Moderate conformational impact of citrate on ovotransferrin considerably increases its capacity to self-assemble at the interface

International audienceWe have compared the behavior of ovotransferrin at the air-solution interface in the presence of a monovalention (acetate), or a divalent ion (citrate), the latter being known to induce conformational changesof this protein upon interaction with its iron-binding sites. We have characterised the adsorption layer atthe air–water interface in terms of homogeneity, surface concentration excess and rheological propertiesat pH 4.0. Besides we have investigated the bulk conformation in the presence of the two anions. In thepresence of citrate only, interfacial layers display well-defined domains of higher overall surface concentrationsuggesting multilayers adsorption. Citrate also induces higher helical content and stabilizes theprotein against thermal denaturation. Hence we propose that these changes are involved in the propensityof ovotransferrin to self-assemble at the air–water interface resulting in thick and heterogeneousinterfacial layer

Comparison of electrospray and matrix-assisted laser desorption ionization on the same hybrid quadrupole time-of-flight tandem mass spectrometer: Application to bidimensional liquid chromatography of proteins from bovine milk fraction

 Recently, two ionization sources, electrospray (ESI) and matrix-assisted laser desorption (MALDI) have been used in parallel to exploit their complementary nature and to increase proteome coverage. In this study, a method using bidimensional (2D) nanoLC coupled online with ESI quadrupole time-of-flight (Q-TOF) with the simultaneous collection of fractions for analyses by LC–MALDI Q-TOF–MS/MS was developed. A total of 39 bovine proteins were identified to a high degree of confidence. To help in differentiating peptide detection following ESI and MALDI with the same mass spectrometer, we compared physico–chemical characteristics of the peptides (molecular mass, charge and size) by principal component analysis (PCA) and analysis of variance on the results of PCA. More hydrophobic peptides with a wider mass coverage were identified when ESI was used, whereas more basic and smaller peptides were identified when MALDIwas used.However, the generally accepted differentiation between ESI and MALDI according to the presence of basic amino acids residues Lys and Arg and the ratio Lys/Arg was not shown as significant in this study. Moreover, we pointed out the importance of the type of mass spectrometer used in complement to both ionization sources for achieving a global increase of proteome coverage

Continuous raw skim milk processing by pulsed electric field at non-lethal temperature: effect on microbial inactivation and functional properties

International audiencePulsed electric field (PEF) is an emerging non-thermal processing technology used to inactivate microorganisms in liquid foods such as milk. The objective of this research was to study the effectiveness of continuous PEF equipment (square wave pulses) on total microorganisms of raw skim milk and on Salmonella enteritidis inactivation under moderate temperatures (T < 50 °C). Processing parameters (electric field and pulse width) were chosen as follows: 45 kV*cm-1/500 ns and 55 kV*cm-1/250 ns, with increasing pulse frequencies from 40 to 120 Hz, that corresponds to an energy input varying from range 0-100 kJ*kg-1. In these conditions, the effectiveness of PEF processing on microbial inactivation was very limited: 1.4-log reduction of total microflora and S. enteritidis was the maximal inactivation ratio obtained. The effect of these PEF treatments on physicochemical and technological properties of the milk was also evaluated. These process conditions had an effect on proteinic components of milk such as casein micelles, since viscosity of PEF-treated milk decreased and coagulation properties were enhanced for high field levels (45-55 kV*cm-1) with 2.1 to 3.5 µs cumulated treatment time and square waves. This study demonstrated that, contrary to numerous previous studies, PEF treatments had an impact on some food constituents.Traitement de lait écrémé cru par champs électriques pulsés à température non létale : efficacité au plan de la destruction microbienne et effet sur les propriétés fonctionnelles. La tendance actuelle de l'industrie alimentaire est de proposer des alternatives aux traitements thermiques de pasteurisation. Ces traitements alternatifs doivent être efficaces sur le plan de la destruction bactérienne, mais moins sévères au plan physico-chimique, afin de garantir la qualité hygiénique des aliments tout en préservant leurs qualités nutritionnelles, sensorielles et fonctionnelles. Les traitements par champs électriques pulsés (CEP) sont souvent reconnus dans la littérature comme un des procédés innovants capables de répondre à ces nouvelles exigences. L'objectif de cette étude était donc ici d'évaluer l'efficacité de traitements en continu par CEP (impulsions électriques rectangulaires) réalisés à température non létale (T < 50 °C) sur la destruction de la flore endogène et des coliformes du lait ainsi que sur du lait préalablement inoculé avec Salmonella enteritidis. Les paramètres de traitements choisis (champs électriques et largeur des impulsions) étaient les suivants : 55 kV*cm-1/250 ns et 45 kV*cm-1/500 ns, avec des fréquences d'impulsion variant de 40 à 120 Hz, ce qui se traduit par un apport d'énergie compris entre 0 et 100 kJ*kg-1. Dans ces conditions, l'efficacité des traitements est très limitée puisque au maximum 1,4 log de réduction microbienne a pu être atteint sur la flore endogène et S. enteritidis. Parallèlement, l'effet des traitements par champs électriques pulsés sur les principales caractéristiques physico-chimiques et techno-fonctionnelles du lait a été évalué. Un effet sur les composants protéiques tels que les micelles de caséine a été constaté suite aux traitements CEP les plus intenses (45-55 kV*cm-1 pendant 2,1 à 3,5 µs) puisque la viscosité du lait était diminuée et ses aptitudes à la coagulation étaient améliorées. En conclusion, ces expériences ont permis de montrer que, contrairement à ce qui est classiquement reporté dans la littérature, les traitements par CEP ont un impact non nul sur des constituants tels que les micelles de caséine

