejas, e pode ser eficientemente associada a outros métodos de controle como a bioproteção. Se por um lado microrganismos Gram negativos foram resistentes aos bioprotetores utilizados, esses se mostraram mais susceptíveis ao tratamento por alta pressão tornando os métodos combinados complementares. Novos trabalhos devem ser realizados com cerveja filtrada e estabilizada físico-quimicamente, para se avaliar se a redução de pressão devido à combinação de tratamentos resultaria em produtos com boa qualidade, principalmente no que se refere à turbidez
Abstract: The use of novel microbial stabilization techniques as alternatives to thermal treatments are promising solutions for a compromise between quality and safety. As such, dynamic high pressure and biopreservation are novel techniques: dynamic high pressure (DHP or ultra high pressure homogenization (UHPH) is a technology that uses fluid compression to reach pressures higher than 100 MPa in order to acceleratethe passage of the fluid through a narrow gap, causing shear and stress to the microorganism cell structure and resulting in cell disruption; biopreservation consists in the employment of naturally occurring or biologically produced substances with antimicrobial activity in order to impart microbiological stability to a food product or food manufacturing process. This work aimed to evaluate the use of dynamic high pressure treatment and biopreservation to achieve microbial stability in lager beer. Minimum inhibitory concentrations (MIC) of nisin, lysozyme, hop aroma extract and sakacin were measured against two wild yeast strains from a brewery. Nisin was able to inhibit the growth of Pediococcus sp (2.0 mg.L-1), L. brevis (3.0 mg.L-1) and L. delbruecki (0.8 mg.L-1), although the wild yeasts and A. aceti were not inhibited up to 0.2 g.L-1. Lysozyme inhibited L. delbruecki (1.0 mg.L-1) and showed a transitory inhibition of L. brevis (50 mg.L-1). None of the biopreservatives inhibited the wild yeasts. High dynamic pressure was applied to the same strains at 60, 150, 100, 250 MPa in beer (pH 4.5, 2.5oP, 4.7oGL). The logarithmic number of reductions rate (Dp) was calculated. A multiple pass experiment was performed at sublethal pressures for up to 3 passes for the most resistant strain. All strains were totally inactivated at pressures of up to 250 MPa even at high initial counts (106CFU/mL). L. delbruecki was the most resistant strain. The Dp was 38.9 MPa. The multiple pass assays showed an additive effect, synergism or the selection of resistant bacteria not being observed. The effect of the inlet temperature (6, 10, 20, 23, 30, 44, 50oC) and CO2 content in beer (0, 0.5, 1.0 and 2.0 volumes at 25oC) on the destruction of Lactobacillus delbruecki by dynamic high pressure at 150 MPa and 170 MPa was evaluated: the inlet temperature showed a significant positive effect on the destruction of the strain and a 3 log destruction difference was observed between 6 and 50oC. The increase in concentration of CO2 showed a negative effect on the destruction. Lactobacillus brevis was used in the combination of high pressure and lysozyme assay, because it showed higher resistance to antimicrobials than L. delbruecki, and was the second microorganism in resistance to the high pressure treatment. The inhibition by lysozyme was transitory. Lysozyme alone, added at 50 mg.L-1 to the suspension, caused a reduction of 1 logarithmic cycle after two hours of contact. The DHP treatment against L. brevis resulted in a reduction of 7 log cycles at 200 MPa. Lysozyme was resistant to DHP, with no loss of muramidase activity until 200 MPa or significant loss of antimicrobial power. A combined treatment was applied using 50 mg.L-1 of lysozyme and a pressure between 150 and 170 Mpa. The somatic effect showed a reduction of about 6 logarithmic cycles and the L. brevis count remained stable after the pressure treatment for a week with the samples stored at room temperature (25oC). L. brevis was inoculated into beer (106 CFU.mL-1), containing 100 mg.L-1 of lysozyme, left for one hour and then treated by dynamic high pressure at 100 or 140 MPa. Samples were taken for survivor enumeration. Lysozyme promoted a destruction of one log cycle in L. brevis after one hour of contact and the subsequent DHP treatment reduced a further one log cycle and 4 log cycles at 100 and 140 MPa, respectively. The treated samples showed complete reduction of the initial load of microorganisms after 10 days of storage. Nisin alone proved to be useful in the control of Lactobacilli in beer, and was shown to be resistant to the high pressure treatment. The effect of the dynamic high pressure treatment was evaluated in the quality parameters of turbidity, colour and redox potential of Lager beer. Lager beer, unfiltered and not physico-chemically stabilized, was treated by dynamic high pressure from 100 to 300 MPa at 25oC. The colour and permanent turbidity were measured using the CieL*a*b* and ASBC/Hunterlab standards. The redox potential was determined by the indicator time test method for the 250 MPa treatments. Samples treated at 250 MPa were stored under ambient conditions for 100 days and the turbidity (ASBC), colour (ASBC) and redox potential value measured periodically. The results showed that the process was effective in retaining the beer colour, altough higher pressures (>200MPa) caused the turbidity to increase up to five fold (300 MPa). The storage results showed good colour stability and redox potential evolution, however the turbidity increased to 140 FTU in one month, and a precipitate formed. DHP proved itself to be a effective the method for microbial stabilization of beer, and can be effectively associated with other control methods, such as biopreservatives. Gram negative strains and wild yeasts although resistant to biopreservatives, were clearly sensitive to pressure treatment. Further work should be performed with filtered beer to evaluate if a reduction in the pressure due to the combined use of treatments would improve the quality with respect to turbidity, which was negatively affected by the dynamic high pressure treatment, and shown to be a major drawback
Doutorado
Doutor em Tecnologia de Alimentos

Orientador: Marcelo Cristianini
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: Diferentes tratamentos não térmicos têm sido estudados nos últimos anos como métodos substitutos ou complementares dos tradicionais tratamentos térmicos na conservação de alimentos. Estes tratamentos apresentam a vantagem de não causar alterações indesejáveis no sabor dos produtos ou perdas de nutrientes. Dentre os tratamentos não térmicos, um dos mais estudados é o processamento a alta pressão, que pode ser aplicado através de dois métodos: o isostático e o de homogeneização. O método isostático têm sido estudado com maior freqüência, no entanto, o método de homogeneização apresenta a vantagem de proporcionar uma produção contínua. Este trabalho visou avaliar o tratamento de homogeneização de alta pressão aplicado à água de coco para eliminação da atividade enzimática e de possíveis contaminações microbiológicas do produto. Os processamentos de Homogeneização a Ultra Alta Pressão (HUAP) foram realizados no equipamento nG7400H:320, fabricado pela Stansted Fluid Power (Stansted, Inglaterra), que atinge pressões de até 320 MPa. Estudou-se a influência da pressão de processo e do pH do produto nas atividades enzimáticas de peroxidase e polifenoloxidase e na destruição da bactéria Lactobacillus fructivorans e da levedura Saccaromyces cerevisiae. Os tratamentos realizados não causaram inativação satisfatória das enzimas presentes na água de coco, porém há evidências de que o tratamento pode ser eficaz se for efetuado em pressões mais elevadas. De maneira geral, a peroxidase apresentou maior resistência ao tratamento do que a polifenoloxidase. A realização de três tratamentos seqüenciais a 300 MPa causou redução parcial da atividade das enzimas, embora não tenha sido verificada uma potencialização do efeito dos tratamentos HUAP quando realizados seqüencialmente. O tratamento mostrou-se eficaz na eliminação de contaminação microbiológica do produto. Para o Lactobacillus fructivorans, a aplicação de tratamentos em pressões maiores que 250 MPa eliminou totalmente a carga de microrganismos inoculada no produto, de 2,0 x 107 UFC/mL. O pH do produto exerceu uma influência muito menor na eliminação dos microrganismos do que a pressão de processo. Em altas pressões (próximas a 300 MPa), o processamento mostrou-se mais efetivo na destruição do Lactobacillus fructivorans quando utilizado em produtos com pH mais elevado. Para baixas pressões, o efeito foi inverso, ou seja, tratamentos em produtos com pHs mais ácidos causaram maior inativação. Para as leveduras (Saccaromyces cerevisiae), a aplicação de tratamentos a pressões acima de 200 MPa eliminou totalmente a carga microbiana inoculada na água de coco, que era de 4,4 x 105 UFC/mL. O estudo da influência do pH não apresentou resultados conclusivos porque a maioria dos tratamentos realizados nesta parte do estudo eliminou todas as células de microrganismos presentes no produto, o que dificultou a análise dos dados. Observou-se uma tendência de que valores menores do pH do produto possam favorecer a eliminação das células das leveduras durante o processamento Nas condições estudadas pode-se concluir que o tratamento de homogeneização a alta pressão foi eficiente na eliminação dos microrganismos estudados mas não foi eficiente para causar a inativação das enzimas presentes no produto (peroxidase e polifenoloxidase). Com isto, espera-se que a água de coco tratada pelo método HUAP apresente boa estabilidade microbiológica, mas não apresente boa estabilidade química, ficando sujeita a alterações devido a reações catalisadas por estas enzimas
Abstract: Different not thermal treatments have been studied in the last years as substitute or complementary methods of the traditional thermal treatments in the food conservation. These treatments present the advantage not to cause alterations undesirable in the flavor of the products or losses of nutrients. Amongst the not thermal treatments, one of the most studied is the processing the high pressure, that can be applied through two methods: the isostático and of homogenization. The isostático method has been studied more frequently, however, the homogenization method presents the advantage to provide a continuous production. This work aimed at to evaluate the applied treatment of high-pressure homogenization to the water of coconut for elimination of the enzymatic activity and of possible microbiological contaminations of the product. The processings of Homogenization Ultra Alta Pressão (HUAP) had been carried through in the equipment nG7400H:320, manufactured for the Stansted Fluid Power (Stansted, England), that it reaches pressures of up to 320 MPa. Studied it influence of the pressure of process and pH of the product in the enzymatic activities of peroxidase and polifenoloxidase and in the destruction of the Lactobacillus bacterium fructivorans and the Saccaromyces leavening cerevisiae. The carried through treatments had not caused satisfactory inativação das enzymes gifts na coconut water, however it has evidences of that the treatment can be efficient will have been effected in raised pressures more. In general way, peroxidase presented greater resistance to the treatment of that polifenoloxidase. The accomplishment of three sequential treatments the 300 MPa caused partial reduction of the activity of enzymes, has even so not been verified a potencialização of the effect of treatments HUAP when carried through sequentially. The treatment revealed efficient in the elimination of microbiological contamination of the product. For the Lactobacillus fructivorans, the application of treatments in 250 bigger pressures that MPa total eliminated the load of microrganismos inoculated in the product, of 2,0 x 107 UFC/mL. PH of the product very exerted a lesser influence in the elimination of the microrganismos of that the process pressure. In high pressures (next the 300 MPa), the processing more revealed more effective in the destruction of the Lactobacillus fructivorans when used in products with pH raised. For low pressures, the effect was inverse, or either, treatments in more acid products with pHs had caused greater inativação. For the leavenings (Saccaromyces cerevisiae), the application of treatments the 200 pressures above of MPa total eliminated the inoculated microbiana load in the coconut water, that was of 4,4 x 105 UFC/mL. The study of the influence of pH it did not present resulted conclusive because the majority of the treatments carried through in this part of the study eliminated all the cells of microrganismos gifts in the product, what it made it difficult the analysis of the data. A trend was observed of that lesser values of pH of the product can favor the elimination of the cells of the leavenings during the processing In the studied conditions can be concluded that the treatment of homogenization the high pressure was efficient in the elimination of the studied microrganismos but it was not efficient to cause the inativação of enzymes gifts in the product (peroxidase and polifenoloxidase). With this, one expects that the water of coconut treated for method HUAP presents good microbiological stability, but does not present good chemical stability, being subjects the alterations due the reactions catalyzed for these enzymes
Mestrado
Mestre em Tecnologia de Alimentos

Orientadores: Marcelo Cristianini, Albert Ibarz
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: A homogeneização a alta pressão (HAP) tem sido estudada por diversos autores como metodologia não térmica para a conservação de alimentos, especialmente produtos de frutas. Trabalhos realizados com esses produtos e testes industriais indicam aumento de consistência devido a HAP. No entanto, poucos trabalhos da literatura estudam alterações físico-químicas em produtos de frutas devido à HAP, especialmente em relação às características reológicas. A avaliação de tais alterações é essencial não só para o entendimento e dimensionamento do processo, mas também permite a utilização do método para provocar alterações intencionais e desejáveis em alimentos, como aumento de consistência. O presente trabalho estudou o efeito da homogeneização a alta pressão (HAP) nas características reológicas de suco de tomate. Na primeira parte do trabalho, avaliaram-se as propriedades reológicas dependentes do tempo, em estado estacionário e de viscoelasticidade linear de sucos não processados, determinando assim as técnicas e os modelos matemáticos mais adequados para utilização nas demais etapas do projeto. Em seguida avaliou-se o efeito da HAP nas características reológicas de um modelo de soro de suco de tomate. A HAP reduziu a viscosidade do modelo, sendo tal redução modelada como função da pressão de homogeneização (PH). Avaliou-se então o efeito da HAP no suco. A HAP reduziu o tamanho das partículas em suspensão, assim como sua distribuição, promovendo maior interação entre partículas e entre partículas e o soro. Como consequência, resultou em aumento da consistência, tixotropía, comportamento viscoso e elástico dos produtos. O efeito da PH se mostrou assintótico, sendo discutidas as razões para tal, modelando-se os parâmetros obtidos como função da PH. Por fim, avaliou-se o efeito da HAP nas propriedades de deformação e relaxação do suco de tomate. Utilizando o modelo de Burger, identificaram-se os componentes estruturais do suco de tomate associados aos comportamentos reológicos. Dessa forma, o presente trabalho descreveu as mudanças estruturais associadas ao processo que afetam a reologia do produto. Conclui-se que a HAP é uma poderosa ferramenta para promoção de alterações físicas em produtos de frutas
Abstract: High pressure homogenization (HPH) has been widely studied as a non-thermal method for food preservation. Industrial assessments and scientific works have demonstrated the consistency increasing in those products due to HPH. However, just a few works in literature have studied the physico-chemical changes in fruit products due HPH processes. The evaluation of rheological changes due to HPH is important not only for process design, but also for using this technology in order to promote desirable changes in food products, such as improvement of its consistency. The present work studied the effect of high pressure homogenization (HPH) on the rheological properties of tomato juice. Firstly, it was evaluated the tomato juice time-dependent, steady-state shear and viscoelastic properties. This first part was carried out in order to evaluate the rheological methods and mathematical models to be used in the work. Then, it was evaluated the effect of HPH on the rheological properties of a tomato juice serum model. The HPH decreased the serum model viscosity, which was modelled as function of the homogenization pressure (PH). The HPH effect on tomato juice was then evaluated. The HPH decreased the suspended particle dimensions and distribution, resulting in higher particle-particle and serum-particle interaction. As a result, it was observed an increasing in product consistency, thixotropy, viscous and elastic behaviour. The effect of PH showed an asymptotic behaviour, which was described and modelled. Finally, it was evaluated the effect of HPH on tomato juice creep and recovery properties. The mechanical Burger model well explained the juice creep compliance, and its parameters were associated to the juice structural components. Therefore, the present work described the tomato juice structural and rheological changes due to HPH processing. It was concluded that the HPH process is a valuable tool to promote physical properties changes in food products
Doutorado
Tecnologia de Alimentos
Doutor em Tecnologia de Alimentos

