El objetivo de la tesis fue evaluar la aptitud tecnológica de la leche tratada por ultra alta presión homogenización (UHPH) como alternativa a la pasteurización en el tratamiento de higienización de la leche para la obtención de queso fresco. Dicho objetivo se llevó a cabo mediante (a) la determinación de las condiciones óptimas de presión y temperatura, estudiando los efectos de la UHPH en las propiedades de coagulación, (b) la identificación de cambios a nivel de las interacciones proteicas de la cuajada así como de la composición proteica de la membrana del glóbulo graso, (c) la evaluación de la aptitud quesera de la leche produciendo quesos frescos a escala piloto, (d) la determinación de la vida útil mediante el seguimiento de la evolución durante la conservación en refrigeración de la microbiota, composición y bioquímica de los quesos, (e) la evaluación de la textura, reología, microstructura y color de los quesos así como la aceptabilidad en su consumo mediante un análisis sensorial.La utilización de la UHPH con una sola etapa y presiones superiores a 200 MPa resultó en una mejora de las propiedades de coagulación. Las condiciones óptimas de tratamiento fueron establecidas como 300 MPa y 30 ºC. La UHPH favoreció las uniones vía calcio de las caseínas y las interacciones hidrofóbicas tanto de caseínas como de proteínas del suero en la cuajada, inhibiendo las interacciones vía puentes de hidrógeno. Al reducir significativamente el tamaño de los glóbulos grasos, la UHPH provocó la adsorción de proteínas de la leche a nivel de la membrana de los glóbulos grasos. La UHPH empeoró en cierta medida el corte de la cuajada produciéndose más finos de quesería por desmenuzado de los granos de cuaja e insuficiente cohesión de los granos. Los quesos frescos producidos con leche tratada por UHPH presentaron mayor vida útil (19 vs. 13 días). El crecimiento de lactococos fue potenciado durante los primeros días de la conservación. Por lo contrario, no se detectaron lactobacilos durante todo el período de almacenamiento. El crecimiento de levaduras y hongos fue parcialmente inhibido resultando en recuentos más bajos al final de la vida útil de los quesos. Durante el almacenamiento, los quesos producidos con leche tratada por UHPH presentaron menor desuerado que aquellos producidos con leches tratadas convencionalmente, resultando en contenidos más altos de humedad y sal. Aunque no se observaron diferencias en el contenido en grasa y proteínas, los quesos UHPH presentaron niveles más bajos de proteólisis y lipólisis. Así mismo, los perfiles en ácidos grasos libres fueron significativamente diferentes. La oxidación lipídica, detectada como el mayor inconveniente, resultó en un gran número de compuestos volátiles tales como aldehídos y cetonas cuya presencia explicó la aparición de flavores defectuosos detectados por algunos de los catadores. Finalmente, la UHPH resultó en quesos más firmes, menos deformables, con menor sensación acuosa en boca y más blancos. Estos cambios podrían atribuirse a los efectos de la UHPH en los glóbulos grasos. A excepción de la escasa sensación acuosa en boca, los catadores calificaron como mejores las características estructurales de los quesos frescos.En conclusión, los resultados obtenidos revelaron que la UHPH es una tecnología prometedora para el tratamiento de la leche de quesería ya que puede alargar la vida útil y mejorar algunas de las características tales como la textura y la microestructura de los quesos frescos. Al solventar los problemas de oxidación mediante la modificación de algunas piezas metálicas del equipo, se espera obtener quesos frescos con una calidad en el sabor similar o incluso superior a la de los quesos producidos con leche tratada convencionalmente. / Among the emerging technologies for minimal food-processing, ultra-high pressure homogenisation (UHPH) is a promising technology for liquid foods