Continuous raw skim milk processing by pulsed electric field at non-lethal temperature: effect on microbial inactivation and functional properties

Pulsed electric field (PEF) is an emerging non-thermal processing technology used to inactivate microorganisms in liquid foods such as milk. The objective of this research was to study the effectiveness of continuous PEF equipment (square wave pulses) on total microorganisms of raw skim milk and on Salmonella enteritidis inactivation under moderate temperatures (T < 50 °C). Processing parameters (electric field and pulse width) were chosen as follows: 45 kV·cm–1/500 ns and 55 kV·cm–1/250 ns, with increasing pulse frequencies from 40 to 120 Hz, that corresponds to an energy input varying from range 0–100 kJ·kg–1. In these conditions, the effectiveness of PEF processing on microbial inactivation was very limited: 1.4-log reduction of total microflora and S. enteritidis was the maximal inactivation ratio obtained. The effect of these PEF treatments on physicochemical and technological properties of the milk was also evaluated. These process conditions had an effect on proteinic components of milk such as casein micelles, since viscosity of PEF-treated milk decreased and coagulation properties were enhanced for high field levels (45–55 kV·cm–1) with 2.1 to 3.5 μs cumulated treatment time and square waves. This study demonstrated that, contrary to numerous previous studies, PEF treatments had an impact on some food constituents.La tendance actuelle de l'industrie alimentaire est de proposer des alternatives aux traitements thermiques de pasteurisation. Ces traitements alternatifs doivent être efficaces sur le plan de la destruction bactérienne, mais moins sévères au plan physico-chimique, afin de garantir la qualité hygiénique des aliments tout en préservant leurs qualités nutritionnelles, sensorielles et fonctionnelles. Les traitements par champs électriques pulsés (CEP) sont souvent reconnus dans la littérature comme un des procédés innovants capables de répondre à ces nouvelles exigences. L'objectif de cette étude était donc ici d'évaluer l'efficacité de traitements en continu par CEP (impulsions électriques rectangulaires) réalisés à température non létale (T < 50 °C) sur la destruction de la flore endogène et des coliformes du lait ainsi que sur du lait préalablement inoculé avec Salmonella enteritidis. Les paramètres de traitements choisis (champs électriques et largeur des impulsions) étaient les suivants : 55 kV·cm-1/250 ns et 45 kV·cm-1/500 ns, avec des fréquences d'impulsion variant de 40 à 120 Hz, ce qui se traduit par un apport d'énergie compris entre 0 et 100 kJ·kg-1. Dans ces conditions, l'efficacité des traitements est très limitée puisque au maximum 1,4 log de réduction microbienne a pu être atteint sur la flore endogène et S. enteritidis. Parallèlement, l'effet des traitements par champs électriques pulsés sur les principales caractéristiques physico-chimiques et techno-fonctionnelles du lait a été évalué. Un effet sur les composants protéiques tels que les micelles de caséine a été constaté suite aux traitements CEP les plus intenses (45-55 kV·cm-1 pendant 2,1 à 3,5 µs) puisque la viscosité du lait était diminuée et ses aptitudes à la coagulation étaient améliorées. En conclusion, ces expériences ont permis de montrer que, contrairement à ce qui est classiquement reporté dans la littérature, les traitements par CEP ont un impact non nul sur des constituants tels que les micelles de caséine