Orientador: Marcelo Cristianini
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: A goma do cajueiro (GC) é um produto isolado do exsudado bruto do cajueiro e tem sido mencionada como uma possível alternativa a substituir a goma arábica na indústria de alimentos. A alta pressão dinâmica (APD) é apresentada como uma opção a modificação física dos polissacarídeos a fim de melhorar suas propriedades tecnológicas. Dessa forma, o trabalho teve como objetivo avaliar o efeito da APD nas características tecnológicas da GC. O exsudado bruto foi purificado e a goma caracterizada quanto à composição centesimal, potencial zeta e reologia. Em seguida GC nativa (sem processamento-controle) e processada a APD foram investigadas quanto suas características tecnológicas (solubilidade e capacidade de absorção de água (CAA) nas temperaturas 30, 60 e 90ºC, capacidade de absorção de óleo (CAO), reologia e propriedades emulsificantes) comparando-as com os resultados obtidos para goma arábica (GA). A GC apresentou 10,5% de umidade, 0,8% de cinzas, 0,9% de proteínas, 0,01% de lipídios e 87,7% de carboidratos. O ponto isoelétrico encontrado foi de 3,2. A CAA para a GC controle foi no mínimo 50% maior que a da GA em todas as temperaturas avaliadas. Entretanto, apresentou CAO 35% menor, solubilidade 4% menor e consistência inferior. A APD aumentou a solubilidade da GC em 5% a 30°C, 4% a 60°C e 12% a 90°C, e reduziu a CAA em 66% a 30°C, 44% a 60°C e 25% a 90ºC. A APD também reduziu a CAO da GC em 63%. Quanto as características reológicas, o processamento da GC por APD a 30 MPa quando comparada com a GC controle resultou em diminuição do índice de consistência (k) em 69% e elevação do índice de fluxo (n) em 16%, aproximando o comportamento ao fluxo da goma ao de um fluido Newtoniano. O efeito da APD nas emulsões produzidas com GC promoveu redução de 14% no diâmetro médio volumétrico (D4,3) e 17% no diâmetro médio de área superficial (D3,2). O potencial zeta permaneceu constante quando a GC foi processada a 20 MPa, mas reduziu quando processada a 40 MPa. A reologia das emulsões permaneceu constante, já o processamento com APD melhorou a estabilidade da emulsão (menor índice de cremeação e maior turbidez). A turbidez das emulsões aumentou em 25%. Por fim, conclui-se que a GC apresenta boa capacidade de absorção de água, alta solubilidade em água (acima de 80%), baixa capacidade de absorção de óleo e baixa viscosidade aparente mesmo em soluções mais concentradas (20% m/v) quando comparada com a goma arábica. A APD é capaz de modificar as características tecnológicas da GC (solubilidade, CAA, CAO e reologia) e melhorar suas propriedades emulsificantes (menor tamanho médio de partículas, maior turbidez e redução no índice de cremeação)
Abstract: The cashew tree gum (CG) is a product isolated by exudate of cashew tree and has been mentioned as possible replacer to arabic gum in food industry. The dynamic high pressure (DHP) is presented as option to physical modification on polysaccharides to improve their technological properties. Thus, this work aimed to evaluate the effect of DHP on technological properties of CG. The exudate was purified and centesimal composition, zeta potential and rheology of the gum were determined. After that, native CG (without processing-control) and processed at DHP were evaluated in terms of its technological properties (solubility and water absorption capacity (WAC) at 30, 60 and 90ºC, oil absorption capacity (OAC), rheology and emulsifying properties) comparing it with the results obtained from arabic gum (AG). CG presented 10,5% of moisture, 0,8% of ash, 0,9% of proteins, 0,01% of fat and 87,7% of carbohydrates. The isoeletric point encountered was 3,2. The WAC for the control CG was at least 50% higher than AG in all temperatures evaluated. However, it presented lower values regarding to OAC (35%), solubility (4%) and consistency. The DHP increased the solubility of CG in 5% at 30ºC, 4% at 60ºC and 12% at 90ºC, decreased the WAC in 66% at 30ºC, 44% at 60ºC and 25% at 90ºC. The DHP decreased the OAC of CG in 63% as well. In terms of rheological characteristics, the processing of CG by DHP at 30 MPa when compared with control CG resulted in decrease of consistency index (k) with a increment of pressure in 69% and elevation of flow index (n) in 16%, making the behavior to the gum flow closer to the Newtonian fluid. The effect of DHP on CG emulsions resulted in a reduction of 14% on average volumetric diameter (D4,3) and 17% on average superficial area diameter (D3,2). The zeta potential remained constant when the GC was processed at 20 MPa, but decreased when processed at 40 MPa. The rheology of the emulsions remained constant, but the processing with APD improved emulsion stability (lower creaming index and higher turbidity). The turbidity of emulsions increased in 25%. Finally, CG presented good WAC, high solubility (up 80%), low OAC and low apparent viscosity even in more concentrated solutions (20% w/v) when compared with arabic gum. The DHP is capable to modify the technological properties of CG (solubility, WAC, OAC and rheology) and improve their emulsifying properties (lower average particle size and creaming index and higher cloudiness)
Mestrado
Tecnologia de Alimentos
Mestra em Tecnologia de Alimentos

Orientador: Marcelo Cristianini
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: O tratamento de homogeneização a ultra alta pressão (HUAP) é uma tecnologia que vem sendo utilizada tanto para inibir o crescimento de microorganismos, como para alterar as propriedades físico-químicas e funcionais do leite e de sua fração proteica. O objetivo deste estudo foi avaliar algumas das principais alterações físico-químicas de leite desnatado submetido a diferentes níveis de HUAP. Além disso, avaliar solubilidade, propriedades de aeração e emulsificação de caseína e proteínas de soro isoladas de leite processado por HUAP. Leite cru desnatado foi submetido a 3 níveis de pressão de homogeneização (100 MPa, 200 MPa e 300 MPa) onde foram avaliadas as alterações físico-químicas do leite (pH, composição centesimal, estabilidade ao álcool, luminosidade, desnaturação de proteínas do soro, hidrofobicidade e viscosidade) e as propriedades funcionais da caseína e proteínas do soro (solubilidade, aeração e emulsificação). O processo apresentou boa repetibilidade e aumento de temperatura de no máximo 55°C. As medidas de pH e nitrogênio não proteico (NNP) foram as únicas variáveis que não apresentaram alteração estatisticamente significativa para nenhum dos níveis de pressão. Estabilidade a precipitação com álcool, luminosidade e hidrofobicidade apresentaram aumento a partir de 100 MPa. A desnaturação de proteínas do soro ocorreu somente a partir de 200 MPa, aumentando ainda mais com uso de 300 MPa. A única variável que apresentou alteração somente no nível de 300 MPa foi a viscosidade. A caseína e as proteínas do soro foram isoladas por acidificação a pH 4,6 e centrifugação, o sobrenadante e precipitado foram neutralizados e liofilizados. Foram avaliadas as seguintes propriedades: solubilidade, capacidade de formação de espuma, estabilidade de espuma, capacidade emulsificante e estabilidade de emulsão. A caseína não sofreu alteração de solubilidade, já as propriedades de aeração e emulsificação apresentaram melhora de performance, sendo estatisticamente significativas a partir de diferentes níveis de pressão de acordo com a propriedade avaliada. As proteínas do soro apresentaram diminuição da solubilidade nos níveis de 100 MPa e 200 MPa, as propriedades de aeração foram melhoradas em 300 MPa, e as propriedades de emulsificação não foram influenciadas. O tratamento de leite fluido por tecnologia de HUAP promove alterações significativas em propriedades funcionais de caseína e proteínas do soro. As alterações estão relacionadas à desestruturação das micelas de caseína, desnaturação de proteínas do soro, interação das micelas entre si e com as proteínas do soro, e com o aumento da hidrofobicidade e capacidade de hidratação das mesmas
Abstract: The ultra high-pressure homogenisation (UHPH) is a recent technology used to inhibit microorganisms¿ growth, as to modify the physical-chemical properties of milk and its protein fraction. The purpose of this study was to evaluate the main physical-chemical characteristics caused by different levels of high-pressure homogenisation in skim milk. Besides to evaluate solubility, foaming properties and emulsifying properties of casein and whey proteins isolated from milk processed by UHPH. Raw skimmed milk was submitted to 3 pressure levels (100 MPa, 200 MPa e 300 MPa), the processing parameters and the modifications on milk physical-chemical properties has been evaluated. The process presented good repeatability and the maximum temperature increase was 55°C. The measurements of pH and non-protein nitrogen (NPN) were the only variables that did not present any statistically significant change. Ethanol stability, lightness (L*) and hydrophobicity showed an improvement or increment from 100 MPa to 300 MPa. Whey protein denaturation occurred only from 200 MPa to 300 MPa. The only variable that presented changes only at 300 MPa was viscosity. Casein and whey proteins were isolated by acidification at pH 4.6 and centrifugation, further they were neutralized and freeze-dried. The following properties were evaluated: solubility, foaming capacity, foam stability, emulsifying capacity and emulsion stability. The freeze-dried casein did not show any modification on solubility, however all foaming and emulsifying characteristics presented improved performance, being statistically significant for different pressure levels according to each analysed property. UHPH decreased the solubility of the whey proteins obtained from milk treated at 100 MPa and 200 MPa, foaming properties increased for the freeze-dried protein obtained from milk treated at 300 MPa, and no influence were noted on the emulsifying properties. The treatment of fluid milk by UHPH was able to promote significant alterations on the functional properties of casein and whey proteins. The modifications were related to casein micelles disruption, whey proteins denaturation, interaction among the micelles and with denaturated whey proteins, and also with increase of molecular hydrophobicty and water retention
Mestrado
Mestre em Tecnologia de Alimentos

L’utilisation des nanoparticules Janus pour la délivrance de médicaments suscite un grand intérêt depuis quelques années. Cependant, beaucoup d’aspects sont encore à comprendre dans la préparation de tels systèmes, notamment en présence de principes actifs.Le but de cette thèse était d’étudier des nanoparticules compartimentées Janus (JNP) constituées d’excipients lipidiques, et principalement de glycérides polyoxyéthylénés. Les objectifs étaient, d’une part, une meilleure compréhension de la structure et de la physico-chimie de ces objets puis, d’autre part, leur utilisation contre Helicobacter pylori (H. pylori).Ces JNP sont produites facilement par un procédé d’homogénéisation haute pression qui a permis de fabriquer des lots allant de 10 mL à 1 L. Elles ont été caractérisées par cryo-microscopie, calorimétrie, diffraction des rayons X, diffusion quasi-élastique de la lumière notamment et ont montrées une très grande stabilité physique sur plus d’un an. Les résultats les plus intéressants ont été obtenus avec une formulation contenant 20% de phase lipidique (Labrafil® M1944CS ou Labrafil® M2125CS) et 3% d’un mélange de tensioactifs (Gelucire® 50/13 – Phospholipon® 90G 2:1 en masse). Diverses expériences autour de la formulation et du procédé ont permis d’identifier certains paramètres critiques, notamment la nature de la phase lipidique, l’ordre d’incorporation des excipients ou le taux de diesters de PEG 1500.Des essais d’encapsulation dans ces JNP et de protection d’un principe actif instable en milieu acide, l’érythromycine, ont également été menés. Les nanodispersions formées, physiquement stable pendant au moins 6 mois, avaient une taille de l’ordre de 150 nm et une distribution de taille unimodale. Des tests in vitro ont montré que l’érythromycine encapsulé gardait son efficacité anti-microbienne et était plus stable en milieu acide. Par contre, les nanoparticules ont perdu leur compartimentation en présence d’érythromycineEnfin, des études ont été menées pour comprendre l’influence de l’incorporation de principes actifs (API) sur le procédé. Ainsi, il a été montré que l’ajout de caféine, chloroxylénol, quercétine et tricloasan dans la formulation n’altérait pas la morphologie des JNP contrairement à l’érythromycine et à la dioxybenzone dans une moindre mesure.L’ensemble de ces résultats a montré que les nanoparticules compartimentées Janus sont des systèmes prometteurs de transport et de protection de principes actifs
The use of Janus nanoparticles (JNPs) as constituent of drug delivery formulations has been a topic of considerable interest for the past several years. However, the formation of vesicles and nanoparticle-associated drug/active are still unclear.The aim of the present study was a physicochemical characterization of lipid nanodispersions based on polyoxylglycerides, especially focused on lipid-based JNPs. The experiments should lead to a better understanding of structure and behavior of the very interesting carrier system on Helicobacter pylori (the abbreviation is H. pylori).The multicompartment lipid-based JNPs were produced easily at small (10 to 50 ml/batch) and large scale (1 liter/batch) by hot high pressure homogenization method. Samples were very well long-time stable for at least over 12 months with respect to particle size, DSC, XRD, and special particle morphology characteristics. Especially for particles containing 20% of lipid phase (Labrafil® M1944CS or Labrafil® M2125CS) and surfactant mixture of Gelucire® 50/13 – Phospholipon® 90G (2:1), strong adhesion could be observed in the studies by cryo-TEM technique. By changing the formulation components and process parameters, data showed that the formation of the multicompartment lipid-based JNPs depended on the using of suitable oil phase with the surfactant mixture and the method to produce the vesicles under the identified conditions.We also successfully prepared the stable nanodispersions to protect a labile antibiotic, erythromycin. The mean diameter of the ERY-loaded nanodispersions was found approximately 150 nm, and the size distribution was unimodal. The system was physically stable at room temperature for over six months. The test of antimicrobial activity in vitro on H. pylori showed that not only the preparation process did not reduce the antimicrobial activity of erythromycin, but also the stability of erythromycin was also improved in acidic environment.Furthermore, the properties of APIs loaded into the blank vesicles also affected their particle morphologies. We achieved the nanoparticles like JNPs when loading caffeine, chloroxylenol, quercetin, and triclosan into the vesicles. These results demonstrated that the multicompartment lipid-based JNPs are a promising carrier to protect unstable APIs and enhance their stability and solubility, despite the changes in the structure of the JNPs when incorporating with erythromycin or dioxybenzone

Orientador: Marcelo Cristianini
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: Leites fermentados probióticos são considerados alimentos funcionais, devendo conferir proteção às culturas. A alta pressão dinâmica (APD) vem sendo investigada como alternativa na produção de leite fermentado, pois altera a funcionalidade de alguns constituintes. Este trabalho objetivou avaliar o uso de leite submetido à APD na cinética de fermentação de S. thermophilus e L. acidophilus, bem como a viabilidade das culturas, as características físico-químicas e reológicas durante a estocagem refrigerada do leite fermentado probiótico.Leite (3% de gordura) foi submetido ao processo de APD (100MPa e 200MPa) e comparados com controle (15/5MPa), e avaliada a cinética de fermentação (pH), atividade proteolítica e a viabilidade durante a fermentação do L. acidophilus (Cap.2). Outro experimento foi realizado com leite (2% de gordura) submetido à APD (50/5MPa, 100/5MPa, 150/5MPa e 180/5MPa) e comparado com o controle (15/5MPa), no qual foi avaliada a cinética de fermentação de S. thermophilus em co-cultura com L. acidophilus (Cap.3), bem como o comportamento reológico (Cap.3) e as características físico-químicas (Cap.4) do leite fermentado probiótico durante a estocagem refrigerada. A cinética de fermentação de Lactobacillus acidophilus foi alterada pela APD (100MPa e 200MPa) em comparação com o controle (15/5MPa), aumentando consideravelmente o tempo de fermentação com o aumento da pressão, sendo que após 24 horas de fermentação as amostras apresentaram valores de pH 4,49 (15/5MPa), pH 4,72 (100MPa) e 4,93 (200MPa). A atividade proteolítica foi alterada, sendo que pressões elevadas reduziram a atividade, atingindo no final da fermentação uma redução de 21,39% (100MPa) e 35,89% (200MPa), quando comparadas com o controle. Além disso, as amostras submetidas à APD apresentaram menor número de células viáveis de L. acidophilus no fim da fermentação em relação ao controle (0,28 ciclos log) (p<0,05). A cinética de fermentação do Streptococcus thermophilus e Lactobacillus acidophilus não foi alterada pela APD. O comportamento reológico durante a fermentação de leite processado à 100/5MPa e 180/5MPa apresentou correlação positiva entre a pressão usada com a consistência do leite fermentado, gerando um produto com maior consistência. Além disso, o processo de APD aumentou a capacidade de retenção de água (CRA) em 9,15% e reduziu a sinérese espontânea do produto em 31%, entre o controle e a amostras processada a 180/5MPa. A viabilidade do S. thermophilus não foi (p<0,05) influenciada pela APD (50/5MPa, 100/5MPa, 50/5MPa e 180/5MPa) em comparação com a homogeneização convencional (15/5MPa) durante a estocagem refrigerada, reduzindo a contagem em aproximadamente 0,2 ciclos log em todos os tratamentos estudados. A viabilidade do L. acidophilus foi maior na amostra processada a 180/5MPa a partir do 14° dia, em comparação ao controle (15/5MPa). O pH apresentou uma pequena redução (p<0,05) somente na amostra processada a 180/5MPa no ultimo dia. A atividade proteolítica das culturas não foi influenciada pela ADP. Os resultados demonstraram que a APD pode melhorar a textura de leite fermentado, aumentar a capacidade de retenção de água, reduzir a sinérese e alterar a cinética de fermentação, dependendo da cultura utilizada. No entanto, a viabilidade das culturas, a redução do pH e a atividade proteolítica não sofreram alterações expressivas pela APD, apesar de apresentar diferenças significativas
Abstract: Probiotics fermented milk are considered functional food, should provide protection to cultures. Dynamic high pressure (DHP) is being investigated as an alternative in the production of fermented milk, because changing the functionality of some constituents. This study evaluated the use of milk submitted to DHP in fermentation kinetics of S. thermophilus and L. acidophilus, as well as the viability of the cultures, the physico-chemical and rheological during refrigerated storage of fermented milk probiotic. Milk (3% fat) was subjected to the process of DHP (100MPa and 200MPa) and compared with control (15/5MPa), and evaluated the kinetics of fermentation (pH), proteolytic activity and viability during fermentation of L. acidophilus (Cap.2). Another experiment was performed with milk (2% fat) subject to DHP (50/5MPa, 100/5MPa, and 150/5MPa 180/5MPa) and compared with the control (15/5MPa), in which was evaluated the kinetics fermentation of S. thermophilus in co-cultured with L. acidophilus (Cap.3), and rheological behavior (Cap.3) and the physico-chemical (Cap.4) of probiotic fermented milk during refrigerated storage. The fermentation kinetics of Lactobacillus acidophilus was modified by DHP (100MPa and 200MPa), comparated to control (15/5 MPa) considerably increasing the fermentation time the higher the pressure used, and that after 24 hours of fermentation samples showed pH 4.49 (15/5MPa), pH 4.72 (100MPa) 4.93 (200MPa). The proteolytic activity was changed, with elevated pressures reducing proteolytic activity at the end of the fermentation, reaching a reduction of 21.39% (100MPa) and 35.89% (200MPa) compared with the control. In addition, the samples submitted to APD had lower number of viable probiotics in the end of fermentation compared to control (0,28 log cycles) (p<0.05). The fermentation kinetics of Streptococcus thermophilus and Lactobacillus acidophilus was not altered by DHP. The rheological behavior during fermentation in milk processed by DHP (100/5 MPa and 180/5 MPa) demonstrated a positive correlation between the pressure and consistency of the fermented milk. Moreover, the process of DHP favored water-holding capacity (WHC) in 10.13% and reduced syneresis spontaneous in 31% between the control and 180/5 MPa. The viability of S. thermophilus was not influenced by DHP (50/5MPa, 100/5MPa, 150/5MPa and 180/5MPa), comparated to conventional homogenization (15/5 MPa) during refrigerated storage, reducing the count in approximately 0.2 log cycles in all treatments. The viability of L. acidophilus was higher in the processed sample 180/5MPa from day 14, compared to the control (15/5 MPa). The pH showed a small reduction in sample subject to 180/5 MPa at the final day of storage. The proteolytic activity of the culture was not influenced by DHP. The results showed that the DHP may improve the texture of fermented milk, increase water-holding capacity, reduce syneresis and alter the kinetics of the fermentation. However, viability of cultures, the decrease in pH and proteolytic activity did not change by DHP, despite showing significant differences
Mestrado
Tecnologia de Alimentos
Mestre em Tecnologia de Alimentos

