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National audience; Les nucléotides sont des molécules constitutives des acides nucléiques comme l’ADN et l’ARN - leur liaison déterminant la succession des bases impliquées, par exemple, dans la formation de la double hélice de l’ADN. Dans les cellules, les nucléotides participent à la régulation de l’expression génétique et bien évidemment à la synthèse des acides nucléiques. Le maintien de la balance du « pool » de nucléotides est très finement régulé tout au long du cycle cellulaire. Par conséquence, toute dérégulation de cette balance peut avoir des conséquences très délétères pour la cellule. Récemment, nous avons montré en utilisant une approche protéomique que des enzymes impliquées dans le métabolisme des nucléotides étaient dérégulées suite à une contamination avec des nanoparticules. Il paraît donc opportun de vérifier ces résultats préliminaires. Le projet consiste donc à développer une méthode analytique permettant le dosage quantitatif des nucléotides intracellulaires, et à appliquer cette méthode pour étudier l’effet d’une contamination, in vitro, de cellules A549 avec des nanoparticules d’argent.

Les nucléotides sont des molécules constitutives des acides nucléiques comme l’ADN et l’ARN - leur liaison déterminant la succession des bases impliquées, par exemple, dans la formation de la double hélice de l’ADN. Dans les cellules, les nucléotides participent à la régulation de l’expression génétique et bien évidemment à la synthèse des acides nucléiques. Le maintien de la balance du « pool » de nucléotides est très finement régulé tout au long du cycle cellulaire. Par conséquence, toute dérégulation de cette balance peut avoir des conséquences très délétères pour la cellule. Récemment, nous avons montré en utilisant une approche protéomique que des enzymes impliquées dans le métabolisme des nucléotides étaient dérégulées suite à une contamination avec des nanoparticules. Il paraît donc opportun de vérifier ces résultats préliminaires. Le projet consiste donc à développer une méthode analytique permettant le dosage quantitatif des nucléotides intracellulaires, et à appliquer cette méthode pour étudier l’effet d’une contamination, in vitro, de cellules A549 avec des nanoparticules d’argent.

Effect of oxygen, polyphenols and metals was studied on degradation of delphinidin and cyanidin 3-O-sambubioside of Hibiscus sabdariffa L. Experiments were conducted on aqueous extracts degassed or not, an isolated polyphenolic fraction and extract-like model media, allowing the impact of the different constituents to be decoupled. All solutions were stored for 2 months at 37 degrees C. Anthocyanin and their degradation compounds were regularly HPLC-DAD-analyzed. Oxygen concentration did not impact the anthocyanin degradation rate. Degradation rate of delphinidin 3-O-sambubioside increased 6-fold when mixed with iron from 1 to 13 mg.kg(-1) but decreased with chlorogenic and gallic acids. Degradation rate of cyanidin 3-O-sambubioside was not affected by polyphenols but increased by 3-fold with increasing iron concentration with a concomitant yield decrease of scission product, protocatechuic acid. Two pathways of degradation of anthocyanins were identified: a major metal-catalyzed oxidation followed by condensation and a minor scission which represents about 10% of degraded anthocyanins.

The mechanisms of main tomato carotenes (phytoene, phytofluene, lycopene and β-carotene) intestinal absorption are still only partly understood. We thus compared carotene bioavailability in mice after gavage with carotene-rich oil-in-water emulsions. We also determined each carotene absorption profile along the duodenal-ileal axis of the intestine to identify their respective absorption sites and compared these profiles with the gene expression sites of their identified transporters, i.e. SR-BI and CD36. Our data show that phytofluene presented a significantly higher bioavailability compared to lycopene and β-carotene (areas under the curve of 0.76 ± 0.09 vs. 0.30 ± 0.05, 0.09 ± 0.05 and 0.08 ± 0.01 μmol/L·h for phytofluene, phytoene, lycopene and β-carotene, respectively). β-Carotene was mostly converted in the proximal and median intestine. Phytoene and phytofluene accumulation tended to be more important in the distal intestine, which did not correlate with the proximal expression of both Scarb1 and CD36. Overall, these results highlight the high bioavailability of phytofluene. Junta de Andalucía CAROTINCO-P12-AGR-1287