such as milk owing to the fact that (a) it is a continuous process, (b) it performs two separated processes at once, i.e., pasteurisation and homogenisation, (c) it can deal with volumes usual at pilot-scale with a view to go for larger volumes such as those at production-scale, (d) it can provoke high temperatures but at such a short time that heat effect can be minimised, and finally, (e) it requires the same equipments as a common homogeniser. UHPH, as a food technology alternative to conventional heat-treatments, is currently investigated in a wide range of liquid foods. Research not only aims at evaluating the effects on the liquid food itself but also on the capacity in obtaining manufactured products and the quality of the final products. The present thesis aimed at assessing the suitability of UHPH as an alternative to conventional processes for the manufacture of starter-free fresh cheeses through (a) determination of optimal pressure and temperature conditions for cheese production, by studying the effects of UHPH on the rennet coagulation properties, (b) identification of changes at the nature of protein interactions and the protein composition of the milk fat globule membrane (MFGM), in order to better understand the overall effect of UHPH on the coagulation and cheese-making properties of milk, (c) evaluation of the suitability of UHPH-treated milk for the manufacture of fresh cheeses, by producing starter-free fresh cheeses at a pilot-scale, (d) determination of the shelf life of produced cheeses, by following their microbiological, compositional and biochemical evolution during cold storage, (e) evaluation of the texture, rheology, microstructure and colour of produced cheeses, and their aptness for consumption through a sensory analysis.Rennet coagulation properties of milk were enhanced by single-stage UHPH over 200 MPa, and optimal UHPH conditions were set to 300 MPa and 30 ºC. UHPH increased calcium-bonding of caseins within curds, impaired hydrogen bonding and favoured hydrophobic interactions. Moreover, by greatly reducing milk fat globule size, UHPH provoked the adsorption of milk proteins onto the MFGM. UHPH impaired in some extent curd cutting due to crumbling, improper matting and poor grain cohesion. Cheeses from UHPH-treated milk showed longer microbiological shelf life (19 vs. 13 days). Lactococci growth was enhanced at an early stage of the storage period but lactobacilli were not detected. The growth of yeasts and moulds was partly inhibited resulting in lower counts at the end of their shelf life. During storage, UHPH cheeses expelled less whey than those from conventionally treated, resulting in higher moisture and salt contents. Although fat and protein contents on a dry basis were not affected, lower levels of proteolysis and lipolysis were observed. Moreover, free fatty acid profiles greatly differed. Oxidation was found to be the major drawback resulting in a great number of volatile compounds, e.g., aldehydes and ketones, which presence explained off-flavours detected by some panellists. Finally, UHPH treatment of milk resulted in firmer, less deformable, with lower water-mouth feeling, and whiter fresh cheeses. Such changes could be attributed to the effects on the fat globules. Except for the dry mouth-feeling, the sensory panel described the structural attributes of cheeses from UHPH-treated milk as their best characteristics.In conclusion, these results show that UHPH is a promising technology as treatment of milk for the manufacture of fresh cheese by increasing their shelf life and improving some of their characteristics such as texture and microstructure. By solving the oxidation problems through the modification of some metal pieces of the UHPH equipment, fresh cheeses with similar or even higher flavour quality are expected to be obtained with UHPH-treated milk than with conventionally treated milk.