Orientadores: Flavia Maria Netto, Ricardo de Lima Zollner
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: O leite bovino contém várias proteínas capazes de induzir resposta alérgica, sendo que a ß-lactoglobulina (ß-Lg) e a a-lactalbumina (a-La), proteínas mais abundantes do soro, estão entre as principais proteínas antigênicas. A homogeneização à alta pressão (HAP) é uma tecnologia emergente que pode alterar a estrutura das proteínas do soro de leite (IPS) e, portanto, seu caráter alergênico. Estudos anteriores apontam que a polimerização com a enzima transglutaminase (TG) reduziu o potencial antigênico da ß-Lg. O presente estudo teve como objetivo avaliar o efeito da HAP associada à polimerização por TG na suscetibilidade à digestão gástrica e gastrointestinal e na resposta antigênica da ß-Lg e a-La antes e após a digestão gástrica. Soluções de IPS (1% m/v), tratadas termicamente (72 °C/22 min) ou não, foram homogeneizadas a diferentes níveis de pressão (0, 100, 180 MPa) ou multiprocessadas por 3 ciclos consecutivos a 180 MPa. As amostras foram caracterizadas por turbidez, pelo teor de grupos sulfidrila reativos e livres e por eletroforese em sistema nativo e em SDS-PAGE em condições redutoras e não-redutoras. As amostras processadas foram polimerizadas com TG (25 U g-1 de proteína) e caracterizadas por eletroforese SDS-PAGE em sistema redutor e densitometria dos géis. Foi avaliada a suscetibilidade da ß-Lg e da a-La à digestão gástrica e gastrointestinal após tratamento térmico, HAP e polimerização, associados ou não. As condições de digestão foram: 182 U de pepsina g-1 de proteína e a relação 1:25 (enzima:proteína) para a pancreatina. A caracterização dos digeridos foi realizada por eletroforese SDS-PAGE/Tricina e cromatografia líquida de alta eficiência de fase reversa (CLAE-FR). A antigenicidade das proteínas foi avaliada pelo método ELISA, utilizando soro anti-ß-Lg ou anti-a-La de camundongos BALB/c imunizados com as proteínas nativas. A homogeneização das soluções de IPS, previamente tratadas termicamente ou não, resultou na diminuição da turbidez e do teor de grupos sulfidrila reativos e livres com o aumento dos níveis de pressão. A polimerização com TG resultou na formação de polímeros com massa molecular (MM) acima de 97,4 kDa, principalmente nas amostras tratadas termicamente e homogeneizadas a 180 MPa por 1 e 3 ciclos, alcançando aproximadamente 20% de polímeros de alta MM. As amostras pré-tratadas e polimerizadas foram mais suscetíveis à digestão gástrica e gastrointestinal, resultando em menor residual de proteína intacta em relação às amostras homogeneizadas pré-tratadas termicamente ou não. Foi observado aumento (P< 0,05) da capacidade de ligação à IgE anti-a-La para a amostra de IPS tratado termicamente e homogeneizado a 180 MPa (IPS-TT-180, 27,88 µg IgE mL-1), em relação ao IPS nativo (IPS-N, 12,80 µg IgE mL-1), sugerindo que a associação do tratamento térmico e HAP aumentou a exposição de seus epítopos. Após a digestão gástrica, não foi observada redução da atividade antigênica da a-La em nenhuma das amostras analisadas. Já para a ß-Lg verificou-se que após a digestão gástrica a amostra de IPS tratado termicamente, homogeneizado a 180 MPa e polimerizado (IPS-TT-180 TG) apresentou diminuição (P< 0,05) da resposta antigênica para IgE anti-ß-Lg (13,59 µg IgE mL-1) em relação ao IPS-N (34,21 µg IgE mL-1). Em conclusão, os tratamentos utilizados alteraram a estrutura das principais proteínas do IPS, resultando na diminuição moderada da resposta antigênica da ß-Lg e no aumento da antigenicidade da a-La
Abstract: Bovine milk contains several proteins, which can induce allergic response. The most abundant whey proteins, â-lactoglobulin (â-Lg) and á-lactalbumin (á-La) are among the major antigenic proteins of cow¿s milk. High pressure homogenization (HPH) is an emergent technology which can alter whey proteins (WPI) structures and possibly WPI antigenicity. Previous studies indicated that polymerization with transglutaminase enzyme (TG) reduced the antigenic potential of â-Lg. The objective of the present study was to evaluate the effect of HPH associated to polymerization by TG on the susceptibility to simulated gastric and gastrointestinal digestion and on antigenic response of â-Lg and á-La before and after gastric digestion. Solutions of WPI (1% w/v) previously heat treated (72 °C/22 min) or without heat treatment were homogenized at several pressure levels (0, 100 and 180 MPa) or at 180 MPa for three consecutive cycles. The samples were characterized by turbidity, content of exposed and free sulfhydryl groups and by electrophoresis on native system and SDS-PAGE under non-reducing and reducing conditions. The processed samples were polymerized with TG enzyme (25 U g-1 of protein) and characterized by SDS-PAGE under reducing conditions followed by densitometry of gels. The susceptibility of â-Lg and á-La to gastric and gastrointestinal digestion was evaluated after heat treatment, HPH and polymerization, associated or not with each other. The digestion conditions were: 182 U pepsin g-1 protein and pancreatin:protein ratio of 1:25. Characterization of the digests was carried out by SDS-PAGE/Tricine and reversed-phase high performance liquid chromatography (RP-HPLC). Protein antigenicity was evaluated by ELISA assay, using serum from BALB/c mouse immunized with native â-Lg and á-La. The homogenization of the WPI solutions, previously heat treated or without heat treatment, resulted in reducing both turbidity and exposed and free sulfhydryl group content as the pressure levels increased. The polymerization with TG enzyme resulted in polymers with molecular mass (MM) above 97.4 kDa, mainly in the samples heat treated and homogenized at 180 MPa for 1 or 3 cycles, forming around 20% of high MM polymers. Either polymerized samples previously treated with heat and/or HPH were more susceptible to gastric and gastrointestinal digestion, resulting in less residual intact protein as compared to the homogenized samples previously heat treated or without heat treatment. It was observed higher (P< 0.05) binding capacity to anti-á-La IgE for the sample heat treated and homogenized at 180 MPa WPI (WPI-TT-180, 27.88 ìg IgE mL-1) than for native WPI (WPI-N, 12.80 ìg IgE mL-1) suggesting that the association of heat treatment and HPH increased the exposition of á-La epitopes. After gastric digestion it was not observed a reduction of á-La antigenic activity. In relation to â-Lg antigenicity, after gastric digestion, the WPI sample heat treated, homogenized at 180 MPa and polymerized (WPI-TT-180 TG) showed reduction (P< 0.05) of anti-â-Lg IgE response (13.59 ìg IgE mL-1) as compared to WPI-N (34.21 ìg IgE mL-1). In conclusion, the treatments studied altered the structure of major protein of WPI, resulting in moderate reduction of â-Lg antigenic response and increased á-La antigenicity
Mestrado
Nutrição Experimental e Aplicada à Tecnologia de Alimentos
Mestra em Alimentos e Nutrição

O objetivo do presente trabalho foi desenvolver e validar um método analítico empregando microextração em fase sólida (SPME) para detecção e quantificação de isoflurano (ISO) na forma volátil e incluso em nanoemulsões intravenosas e, ainda, avaliar o efeito biológico destas. A detecção do ISO foi realizada através de cromatografia em fase gasosa com detector de ionização de chama (CG/DIC). As condições ideais para realização da pré-concentração e extração de ISO através da técnica de SPME foram temperatura ambiente, agitação constante, 30 min de extração e 2 min de dessorção no injetor do CG. O método desenvolvido foi validado avaliando os parâmetros de especificidade, linearidade, limites de detecção e quantificação, precisão, exatidão e robustez. As nanoemulsões contendo ISO foram desenvolvidas através da homogeneização à alta pressão, e apresentaram diâmetro médio, índice de polidispersão, potencial zeta e pH de 150 ± 0,78 nm, 0,08 ± 0,01, - 18 ± 2,4 mV e 6,03 ± 0,04, respectivamente. O pH foi ajustado para 7,4 (valor fisiológico). O teor de ISO nas formulações foi de 98,4 %. Não houve modificação das características físico-químicas das nanoemulsões após 30 dias de armazenamento a 8 ºC. Análises de espalhamento de luz múltiplo não demonstraram tendência a fenômenos de instabilidade física para as formulações. Os estudos do efeito anestésico das nanoemulsões intravenosas contendo ISO em cães evidenciaram uma redução significativa (p < 0,05) na dose comparada com a administração de ISO volátil. Não houve alterações no débito cardíaco, saturação de oxigênio na hemoglobina e nos biomarcadores das funções renal, hepática e muscular. Uma queda na pressão arterial dos cães foi observada em todos os tratamentos devido ao efeito hipotensor do ISO. Após administração das nanoemulsões contendo ISO e branca, observou-se taquipnéia, edema, eritema, e baixas concentrações de dióxido de carbono expiradas. Assim, a nanoemulsificação do ISO foi realizada com sucesso e a aplicação na anestesia geral intravenosa foi demonstrada.
The aims of this work were to develop and validate an analytical method using solidphase microextraction (SPME) to detect and quantify isoflurane (ISO) inhalation liquid and loaded in intravenous nanoemulsions, and also evaluate the biological effect of the formulations. ISO detection was made by gas chromatography with flame ionization detector (GC/FID). The ideal conditions setting for the pre concentration and extraction of ISO through SPME were environmental temperature, constant stirring, 30 min for extraction and 2 min for analyte desorption in the GC inlet port. The developed method was validated by means of specificity, linearity, detection and quantification limits, precision, accuracy and robustness. The ISOloaded nanoemulsions were formulated by high-pressure homogenization, and presented average diameter, polydispersity index, zeta potential and pH of 150 ± 0.78 nm, 0.08 ± 0.01, -18 ± 2.4 mV and 6.03 ± 0.04, respectively. The pH was adjusted to 7.4 (physiological value). The drug content on the formulations was 98.4 %. After 30 days of storage at 8 ºC no changes on nanoemulsion’s physicalchemical characteristics were observed. Multiple light scattering analysis did not demonstrate any physical destabilization phenomena for the formulations. The anesthetic effect study for the intravenous ISO-loaded nanoemulsions in dogs highlighted a significant reduction (p < 0.05) in dosage regimen when compared to the volatile ISO administration. There were no alterations on cardiac rate, oxygen hemoglobin saturation and on biomarkers of the renal, hepatic and muscle functionalities. A decrease in dog’s arterial blood pressure in all treatments due the hypotensive effect caused by ISO was observed. After the administration of the nanomulsions, ISO-loaded and unloaded, occurred tachypnea, edema, erythema and low end tidal concentrations of carbon dioxide. Taking all above into account, the method was considered easy on execution and suitable for laboratory routines, the ISO nanoemulsification was made successfully and its application on general anesthesia was demonstrated.

Estudos in vitro de absorção percutânea são uma importante ferramenta para avaliação de formulações semissólidas e transdérmicas. Embora haja um grande número de agências reguladoras preocupadas com a harmonização metodológica, em muitos parâmetros elas permanecem flexíveis e isto é possível verificar na ampla variedade e divergências encontradas na literatura. O objetivo do trabalho foi avaliar parâmetros ainda flexíveis a respeito dos estudos in vitro como modo de separação das camadas da pele, permeabilidade da pele congelada (tempo de armazenamento) e diferença de permeabilidade dos locais anatômicos. Os estudos foram conduzidos utilizando células de Franz, pele suína como membrana e formulações (comercializada e inovadora) contendo penciclovir. Inicialmente, nanoemulsões foram preparadas utilizando técnica de homogeneização a alta pressão, caracterizadas, incorporadas em gel de carbômero 940 e avaliadas quanto à liberação tópica em pele suína. Simultaneamente ao desenvolvimento da formulação foi desenvolvido e validado método analítico para quantificação do fármaco nas formulações e nas camadas da pele suína. As nanoemulsões apresentaram-se monodispersas com diâmetro de gotícula em torno de 180-200 nm, potencial zeta de -27 mV e teor de penciclovir de 98% mantendo sua estrutura após a incorporação em carbômero 940. A metodologia analítica demonstrou ter alta sensibilidade (LoQ 0,05 μg/mL), especificidade e uma adequada recuperação do fármaco a partir das matrizes biológicas (90 – 104%). Quanto aos estudos in vitro de comparação de metodologias, foi possível observar que, dependendo da solubilidade do fármaco em água e das características da formulação, o método clássico de separação das camadas da pele por imersão em água não é o mais indicado. Já para permeabilidade da pele suína congelada, os resultados obtidos indicam um aumento significativo na penetrabilidade e permeabilidade após um mês de congelamento. Dentre os locais anatômicos testados, não houve diferença entre abdômen e orelha suína desde que obtidos antes do procedimento de escaldo.
In vitro percutaneous absorption studies are an important tool for evaluation of semisolid and transdermal formulations. Although there are a large number of official guides concerned with methodological harmonization in many parameters they remain flexible and it is possible to see the wide variety and differences reported in the literature. The aim of study was to evaluate some parameters regarding the in vitro studies as the mode of skin layers separation, skin frozen stability and permeability difference of anatomical sites. These studies were conducted with porcine skin and formulations (conventional and novel) using penciclovir as model drug. Initially, nanoemulsions were prepared using high pressure homogenization, characterized and incorporated into carbomer 940 gel and evaluated for topical delivery using porcine skin. Simultaneously with the development of the formulation, analytical method for quantification of the drug in the formulations and porcine skin layers was developed and validated. The nanoemulsions presented themselves monodisperse with droplet diameter of 180-200 nm, zeta potential of about -27 mV and penciclovir content of 98% maintaining their structure after incorporation into carbomer 940. The analytical methodology was shown to have high sensitivity (LOQ 0.05 μg/mL), specificity and adequate recovery of drug from the biological matrices (90-104%). Regarding the in vitro comparison methodologies, it was observed that, depending on the solubility of the drug in water and the characteristics of the formulation, the classical separation is not the most suitable for separation of the skin layers. For the stability of frozen porcine skin, the results indicate a significant increase in permeability and penetrability after one month of freezing. Within the anatomical sites tested, there was no difference between the abdomen and ear porcine skin since obtained before the scald procedure.