International audience; Though many different methods have been applied to protein-tannin interactions, divergent results are often reported. To better understand the origin of these differences, we compare here haze/aggregates formation and thermodynamic parameters occurring for protein-procyanidin interactions. Proteins well referenced for interaction with polyphenols, namely a polypeptide of extended structure that resembles salivary proteins (poly-L-proline (PLP) and a standard globular protein (bovine serum albumin (BSA)) were used. Flavan-3-ols of three degrees of polymerization were tested, (-)-epicatechin (EPI), a dimer (DP2) and a procyanidin (DP8). The interactions were tested in identical conditions for both methods. The association constants determined by isothermal titration calorimetry (expressed as constitutive unit) varied with the following scale: PLP-DP8 >> BSA-DP8 z BSA-DP2 z PLP-DP2 > BSA-EPI (no affinity detected for PLP-EPI). However aggregates formed more readily for highly polymerized procyanidins DP8 with BSA thanwith PLP, and the scale (with 10 mmol/L of constitutive unit of polyphenol and 0.07 mmol/L of proteins) was: BSA-DP8 > BSA-DP2 >> PLP-DP8 > PLP-DP2 z PLP-EPI, with no haze formation for BSA-EPI. However the impact of polyphenol concentration on haze formation and precipitation was different for the two proteins.

The quantification of lactoferrin in dairy products is mainly performed using immunological methods (ELISA, radio-immunodiffusion and optical biosensing). These methods are species-specific and considered to be sensitive (ng on a diluted sample basis) but are expensive and reproducibility is average. Separative techniques such as electrophoresis and HPLC can interestingly discriminate different forms of lactoferrin but appeared disappointing for quantification. Indeed, caseins and fat should be removed before analysis, which results in the dilution and loss of lactoferrin, and critical resolution from whey proteins was often observed. In the proposed method, skimmed cow’s milk sample (1 mL) is deposited as is in a microtube containing 100 mg of macroporous cation-exchange sulfonated resin and stirred rotationally for 90 minutes at 20-25 °C. Lactoferrin which is positively charged at the pH of native milk (6.8) is quantitatively adsorbed while the other non-adsorbed negatively-charged whey proteins and caseins are removed by centrifugation. After washing 4 times the resin with 1 ml of water, lactoferrin is quantitatively desorbed after 30 minutes stirring with 1 mL 2M NaCl containing phenylalanine as a dilution marker. Lactoferrin is then completely separated from the co-desorbed cationic peptides and lactoperoxidase using RP-HPLC at 220 nm. This cheap, universal and flexible method improves selectivity (no protein interference) and sensitivity (no dilution) over previous separative methods. Its intra-laboratory validation (six concentrations in triplicate over five days) demonstrated linearity up to 500 μg.mL-1, accuracy (98-110% recovery), sensitivity (LOQ=25 μg.mL-1 milk) and precision (< 4%) which challenge most immunochemical methods. MBRE-HPLC and monoclonal ELISA-sandwich gave similar results for individual raw cow’s milks. Nevertheless different evolutions were observed with milk heated at increasing temperature (60-87° C, 30 s) indicating that the epitope sensitive in ELISA and cationic part implicated in MBREHPLC are situated in different portions of the molecule that are affected differently by heating.

Degradation parameters of two main anthocyanins from roselle extract (Hibiscus sabdariffa L.) stored at different temperatures (4-37 degrees C) over 60 days were determined. Anthocyanins and some of their degradation products were monitored and quantified using HPLC-MS and DAD. Degradation of anthocyanins followed first-order kinetics and reaction rate constants (k values), which were obtained by non-linear regression, showed that the degradation rate of delphinidin 3-O-sambubioside was higher than that of cyanidin 3-O-sambubioside with k values of 9.2.10(-7) s(-1) and 8.4.10(-7) s(-1) at 37 degrees C respectively. The temperature dependence of the rate of anthocyanin degradation was modeled by the Arrhenius equation. Degradation of delphinidin 3-O-sambubioside (Ea = 90 kJ mol(-1)) tended to be significantly more sensitive to an increase in temperature than cyanidin 3-O-sambubioside (Ea = 80 kJ mol(-1)). Degradation of these anthocyanins formed scission products (gallic and protocatechuic acids respectively) and was accompanied by an increase in polymeric color index.

This study was designed to evaluate the applicability of a new liquefied gas, HFO-1234ze, as a safe alternative for the substitution of usual solvents such as n-hexane, acetone or ethanol, for the extraction of natural products. The performance of this new solvent was assessed using several plant materials: rapeseeds, carrots, olive leaves and rosemary leaves. An in-silico evaluation of the relative performance of each solvent was performed using Hansen solubility parameters and COSMO-RS model. Extraction experiments were also performed at laboratory scale on several plant materials, in order to determine qualitative and quantitative characteristics of the extracts. Finally, safety considerations, as well as energetic and environmental impacts using this new liquefied gas solvent were evaluated in a scale-up perspective.