Continuous raw skim milk processing by pulsed electric field at non-lethal temperature: effect on microbial inactivation and functional properties

International audiencePulsed electric field (PEF) is an emerging non-thermal processing technology used to inactivate microorganisms in liquid foods such as milk. The objective of this research was to study the effectiveness of continuous PEF equipment (square wave pulses) on total microorganisms of raw skim milk and on Salmonella enteritidis inactivation under moderate temperatures (T < 50 °C). Processing parameters (electric field and pulse width) were chosen as follows: 45 kV*cm-1/500 ns and 55 kV*cm-1/250 ns, with increasing pulse frequencies from 40 to 120 Hz, that corresponds to an energy input varying from range 0-100 kJ*kg-1. In these conditions, the effectiveness of PEF processing on microbial inactivation was very limited: 1.4-log reduction of total microflora and S. enteritidis was the maximal inactivation ratio obtained. The effect of these PEF treatments on physicochemical and technological properties of the milk was also evaluated. These process conditions had an effect on proteinic components of milk such as casein micelles, since viscosity of PEF-treated milk decreased and coagulation properties were enhanced for high field levels (45-55 kV*cm-1) with 2.1 to 3.5 µs cumulated treatment time and square waves. This study demonstrated that, contrary to numerous previous studies, PEF treatments had an impact on some food constituents.Traitement de lait écrémé cru par champs électriques pulsés à température non létale : efficacité au plan de la destruction microbienne et effet sur les propriétés fonctionnelles. La tendance actuelle de l'industrie alimentaire est de proposer des alternatives aux traitements thermiques de pasteurisation. Ces traitements alternatifs doivent être efficaces sur le plan de la destruction bactérienne, mais moins sévères au plan physico-chimique, afin de garantir la qualité hygiénique des aliments tout en préservant leurs qualités nutritionnelles, sensorielles et fonctionnelles. Les traitements par champs électriques pulsés (CEP) sont souvent reconnus dans la littérature comme un des procédés innovants capables de répondre à ces nouvelles exigences. L'objectif de cette étude était donc ici d'évaluer l'efficacité de traitements en continu par CEP (impulsions électriques rectangulaires) réalisés à température non létale (T < 50 °C) sur la destruction de la flore endogène et des coliformes du lait ainsi que sur du lait préalablement inoculé avec Salmonella enteritidis. Les paramètres de traitements choisis (champs électriques et largeur des impulsions) étaient les suivants : 55 kV*cm-1/250 ns et 45 kV*cm-1/500 ns, avec des fréquences d'impulsion variant de 40 à 120 Hz, ce qui se traduit par un apport d'énergie compris entre 0 et 100 kJ*kg-1. Dans ces conditions, l'efficacité des traitements est très limitée puisque au maximum 1,4 log de réduction microbienne a pu être atteint sur la flore endogène et S. enteritidis. Parallèlement, l'effet des traitements par champs électriques pulsés sur les principales caractéristiques physico-chimiques et techno-fonctionnelles du lait a été évalué. Un effet sur les composants protéiques tels que les micelles de caséine a été constaté suite aux traitements CEP les plus intenses (45-55 kV*cm-1 pendant 2,1 à 3,5 µs) puisque la viscosité du lait était diminuée et ses aptitudes à la coagulation étaient améliorées. En conclusion, ces expériences ont permis de montrer que, contrairement à ce qui est classiquement reporté dans la littérature, les traitements par CEP ont un impact non nul sur des constituants tels que les micelles de caséine