KBS-3 and very deep borehole (VDH) concepts are two major types of long-term geologicaldisposal methods for high-level radioactive waste (HLW) isolating from the biosphere. TheKBS-3V concept for isolating the HLW at the depth of 400-500 m, is the officially proposedoption in Sweden and has been the subject of considerable research in the past few decades,while the VDH concept was considered as an option in the 1950s but later became discouragedbecause of insufficient experience in drilling technology. The greatest merit of the VDHconcept is that the almost stagnant groundwater in the deep boreholes prevents the transport ofthe possible release of radionuclides into the rock or up to the ground level. Since variousdisadvantages of the KBS-3V concept were found in previous research, the superiority of VDHconcept attracted the researchers to continue studying it into the late 1980s.The geological repositories of both of KBS-3V and VDH types primarily consist of a naturalbarrier (host rock) and of an engineering barrier (also known as a buffer/backfill barrier).According to the principle of IAEA and national relative research organizations, thebuffer/backfill material should have low permeability and good expandability, as well assuitable physical and sealing properties.The thesis concerns the VDH concept and is focused on the construction and performance ofthose parts of the sealed repository that are not affected by high temperature or gamma radiation.In the lower part of a VDH repository, the clay packages containing HLW will be exposed tohigh temperature (100-150 􀄇 ) in the borehole and to highly saline groundwater. In theinstallation phase of HLW, the groundwater will be pumped out and replaced by medium-softsmectite clay mud in which the HLW packages are installed vertically. During the hydrationand maturation of the clay components, the microstructural reorganization, water transport,migration of clay particles and redistribution of the density of the components take place. Thematuration determines the transient evolution of the clay seals and influences the rheologicaland soil mechanical behavior in the installation phase. The maturation of clay system alsodetermines their ultimate sealing potential of VDH repositories.This study presents the work carried out for investigating the maturation of the buffer-backfillclay in the HLW deep borehole. Initially in the study three types of clays, the Namontmorillonite,magnesium-rich and illite-smectite mixed layer clays, were examined for estimating their performance as the barrier candidate material. This is mainly presented in theliterature review. The experimental study was conducted on montmorillonite GMZ clays andI/S mixed-layer Holmehus clay. The expandability and permeability tests were carried out forinterpretation of the recorded swelling development and assessment of the effect of the salineconditions, with the goal of deriving a relationship between swelling pressure and hydraulicconductivity for different dry densities. The maturation tests of initially fully-saturatedHolmehus clay and partly saturated GMZ clay were performed. During the tests, the shearstrength mobilised by the relative movement of densified mud and migrated dense clay -contained in a perforated central tube - were determined. According to the results of shearstrength tests, the maximum operation time or the number of clay packages to be placed in asingle operation was evaluated, whilst the suitable saturation degree of the dense clay wasdiscussed as well.A model of the maturation of initially water-saturated clay seals based on Darcy’s law wasworked out and the evolution of the clay components in a lab-scale borehole using Holmehusclay were performed and compared with the experimental recordings. Good agreementsbetween the physical behaviors of the theoretical simulations and the measurements wasachieved by which the validity of the model was verified. Using the results, the hydration andsoil migration in the entire maturation process were presented in diagram. The model was alsoused for preliminary evaluation of the maturation products in real boreholes by assuming thesame Holmehus clay as used in the tests. Two constellation of borehole and dense clay withdifferent diameters, 80 cm borehole /60 cm clay and 80cm/50cm, were assumed. The resultsrespecting dry density and hydraulic conductivity of the ultimate maturation products, and thedegree of homogeneous of the buffer and backfill clay system in the assumed boreholes, arepresented and discussed. The options of different mineral types and initial physical propertiesof the candidate buffer clays provide a reference for engineering barrier design of HLW disposalin VDH.

Cette thèse porte sur la mise au point de formulations de nanoparticules lipidiques à base de polyoxylglycérides afin d’assurer la protection de principes actifs instables chimiquement et physiquement, la quercétine (un flavonoïde antioxydant fragile) dans le cas présent. Différents systèmes dispersés ont été préparés par homogénéisation haute pression à chaud avec une taille des particules blanches entre 100 - 200 nm. Ces nanodispersions sont très stables sur plusieurs années à température ambiante. L’encapsulation de la quercétine, dans les nanoparticules lipidiques multicompartimentées et la préparation de nanocristaux ont permis d’augmenter fortement sa teneur dans la dispersion et d’améliorer effectivement sa stabilité physico-chimique
This thesis focuses on the development of polyoxylglycérides-based lipid nanoparticles to protect labile APIs, quercetin (a fragile antioxidant flavonoid) in this case. Different nanoparticulate systems were prepared by high pressure homogenization with particle size between 100 to 200 nm. These nanodispersions are very stable over several years at room temperature. Encapsulation of quercetin in compartmented lipid nanoparticles and preparation of nanocrystals have increased significantly its content in the dispersion and effectively improve its physical and chemical stability

Les nanoémulsions (NEs) huile/eau peuvent être utilisées en tant que systèmes de délivrance des médicaments pour l’encapsulation des substances actives hydrophobes afin d’améliorer leur stabilité et leur biodisponibilité. Néanmoins, leur stabilisation nécessite l’utilisation de concentrations plus importantes de tensioactifs par rapport aux émulsions conventionnelles en raison de l’augmentation de la surface spécifique. La plupart des tensioactifs synthétiques couramment utilisés dans la formulation des émulsions sont potentiellement irritants, voire toxiques. Cela entrave l'application thérapeutique des NEs en particulier pour les traitements à long terme. L'objectif de cette thèse est alors de formuler des NEs pharmaceutiques huile/eau stabilisées par un biopolymère, la beta-lactoglobuline (beta-lg), à la place des tensioactifs synthétiques.Les NEs ont été préparées par homogénéisation à haute pression (HHP). La composition de la formulation et les conditions du procédé ont été optimisées afin d’obtenir des gouttelettes nanométriques dans des NEs stables. Les résultats ont montré que les NEs les plus stables, avec une taille de gouttelettes < 200 nm, ont été obtenues quand 5 m/m% de l’huile ayant la viscosité la plus faible ont été utilisés en tant que phase huileuse, 95 m/m% de la solution de beta-lg à une concentration de 1 m/m% ont été utilisés en tant que phase aqueuse et 4 cycles d’HPH de 100 MPa ont été appliqués. Cette formulation a été stable contre les phénomènes de croissance de gouttelettes pendant au moins 30 jours grâce à un film interfacial quasiment purement élastique. La gomme xanthane, un polysaccaride naturel, a été ajoutée à la formulation optimale à une concentration de 0,5 m/m% en tant qu’agent épaississant. Cela a permis d’obtenir une texture crémeuse avec un comportement rhéofluidifiant. Dans cette dernière formulation, la vitesse de migration des gouttelettes a été considérablement réduite et la stabilité des NEs a été améliorée.Les effets du procédé d’HPH sur les différents niveaux de structure de la protéine ont été évalués à l’aide de méthodes spectroscopiques, chromatographiques et électrophorétiques. L’influence de ce traitement sur ses propriétés interfaciales et émulsionnantes a également été étudiée. L’efficacité émulsionnante optimale a été obtenue quand les conditions d’HPH n’ont pas altéré la structure de la beta-lg, ni ses propriétés interfaciales. Néanmoins, un traitement d’HHP excessif (300 MPa/5 cycles) a induit des modifications structurelles, principalement une transformation des feuillets beta en structures désordonnées, une large perte dans le cœur hydrophobe, et une agrégation importante par des liaisons disulfure intermoléculaires. La beta-lg modifiée par l’HHP a montré une hydrophobie de surface plus importante conduisant à une vitesse d’adsorption à l’interface huile/eau plus élevée et une formation plus précoce d’un film interfacial. La dénaturation de la protéine par ce traitement à haute pression, qui a été effectuée avant le processus d’émulsification, n'a pas modifié de façon significative l'efficacité émulsionnante. La réduction de l’efficacité a été probablement plutôt induite par la dénaturation simultanée avec l’émulsification sous conditions d’écoulement très turbulent.L’intérêt de la formulation développée en tant que véhicule pour un modèle de substance active hydrophobe a été étudié avec l’isotrétinoïne (IT), usuellement utilisé pour le traitement de l’acné sévère. La formulation développée a permis d’encapsuler 0,033 m/m% d’IT sans aucune modification de la stabilité du système. Environ 10 % de l’IT ajoutée ont été solubilisés dans la phase aqueuse en association avec la protéine libre en excès. L’IT encapsulée dans les gouttelettes huileuses a été plus stable contre la photo-isomérisation que celle associée à la protéine libre. La formulation développée apparait prometteuse en tant que système de délivrance de l’IT pour une application cutanée
Oil-in-water nanoemulsions can be used as drug delivery systems for the encapsulation of hydrophobic active substances in order to increase their solubility and their bioavailability. However, due to their higher specific area, their stabilization requires higher surfactant concentrations compared to conventional emulsions. Most of the synthetic surfactants commonly used in emulsion formulation are potentially irritant and even toxic, which hinders the therapeutic application of nanoemulsions especially during long-term treatment. The objective of this thesis is thus to formulate pharmaceutical oil/water nanoemulsions stabilized by a biopolymer, beta-lactoglobulin (beta-lg), instead of synthetic surfactants. Nanoemulsions were prepared by high pressure homogenization (HPH). The formulation composition and the process conditions were optimized in order to obtain nanometric droplets within stable nanoemulsions. The results showed that the most stable nanoemulsions, with droplet size inférieure à 200 nm, were obtained when 5 w/w% of the oil with the lowest viscosity value was used as the oily phase, 95 w/w% of beta-lg solution at a concentration of 1 w/w% was used as the aqueous phase, and 100 MPa of homogenization pressure was applied for 4 cycles. This formulation was stable against droplet growth phenomena during 30 days at least, thanks to a quasi purely elastic interfacial film. Xanthan gum, a natural polysaccharide, was added to the optimal formulation as a texturizing agent at a concentration of 0.5 w/w%. This allowed obtaining a cream texture with a shear thinning behavior. In this formulation, the migration rate of droplets was considerably reduced and the nanoemulsions stability was enhanced.The effects of the homogenization process on the different levels of the protein structure were assessed by spectroscopic, chromatographic and electrophoretic methods. The influence of this treatment on its interfacial and emulsifying properties was also investigated. The optimal emulsifying efficiency was obtained when the homogenization conditions did alter neither the structure of beta-lg nor its interfacial properties. However, an excessive HPH treatment (300 MPa/5 cycles) introduced structural modifications, mainly from beta-sheets into random coils, wide loss in lipocalin core, and protein aggregation by intermolecular disulfide bridges. HPH modified beta-lg displayed higher surface hydrophobicity inducing a higher adsorption rate at the O/W interface and an earlier formation of an elastic interfacial film. Structural and interfacial properties modifications by HPH denaturation appeared qualitatively similar to that of the heat denaturation with, however, differences in extent. Protein denaturation by a high pressure treatment that was performed before the emulsification process did not alter significantly its emulsifying efficiency. The reduction in the efficiency was rather induced by the simultaneous denaturation with the emulsification under high turbulent flow.The efficiency of the developed formulation as a vehicle for a model hydrophobic active substance was studied using isotretinoin, usually used for the treatment of severe acne. The developed formulation was able to encapsulate 0.033 w/w of isotretinoin without any modification on the system stability. About 10 % of the added isotretinoin was solubilized in the aqueous phase associated with the free protein in excess. Isotretinoin encapsulated in the oily droplets was more stable against photo isomerization than the one associated to the excess protein in the aqueous phase. The developed formulation seems promising as a drug delivery system of isotretinoin for a dermal application

El objetivo principal de esta tesis fue estudiar los efectos de la tecnología de UHPH sobre la fase grasa de la leche (nata) y la incorporación de ésta en la leche de quesería para la elaboración de quesos reducidos en grasa. Para este propósito, se realizó un estudio preliminar del efecto de incorporación de natas tratadas por UHPH en leche desnatada con el fin de determinar las propiedades de coagulación enzimática, rendimiento potencial quesero y microestructura de los geles. Se elaboraron 4 tipos de leches a partir de leche desnatada pasteurizada y nata pasteurizada a 65 ºC por 30 min (LP), nata homogeneizada-pasteurizada a 15 MPa, 65 ºC por 30 min (LPH), nata tratada por UHPH a 300 MPa con temperatura de entrada (Ti) = 40 ºC (LUH) y nata adicionada de 1,5% de caseinato de sodio y tratada por UHPH a 300 MPa y Ti = 40 ºC (LUHC). Los tratamientos de homogeneización modificaron las propiedades de coagulación enzimática de la leche, disminuyendo la velocidad de agregación micelar y la firmeza del gel, en comparación a la leche añadida de nata pasteurizada. El tratamiento de UHPH de las natas mejoró el rendimiento quesero potencial de las leches en comparación al tratamiento de homogeneización convencional. Este rendimiento fue mayor con nata tratada por UHPH adicionada de caseinato de sodio, alcanzando rendimientos similares a los obtenidos con la leche entera. La siguiente fase fue evaluar el efecto de incorporación de natas tratadas mediante UHPH en leche de quesería sobre las características fisicoquímicas, microbiológicas, sensoriales, color y textura de quesos frescos reducidos en grasa, en comparación con quesos elaborados a partir de natas tratadas por métodos convencionales (homogeneización convencional y/o pasteurización). Se fabricaron 5 tipos diferentes de quesos frescos a partir de las leches con 1,5% de grasa LP, LPH, LUH y LUHC para obtener los quesos reducidos en grasa QPf, QPHf, QUHf y QUHCf, respectivamente. También se elaboró un queso a partir de leche al 3,2% de grasa por adición de nata pasteurizada (QEf). Los tratamientos de homogeneización de la nata produjeron quesos con una matriz proteica más compacta. Sin embargo, la adición de caseinato a la nata antes del tratamiento UHPH produjo quesos reducidos en grasa (QUHCf) con propiedades texturales y sensoriales similares a los elaborados con leche entera, en comparación al queso elaborado a partir de nata tratada por homogeneización convencional que fue descrito por los panelistas como quesos más duros y de textura granulosa. Asimismo, la lipólisis y oxidación lipídica fue menor en el QUHCf en comparación al QPHf. Del estudio anterior se seleccionaron los mejores tratamientos aplicados en la elaboración de quesos frescos, y se realizó un ensayo de incorporación de nata tratada por UHPH en leche de quesería estudiando las características fisicoquímicas, bioquímicas, sensoriales, de color y textura de quesos madurados y reducidos en grasa, en comparación con quesos elaborados a partir de nata pasteurizada durante 60 días de maduración. Se fabricaron 3 tipos diferentes de quesos madurados: un queso a partir de leche al 3,2% de grasa por adición de nata pasteurizada (QEm) y 2 quesos reducidos en grasa elaborado con LP (QPm) y LUHC (QUHCm), respectivamente. La reducción de grasa de la leche de quesería produjo disminuciones del rendimiento quesero, incrementando el pH y humedad de los quesos en comparación a los elaborados con leche entera. Todos los quesos presentaron similitudes en los niveles de oxidación lipídica y proteólisis secundaria. Los quesos elaborados con nata tratada por UHPH más caseinato presentaron menores niveles de lipólisis y compuestos volátiles y mayor dureza y luminosidad en comparación a los quesos elaborados con el tratamiento de pasteurización.
The aim of this study was to evaluate the effect of UHPH on cream milk and their inclusion as ingredient in the elaboration of reduced-fat cheeses. For this purpose, a preliminary study of the effect of the incorporation of creams treated by UHPH in the skimmed milk was carried out in order to determine the properties of enzymatic coagulation, potential cheese yield and microstructure of gels. Four types of milk were elaborated from pasteurized skimmed milk and pasteurized cream at 65 ºC for 30 min (LP), homogenised-pasteurized cream at 15 MPa, 65 ºC for 30 min (LPH), cream treated by UHPH at 300 MPa and inlet temperature (Ti) = 40 °C (LUH) and cream added with 1,5% sodium caseinate and treated by UHPH at 300 MPa and Ti = 40 °C (LUHC). The homogenisation treatments modified the enzymatic coagulation properties of milk, reducing the micellar aggregation rate and the gel firmness, in comparison to milk added with pasteurized cream. The UHPH treatment of creams improved the potential cheese yield of milks compare to conventional homogenisation. This yield was higher in cream treated by UHPH added with 1.5% of sodium caseinate, reaching similar yields to those obtained with whole milk. The next phase was to evaluate the effect of the incorporation of creams treated by UHPH in cheese milk on the physicochemical, microbiological, sensory, colour and texture characteristics of reduced-fat fresh cheeses, compared to cheeses made with creams treated by conventional methods (conventional homogenisation and/or pasteurization) during 7 days of storage at 4 ºC. Five different types of fresh cheeses were elaborated from milks at 1,5% of fat, LP, LPH, LUH and LUHC to obtained cheeses reduced in fat QPf, QPHf, QUHf and QUHC, respectively. A cheese with milk at 3,2% of fat was also made by the addition of pasteurized cream (QEf). The homogenisation treatments (conventional and UHPH) of cream produced fresh cheese with a compact protein matrix. However, the addition of sodium caseinate before UHPH treatment produced reduced-fat cheeses (QUHCf) with textural properties and sensorial characteristics similar to whole milk cheeses, compared to cheese made from treated cream by conventional homogenisation, which was described by the panellist as hardest and with granular texture. Likewise, lipolysis and lipid oxidation was lower in the QUHCf compared to the QPHf. From the previous study, the best treatments applied in the preparation of fresh cheeses were selected, and a trial of incorporation of cream treated by UHPH in cheese milk was carried out studying the physicochemical, biochemical, sensory, colour and texture characteristics of reduced-fat ripened cheeses, compared to cheese made from pasteurized cream during 60 days of ripening. Three different types of ripened cheeses were elaborated: cheese with milk at 3,2% of fat with NP (QEm) and two reduced-fat cheeses made with LP (QPm) and LUHC (QUHCm), respectively. The reduction of fat content in cheese-making milk produced a decrease in cheese yield, increasing pH and moisture of ripened cheeses compared to those made with whole milk. All matured cheese showed similar lipid oxidation and secondary proteolysis. Cheeses made with cream treated by UHPH more caseinate showed lower levels of lipolysis and volatile compounds and greater hardness and luminosity compared to those made with pasteurization treatment. These results are in accordance with the described by the panellist as cheeses with less aroma and greater hardness and whiteness.