International audience; Recent trends in food extraction techniques have largely focused on finding solutions that minimize the use of toxic solvents or to find alternatives for n-hexane. As an original solution, a new process for extraction of natural product using liquefied gases has been developed at laboratory scale. The applicability of this new process was demonstrated using n-butane as alternative solvent to n-hexane. Extraction of lipophilic natural products such as carrot oleoresin, caraway aromas and sunflower oil was assessed using liquefied n-butane. COSMO-RS simulations allowed determination of the relative solubility of the lipophilic compounds in liquefied n-butane and n-hexane. This theoretical approach was validated with extraction experiments on carrots, caraway seeds and sunflower seeds. Compositions of the extracts were comparable but the extract yields were lower using n-butane. For example the extraction yields were, with n-butane and with n-hexane respectively, 36.9% and 53.4% for sunflower seeds oil, 10.9% and 15.0% for caraway seed essential oil, 25.7% and 55.8% for carrots oleoresin. However the extracts obtained by using n-butane are directly free of solvents and also food grade, as it is an authorized solvent for foodstuff production.

Background: During must fermentation thousands of volatile aroma compounds are formed, with higher alcohols, acetate esters and ethyl esters being the main aromatic compounds contributing to floral and fruity aromas. The action of yeast, in particular Saccharomyces cerevisiae, on the must components will build the architecture of the wine flavour and its fermentation bouquet. The objective of the present work was to better understand the molecular and metabolic bases of aroma production during a fermentation process. For such, comparative transcriptomic and metabolic analysis was performed at two time points (5 and 50 g/L of CO2 released) in fermentations conducted by four yeast strains from different origins and/or technological applications (cacha double dagger a, sake, wine, and laboratory), and multivariate factorial analyses were used to rationally identify new targets for improving aroma production.Results: Results showed that strains from cacha double dagger a, sake and wine produced higher amounts of acetate esters, ethyl esters, acids and higher alcohols, in comparison with the laboratory strain. At fermentation time T1 (5 g/L CO2 released), comparative transcriptomics of the three S. cerevisiae strains from different fermentative environments in comparison with the laboratory yeast S288c, showed an increased expression of genes related with tetracyclic and pentacyclic triterpenes metabolism, involved in sterol synthesis. Sake strain also showed upregulation of genes ADH7 and AAD6, involved in the formation of higher alcohols in the Ehrlich pathway. For fermentation time point T2 (50 g/L CO2 released), again sake strain, but also VL1 strain, showed an increased expression of genes involved in formation of higher alcohols in the Ehrlich pathway, namely ADH7, ADH6 and AAD6, which is in accordance with the higher levels of methionol, isobutanol, isoamyl alcohol and phenylethanol observed.Conclusions: Our approach revealed successful to integrate data from several technologies (HPLC, GC-MS, microarrays) an, Ines Mendes is recipient of a fellowship from the Portuguese Science Foundation, FCT (SFRH/BD/74798/2010). This work was supported by FCT through grant (PTDC/AGR-ALI/121062/2010) and by the strategic programme UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) funded by national funds through the FCT I.P. and by the ERDF through the COMPETE2020 - Programa Operacional Competitividade e Internacionalizacao (POCI)., info:eu-repo/semantics/publishedVersion

Decreasing the complexity of UHPLC-HR/AM-MS data by targeted Kendrick mass defect filtering and 3D Van Krevelen representation strategies to monitor wine polyphenol polymerisation. JFSM 2016 33. journées françaises de spectrométrie de masse

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International audience; Microwave hydrodiffusion and gravity (MHG) technique has been applied to pilot-scale solvent-free microwave extraction (SFME) of polyphenols from Lettuce sativa. Following the dictates of green extraction and with the aim to save time and energy, the lab-scale knowledge on SFME was exploited for the development of a pilot-scale process. The investigation entailed the optimization of all main parameters (temperature, time, extracted water volume, etc.) and we showed that the polyphenols composition profile under SFME was similar to the classic methods though a bit lower in total content. The energy consumption in the optimized procedure (30 min) was 1W/g of fresh matrix.