<p>Abstract</p><p>The aim of the present study was to increase the understanding of how the cell surface properties affect the performance of unit operations used in primary protein purification. In particular, the purpose was to develop, set up and apply methods for studies of cell surface properties and cell interactions.</p><p>A method for microbial cell surface fingerprinting using surface plasmon resonance (SPR) is suggested. Four different <em>Escherichia coli </em>strains were used as model cells. Cell surface fingerprints were generated by registration of the interaction between the cells and four different surfaces, with different physical and chemical properties, when a cell suspension was flown over the surface. Significant differences in fingerprint pattern between some of the strains were observed. The physical properties of the cell surfaces were determined using microelectrophoresis, contact angle measurements and aqueous two-phase partitioning and were compared with the SPR fingerprints. The generated cell surface fingerprints and the physical property data were evaluated with multivariate data analysis that showed that the cells were separated into individual groups in a similar way using principal component analysis plots (PCA).</p><p>Studies of the behaviour of the model cells on stirred cell filtration and in an interaction test with different expanded bed adsorption (EBA) adsorbents were performed. It could be concluded that especially one of the strains behaved differently. Differences in the properties of the model cells were indicated by microelectrophoresis and aqueous two-phase partitioning which to some extent correlated with observed differences in behaviour during filtration and in an interaction test with EBA adsorbents.</p><p>The impact of high-pressure homogenisation of <em>E. coli </em>cell extract was examined, with a lab scale and a pilot scale technique. The DNA-fragmentation, visualised with agarose gel electrophoresis, and the resulting change in viscosity was analysed. A short homogenisation time resulted in increased viscosity of the process solution that correlated with increased concentration of released non-fragmented DNA. With longer homogenisation time the viscosity decreased with increasing degree of DNA-fragmentation.</p><p>The results show that strain dependant cell surface properties of<em> E. coli</em> may have an impact on several primary steps in downstream processing.</p>

Abstract The aim of the present study was to increase the understanding of how the cell surface properties affect the performance of unit operations used in primary protein purification. In particular, the purpose was to develop, set up and apply methods for studies of cell surface properties and cell interactions. A method for microbial cell surface fingerprinting using surface plasmon resonance (SPR) is suggested. Four different Escherichia coli strains were used as model cells. Cell surface fingerprints were generated by registration of the interaction between the cells and four different surfaces, with different physical and chemical properties, when a cell suspension was flown over the surface. Significant differences in fingerprint pattern between some of the strains were observed. The physical properties of the cell surfaces were determined using microelectrophoresis, contact angle measurements and aqueous two-phase partitioning and were compared with the SPR fingerprints. The generated cell surface fingerprints and the physical property data were evaluated with multivariate data analysis that showed that the cells were separated into individual groups in a similar way using principal component analysis plots (PCA). Studies of the behaviour of the model cells on stirred cell filtration and in an interaction test with different expanded bed adsorption (EBA) adsorbents were performed. It could be concluded that especially one of the strains behaved differently. Differences in the properties of the model cells were indicated by microelectrophoresis and aqueous two-phase partitioning which to some extent correlated with observed differences in behaviour during filtration and in an interaction test with EBA adsorbents. The impact of high-pressure homogenisation of E. coli cell extract was examined, with a lab scale and a pilot scale technique. The DNA-fragmentation, visualised with agarose gel electrophoresis, and the resulting change in viscosity was analysed. A short homogenisation time resulted in increased viscosity of the process solution that correlated with increased concentration of released non-fragmented DNA. With longer homogenisation time the viscosity decreased with increasing degree of DNA-fragmentation. The results show that strain dependant cell surface properties of E. coli may have an impact on several primary steps in downstream processing. / QC 20101129