En aquest treball es va estudiar l’efecte dels tractaments UVC (llum ultravioleta C) en continu, utilitzant un reactor UVC amb unes característiques intrínseques específiques (Patent nº 2.965.766 B2), a la supervivència de Bacillus subtilis i altres microorganismes inoculats en diferents matrius alimentàries líquides de pH neutre amb diferent coeficient d’absorció (254 nm). A més, es van combinar els tractaments de UVC amb tractaments d’UHPH (Ultra Alta Homogeneïtzació per Pressió) a diferents pressions (100 i/o 200 MPa) i temperatures (20 i/o 60 ºC) per poder avaluar el possible efecte sinèrgic, additiu o complementari de les dues tecnologies (UHPH-UVC). Es van aplicar tres tractaments UVC (T1, T2 i T3) que diferien en el nombre d’entrades al reactor (valor NET), tot i que les dosis UVC aplicades van ser les mateixes (entre 10 i 160 J/ml). A les espores de B. subtilis inoculades en llet sencera, llet desnatada, liquat de soja i te verd els millors resultats es van obtenir en el tractament amb major valor NET (T3) i a la major dosi (160 J/mL), amb una letalitat en tots els casos igual o superior de 5 log. No obstant, la major eficàcia (p <0,05) es va obtenir en la matriu de menor coeficient el te verd, on no es van apreciar diferències en la letalitat en funció de la temperatura del reactor (4, 20 i 55 ºC). En el te verd i el liquat de soja, a més de B. subtilis, es varen inocular conidiospores d’Aspergillus niger, Escherichia coli i Listeria monocytogenes. El microorganisme més resistent als tractaments UVC va ser A. niger, la letalitat fins i tot amb el tractament T3 i a la màxima dosi (160 J/mL) no es va arribar a reduir 5 log. Les formes vegetatives d’E. coli i especialment L. monocytogenes van mostrar una major sensibilitat. Al liquat de soja, es va aconseguir una reducció de 5 log en el tractament T3 i a dosis de 120 i 80 J/mL, respectivament. Mentre que en el te verd, la dosi necessària amb el tractament T3 va ser de només de 10 J/mL en els tractaments T1 i T2 de 80 J/mL sobre E. coli. En el cas de L. monocytogenes, el te va mostrar per se mostrar activitat antilisteria. En els tractaments UHPH de 100 i/o 200 MPa a 20 i/o 60 ºC la letalitat obtinguda en la espores de B. subtilis inoculades en llet sencera i desnatada i liquat de soja va ser molt reduïda, però, en el cas de les conidiospores de A. niger en liquat de soja tractat a 200 MPa a 20 ºC i a 60 ºC es va aconseguir una reducció completa. Considerant l’efecte de la UVC en B. subtilis i de la UHPH en A. niger, es van aplicar de forma combinada tractaments UHPH i UVC. Tant per a les conidiospores d’A niger inoculades en liquat de soja com les espores de B. subtilis inoculades en liquat de soja, llet sencera i llet desnatada no es va observar efecte sinèrgic o additiu, sent la letalitat obtinguda similar als tractaments UVC en el cas del liquat de soja i llet sencera. En el cas de la llet desnatada va ser inferior. Només es va obtenir una letalitat més gran en la combinació UHPH i UVC en els tractaments T1 de la llet sencera, però la letalitat obtingudes va ser inferior a 2 log. En les matrius amb elevat coeficient d’absorció és difícil aconseguir una esterilització comercial ja que la dosi UVC estimades en els models van ser molt elevades, tot i que sí que seria factible en el te verd, especialment si es combinés amb UHPH.
En este trabajo se estudió el efecto de los tratamientos UVC (Ultravioleta C) en continuo, utilizando un reactor UVC con unas características intrínsecas específicas (Patente nº 2 965 766 B2), en la supervivencia de Bacillus subtilis y otros microorganismos inoculados en distintas matrices alimentarias líquidas de pH neutro con diferente coeficiente de absorción (254 nm). Además, se combinaron los tratamientos de UVC con tratamientos de UHPH (Ultra Alta Homogeneización por Presión) a diferentes presiones (100 y/o 200 MPa) y temperaturas (20 y/o 60 ºC) para poder evaluar el posible efecto sinérgico, aditivo o complementario de ambas tecnologías (UHPH-UVC). Se aplicaron tres tratamientos UVC (T1, T2 y T3) que diferían en el número de entradas en el reactor (valor NET), aunque las dosis UVC aplicadas fueron las mismas (entre 10 y 160 J/mL). En las esporas de B. subtilis inoculadas en leche entera, leche desnatada, licuado de soja y té verde los mejores resultados se obtuvieron en el tratamiento con mayor valor NET (T3) y a la mayor dosis (160 J/mL), con una letalidad en todos los casos igual o superior de 5 log. Sin embargo, la mayor eficacia (p < 0,05) se obtuvo en la matriz de menor coeficiente el té verde, no apreciándose diferencias en la letalidad en función de la temperatura del reactor (4, 20 y 55 ºC) . En el té verde y el licuado de soja inoculadas además de B. subtilis, con conidiosporas de Aspergillus niger, Escherichia coli y Listeria monocytogenes el microorganismo más resistente a los tratamientos UVC fue A. niger, cuya letalidad incluso con el tratamiento T3 y a la máxima dosis (160 J/mL) no se llegó a reducir 5 log. Las formas vegetativas de E. coli y especialmente L. monocytogenes mostraron una mayor sensibilidad. En el licuado de soja, se alcanzó una reducción de 5 log en el tratamiento T3 ya dosis de 120 y 80 J/mL, respectivamente. Mientras que en el té verde, la dosis necesaria con el tratamiento T3 fue de sólo10 J/mL y en los tratamientos T1 y T2 de 80 J/mL sobre E. coli. En el caso de L. monocytogenes el té mostró per se mostró actividad antilisteria. En los tratamientos UHPH de 100 y/o 200 MPa a 20 y/o 60 ºC la letalidad obtenida en la esporas de B. subtilis inoculadas en leche entera y desnatada y licuado de soja fue muy reducida, sin embargo, en el caso de las conidiosporas de A. niger en licuado de soja tratado a 200 MPa a 20 ºC y a 60 ºC se consiguió una reducción completa. Considerando el efecto de la UVC en B. subtilis y de la UHPH en A. niger, se aplicaron de forma combinada tratamientos UHPH y UVC. Tanto para las conidiosporas de A. niger inoculadas en licuado de soja como las esporas de B. subtilis inoculadas en licuado de soja, leche entera y leche desnatada no se observó efecto sinérgico o aditivo, siendo la letalidad obtenida similar a los tratamientos UVC en el caso del licuado de soja y leche entera y en el caso de la leche desnatada fue inferior. Solo se obtuvo una letalidad mayor en la combinación UHPH y UVC en los tratamientos T1 de la leche entera, sin embargo la letalidad obtenidas fue inferior a 2 log. En las matrices con elevado coeficiente de absorción es difícil conseguir una esterilización comercial ya que la dosis UVC estimadas en los modelos fueron muy elevadas, aunque sí que sería factible en el té verde, especialmente si se combinara con UHPH.
In this work we studied the effect of UVC (Ultraviolet C) treatments in continuous, using a UVC reactor with specific intrinsic characteristics (Patent No. 2 965 766 B2), on the survival of Bacillus subtilis and other microorganisms inoculated in different food matrices with pH neutral liquids over different absorption coefficient (254 nm). In addition, UVC treatments were combined with UHPH (Ultra High Pressure Homogenization) treatments at different pressures (100 and / or 200 MPa) and temperatures (20 and / or 60 ° C) in order to evaluate the possible synergistic, additive or complementary effect of both technologies (UHPH-UVC). Three UVC treatments (T1, T2 and T3) were applied that differed in the number of entries in the reactor (NET value), although the UVC doses applied were the same (between 10 and 160 J / mL). In B. subtilis spores inoculated in whole milk, skim milk, soy smoothie and green tea the best results were obtained in the treatment with the highest NET value (T3) and at the highest dose (160 J / mL), with lethality in all cases equal to or greater than 5 log. However, the highest efficacy (p <0.05) was obtained in the lowest coefficient matrix of green tea, with no differences in lethality depending on the reactor temperature (4, 20 and 55 ° C). In green tea and soy smoothie inoculated in addition to B. subtilis, with conidiospores of Aspergillus niger, Escherichia coli and Listeria monocytogenes the microorganism most resistant to UVC treatments was A. niger, whose lethality even with T3 treatment and maximum dose (160 J / mL) 5 log was not reduced. Vegetative forms of E. coli and especially L. monocytogenes showed greater sensitivity. In the soy smoothie, a reduction of 5 log was achieved in the T3 treatment and at doses of 120 and 80 J / mL, respectively. While in green tea, the necessary dose with the T3 treatment was only 10 J / mL and in the T1 and T2 treatments 80 J / mL on E. coli. In the case of L. monocytogenes, tea showed per se antilisteria activity. In the UHPH treatments of 100 and / or 200 MPa at 20 and / or 60 ° C the lethality obtained in the B. subtilis spores inoculated in whole and skimmed milk and soy liquefied was very reduced, however, in the case of A. niger conidiospores in soybean smoothie treated at 200 MPa at 20 ° C and at 60 ° C a complete reduction was achieved. Considering the effect of UVC in B. subtilis and UHPH in A. niger, UHPH and UVC treatments were applied in combination. For both A. niger conidiospores inoculated in soybean smoothie and B. subtilis spores inoculated in soybean smoothie, whole milk and skim milk no synergistic or additive effect was observed. The lethality obtained was similar to UVC treatments in the case of soy and whole milk smoothie and in the case of skim milk it was lower. Only a higher lethality was obtained in the UHPH and UVC combination in the T1 treatments of whole milk, however the lethality obtained was less than 2 log. In matrices with high absorption coefficient it is difficult to achieve commercial sterilization since the UVC doses estimated in the models were very high, although it would be feasible in green tea, especially if combined with UHPH.

La bebida de almendra ha experimentado en los últimos años un notable aumento en su consumo, y por tanto, se ha expandido el área de comercialización de este producto, en casi todo el mundo. Esta bebida, además de ser una alternativa a la leche (aunque no son comparables) para personas con intolerancia a los productos lácteos, se demanda por sus cualidades funcionales que pueden contribuir a reducir o prevenir enfermedades, que aquejan en la actualidad a la sociedad; esto debido principalmente al contenido de ácidos grasos poliinsaturados y monoinsaturados, y a las fracciones con propiedades antioxidantes, como vitamina E, compuestos fenólicos, flavonoides y poliaminas. El principal propósito de este estudio fue aplicar una tecnología emergente como la Ultra Alta Presión de Homogeneización (UHPH) y compararla con tecnologías que actualmente se utilizan en la industria para procesar este tipo de bebidas tales como los tratamientos térmicos (pasteurización y UHT). Dicha tecnología UHPH, utiliza homogeneizadores con un diseño específico capaces de soportar presiones hasta de 400 MPa, lo cual ayuda a producir alimentos de excelente calidad, ya que al aplicar tan elevadas presiones, se producen una serie de fenómenos que conllevan a la disminución en el tamaño de partículas, que conduce a la mejoría de factores como la estabilidad coloidal, la destrucción microbiana, etc. Por todo ello, la hipótesis de partida es que los tratamientos UHPH pueden constituir una alternativa mejorada a los tratamientos térmicos convencionales. Esta investigación se realizó a través de cuatro estudios sucesivos. El primero, sirvió para optimizar las condiciones de elaboración de la bebida de almendra en planta piloto, ya que será el producto de partida para los posteriores estudios. Asimismo se estudió la influencia de la adición de lecitina como emulgente en la estabilidad de los licuados pasteurizados, evaluándose los análisis más relevantes relacionados con la calidad global del alimento. En el segundo estudio se evaluó la influencia de las condiciones de tratamientos UHPH (200 y 300 MPa, 55, 65 y 75 °C) en las características de los licuados de almendra y se compararon con tratamientos convencionales de pasteurización y UHT y con el producto base crudo. Los tratamientos que se revelaron como alternativos de la pasteurización fueron 200 MPa, tanto a 55 como a 75 ºC de temperatura de entrada. Mientras que 300 MPa a 65 y 75ºC fueron los alternativos a la UHT, en los que se pudo conseguir bebidas de almendra comercialmente estériles. El tercer estudio consistió en la identificación de los microorganismos potencialmente alterantes en la bebida de almendra tratada por UHPH, para posteriormente evaluar la letalidad producida por los tratamientos de UHPH de 300 MPa, 55, 65, 75 y 85 °C en los microorganismos que fueron más frecuentes aislados y que habían mostrado previamente, cierta resistencia a este tipo de tratamientos, que fueron Bacillus cereus, Bacillus subtillis y Lysinibacillus spp. La letalidad fue completa cuando se utilizó el tratamiento de 300 MPa con 75 ºC y 85 °C de temperatura de entrada. Por último, con las condiciones seleccionadas de los tratamientos UHPH, alternativos a la pasteurización (200 MPa, 55 y 75 °C), se estudió la calidad microbiológica, física y química de la bebida de almendra durante el almacenamiento a 4ºC y se comparó con la bebida de almendra pasteurizada. Se realizó un seguimiento, analizando los licuados almacenados en refrigeración cada 7 días durante tres semanas. Los resultados demostraron que durante el almacenamiento a 4ºC las bebidas de almendra tratadas por UHPH a ambas temperaturas de entrada, tuvieron en general, mejores características que las pasteurizadas.
In the last years the almond beverage has experimented an increase on the consume, therefore, the commercialization area has been expanded, almost all around the world. This beverage further than be an alternative to milk for people with lactose intolerance, it is claimed because of its functional qualities that can contribute to reduce or prevent diseases, which are besetting society; this mainly due thanks to the content of the fatty acids, monounsatured and polyunsatured, and the antioxidants just like E vitamin, phenolic compounds and polyamines. This thesis project was focused on the application of an emerging technology, Ultra High Pressure Homogenization (UHPH), in the production of almond beverage. This non-thermal technology consists of a high pressure machine capable of applying pressures of up to 400 MPa using a special homogenizing system designed to produce a conserving effect, improving the colloidal and microbial stability while maintaining good nutritional and sensory qualities. Considering this hypothesis, UHPH could be an alternative technology to those commonly applied in the food industries. For that, a comparative study of UHPH with thermal treatments (pasteurization) was carried out in this work. This investigation has been conducted through four successive studies. The first study was aimed to optimize processing conditions of the elaboration of a base product of almond beverage for being used in subsequent studies. Moreover, influence of the addition of lecithin as an emulsifier in the stability of pasteurized beverage was studied and the most relevant analytical parameters related to the overall quality of the food were analyzed. In the second study, the influence of the UHPH treatments (200 and 300 MPa, 55, 65 and 75 ºC) were evaluated in the almond beverage characteristics, and they were compared with samples treated with convectional pasteurization, UHT and with based crude product. UHPH at 200 MPa 55 and 75 ºC of inlet temperature were revealed as alternative treatments to pasteurization, and 300 MPa 65 y 75ºC as alternative to UHT. The UHPH condition of 300 MPa and 65 and 75ºC inlet temperature was able to produce commercial sterile almond beverages. The third study was the identification of potentially contaminating spoilage microorganisms in treated almond beverage, to evaluate subsequently the lethality produced by UHPH treatments 300 MPa, 55, 65, 75 and 85 °C, in microorganisms with higher resistance to UHPH and most frequently isolated. These microorganisms were Bacillus cereus, Bacillus subtilis and Lysinibacillus spp. Lethality was complete when treatment was used 300 MPa and 75 and 85°C inlet temperatures. The second part consisted in the shelf-life evaluation of soymilk treated by UHPH using the selected optimal conditions determined in the previous step Finally, with the selected conditions of UHPH treatments (200 MPa, 55 and 75 ºC), was studied the microbiological, physical and chemical quality of the almond beverage during cold storage time and it was compared with pasteurized almond beverage. Monitoring was conducted every 7 days for 3 weeks. During cold storage, almond beverage samples treated by UHPH at both inlet temperatures had generally better characteristics than pasteurized ones.