International audience; The seasonal variations of the content and diversity of phenolic compounds, as well as the antioxidant activity of leaves, stems and fruits of bilberry collected in May, July and September, were evaluated for two consecutive years. UPLC/MSn analyses showed the predominance of anthocyanins in fruits, caffeic acid derivatives in leaves whereas flavanol oligomers represented more than half of the phenolic compounds in stems. Thioacidolysis revealed degrees of polymerization between 2 and 4 and (-)-epicatechin as the main flavanol unit. The sum of the phenolic compounds by UPLC was highly correlated with the total polyphenol content and the antioxidant activity in the DPPH test for all the extracts except for May leaves. The latter were relatively rich in p-coumaric acid derivatives. Seasonal effects were more marked for leaves, which exhibited higher antioxidant activities and phenolic contents in July and September when these parameters were at their highest in July for stems.

The phenolic compounds are involved in the main organoleptic properties of plant-derived foods. They are especially responsible for the bitterness, the astringency and the color of apple cider products. The browning observed during fruit processing is related to the formation of yellow-orange pigments. By its catalytic activities, the polyphenol oxidase, a plastidial enzyme, initiates the oxidation of polyphenols in the presence of oxygen. It leads to the formation of quinones, very reactive species which are quickly involved in further chemical reactions. Some of the neoformed products are colored. Four apple juices, showing contrasted colors, were analyzed by reversed-phase HPLC-DAD-MS in order to obtain their profiles in colored compounds. The presence of yellow-orange oxidized tanning and non-tanning compounds was highlighted. Within the latter compounds, some phloridzin oxidation products, deriving from dihydrochalcones, were clearly identified (POPj, XGPOPj). In parallel, coupled approaches in real media and model solutions led to the identification of a new colored structure deriving from an oxidative coupling between a dihydrochalcone and a flavanol monomer. Finally, catechin trimers were also characterized in synthetic medium but they were not detected directly in apple juices. Moreover, the color expressed by the yellow-orange pigments can suffer some variations as a function of the physical-chemical environment. For instance, the cider fermentation results generally in a loss of color in apple juices; but the composition of the fermentative medium evolves under the influence of several parameters, such as pH and redox potential. The impact of reducing conditions on the color of POPj and A-type dehydrotriepicatechin, both selected as model pigments of apple juices, was explored by cyclic voltammetry and UV-visible spectroelectrochemistry. Besides, the changes in spectral properties of POPj, related to its protonation state, were rationalized by TD-DFT calculations., Les composés phénoliques sont impliqués dans les propriétés organoleptiques majeures des aliments dérivant d’une matrice végétale. Ils sont notamment responsables de l’amertume, de l’astringence et de la couleur des produits cidricoles. Le brunissement observé lors de la transformation des fruits est lié à la formation de pigments jaunes-orangés. Il est initié par l’activité d’une enzyme plastidiale, la polyphénol oxydase, qui catalyse l’oxydation des polyphénols en présence d’oxygène. Cela aboutit à la formation de quinones, espèces chimiques très réactives qui sont rapidement impliquées dans des voies réactionnelles ultérieures. Certains produits néoformés sont colorés. Un profilage des molécules colorées présentes dans quatre jus de pomme monovariétaux aux couleurs contrastées a été réalisé en CLHP-DBD-SM. Il a mis en évidence l’existence de tannins oxydés colorés et de composés jaunes-orangés non tannants. Parmi ces derniers, des produits d’oxydation de la phloridzine, dérivant de la classe des dihydrochalcones, ont été clairement identifiés (POPj, XGPOPj). Des approches couplées en milieu réel et solution modèle ont en outre permis d’identifier une nouvelle structure colorée dérivant d’un couplage oxydatif entre une dihydrochalcone et un flavanol monomère. Enfin, des déhydrotrimères d’épicatéchine ont pu être caractérisés en solution synthétique mais n’ont pas été détectés directement dans les jus. Par ailleurs, la couleur exprimée par les pigments formés peut subir des modulations en fonction de l’environnement physico-chimique. A titre d’exemple, la fermentation cidricole génère classiquement une décoloration des moûts ; or la composition du milieu fermentaire évolue sous l’influence de différents paramètres tels que le pH ou le potentiel redox. L’impact d’un milieu réducteur sur la couleur du POPj et d’une déhydrotriépicatéchine de type A, sélectionnés comme pigments modèles des jus de pomme, a été exploré par voltammétrie cyclique et spectroélectrochimie UV-visible. En outre, les changements de propriétés spectrales du POPj liés à son état de protonation ont été rationalisés par des approches de TD-DFT.

