Chromatography and intrinsic fluorescence studies of possible structural evolution of globular proteins during ultrafiltration.

International audienceUltrafiltration (UF) is a sepn. process widely used for filtration of proteins. For applications with high added value we should ensure that they are not denatured, which could lead to a loss of functional properties. We propose a study methodol. based on model expts. able to voluntary denature proteins in a mech./thermal way and the comparison of final states with those obtained in UF retentate after long time filtration. The approach is validated by two complementary anal. techniques: intrinsic fluorescence and reverse phase HPLC (RP-HPLC). [on SciFinder(R)

Chromatography and intrinsic fluorescence studies of possible structural evolution of globular proteins during ultrafiltration.

International audienceUltrafiltration (UF) is a sepn. process widely used for filtration of proteins. For applications with high added value we should ensure that they are not denatured, which could lead to a loss of functional properties. We propose a study methodol. based on model expts. able to voluntary denature proteins in a mech./thermal way and the comparison of final states with those obtained in UF retentate after long time filtration. The approach is validated by two complementary anal. techniques: intrinsic fluorescence and reverse phase HPLC (RP-HPLC). [on SciFinder(R)

Sur l intégrité des protéines et la valorisation des effluents pour une production durable par membrane d ultrafiltration (application à l industrie laitière)

Les protéines solubles du lait de vache ont un intérêt reconnu dans les industries agro-alimentaire en raison de leur valeur nutritionnelle et de leurs propriétés techno-fonctionnelles. A condition de valider que les protéines ne sont pas dénaturées par les procédés membranaires, ceci pourraient être utilisés à l échelle industrielle pour la préparation de fractions protéiques à fonctions ciblées. Cette thèse propose une première partie sur la mise au point d une méthodologie pour étudier la possibilité de dénaturation des protéines par des membranes fortement rétentives en comparant avec des expériences modèles de dénaturation mécanique et thermique (mixeur, agitateur magnétique et bain marie). L analyse par fluorescence intrinsèque et HPLC en phase inverse pour laquelle de nouveaux gradients ont été mis au point pour chaque protéine, s avèrent être deux outils pertinents pour la mise en évidence de la dénaturation ciblées. Par ailleurs, les procédés à membranes génèrent des volumes d effluents qui doivent être minimisés pour une production éco-compatible et durable. La deuxième partie de cette thèse propose une démarche pour une valorisation des effluents globaux après traitement par ultrafiltration pour le nettoyage de membranes PES spirales colmatées par du lait écrémé.The soluble proteins of cow milk have an interest recognized in the food industry because of their nutritional values and their techno-functional properties. Provided to validate that the proteins are not denatured by membrane processes, this can be used at industrial scale for preparation of proteins fractions with targeted functions. The first part of this thesis is focused on a methodology to study the possibility of the denaturation of proteins by highly retentive membranes in comparison with model experiments of mechanical and thermal denaturation (blender, magnetic stirrer and Marie bath). The analysis of intrinsic fluorescence and reversed phase HPLC for which new gradients were proposed for each protein, prove to be two relevant tools for highlighting targeted denaturation. Moreover, the membranes processes generates large volumes of effluents that must be minimized for an eco-friendly and sustainable production. The second part for this thesis proposes a step for a valorization of global effluents after ultrafiltration for the cleaning of spiral wounded PES membranes fouled by skim milk.RENNES1-BU Sciences Philo (352382102) / SudocSudocFranceF

Structural modifications of globular proteins in an ultrafiltration loop as evidenced by intrinsic fluorescence and reverse-phase liquid chromatography.

International audienceOccurrence of denaturation of proteins during ultrafiltration (UF) was already discussed in literature but it must be underlined that no general trends were drawn explaining the versatile obsd. behaviors. This paper aims at proposing a complementary approach focused on slight denaturation of proteins due to mech. stress in a UF loop. This methodol. has required powerful anal. tools as it aims at revealing slight denaturations leading to proteins remaining in sol. states and not to pptn. that is quite easily shown. Intrinsic fluorescence and Reverse Phase HPLC (RP-HPLC) techniques were selected to highlight subtle changes in protein structure. Mainly, only highly enriched fractions of proteins were available (with a more than 95% purity), but it is a limitation to the use of intrinsic fluorescence that is very sensitive to occurrence of minor protein contaminants. Then RP-HPLC was used not only for quantification of remaining sol. proteins but also to show slight changes in the protein peak shape, only evidenced with elution gradients specially established for this study to reach the goal. The main novelties of this paper deal with: (i) the use of a wide variety of globular proteins. A set of five proteins with different structural characteristics (mol. wt., overall hydrophobicity, occurrence of quaternary structures or not) was used for the demonstration, and (ii) the selection of specific RP-HPLC elution conditions able to highlight protein structural evolution with respect to UF requirement, esp. concerning the use of low concns. (1 g L-1) to avoid denaturation due to highly conc. media. An in-depth discussion based on protein peak shape was proposed and (iii) the establishment of model expts. that voluntary led to denatured proteins only obtained by mech. stress at 45 °C, conceptually different than denaturation obtained by external physico-chem. variations such as addn. of chem. compds. modifying protein structure as commonly used to denature proteins for anal. purpose. These model expts. were based on establishment of various shear stresses obtained by more or less quick stirring from zero to several thousand rotations per min allowing establishing a ref. scale of protein denaturation states with a Reynolds no. varying in a wide range. Cross-flow UF of each single protein soln. was performed in batch mode during 5-6 h at 45 °C, retentates were analyzed and compared to the ref. scale of mech. denaturation obtained from the model expts. As a general conclusion, it seemed that proteins can be classified according to a "soft/hard" criterion depending on the protein own ability to be denatured by contact with a surface. The "soft" proteins were partly denatured by the combination of shear stress and temp. in model expts. and in UF, whereas the "hard" proteins of quite stable structure were not modified. The behavior during UF was sometimes much more complex, esp. when the protein were capable of crossing the membrane. [on SciFinder(R)