Les propriétés émulsifiantes et interfaciales des protéines du lait de chamelle ont été étudiées seules, en mélange ou dans leur environnement naturel (dans le lactosérum sérum, sous forme de caséinates ou directement dans le lait de chamelle). L’impact de certaines conditions physico-chimiques a été étudié, notamment l’impact du pH, du traitements thermiques, de la concentration et de l’homogénéisation hautes pressions, afin de comprendre le rôle de chacune des dénaturations appliquées et leur impact sur les propriétés émulsifiantes. Cette étude a été réalisée en parallèle sur le lait de vache, dans des conditions identiques. Les principaux résultats suggèrent que le lait de chamelle est plus émulsifiant que le lait de vache quelle que soit la température du traitement, cependant, la stabilité des émulsions préparées par le lait de vache non traité est plus importante. L’effet d’homogénéisation hautes pressions permet d’avoir des émulsions très stables comparées au lait de vache. Les effets du pH et de la température sur les fractions caséinique et sérique du lait de chamelle influencent significativement leurs comportements à l’interface huile-eau et leurs pouvoirs à créer des émulsions. Le recouvrement protéique des caséinates de sodium de lait de chamelle à la surface des gouttelettes d’huile augmente en fonction du pH (de 3,0 à 9,0) et ceci quelle que soit la température de traitement appliqué (entre 25°C et 95°C). Ce comportement vis-à-vis le pH est conséquent d’une dénaturation et une perte de la structure micellaire des caséinates de sodium à l’interface huile-eau en facilitant ainsi leurs adhésions et adsorption à l’interface. Cependant, la fraction sérique du lait de chamelle a exprimé une activité émulsifiante importante à pH acide contrairement à la fraction des caséinates de sodium ainsi qu’une meilleure stabilité émulsifiante des gouttelettes crées. Le lactosérum du lait de chamelle est moins sensible à l’effet de la température que le lactosérum du lait bovin riche en β-lactoglobuline (plus sensible au traitement thermique). Ces aspects sont dus à la petite taille moléculaire des protéines sériques relative à la taille des caséines ce qui leurs permet de s’adsorber rapidement et efficacement à l’interface des gouttelettes huileuses et de former un film viscoélastique stable tout autour. Des réarrangements structuraux de l’α-lactalbumine cameline (purifiée) ainsi que des répulsions électrostatiques entre les gouttelettes d’huile stabilisées par ces protéines sont les deux facteurs clés pour expliquer l'effet de la variation du pH et du traitement thermique. Les propriétés de surface de l’α-lactalbumine cameline sont significativement améliorées à la suite des traitements thermiques élevés (95°C) ce qui maintiennent des propriétés émulsifiantes potentielles. Quant à la β-caséine purifiée, son activité émulsifiante est plus importante pour le lait de chamelle que le lait de vache. Une structure micellaire des β-caséines se forment à partir de la concentration micellaire critique (CMC) qui maintient une structure compacte proche du pH 6,0 et se relâche à pH alcalin ou les forces électrostatiques sont maximales. Le comportement rhéologique des émulsions est non-newtonien où la viscosité est plus importante à faible vitesse de cisaillement. Elle dépend fortement de la valeur du pH appliqué. Ainsi, les émulsions stabilisées à pH acide sont les plus visqueuses indépendamment de la température du traitement thermique appliquée pour la plupart des fractions étudiées. (...)
The emulsifying and interfacial properties of camel milk proteins were studied alone, in mixture or in their natural environment (in serum whey, as caseinates or directly in camel milk). The impact of certain physico-chemical conditions was studied, the impact of pH, heat treatment, concentration and homogenization at high pressures. This study was carried out in parallel on cow's milk, under identical conditions. The main results suggest that camel milk is more emulsifying than cow's milk regardless of the processing temperature, however, the stability of emulsions prepared by cow's milk is more important. The homogenizing effect at high pressures allows very stable emulsions compared to cow's milk. The effect of pH and temperature on the caseinic and serum fractions of camel milk is very significant on their behaviour at the oil-water interface and their ability to create emulsions. The coating of oil droplets by sodium caseinates protein of camel milk increases with incrising pH (from 3.0 to 9.0) regardless of the heat treatment (between 25°C and 95°C). This behaviour is the result of denaturation and loss of the micellar structure of sodium caseins at the oil-water interface, thus facilitating their adhesions and adsorption at the interface. However, the whey fraction of camel milk expressed significant emulsifying activity at acid pH as opposed to the sodium caseinate fraction and better emulsifying stability of the created oil droplets. Camel milk whey proteins are found to be less sensitive to the effect of temperature in emulsion creation than bovine milk whey (richer in β-lactoglobulin which are more sensitive to heat treatment). These aspects are due to the small molecular size of serum proteins relative to casein size, which allows them to adsorb quickly and efficiently at the interface of oil droplets and form a stable viscoelastic film all around which suggest that camel milk is more emulsifying active than cow's milk regardless of the processing temperature, however, the stability of emulsions prepared by cow's milk is found to be more important. The homogenizing effect at high pressures allows very stable emulsions compared to cow's milk. The main factor governing the stability of camel milk α-lactalbumin emulsions is its conformational flexibility at the oil-water interface. Structural rearrangements of camel milk α-lactalbumin as well as electrostatic repulsions between oil droplets stabilized by these proteins are the two key factors to explain the effect of pH variation and heat treatment. The surface properties of α-lactalbumin from camel milk are significantly improved after high heat treatments (95°C) which maintain potential emulsifying properties. As for the purified β-casein, its emulsifying activity is more important for camel milk than cow's milk. A micellar structure of caseins is formed from the critical micellar concentration (CMC) which maintains a compact structure close to pH 6.0 and is released at alkaline pH where electrostatic forces are at their highest. This induced a high stability of emulsions at pH 9.0 as well as a better emulsifying activity of β-caseins. The rheological behaviour of emulsions is non-Newtonian where viscosity is higher at low shear rates which depends strongly on the applied pH. Thus, stabilized emulsions at acid pH are the most viscous regardless of the heat treatment temperature applied for most of the studied fractions. This is mainly due to hydrophobic interactions between proteins of the different fractions and to high cohesion forces between larger droplets. However, the viscosity of emulsions stabilized by the protein fractions of cow's milk is often more viscous than camel milk as a result of their higher emulsifying stability. (...)