Unravelling the color of Rosé wine: Polyphenol profiling by UHPLC-MRM-MS gives new insights into the color diversity of worldwide Rosé wines. 2. International symposium on profiling

Grape anthocyanins from rose wine pomace and grape skins were fractionated by multilayer coil countercurrent chromatography (MLCCC). Tert-butyl methyl ether/n-butanol/acetonitrile/water acidified with trifluoroacetic acid (2/2/x/5) was chosen as the solvent system because of its demonstrated efficiency in separating anthocyanins. A method combining MLCCC and step gradient elution has been developed. This method enabled the fractionation of anthocyanins as series of glucosides, and the corresponding acetylated, coumaroylated, and caffeoylated derivatives. The different anthocyanins were identified on the basis of their UV spectra, their elution time on reversed-phase HPLC as well as by mass spectrometry. A polar-colored fraction free of anthocyanin monomers and corresponding to the material remaining in the stationary phase after the completion of the gradient was recovered. Keywords: Grape; anthocyanins; liquid−liquid separation; multilayer coil countercurrent chromatography; step gradient elution

International audience; Red sorghum is a source of phenolic compounds (PCs), including 3-deoxyanthocyanidins that may protect against oxidative stress related disease such as atherosclerosis. HPLC was used to characterise and quantify PCs extracted from red or white sorghum whole grain flour. Antioxidant activity was measured by an oxygen radical absorbance capacity assay and against LDL-oxidisability, and further compared to that of synthesised 3-deoxyanthocyanidins (i.e., luteolinidin and apigeninidin). Phenolic content of red and white sorghums was evaluated as 3.90 ± 0.01 and 0.07 ± 0.01 mmol gallic acid equivalents L- 1, respectively. Luteolinidin and apigeninidin were mainly found in red sorghum. Red sorghum had almost 3 and 10 times greater specific antioxidant activity compared to luteolinidin and apigeninidin, respectively. Red sorghum PCs and the two 3-deoxyanthocyanidins were also effective at preventing LDL vitamin E depletion and conjugated diene production. Red sorghum flour exhibits antioxidant capacity suggesting that it may be a valuable health-promoting food.

International audience; This present study was designed to evaluate the performances of five alternative solvents (alcohols: ethanol, isopropanol and terpenes:ρ-limonene, α-pinene, p-cymene) compared to η-hexane in rapeseed oil extraction. The extracted oils were quantitatively and qualitatively analyzed to compare the solvents’ performances in terms of kinetics, fatty acid compositions, lipid yields, and classes. Moreover, micronutrients in extracted oils were also respectively quantified by high-performance liquid chromatography (HPLC) and gas chromatography (GC). In addition, the interactions between alternative solvents and rapeseed oil have been theoretically studied with the Hansen solubility methodology to get a better comprehension of dissolving mechanisms. The results indicated that pcymene could be the most promising solvent for n-hexane substitution with higher lipid yield and good selectivity, despite the micronutrient contents were relatively low.

Though folates are sensitive to heat treatments, leaching appears to be a major mechanism involved in folate losses in vegetables during processing. The aim of our study was to study folate diffusivity and degradation from spinach and green beans, in order to determine the proportion of each mechanism involved in folate losses. Folate diffusivity constant, calculated according to Fick's second law (Crank, 1975), was 7.4×10(-12) m(2)/s for spinach and 5.8×10(-10) m(2)/s for green beans, which is the same order of magnitude as for sugars and acids for each vegetable considered. Folate thermal degradation kinetics was not monotonous in spinach and green beans especially at 45 °C and did not follow a first order reaction. The proportion of vitamers changed markedly after thermal treatment, with a better retention of formyl derivatives. For spinach, folate losses were mainly due to diffusion while for green beans thermal degradation seemed to be preponderant.