Résumé : L'interaction entre la β-Lactoglobuline (β-Lg) et le rétinol ou entre les micelles de phosphocaséines (PC) et le rétinol à un pH proche de la neutralité, a été étudiée à pression atmosphérique et sous pression isostatique jusqu'à 400 MPa. Les constantes de dissociation et le nombre de sites de liaison ont été calculés indiquant des différences d'affinité en fonction de la structure protéique. A 25°C, des pressions inférieures à 150 MPa favorisent l'association β-Lgrétinol (rapport molaire β-Lg/rétinol : 1/1). A ≥ 150 MPa le complexe se dissocie. A 350 MPa, la β-Lg est dénaturée et le complexe irréversiblement dissocié. Le complexe PCrétinol (rapport molaire PC/rétinol : 1/1) reste au contraire formé après un traitement à 400 MPa et 25°C, bien que la pression induise des phénomènes de dissociation/réassociation des assemblages micellaires à ≥ 100 MPa. L'interaction PCrétinol stabiliserait plutôt les micelles de PC vis-à-vis de la pression, de même qu'une température de pressurisation modérée (35°C) comparativement à une température plus basse (15°C). Un isolat protéique de lactosérum (IPL) en dispersion dans l'eau à 10% (p/p) de protéines (pH 6,5) et en présence d'acétate de rétinol (AcRet) (rapport molaire β-Lg/AcRet : 10/1) a été traité par (i) haute-pression isostatique (HP) (350 MPa, 25°C, 15 min), (ii) traitement thermique de courte durée (TTCD) (75°C, 4 s) ou (iii) homogénéisation à ultra-haute pression (UHPH) (300 MPa, Tin = 24°C). Les trois traitements permettent de former des agrégats de β-Lg capables de retenir l'acétate de rétinol, mais avec une efficacité différente dépendant probablement des mécanismes d'agrégation induit par le chauffage (TTCD), la pression isostatique (HP) ou dynamique (UHPH). Des dispersions à 2,38% (p/p) en phosphocaséines (pH 6,6) en présence d'acétate de rétinol (rapport molaire PC/AcRet : 5/1) ont été traitées par (i) HP (300 MPa, 14°C ou 34°C, 15 min), ou (ii) UHPH (300 MPa, Tin = 14°C). Ces deux traitements favorisent la rétention de l'acétate de rétinol par les micelles de PC pouvant ainsi servir de cargo pour véhiculer des molécules bioactives. Mots clefs : haute pression isostatique, haute pression dynamique, homogénéisation à ultra-haute pression, fluorescence, β-Lactoglobuline, micelles de phosphocaséines, rétinol, acétate de rétinol, agrégats protéiques, interaction protéine-ligand. / Abstract: The binding of retinol to native β-Lactoglobulin (β-Lg) or phosphocasein (PC) micelles at pH close to neutral was studied at atmospheric pressure or under isostatic high-pressure. The dissociation constants and number of binding sites were calculated indicating that difference in retinol affinity depended on protein structure. At 25°C, pressure level < 150 MPa promoted β-Lgretinol association (β-Lg/retinol molar ratio: 1/1). At ≥ 150 MPa, the complex dissociated. At 350 MPa, β-Lg was denatured and the complex irreversibly dissociated. PC and retinol (PC/retinol molar ratio: 1/1) remained associated after pressurisation at 400 MPa and 25°C, while pressure induced dissociation/reassociation phenomena of micelle assemblies. The binding of retinol to PC stabilised micelles towards pressure, as well as moderate temperature of pressurisation (35°C) compared to lower temperature (15°C).A whey protein isolate (WPI) dispersed in water at 10% (w/w) proteins (pH 6.5) in the presence of retinyl acetate (RetAc) (β-Lg/RetAc molar ratio: 10/1) was processed by (i) isostatic high-pressure (HP) (350 MPa, 25°C, 15 min), (ii) short-time thermal treatment (STTT) (75°C, 4 s) or (iii) ultra-high pressure homogenisation (UHPH) (300 MPa, Tin = 24°C). All processing produced β-Lg aggregates able to retain RetAc, but with different efficiency depending on aggregation mechanisms induced by heating (STTT), isostatic high-pressure (HP) or dynamic high-pressure (UHPH). Phosphocaseins dispersed at 2.38% (w/w) proteins (pH 6.6) in the presence of RetAc (PC/RetAc molar ratio: 5/1) were processed by (i) HP (300 MPa, 14°C or 34°C, 15 min), or (ii) UHPH (300 MPa, Tin = 14°C). Both treatments promoted RetAc retention by phosphocasein micelles that can be used as cargoes to transport bioactive molecules.Keywords: isostatic high-pressure, dynamic high-pressure, ultra-high pressure homogenisation, fluorescence, β-Lactoglobulin, phosphocasein micelles, retinol, retinyl acetate, protein aggregates, protein-ligand binding.Discipline: Biochimie, Chimie et Technologie des Aliments.Thèse préparée à : Université Montpellier 2 – Equipe de Biochimie et Technologie Alimentaire – UMR IATE 1208 – Pôle EVAP – Place E. Bataillon, 34095 Montpellier, France.