Lung cancer is the deadliest cancer in the world, with a global 5-year survival rate of about 15%. Despite a notable impact of the latest improvements in prevention, screening, detection and staging, the efficacy of conventional treatments is not sufficient and has reached a therapeutic plateau. These conventional treatments involve a combination of surgery, radiotherapy (RT) and chemotherapy (CT). CT is used in almost all stages: in operable and inoperable stages to limit tumour cell invasion and in latest stages as a palliative treatment. Cisplatin is one of the most frequently used and most potent drugs available. It is administered by parenteral route at doses limited by its high and cumulative nephrotoxicity but also by other systemic toxicities (e.g. ototoxicity). Its administration therefore requires many precautions (long hydration procedure, surveillance of the renal function), which mobilize medical personnel. A major limitation of parental CT is the low concentration of drug that successfully reaches the tumour or the metastases. A potential additional modality could be aerosolized CT to localize lung cancer treatment. It has shown a relative local tolerance for cisplatin through preclinical and clinical studies in humans by means of nebulized solutions or liposomal formulations. As a local treatment, aerosolized CT has a clear pharmacokinetic (PK) advantage, as it can increase local exposure while decreasing systemic exposure. However, because CT drugs, such as cisplatin, are active at rather high doses (in the mg range), the duration of administration from nebulizers is very long as it depends on the drug solubility or on drug encapsulation into liposomes. They also pose a high risk of environmental contamination and require HEPA-filtrated hoods during the nebulization procedure. Of all the inhalation devices available to deliver high drug doses, dry powder inhalers (DPIs) were chosen in this work. These were chosen to circumvent the above issues by providing higher deposited doses, in very short timeframes, using a patient-driven device that could help limit environmental exposure to only very low levels of drug. DPI in general also have the advantage of being applicable to both poorly-water-soluble and to water-soluble anticancer drugs. However, because direct deposition of high quantities of anticancer drugs to the lung parenchyma could pose a high risk of local irritation and pulmonary adverse effects, controlled release (CR) of cisplatin from deposited particles in the lung parenchyma was needed. However, in the lungs, foreign undissolved particles are rapidly eliminated by means of naturally occurring clearance mechanisms, in particular macrophage uptake in the alveoli. Therefore, formulation strategies able to limit the particles clearance are needed to assure high lung residence of these CR particles. The formulation strategy of this work was to develop DPI formulation based on solid-lipid microparticles (SLM) able to (i) be deposited into the lung, (ii) control the release of cisplatin and (iii) escape macrophage uptake in order to remain in the lung long enough and at a concentration able to optimize the therapeutic index (i.e. increase the potential therapeutic effect and decrease the potential side effects).The primary objectives of the SLM-based DPI formulations were to (i) exhibit aerodynamic properties compatible with lung cancer patients abilities and cisplatin requirements (e.g. a high deposited fraction, high deagglomeration abilities under low airflow within a low-resistance DPI, deposition in the mg range), (ii) provide a CR matrix for cisplatin in vitro, (iii) be able to be retained into the lung long enough in vivo, (iv) using scalable production techniques and (v) using only potentially well-tolerated excipients.Cisplatin was initially reduced to microcrystals under high-pressure homogenization (HPH) cycles up to 20 000 psi. This procedure permitted uncoated particles with mean diameters below 1.0 μm to be obtained. To assess the cisplatin release abilities of the DPI formulations on the deposited fraction only, a new dissolution test was adapted. This test used a classical paddle apparatus from the pharmacopoeia and a Fast Screening Impactor (FSI). An excipient-free formulation, obtained from the spray dried suspension of cisplatin microcrystals (100% cisplatin) was initially produced. It was compared to a 95:5 cisplatin/tocopheryl polyethylene glycol succinate (TPGS) formulation, which exhibited a higher deposition ability (fine particle fraction (FPF) of 24.2 vs. 51.5% of the nominal dose, respectively). Both exhibited immediate release (IR), with 90% dissolved under 10 minutes.Solid lipid microparticle (SLM)-based formulations were then produced using the cisplatin microcrystalline suspension and various lipid excipients. Those had previously been screened for their ability to be spray dried following their solubilisation in heated isopropanol. The addition of a triglyceride, tristearin (TS), as the main lipid component and if necessary a polyethylene glycol (PEG) excipient-comprising fraction with TPGS or distearoyl phosphoethanolamine polyethylene glycol 2000 (DSPE-mPEG-2000) as a surface modifier, provided spray dried particles with interesting characteristics. These formulations, comprised of at least 50% cisplatin, exhibited high CR abilities in simulated lung fluid at 37°C for more than 24 h (as low as 56% released after 24 h) and a low burst-effect (as low as 24% and 16% after 10 minutes with and without PEGylated excipients, respectively). They also showed high aerodynamic properties, with a high FPF ranging from 37.3 to 50.3% w/w of the nominal dose and a low median mass aerodynamic diameter (MMAD) between 2.0 and 2.4 μm. The process also offered high production yields (> 60%).The best IR DPI formulation (evaluated on the FPF, i.e. cisplatin/TPGS 95:5) and the most promising CR formulations without (i.e. cisplatin/TS 50:50) and with PEGylated excipients (evaluated on CR abilities, i.e. cisplatin/TS/TPGS 50:49.5:0.5) were then administered to CD 1 mice, concurrently to endotracheal nebulization (EN) of a cisplatin solution. This was done using specific endotracheal devices, the Penn-Century Inc. DP-4M© Dry Powder Insufflatorn and for the cisplatin solution, the Microsprayer™ IA-1C©. They were compared to intravenous (IV) injection during a PK study over 48 hours. The administration of DPI formulations required the development of a spray dried diluent (Mannitol:Leucine 10:1) and specific dilution method (3D mixing for 4 hours and double-sieving) to be able to deliver precise and repeatable quantities of powder into the lungs of mice at 1.25 mg/kg dose. A PK study was carried out of the lungs, blood, kidneys, liver, mediastinum and spleen of the mice. The study used a developed and validated electrothermal atomic absorption spectrometry (ETAAS) method. Results showed that endotracheal administration of DPI formulations permitted the exposure of the lungs to cisplatin, expressed as the area under the curve (AUC) to be greatly increased while decreasing the systemic exposure. More precisely, the only formulation that exhibited prolonged lung retention was the one comprising PEGylated excipient (cisplatin/TS/TPGS 50:49.5:0.5), which was observed for ~7 hours. This lung retention was associated with smoother concentration vs. time profiles in blood (higher tmax and lower Cmax), which also confirmed its CR abilities in vivo as dissolved cisplatin is a highly permeable drug. The overall exposure, established by the AUC, helped calculate the target efficiency (Te: the ratio of AUC in the lungs to the sum of AUC in non-target organs) and the target advantage (Ta: ratio of AUC in the lungs by the tested route to the AUC in the lungs by the IV route). For instance, the Ta of the aforementioned formulation (cisplatin/TS/TPGS 50:49.5:0.5) was of 10.9, as compared to 1 for IV, 3.3 for EN, 2.6 for the IR DPI formulation (cisplatin/TPGS 95:5) and 3.7 for the non-PEGylated CR DPI formulation (cisplatin/TS 50:50). In the meantime, the Te for the same formulations were 1.6, 0.09, 1.1, 0.4 and 0.9, respectively, showing again the great efficiency of the inhaled route vs. the IV route in targeting the lungs. More importantly, it showed the added efficiency of the CR DPI formulation with lung retention abilities, provided by the addition of PEGylated excipients. In the last part of the work, maximum tolerated doses (MTD) of formulations were established. These showed that the best candidate, selected based on the PK results (CR DPI with lung retention abilities composed of cisplatin/TS/TPGS 50:49.5:0.5) had better overall tolerance than IR approaches (DPI formulation at cisplatin/TPGS 95:5 and EN of a cisplatin solution). More precisely, it was possible to double the administered dosage for the CR formulation (1.0 mg/kg) vs. the IR DPI and EN (both at 0.5 mg/kg) under a repeated administration scheme (3 times a week for 2 weeks).Moreover, an assessment of the lung tolerance of this best candidate was realized and compared to the IR DPI, EN and the IV route. It was done through analysis of the broncho-alveolar lavage fluid (BALF) 24 hours following a single administration at the pre-determined MTD. IL-1β, IL-6 and TNF-α cytokines were not increased following the administrations. No evidence of tissue damage or cytotoxicity could be observed through quantification of the protein content and of lactate dehydrogenase (LDH) activity. The only observations were a decrease in total cells and an increase in polynuclear neutrophils (PN) cells in the BALF, which was not observed by IV or following the administration of the vehicle of the CR formulation alone (i.e. PEGylated SLM and dry diluent). This increase was not directly linked to the formulation but rather to cisplatin, as it was observed in each cisplatin inhalation experiments, and not with the vehicle of the CR formulation, which was comparable to the non-treated mice.In parallel, we realized a survival study following the administration of the best DPI formulation candidate (cisplatin/TS/TPGS 50:49.5:0.5) vs. the IR DPI candidate (cisplatin/TPGS 95:5), both at their respective MTD under the aforementioned repeated dosing scheme. Cisplatin was administered to mice bearing a grafted orthotopic M109-HiFR lung tumour model, previously developed in the laboratory. The DPI formulations were evaluated against IV administration at each dose (0.5 and 1.0 mg/kg, respectively). This study first confirmed the lower toxicity of the CR approach, as the IR DPI formulation caused a much higher number of deaths during treatment of the grafted mice. The CR formulation administered at 1.0 mg/kg showed a higher survival than the negative control but a tumour response comparable to IV administered at half this dose (0.5 mg/kg). This unexpected outcome with regard to the PK results is explained by the fact that the tumour model is highly metastatic. Mice treated with inhaled formulations died due to distant tumour involvement, while those treated systemically died due to pulmonary tumour involvement. This led us to believe that this kind of treatment may have greater potential in combination, adjuvant to the parenteral route.This work helped establish the proof-of-concept of a cisplatin CR DPI formulation with an up-scalable process. The SLM approach confirmed that encapsulation of drugs exhibiting low solubility, such as cisplatin, was possible using highly hydrophobic excipients and that surface modification was mandatory to provide notable lung retention in vivo. The SLM approach showed good signs of tolerance during the exploratory study but still needs to be confirmed under a chronic scheme using other determinants such as histopathological analyses of the lung tissue. Moreover, comparison of the nephrotoxicity of formulations against that of the IV route should be conducted with appropriate and sensitive methods. Finally, the survival study of the CR DPI formulation showed mitigated results, partly because of the orthotopic model characteristics. This could be proof that inhaled CT has a role to play combined with classical systemic CT. This needs to be assessed in a further study.Le cancer du poumon est le cancer ayant le taux de mortalité le plus élevé au monde, avec un taux de survie global à 5 ans d'environ 15%. Malgré un impact notable des dernières améliorations en matière de prévention, de dépistage, et de classification du cancer du poumon, l'efficacité des traitements classiques n'est toujours pas suffisante et semble avoir atteint un plateau thérapeutique. Ces traitements classiques comprennent de la chirurgie, de la radiothérapie et de la chimiothérapie, le plus souvent en combinaison. La chimiothérapie est utilisée à presque tous les stades: dans les stades opérables et inopérables afin de limiter l'invasion par les cellules tumorales jusqu’aux derniers stades en tant que traitement palliatif. Le cisplatine est l'un des médicaments anticancéreux les plus fréquemment utilisés et les plus puissants actuellement disponibles. Il est administré par voie parentérale à des doses qui sont limitées par sa néphrotoxicité élevée et cumulative mais également par d'autres toxicités systémiques (par exemple, de l'ototoxicité). Son administration nécessite donc de nombreuses précautions (longue procédure d'hydratation, surveillance de la fonction rénale), ce qui mobilise fortement le personnel médical. Une limitation importante de la chimiothérapie parentérale est la faible concentration d’actif qui atteint avec succès la tumeur ou les métastases. Une autre voie d’accès potentielle pourrait être la chimiothérapie inhalée pour traiter le cancer du poumon. Cette approche a montré une relativement bonne tolérance locale pour le cisplatine à travers différentes études précliniques et cliniques chez l'homme au moyen de solutions ou de formulations liposomales nébulisées. En tant que traitement via la voie pulmonaire, la chimiothérapie inhalée présente un avantage pharmacocinétique évident, car elle permet d’augmenter l'exposition locale tout en diminuant l'exposition systémique. Cependant, du fait que les médicaments chimiothérapeutiques, tels que le cisplatine, soient actifs à des doses relativement élevées (dans la gamme du mg), la durée d'administration à partir des nébuliseurs s’avère en pratique très longue car elle dépend principalement de la solubilité de l’actif ou de son encapsulation dans les liposomes. Les nébuliseurs présentent également un risque élevé de contamination de l'environnement et nécessitent de lourds appareillages (hottes filtrantes en particulier) pendant la procédure d’administration.Parmi tous les dispositifs d'inhalation existants, capables de délivrer des doses élevées de médicaments, les inhalateurs de poudre sèche (DPI) semblent être de bons candidats. Ceux-ci ont été choisis dans ce travail afin de contourner les problèmes énumérés ci-dessus, en fournissant des doses pulmonaires plus élevées, dans des délais très courts. De plus, ces dispositifs sont activés par le flux inspiratoire du patient, ce qui pourrait aider à limiter l'exposition environnementale à des niveaux très faibles. Les inhalateurs à poudre sèche présentent également l'avantage d'être utilisables à la fois avec des médicaments solubles et des médicaments peu solubles dans l’eau. Malgré tout, étant donné que la déposition directe de quantités élevées de médicaments chimiothérapeutiques dans le parenchyme pulmonaire pourrait présenter un risque élevé d'irritation et d'effets indésirables locaux, une libération contrôlée du cisplatine à partir de particules déposées dans le parenchyme pulmonaire s’avère nécessaire. Cependant, dans les poumons, ces particules non dissoutes d’origine étrangère sont rapidement éliminées par les mécanismes d’élimination, en particulier par la clairance par les macrophages au niveau des alvéoles. Par conséquent, des stratégies de formulation capables de limiter la clairance des particules sont nécessaires pour assurer une résidence pulmonaire élevée de ces particules à libération contrôlée.La stratégie de formulation de ce travail a donc consisté à développer une formulation pour inhalateur à poudre sèche à base de microparticules lipidiques solides capable de (i) être déposées dans le poumon, (ii) de contrôler la libération du cisplatine et (iii) de rester dans le poumon suffisamment longtemps dans le but d’optimiser l'indice thérapeutique (c'est-à-dire augmenter le potentiel thérapeutique du cisplatine et diminuer ses potentiels effets secondaires).Les objectifs principaux des formulations basées sur les microparticules lipidiques solides étaient (i) de présenter des hautes charges en cisplatine au sein des microparticules lipidiques tout en présentant des propriétés aérodynamiques compatibles avec la capacité pulmonaire des patients atteints de cancer du poumon (par exemple, une fraction déposée élevée et une capacité élevée à la désagglomération sous faible débit d'air dans un inhalateur de faible résistance), (ii) de fournir une matrice capable de libérer le cisplatine de manière contrôlée in vitro, (iii) d’être capable de rester dans le poumon suffisamment longtemps in vivo, tout cela (iv) en utilisant des techniques de production ayant une bonne capacité d’augmentation d’échelle et (v) de n’utiliser que des excipients potentiellement bien tolérés au niveau du poumon.Le cisplatine a été initialement réduit sous forme microcristalline à l’aide de cycles d'homogénéisation à haute pression jusqu'à 20 000 psi. Cette procédure a permis d'obtenir des particules non enrobées ayant un diamètre moyen inférieur à 1.0 μm. Afin d’évaluer les capacités de libération du cisplatine des formulations à partir de la fraction capable théoriquement de se déposer dans les poumons, un nouveau test de dissolution a été adapté à partir d’un appareil à palettes classique de la pharmacopée et d’un impacteur à cascade « Fast Screening Impactor ». Une formulation sans excipient, obtenue à partir de la suspension de cisplatine, soumise à la technique de séchage par l’atomisation (100% de cisplatine) a été produite comme point de départ. Celle-ci a ensuite été comparée à une formulation de cisplatine/tocophéryl polyéthylène glycol succinate (TPGS) (95:5), qui présentait une capacité de déposition pulmonaire in vitro (fraction de particules fines (FPF) de 24.2% pour la première et de 51.5% pour la deuxième, exprimée par rapport à la dose nominale). Toutes deux ont démontré des capacités de libération immédiate, avec 90% du cisplatin dissous en moins de 10 minutes.D’autres formulations, cette fois élaborées sous la forme de microparticules lipidiques solides ont ensuite été produites à partir de la suspension microcristalline de cisplatine et de divers excipients lipidiques. Ces microparticules avaient préalablement été testées pour leur aptitude à être séchées par atomisation après solubilisation des excipients dans de l'isopropanol chaud. L’ajout d’un triglycéride, la tristéarine (TS), comme excipient lipidique principal et également d’une fraction comprenant un excipient contenant du polyéthylène glycol (PEG), à l’aide de TPGS ou de distéaroyl phosphoéthanolamine polyéthylène glycol 2000 (DSPE-mPEG-2000) a montré des résultats intéressants. Ces formulations, ayant une teneur en cisplatine d’au moins 50%, ont présenté des aptitudes élevées pour la libération contrôlée dans le fluide pulmonaire simulé in vitro à 37 °C, et ce, pendant plus de 24 h (jusqu'à 56% libérées après 24 h) ainsi qu’un faible « burst-effect » (de seulement 24% et 16% après 10 minutes avec et sans excipients PEGylés, respectivement). Elles ont également montré des propriétés aérodynamiques élevées, avec une FPF élevée allant de 37.3 à 50.3% m/m par rapport à la dose nominale et un diamètre aérodynamique compris entre 2.0 et 2.4 μm. Le meilleur candidat à libération immédiate (évaluée sur base de la FPF, soit la formulation cisplatine/TPGS 95:5 m/m) et les formulations à libération contrôlée les plus prometteuses n’incluant pas d’excipients PEGylés (cisplatine/TS 50:50 m/m) et incluant des excipients PEGylés (évalués sur les capacités de libération contrôlée, c'est-à-dire la formulation cisplatin/TS/TPGS 50:49.5:0.5 m/m/m) ont ensuite été administrées à des souris CD-1, en comparaison d’une nébulisation endotrachéale d'une solution de cisplatine. Ceci a été fait à l’aide de dispositifs endotrachéaux dédiés aux poudres pour le DP-4M© « Dry Powder Insufflator » et aux solutions pour le Microsprayer™ IA-1C© de Penn-Century. Ces formulations ont été comparées à l'injection intraveineuse (IV) au cours d’une étude pharmacocinétique étendue sur 48 heures.L'administration de formulations de poudres sèches pour inhalation a nécessité le développement préalable d'un diluant par atomisation (Mannitol:Leucine 10:1 m/m) ainsi que d’une méthode de dilution des poudres (mélange tridimensionnel pendant 4 heures et suivi d’un double-tamisage) afin de pouvoir délivrer des quantités précises et répétables de poudre dans les poumons de souris à la dose d’1.25 mg/kg. Le suivi des paramètres pharmacocinétiques a ainsi pu être réalisé au niveau des poumons, du sang, des reins, du foie, du médiastin et de la rate des souris. Ceci a été fait à l’aide d’une méthode de spectrométrie d'absorption atomique électrothermique, qui a été préalablement développée et validée. Les résultats obtenus ont montré que l'administration endotrachéale de formulations de poudres sèches permettait d’augmenter fortement l'exposition des poumons par le cisplatine, exprimée en aire sous la courbe (AUC) tout en diminuant l'exposition systémique. Plus précisément, la seule formulation présentant une rétention pulmonaire prolongée était celle qui comprenait un excipient PEGylé (cisplatine/TS/TPGS 50:49.5:0.5 m/m/m), ce qui a été observé pendant environ 7 heures. Cette rétention pulmonaire a été associée à des profils de concentration en fonction du temps plus réguliers dans le sang (tmax supérieur et Cmax inférieur), ce qui a également confirmé ses capacités de libération contrôlée in vivo car la perméabilité de l’épithélium pulmonaire pour le cisplatine dissous s’est avérée très élevée. L'exposition globale établie à partir de l’AUC a permis de calculer l’efficacité de ciblage (Te: rapport de l'AUC mesurée dans les poumons et de la somme des AUC mesurées dans les organes non cibles) et l’avantage du ciblage (Ta: rapport de l’AUC mesuré dans les poumons suite à l’administration pulmonaire et de l'AUC mesurée dans les poumons suite à l’administration par la voie IV). Par exemple, le Ta de la formulation décrite ci-dessus (cisplatine/TS/TPGS 50:49.5:0.5 m/m/m) était de 10.9, comparativement à 1 pour l’IV, 3.3 pour la nébulisation endotrachéale, 2.6 pour la formulation de poudre sèche à libération immédiate (cisplatine/TPGS 95:5 w/w) et 3.7 pour la formulation de poudre sèche à libération contrôlée ne comprenant pas d’excipient PEGylé (cisplatine/TS 50:50). Dans le même temps, le Te mesuré pour les mêmes formulations était de 1.6, 0.09, 1.1, 0.4 et 0.9, respectivement, démontrant également le rendement élevé de la voie inhalée par rapport à la voie IV dans sa capacité à cibler les poumons. Plus important encore, ceci a démontré le grand avantage des capacités de rétention pulmonaire de la formulation à libération contrôlée comprenant un excipient PEGylé.Dans la dernière partie de ce travail, les doses maximales tolérées (DMT) des formulations ont été déterminées. Le meilleur candidat, choisi en fonction des résultats de pharmacocinétique (formulation à libération contrôlée ayant des capacités de rétention pulmonaire composé de cisplatine/TS/TPGS 50:49.5:0.5 m/m/m), avait une meilleure tolérance globale que les deux approches à libération immédiate testées (formulation de poudre sèche cisplatine/TPGS 95:5 et la nébulisation endotrachéale d'une solution de cisplatine). Plus précisément, il s’est avéré possible de doubler le dosage administré pour la formulation à libération contrôlée (1.0 mg/kg) par rapport à la poudre sèche à libération immédiate et à la nébulisation endotrachéale (toutes les deux à 0.5 mg/kg) suivant un schéma d'administration chronique (3 fois par semaine pendant 2 semaines). De plus, une évaluation de la tolérance pulmonaire de cette formulation à libération prolongée a été réalisée et comparée à la poudre sèche à libération immédiate, à la nébulisation endotrachéale et à la voie IV. Elle a été réalisée par analyse du liquide provenant du lavage broncho-alvéolaire, 24 heures après une administration unique à la dose maximale tolérée préalablement déterminée pour chaque formulation. Aucune augmentation des cytokines IL-1β, IL-6 et TNF-α n’a pu être détectée à la suite des administrations. Aucunes preuves de lésion tissulaire ou de cytotoxicité n'ont pu être observées au travers du dosage de la teneur en protéines totale et de l'activité de la lactate déshydrogénase. Les seules observations qui ont pu être faites ont été une diminution des cellules totales et une augmentation des polynucléaires neutrophiles dans le lavage broncho-alvéolaire, ce qui n'a pas été observé suite à l’administration IV ou après l'administration du véhicule de la formulation à libération contrôlée seul (c'est-à-dire les microparticules lipidiques solides PEGylées et le diluant). Cette augmentation ne semble pas liée aux microparticules lipidiques solides ou au diluent mais probablement à l’exposition pulmonaire au cisplatine, car cette augmentation a été observée pour chaque groupe inhalé contenant du cisplatine. Le cisplatine a ensuite été administré à des souris qui ont été greffées de manière orthotopique par une lignée murine de carcinome pulmonaire M109-HiFR, modèle préclinique préalablement développé au sein de notre laboratoire. Les formulations de poudres sèches ont été évaluées par rapport à l'administration IV à chaque dose testée (0.5 et 1.0 mg/kg, respectivement). Cette étude a d'abord confirmé la toxicité plus faible de l'approche à libération contrôlée, car la formulation à libération immédiate a causé un nombre beaucoup plus élevé de décès pendant le traitement des souris greffées. La formulation à libération contrôlée administrée à 1.0 mg/kg, a montré une survie plus élevée que le contrôle négatif, mais une réponse comparable à la dose IV administrée à la moitié de la dose (0.5 mg/kg). Ce résultat inattendu par rapport aux résultats de l’étude pharmacocinétique s'explique probablement par le fait que le modèle de tumeur utilisé est hautement métastatique. Les souris traitées avec des formulations inhalées sont mortes en raison de tumeurs secondaires distantes par rapport à la tumeur primaire implantée au niveau du poumon, alors que celles traitées par la voie systémique sont mortes en raison d’un envahissement tumoral pulmonaire. Cela nous amène à penser que ce type de traitement inhalé pourrait avoir un plus grand potentiel en combinaison à la voie parentérale. Ce travail a ainsi permis d’établir la preuve du concept de formulation à base de poudre sèche de cisplatine à libération contrôlée, en utilisant un processus de fabrication capable de subir une mise à l’échelle industrielle. L’utilisation de microparticules lipidiques solides a confirmé que l'encapsulation d’actifs présentant une certaine hydrophilie, comme le cisplatine, était possible en utilisant des excipients hautement hydrophobes et qu'une modification de leur surface était cependant obligatoire pour obtenir une rétention pulmonaire intéressante in vivo. Les microparticules lipidiques solides ont montré de bons signes de tolérance au cours de l'étude exploratoire, mais celle-ci doit encore être confirmée avec une administration chronique des poudres. Ceci doit être fait en suivant des paramètres supplémentaires, tels que des analyses histologiques du tissu pulmonaire. De plus, la comparaison de la néphrotoxicité des formulations avec celle mesurée par la voie IV doit être effectuée avec des méthodes appropriées et sensibles. Enfin, l'étude de survie de la formulation à libération prolongée a montré des résultats mitigés, en partie à cause des caractéristiques du modèle orthotopique de tumeur pulmonaire. Cependant, il semblerait que la chimiothérapie inhalée à un rôle important à jouer en combinaison avec la chimiothérapie systémique classique. Ceci doit être évalué dans une étude future.
Doctorat en Sciences biomédicales et pharmaceutiques (Pharmacie)
info:eu-repo/semantics/nonPublished

碩士
東海大學
食品科學系
98
Chitin was treated with NaOH solution (57%, w/v) under high temperature treatment (100℃) for 160 min to cleave acetyl group and became 80~90 % deacetylated chitosan. Pre-emulsions that were carried out by adding oil (10, 30, 50, 70, 90 and 120 mL oil/100 mL chitosan solution, 3 mL/interval) into chitosan solution (1.5 % concentrations, pH 6.0) and homogenizing with polytron homogenizer (9500 rpm), then, were passed through the high pressure homogenizer at different pressures (500, 1000, 1500 and 1800 bar) and recycled in some passes (1 to 7 passes). The emulsions were determined on the droplet size and stability for four weeks. From the above result, emulsions that had nano-droplets and good stability were chosen for tests of thermal and freeze-thawing stabilities. Thermal stability was treated at 5℃, 25℃, 60℃, 100℃ and 121℃ for 30 min, then stored at room temperature for 4 weeks. Freeze-thawing treatment was carried out by freezing at -18℃ and thawing at 25℃ for 1 hr, individually; five freeze-thawing cycles were made. The treated emulsions were determined the droplet size, viscosity and stability. As result, emulsions that were emulsified under suitable conditions showed nano size droplets and good stability. Lower oil/chitosan solution ratio, higher homogenizing pressure and more number of passes gave smaller droplet-sizes and more stable emulsions, group A: 1500 bar, 30 mL oil/100 mL chitosan solution, 3 passes; group B: 1500 bar, 50 mL oil/100 mL chitosan solution, 7 passes; group C: 1500 bar, 70 mL oil/100 mL chitosan solution, 7 passes; group D: 1500 bar, 90 mL oil/100 mL chitosan solution, 6 passes and group E: 1500 bar, 120 mL oil/100 mL chitosan solution, 7 passes. On the thermal test, increasing treatment temperature showed the emulsions slightly increasing in droplet-size and decreasing in viscosity, but still remaining in nano droplets and stable above 26 days. Especially, group A and B appeared no separation after any temperature treatment during four weeks. On the freeze-thawing resistance, all emulsions were stable and gave no separation in 5 freeze-thawing cycles. Droplets of group A, B and C still remained in nano size.

The focus of this study was to look at relationship between polydispersity caused by high pressure homogenization and molecular weight dependent antimicrobial activity of chitosan. It has been shown that chitosan has antimicrobial activity against bacteria, fungi, and viruses. Chitosan is obtained by partial de-N-acetylation of chitin which, consists of a ß 1-4 copolymer of glucosamine and N-acetylglucosamine residues. In this experiment we compared chitosan of sixteen different molecular weights after being processed through a high pressure homogenizer. Processed chitosan (420 kDa average molecular weight, 30% of acetylation) was dissolved in a 1% (v/v) acetic acid in water to a final concentration of 1% (w/v) and apparent viscosity of 183 MPa. The chitosan solution was passed through a high pressure homogenizer with 0-5 passes at pressure levels 0, 100, 200, and 300 MPa. After processing, the chitosan acetate was investigated to determine the effect on polydispersity in terms of molecular weight and the antimicrobial properties of chitosan at different molecular weights. All compounds were tested against Escherichia coli K-12 to determine antimicrobial activity. There is growing interest in the application of chitosan in food industry due to its wide range of desirable properties including being non-toxic and biodegradability. However, as a hydrophobic material, it is very challenging to work with. Though chitosan is a challenge to work my findings indicated a strong antimicrobial relationship with chitosan at 1% concentration with a molecular weight of 200 kDa and lower against E. coli. In conclusion chitosan has viable application with a variety of foods and can be used as a preservative that decrease bacterial activity below detection level’s and helps to prolong shelf-stable products.