L’industrie laitière rencontre parfois des problèmes d’instabilité du lait UHT (Ultra Haute Température) au cours de son stockage. Cette instabilité se traduit généralement par une gélification ou par une séparation de phases. Parmi les causes qui sont multifactorielles (zoologique, enzymatique, physico-chimique...) la protéolyse des micelles de caséines des laits UHT au cours de leur stockage par des protéases thermorésistantes appartenant au genre Pseudomonas est souvent évoquée. Ce travail a donc été effectué dans le but de mieux comprendre la déstabilisation du lait UHT dont le lait cru a été préalablement contaminé par des bactéries protéolytiques du genre Pseudomonas. La spectroscopie infrarouge à transformée de Fourier a été explorée afin de détecter cette cause et de prédire la stabilité des laits avant leurs transformations technologiques. Dans un premier temps, 32 souches du genre Pseudomonas ont été sélectionnées en fonction de leur caractère protéolytique et de la présence de la protéase AprX (seule protéase extracellulaire identifiée à partir de Pseudomonas isolée du lait). Toutes les souches des espèces P. fluorescens et P. tolaasii ainsi que quelques souches des espèces P. chlororaphis, P. fragi et P. aeruginosa sécrétaient cette protéase. Parmi les espèces P. aeruginosa et P. fragi, certaines souches sécrétaient une protéase de masse moléculaire différente de celle de la protéase AprX. Indépendamment de l’espèce étudiée et de la protéase secrétée, il existait une variabilité de protéolyse au sein des 32 souches. Certaines souches étaient protéolytiques alors que d’autres ne l’étaient pas. Dans un second temps, le suivi de la stabilité de laits UHT préalablement et indépendamment inoculés par 9 souches de P. fluorescens, sécrétant AprX mais au caractère protéolytique différent, a montré que cette hétérogénéité de protéolyse existait aussi dans le lait UHT. Certaines souches déstabilisaient le lait UHT avant la fin des 90 jours de stockage alors que d’autres non. L’addition de la protéase AprX, dans le lait cru avant traitement UHT a également engendré une déstabilisation du lait UHT avant la fin des 90 jours de stockage confirmant le rôle de cette enzyme dans le phénomène de déstabilisation. L’étude réalisée avec la souche P. fragi 9.12 qui semblait sécréter une protéase différente d’AprX nous a montré qu’elle était capable de déstabiliser le lait UHT au cours du stockage. Ce résultat nous a conforté sur le fait qu’AprX ne serait pas la seule protéase extracellulaire des bactéries du genre Pseudomonas ayant un effet sur la déstabilisation du lait UHT. Pour chaque expérimentation effectuée, la déstabilisation se traduisait par une apparition d’agrégats, des augmentations des teneurs en azote non protéique et non caséinique (NPN et NCN) et des diminutions de l’hydratation micellaire et du potentiel zêta, nous indiquant une protéolyse des micelles de caséines. Les résultats obtenus par l’analyse HPLC couplée à la spectrométrie de masse des fractions NCN des laits déstabilisés par les 5 souches de P. fluorescens déstabilisantes et la protéase AprX, ont montré qu’AprX avait un large spectre d’action et était capable d’hydrolyser les caséines β, s1, s2 et κ. Dans un troisième temps, la spectroscopie IRTF a été utilisée dans le but de détecter la présence de bactéries du genre Pseudomonas protéolytiques. Cette phase exploratoire nous a permis de déterminer le seuil de détection d’un lait contaminé par une bactérie du genre Pseudomonas ainsi que le seuil de discrimination d’un lait contenant des Pseudomonas protéolytiques d’un lait contenant des Pseudomonas non protéolytiques (5.102 UFC/mL). Cependant, l’analyse d’un lait contenant minoritairement des Pseudomonas protéolytiques nous a montré que le caractère protéolytique ne ressortait pas sur le spectre IR alors que le risque de protéolyse était bien présent. Des travaux supplémentaires sont donc à réaliser afin que la spectroscopie IRTF soit utilisée comme un outil de prédiction de la stabilité des laits., Dairy industries have sometimes problems of instability of UHT milk (Ultra High Temperature) during storage. This instability usually results in gelation or phase separation. Among the multifactorial causes (Zoological enzyme, Physico-chemical ...), proteolysis of casein micelles of UHT milk by heat-resistant proteases belonging to the genus Pseudomonas during storage, is often mentioned. This work has been conducted to understand the destabilization of UHT milk with raw milk previously contaminated by proteolytic Pseudomonas. Infrared spectroscopy Fourier transform has been explored in order to detect the cause and predict the stability of milk before their technological transformations. Initially, 32 strains of the genus Pseudomonas were selected according to their proteolytic potential and the presence of protease AprX (unique extracellular protease identified from Pseudomonas isolated from milk). All strains of the species P. fluorescens and P. tolaasii and some strains of the species P. chlororaphis, P. fragi and P. aeruginosa secreted this protease. Among the species P. aeruginosa and P. fragi, some strains secreted a protease with a molecular mass different from the one of the protease aprx. Regardless of the species studied and protease secreted, there was a proteolysis variability of the 32 strains. Some strains were proteolytic, while others were not. In a second step, the monitoring of the stability of UHT milk previously and independently inoculated by nine strains of P. fluorescens, secreting AprX but with different proteolytic potential, showed that this heterogeneity of proteolysis also happened in UHT milk. Some strains destabilized UHT milk before the end of 90 days of storage, while others did not. The addition of protease AprX in raw milk before UHT treatment also resulted in a destabilization of UHT milk before the end of 90 days of storage, confirming the role of this enzyme in the phenomenon of destabilization. The study with the strain P. fragi 9.12 that secreted a protease different from AprX, showed that it was able to destabilize UHT milk during storage. This result confirmed the fact that AprX was not the only extracellular protease bacteria of the genus Pseudomonas having an effect on the destabilization of UHT milk. For each experiment performed, the destabilization resulted in a formation of aggregation, in increases of non-protein and non-casein nitrogen contents (NPN and NCN) and in decreases of hydrations and zeta potentials of caseins micelles, indicating proteolysis of casein micelles. The results obtained by HPLC analysis coupled with mass spectrometry of NCN fractions of milk destabilized by 5 proteolytic strains of P. fluorescens and the protease AprX showed that this enzyme had a large action spectrum and was able to hydrolyze β, s1, s2 and κ casein. In a third step, FTIR spectroscopy was used to detect the presence of proteolytic bacteria of the genus Pseudomonas. This exploratory phase allowed us to determine the threshold for detection of milk contaminated with bacteria of the genus Pseudomonas and the discrimination threshold of milk containing proteolytic Pseudomonas from milk containing non-proteolytic Pseudomonas (5.102 CFU / mL). However, the analysis of milk containing a smaller quantity of proteolytic Pseudomonas than non proteolytic Pseudomonas, showed us that the character proteolytic dit not appears in the IR spectrum, while the risk of proteolysis was present. Further works have to be undertaken to use FTIR spectroscopy as a tool for predicting the stability of milk.