Evidence for synergy in the denaturation at the air-water interface of ovalbumine, ovotransferrin and lysozyme in ternary mixture

The conformational changes of egg-white proteins, in a ternary-protein system, at the air–water interface have been studied. Three of the major egg-white proteins, ovalbumin, ovotransferrin and lysozyme, were studied with concentration ratios reflecting those in egg-white. Results were compared to those obtained in a previous work on protein denaturation at the air–water interface in single-protein systems (Lechevalier, V., Croguennec, T., Pezennec, S., Guérin-Dubiard, C., Pasco, M., & Nau, F. (2003). Ovalbumin, ovotransferrin, lysozyme: Three model proteins for structural modifications at the air–water interface. Journal of Agricultural and Food Chemistry 51, 6354–6361). Foaming altered the protein structure more profoundly in the mixture than in single-protein systems. Strong electrostatic interactions were observed between the three proteins. Their existence at the air–water interface could ease intermolecular sulfhydryl–disulfide exchange reactions between ovalbumin and both ovotransferrin and lysozyme. This study highlighted the fact that results obtained on single-protein systems were not easily extrapolable to complex systems, such as egg-white

Importance of water flux through protein interfacial films as revealed by neutron reflectometry on lysozyme and ovalbumin layers

International audienceIn food technology, proteins are often used to stabilize dispersed systems through the formation of dense interfacial films (foaming, emulsification, encapsulation). Those films are of particular importance in many products (bread, beer, ice-cream, dressings...) as they partly determine their functionality. Egg white proteins are known for their ability to form interfacial films at air-water interfaces. Among them, lysozyme and ovalbumin were studied by ellipsometry at pH 7, 40mM ionic strength and showed very different behaviors [1]. Indeed, ovalbumin, which was negatively charged (-10e), formed monolayers at the interface, whereas lysozyme, which was positively charged (+8e), formed multilayers. Further studies by ellipsometry on ovalbumin at pH 3.6 showed that in these conditions of positive charge (+31e), this protein formed multilayers. Neutron reflectometry was performed to get information about the concentration profiles inside the interfacial layers generated by lysozyme and ovalbumin, at pH 7 and 3.6, and different ionic strengths. Surprisingly, the results did not show any formation of multilayers for lysozyme at pH 7 or ovalbumin at pH 3.6. The main difference between our ellipsometry and neutrons reflectivity experiments being the presence and absence, respectively, of an evaporation flux, we propose that the first layer is created by hydrophobic interactions with air, but that the supplementary layers require water transfer through the interface as a driving force. Then, after being carried by advection to the first monolayer, the nature of the interactions (repulsive/attractive) between the protein monomers determines the creation –or not– of a multilayer. As the concentrations in interfacial films of proteins can be very high (between 300 and 700 g/L ; i.e., 0.2–0.5 volume fraction), our results suggest that studies, in bulk, of interactions in highly crowded protein solutions is a necessary complement to fully understand the interfacial behaviour

Effects of dry heating on the progression of in vitro digestion of egg white proteins: contribution of multifactorial data analysis

The impact of dry heating on the progression of in vitro digestion of egg white proteins was investigatedthrough application of multiple factor analysis (MFA) to electrophoresis data. Dry heating (from 1 to 10days between 60 and 90 °C) enhanced protein unfolding and aggregation, thus generating differentSDS-PAGE patterns for each sample before digestion. The progression of in vitro digestion was thenmodified according to the degree of protein unfolding and/or aggregation. In vitro digestion tended todecrease the heterogeneity of sample electrophoretic patterns overall but it occurred either at the verybeginning of the gastric stage or throughout the gastric stage or again during the duodenal stage,depending on the heat treatment to which the sample had been subjected. At the end of digestion, threegroups of samples were obtained: all samples dry heated at 60 °C and one sample dry heated for 1 day at70 °C that were more hydrolysed than the control, samples dry heated for more than 2 days at 80 °C or90 °C that were less hydrolysed than the control, and samples dry heated for more than 2 days at 70 °C or1 day at 80 or 90 °C that were as hydrolysed as the control

Ovalbumin aggregation due to heating in solution increases its in vitro digestibility, compared to the native and dry-heated protein

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