Alicyclobacillus acidoterrestris is a spore-forming food spoilage bacterium. Its spore is problematic to the juice industry because of its ability to grow in low pH environments and survive pasteurization processes. The purpose of this study was to investigate the effect of the non-thermal technology, high pressure homogenization (HPH) and the antimicrobial compound, dimethyl dicarbonate (DMDC), on inactivation of A. acidoterrestris, in a broth system. Vegetative cells and spores of five strains of A. acidoterrestris (N-1100, N-1108, N-1096, SAC and OS-CAJ) were screened for their sensitivity to HPH (0, 100, 200 and 300 MPa) in Bacilllus acidoterrestris thermophilic (BAT) broth. Strain SAC (most resistant) and OS-CAJ (least resistant) were further tested for their sensitivity to 250 ppm DMDC. This was followed by evaluation of combined effects of HPH and DMDC against strain SAC. Effects of HPHand DMDC treatment combinations (no DMDC, 250 ppm DMDC added 12 h before, 2 h before, immediately before, and immediately after 300 MPa HPH treatment) on spores of SAC over a 24-h period were evaluated. After all treatments, samples were serially diluted and surface plated onto BAT agar, and the populations were determined after incubation at 44 °C for 48 h. All HPH and DMDC treatments significantly (P<0.05) inhibited growth of vegetative cells, spores were less affected by these treatments. HPH caused a 1-to 2-log reduction in vegetative cell populations at 300 MOa for four strains, but only about 0.5-log reduction of SAC strain. Spores of all five strains were not significantly reduced by HPH. DMDC also slowed growth of vegetative cells significantly. For vegetative cells of SAC and OS-CAJ, 250 ppm DMDC reduced the population by about 2 log whereas spore population was reduced by less than 0.5 log. The addition of DMDC together with HPH slightly enhanced the inactivation effect over a 24-h period as compared with treatment with HPH alone. These results demonstrate that HPH and DMDC show promise for aiding in control of growth of vegetative cells of A. acidoterrestris. However, neither treatment alone or in combination, is very effective against spores.

碩士
輔仁大學
食品科學系
98
High pressure homogenization is a novel non-thermal processing technique for improving surface tension of emulsifier and modifying the particle size distribution of the ingredients. Tofu is rich in various nutrients and functional components. Thus, it is one of the popular soy products in the world. During tofu making, soymilk is heated for protein denaturation and the heated soymilk is further gelled with the addition of coagulant. However, thermal processing in tofu making is energy and time consuming. It also affects the nutrients and flavor of tofu. The objective of this research was to study the effect of high pressure homogenization on the physicochemical property and microstructure of soymilk and tofu. Two types of high pressure homogenizer, vale mode (VHPH) and microfluidized mode (MHPH) were used in this study and the pressure was set at 150 MPa. The results showed that the hydrophobicity of VHPH soymilk was higher than that of MHPH soymilk, while the difference in surface free sulfhydryl content between the VHPH soymilk and the MHPH soymilk was contrary. Thus, the VHPH was more suitable for tofu making than MHPH. The rheological property and microstructure of the tofu prepared with VHPH soymilk and heated soymilk were different significantly. A possible gelation mechanism of VHPH soymilk was proposed and compared with the mechanism of heated soymilk based on the observed results. However, the higher expressible water and lower total isoflavone content in the tofu prepared with VHPH soymilk need to be improved in the future. In conclusion, high pressure homogenization of soymilk with a vale mode homogenizer was a suitable non-thermal processing technique for manufacturing the tofu with unique microstructure and texture.

碩士
大葉大學
生物產業科技學系
98
Nanoemulsion droplets size is in the range of 10-100 nm. Nanoemulsion was stable and easily absorbed through skin due to its miniaturized size. High pressure homogenization may be applicable to manufacture nanoemulsion products by providing great energy in a short time with a homogeneous flow to decrease particle size. In this study, the effects of oil (2-6%, w/w), surfactant (3-7%, w/w), and the homogenization pressure (70-130 MPa) on the particle size of emulsion were investigated using response surface methodology (RSM) with 3-factor-3-level Box-Behnken design. Based on the analysis of canonical, the effect of the number of passes of homogenization on particle size to obtain emulsion with low particle size and high stability was determined.Then, glabridin was added to the nanoemulsion as a functional ingredient, and effectiveness of the product was evaluated. Results showed that a good nanoemulsion was obtained by mixing oil 3.65% with emulsifier 5.3% and then homogenized at 129 MPa. The actual minimum particle size (58 nm) was closed to the predicted value (40.4 nm). Homogenization pressure was the key factor affecting particle size from the analysis of variance for joint test (p < 0.005). The particle size was proportionally reduced as the pressure increased. Moreover, the number of passes of homogenization could also affect the particle size, and stable particle size at about 28 nm was resulted after 3 passes of homogenization. Glabridin had a high DPPH radical scavenging activity (80%) of 1 mg/mL. Activity of tyrosinase was inhibited by 80% using 1 μg/mL of glabridin. The nanoemulsion containing glabridin retained tyrosinase inhibitory activity even after high pressure homogenization. The cumulative amount (0.086 g/mL) for the product was better than that that of non-high pressure homogenization (0.057 mg/mL) after transdermal diffusion for 24 hours. Furthermore, the glabridin nanoemulsion did not cause any irritation or allergy during the safety and stability test. In conclusion, this study showed that an optimal condition was developed for the preparation of a stable nanoemulsion, and glabridin is a highly valuable active ingredient for whitening the skin. Results also showed that the miniaturized particle of this product could be absorbed without irritation and allergy through skin while providing an effective high value skin care.

碩士
國立宜蘭大學
食品科學系碩士班
105
Rice pasta offers a good choice for coeliac patients because of gluten free. However, rice pasta cannot offer the desirable texture similar to wheat noodle. The purposes of this study were to improve the texture of rice pasta through the modification of rice dough patterns and process conditions. According the investigation of the physicochemical properties of miniaturized rice flour prepared by high-pressure homogenization (HPH) as well as the evaluation of the texture, cooking loss and sensory evaluation of rice pasta made from desirable combination ratio of HPH treated rice flour (HPHRF), wet milled rice flour (WRF) and gelatinized rice dough (GRD), the forming, rolling and gum addition conditions were studied. The volume mean diameter (D(4,3)) of HPHRF under 100-180 MPa for 2 passes HPH decreased from 29.1 to 5.61-5.52 μm. The ultimate particle size (5.52 μm) of HPHRF was obtained under 180 MPa. However, the D(4,3) of the HPHRF increased to 6.49 μm under 220 MPa for 2 passes. Such results may be due to rice starch were partially gelatinized and aggregated, which can be proved by the gel-like structures in SEM images and the peaks were weakened in X-ray patterns for the HPHRF at pressures of 220 MPa for 2 passes. HPH treatment also decreased crystallinity and damaged starches which caused the decrease in gelatinization enthalpies due to the increase in solubility and swelling power of HPHRF. HPHRF also exhibited lower peak, hot paste, breakdown, setback and cool pasta viscosity than those in WRF. High firmness (4.58N) and elongation at break (120.3%) were obtained in rice pasta prepared from the formula of WRF/HPHRF (100 MPa for 2 passes)/GRD ratio under 60/15/25 prepared by forming extruder. Comparison with commercial products, the rice pasta showed higher cooking loss (16.8%) and lower hedonic score (4.5) in sensory evaluation. The flat rice noodle, prepared by a sheeting machine through premix of rice dough with forming extruder and rolling 10 times by passing through the reduction rolls, exhibited lower cooking loss (11.1%) and higher mechanical strength. The cooking loss further reduced to 7.9% and the firmness (7.1 N) and tensile properties increased for the rice pasta added with gums, which resulted in the hedonic score of sensory evaluation increased to 6.1. Overall, appropriate modification of rice dough patterns, including miniaturization of rice flour by HPH and partial gelatinization of rice dough, and rolling process as well as addition of gums could effectively improve the texture of rice pasta.

Emulsions constitute a wide range of natural as well as processed products. Pharmaceutical applications of emulsions include oral administration, parenteral delivery, ophthalmic medicine, topical and transdermal creams, and fluorocarbon-in-water emulsions for blood oxygenation. In the foods area many of the products like mayonnaise, margarine, ice-creams are emulsions by nature and some products can also be used for delivery of active ingredients (e.g. nutraceuticals) with potential health benefits. Emulsions are also encountered at many stages of petroleum recovery, transportation, and processing. Typically, emulsions are manufactured in a two-step process. First a coarse emulsion called a premix is made which is passed through a high-pressure homogenizer. Intense energy supplied in the high pressure homogenizer causes breakage of the coarse emulsion to a fine one with a tighter distribution. Population balance equation (PBE) models are useful for emulsions since they allow prediction of the evolution of the drop size distribution on specification of the two rate processes i.e., breakage of drops due to the flow field and coalescence of colliding drops. In our work, we developed a PBE model to describe emulsion breakage in a high pressure homogenizer. The focus of the work was breakage and conditions to keep coalescence to minimum were implemented. Two breakage rates representing two mechanisms i.e., turbulent inertial and turbulent viscous breakage were necessary for reproducing the bimodal nature of the distributions. We used mechanistic functions in the PBE model to develop a predictive model which could be extended to changes in formulation variables as well as process variables. Starting with the assumption of binary breakage, the model was refined to include multiple drop breakage. The developed model was found to be extensible to reasonable changes in oil concentration, surfactant concentration, continuous phase viscosity and constant ratio of oil to surfactant. Anomalies in pressure prediction encountered earlier were also corrected for by including some additional features like heating, maximum stable diameter, and number of daughter drops. A preliminary attempt was also made to use the developed model for designing experiments for making target emulsions with pre-specified properties.

碩士
輔仁大學
食品科學系
99
Abstract Sweet potato is the sixth largest crop in the world and it contains a great amount of nutrients. The newly developed purple sweet potato cultivar is rich in anthocyanins with antioxidant, antimutagenic and anti-inflammatory activities. Thermal processing is often applied to preparing the sweet potato flour for the preservation and development of diverse products in food industry. However, study on the physicochemical properties and anthocyanins content of thermal processed purple sweet potato is rare. Furthermore, the granule size of sweet potato starch is large resulting in higher viscosity during processing. High-pressure homogenization ( HPH ) is a novel non-thermal processing technology for the modification of physicochemical properties of foods. Therefore, the objectives of this research were to study the effect of thermal processing on the physicochemical properties and anthocyanins content of purple sweet potato flour, and to investigate the effects of HPH pressure and cycle on the antioxidant activity and rheological properties of purple sweet potato flour. The results showed that the purple sweet potato skin did not effect pasting properties and water absorption of its flour. The color lightness of steamed ( 100℃, 20 min ) sweet potato flour was better and the amylose content was higher than cooked flour resulting in higher pasting properties. The steamed sweet potato flour had organized and loose structure leading to a better swelling power. Increase the pressure of 1-cycle HPH decreased the anthocyanins and total phenol contents of purple sweet potato flour, but 2-cycle HPH showed an opposite trend. Furthermore, the antioxidant activity of HPH treated purple sweet potato flour increased up to 90 % with increasing the homogenization pressure. The purple sweet potato puree exhibited shear thinning behavior by fitting the Power law model. The HPH decreased the viscoelasticity of purple sweet potato puree. In addition, the particle size of sweet potato flour was not affected by HPH. However, the network structure of flour particles showed the flat shape and the surface of flour became smooth after HPH. The changes of microstructure consequently increased the extraction efficiency of functional composition of flour and increase the flow behavior of purple sweet potato puree. In conclusion, the combination of steam treatment and HPH could increase the antioxidant activity and successfully reduce the viscoelasticity of purple sweet potato puree.

碩士
東海大學
食品科學系
103
Chitosan (CS,≧85% deacetylation) was dissolved in 0.1 M acetic acid at 0.5% concentration and adjusted to pH 6.0. Linolenic acid (LNA, ω-3)and vitamin E (Vit. E) were mixed at the ratio 1000: 5, 1500:7.5, 2000:10, 2250:11.25 and 2500:12.5 (w/w,mg) in hexane before adding into 100 mL chitosan solution. These mixtures were pre-emulsified by a Polytron homogenizer (13,500 rpm, 2 min) before passed through a high pressure homogenizer (APV) at different pressure (500,1000 and 1500 bar) and recycled in different passes (1~10 passes).The emulsions were determined on the droplet size and electrical charge (Zeta potential). Suitable eemulsifying conditions (i.e., LNA/Vit. E concentration, homogenizing pressure and recycling pass) showed a small droplet and high zeta potential emulsions were applied to prepare the CS-LNA-Vit. E emulsions that were then determined on the emulsion stability during (i.e., oil-water separation, droplet size and peroxide value POV change) during 28 day storage at 25℃. Such homogenizing conditions were also applied for emulsifying with dual emulsifiers of chitosan and lecithin(Le) into CS-Le- LNA-Vit. E nano droplet emulsions which were then determined on the droplet size, Zeta potential and emulsion stability. Nano droplets of CS-LNA-Vit. E and CS-Le-LNA-Vit. E emulsions were also observed by transmission electron microscopy (TEM). As result, on the CS-LNA-Vit. E emulsion : a higher homogenizing pressure and more number of recycling pass gave smaller droplet size under LNA-Vit. E conditions of 1000: 5- 2250:11.25 (w/w,mg) , but showed a larger droplet size at a conditions of 2500:12.5 (w/w,mg). Zeta potential of the emulsions increased as the LNA-Vit. E concentration increased but not affected by both the homogenizing pressure and more recycling pass (+33~36 mV). Emulsions that were carried out by the following conditions gave a comparation small droplet and high Zeta potential : “A emulsion” (1000:5 w/w,mg LNA-Vit. E, 1500 bar, 10 passes), ” B emulsion” (1500:7.5 w/w,mg LNA-Vit. E, 1500 bar, 10 passes), “ C emulsion” (2000:10 w/w,mg LNA-Vit. E, 1500 bar, 10 passes), “ D emulsion” (2250:11.25 w/w,mg LNA-Vit. E, 1500 bar, 10 passes), “ E emulsion” (2500:12.5 w/w,mg LNA-Vit. E, 1500 bar, 9 passes).These emulsions were determined on their stability. On the oil-water separation, A, B and C emulsions were stable during 28 day storage ; D emulsion showed separation at 20th day, and E emulsion showed separation at the 12th day. On the droplet-size change, A, B and C emulsions showed a decreasing during the storage (A:880 nm→667 nm ; B:941 nm→723 nm ; C:853 nm→754 nm), D and E emulsions showed a gradual larger during the storage. On the POV change, all emulsions gave a increasing POV , about 710-725 meq/Kg at the 5nd day storage at 50℃ while pure LNA-Vit. E showed about 820-860 meq /Kg. The CS-Le-LNA-Vit. E nano droplet emulsions showed a droplet size in a range of 790-992 nm and zeta potential almost +46 mV.On the emulsion stability, they had no separation and showed a gradually decreasing in droplet size and more stable POV(200 meq/Kg) during the storage. Thus, under the same emulsified conditions, CS-Le-LNA-Vit. E had larger droplet size but higher Zeta potential and more stable in the storage than that of the CS-LNA-Vit. E emulsion . Both CS-Le-LNA-Vit. E droplets and CS-LNA-Vit. E droplets were spherical or oval shapes under TEM observation.

碩士
大葉大學
生物產業科技學系碩士在職專班
99
Polysaccharopeptides (PSP), isolated from the mushroom Coriolus versicolor (syn. Trametes versicolor), has known effects for immunestimulatory, anti-tumor and analgesic. PSP has been used in both health food and pharmaceuticals, however, it has not been studied for using in cosmetics yet. This study aimed to evaluate the effects on whitening and moisture retention. The results showed good ability ( IC50, 133.90 μg/mL) on inhibitory activity of mushroom tyrosinase (in vitro). The determination of hydration showed good ability on water retention capacity and moisture absorption. Another study was PSP W/O (water in oil) nano-emulsion formation by collision-high pressure homogenization. The effects of 5 mg/mL PSP solution (35-45 %, w/w), surfactant (4-8 %, w/w) and the homogenization pressure (100-160 Mpa) on the particle size of emulsion were investigated using response surface methodology (RSM) with three-factor-three-level Box-Behnken design. Base on the canonical analysis, the effect of the number of passes of homogenization on particle size to RSM analysis results showed that an optimization conditions of making nano-emulsion as the condition of mixing water 39.03 % with emulsifier 6.54 % and then homogenized at 178.11 Mpa. The actual minimum particle size 82 nm was closed to the predicted value 64 nm. Effect of homogenization pressure was the most important factor for particle size from the analysis of variance for joint test ( p < 0.05). Moreover, the number of passes of homogenization could also affect the particle size. The result after 5 passes of homogenization was about 22 nm. This study showed that using of collision high pressure homogenization, cosmetics can be made of nano size molecular smaller than skin pores to effectively increase skin permeability and transfer rate that greatly enhance utilization of the material. PSP have good moisture retention and water-absorbing ability with good whitening function. It is also affordable and easily to get by using the method of submerged fermentation culture of Coriolus versicolor, this gives PSP great potential in developing cosmetic materials in modern biotechnology and applications.