β-lactoglobulin (β-lg) is a major whey protein in bovine milk, which has the ability to bind hydrophobic ligands such as fatty acids. It is well known that the whey protein alpha-lactalbumin also forms a stable complexe with fatty acid, which is cytotoxic to a wide variety of tumour cells. The aim of this project was to investigate the binding stoichiometry of β-lg and linoleic acid (LA; cis,cis-9,12-octadecadienoic acid) and investigate the impact of this complex on viability of intestinal cancer cells in vitro. The interaction between β-lg and fatty acids was studied by HPLC and GC. Complexes with a binding stoichiometry of 1, 2 and 3 LA:β-lg were produced. The effect of the complexes on cell viability was measured by exposing the human colorectal adenocarcinoma epithelial cell line, Caco-2, to various concentrations of complexes from 0 to 72 hours. LA on its own exhibited a cell viability effect at 58 M as measured by MTS assays. A cytotoxic effect at 35 M was measured by an impedance-based biosensing microelectronic real-time cell analyzer system (XCELLigence RTCA system, Roche Diagnostics). Neither the β-lg (0 to 75 μM) nor β-lg/LA complexes (LA concentration from 0 to 200 μM) had an adverse effect on Caco-2 cells. In vivo it is assumed that β-lg gradually releases LA during gut transit which, in turn, may modulate fatty acid uptake by the gut and/or have a satiating effect. Future work will investigate fatty acid uptake and satiety signaling in the gut in response to β-lg/LA complexes.

The effect of 2 isoflavonoids (the soybean phytoalexin, glyceollin I, and the coumestan, coumestrol) and 1 flavonoid, quercetin, were tested on in vitro spore germination of Gigaspora margarita. Glyceollin I and coumestrol were tested at 0, 0.05, 0.5, 5 and 50 μM in water agar containing 0.5% ethanol. Quercetin was tested at 0, 0.1, 1 and 10 μM in pure water agar. All germination parameters were measured after 5 and 7 d. Germination rate was not significantly affected by any of these compounds. The number of germ tubes per spore was slightly increased by glyceollin I; mycelium length from germinated spores was increased by low concentrations of glyceollin I but was significantly decreased at the highest concentration. A positive correlation was found between coumestrol concentration and mycelium length, and vesicle number was decreased by coumestrol, quercetin and the highest concentration of glyceollin but was increased by glyceollin at 0.5 μM. Results are discussed in relation to the potential of flavonoids and isoflavonoids in acting as regulatory factors in plant-AM fungus interactions., Deux isoflavonoïdes (la glycéolline I et le coumestrol) et un flavonoïde (la quercétine) ont été éprouvés vis-à-vis de la germination in vitro de Gigaspora margarita. Les expériences ont été faites en utilisant les concentrations de 0, 0,05, 0,5, 5 et 50 μmol.l-1 dans l’eau gélosée contenant 0,5% d’éthanol pour les premiers et de 0, 0,1, 1, et 10 μmol.l-1 dans l’eau gélosée pure pour la quercétine. Les mesures, réalisées après 5 et 7 j de croissance ont montré que le taux de germination n’a été affecté par aucun des composés, que le nombre de tubes germinatifs a été légèrement augmenté par la glycéolline I et que la longueur du mycélium par spore germée a été augmentée par la glycéolline I aux faibles concentrations alors qu’elle a été significativement diminuée à concentration supérieure. Une corrélation positive a été trouvée entre la teneur en coumestrol et la longueur du mycélium des tubes germinatifs. Les résultats sont discutés en relation avec la potentialité des flavonoïdes et isoflavonoïdes d’agir comme régulateurs dans le processus infectieux du champignon mycorhizien.

The reaction between procyanidin dimer Ec-EcG (B2 3'-O-gallate) and malvidin 3-O-glucoside (Mv3glc) was studied in a model solution system at two different pH values, 2.0 and 3.8. Disappearance of both species was much faster at pH 3.8 than at pH 2.0. That of Mv3glc was increased in the presence of Ec-EcG, whereas that of Ec-EcG was the same in the presence or absence of the anthocyanin. Values of absorbance at 520 nm measured at pH 2.0 were correlated with the amount of residual Mv3glc. Those measured at pH 3.8 hardly changed during the incubation, but absorbance values at 420 and 620 nm as well as resistance to sulfite bleaching were much increased, confirming that Mv3glc was converted to other pigments. Anthocyanin-flavanol adducts were observed at both pH values, but their structures were different. At pH 2.0, cleavage of the procyanidin linkage followed by nucleophilic addition of flavanol or anthocyanin moieties led to (Ec)(eta)-EcG and (Ec)(eta)-Mv3glc, respectively. At pH 3.8, nucleophilic addition of Ec-EcG onto the anthocyanin yielded Mv3glc-(Ec-EcG).

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42 ref.; International audience

The composition of whey proteins from ruminant Mongolian domestic animals was analyzed and a comparative study between camel (Camelus bactrianus) and dromedary (Camelus dromedarius) was made. Whey proteins were separated by ion-exchange chromatography and identified by polyacrylamide gel electrophoresis, amino acid composition and N-terminal sequence determination. The main components of wheys of yak and khainak were nearly identical with their bovine counterparts. Three different forms of α-lactalbumin were isolated in the whey of Camelus bactrianus and two from Camelus dromedarius. As shown by classical biochemical and immunological studies, β-lactoglobulin was absent from whey of both Camelus. Camel whey basic protein (CWBP), having no analogy with known milk and nonmilk proteins, was identified in the whey of Camelus bactrianus and Camelus dromedarius and its N-terminal sequence was determined.

Not available, Depuis la mise au point des techniques de formation des lignées d'hybridomes, des efforts importants sont fournis pour améliorer la productivité des anticorps monoclonaux et pour mieux comprendre le métabolisme cellulaire. Les méthodes analytiques jouent un rôle important dans le contrôle des procédés en permettant la détermination quantitative et qualitative de plusieurs paramètres et variables. L'objectif général de cette étude consiste à contribuer à l'amélioration de la maitrise des bioprocédés en proposant une méthode analytique capable de quantifier les protéines présentes dans les cultures d'hybridomes. Afin d'atteindre cet objectif, les potentialités de l'HPLC ont été testées. Les protéines considérées dans cette étude sont d'une part, les composes protéiques propres au milieu de culture et d'autre part, les anticorps monoclonaux secrétés par les cellules. La première partie de cette étude présente l'optimisation des conditions opératoires de la méthode chromatographique. Les deux modes chromatographiques mis à profit sont l'HPLC d'affinité et la RP-HPLC. Une étude comparative des performances de la méthode par HPLC et du test ELISA est exposée dans une seconde partie. La troisième partie concerne l'application de la méthode chromatographique aux suivis des protéines de cultures cellulaires d'hybridomes. Enfin, la réalisation couplage automatise réacteur-échantillonneur-HPLC et son application à la détermination en ligne de la concentration des IgG d'une culture discontinue, constituent la dernière partie de ce travail

Chiral gamma-lactones from Fusarium poae: enentiomeric ratios and sensory differentiation of cis-6-gamma-dodecenolactone enantiomers

23 ref.; Cytokinins in vegetative and reproductive buds of Pseudotsuga menziesii

Study of endogenous plant growth sustances in Douglas-fir. II. Gibberellin analysis

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Changes in endogenous cytokinins during flowering induction in Douglas fir : effects of exogenous applications

Dosage des polyphenols présents dans des cotyledons de noix, cultivés in vitro, au cours des differents stades de l'organogenèse, par chromatographie liquide haute performance