Protection of yeast cells in micro-organized shells of natural polyelectrolytes during drying process

The encapsulation techniques are applied in various fields and for various applications. The layer-by-layer self-assembly (LbL), one of the encapsulation techniques, is built by the electrostatic attraction between oppositely charged polyelectrolytes, and the topmost layer determines essentially the sur-face properties of the edifice. This technique offers several advantages (low cost, simplicity of process and equipment, biocompatibility and biodegra-dation, etc.). In this present paper, results of the protection of Saccharomyces cerevisiae yeast cells in microorganized shell of natural polyelectrolytes during dehydration process are reported. To apply the LbL method to individually encaps…

Native-state pea albumin and globulin behavior upon transglutaminase treatment

International audience; The behavior of pea albumin (Alb) and globulin (Glob) in their native state upon microbial transglutaminase (MTGase) treatment was studied. Only Glob was able to form a gel, at up to a 10% (w/w) concentration, with a minimum gelling concentration of 6% (w/w), and with a cross-linking degree of 25%. The most affected Glob subunits were convicilin (71 kDa), vicilins (55, 50, and 35 kDa), and legumin acidic subunit (40 kDa). In contrast, the legumin basic subunit (20 kDa) and vicilins of molecular weight less than 20 kDa remained mostly intact in all studied conditions. The cross-linking degree of Alb was 12%, which was not sufficient to form MTGase-induced gel. Major a…

Protection of living yeast cells by micro-organized shells of natural polyelectrolytes

International audience; Saccharomyces cerevisiae, a eukaryotic model organism, plays a key role in the oxidative stability of fermented products. In order to protect cells against environmental stresses, we report a method of modifying the cell surface architecture while maintaining the internal working properties of the system. The objective was to encapsulate living yeast cells in micro-organized polyelectrolyte shells using layer-by-layer (LbL) assembly. For the first time, the natural polyelectrolytes, β-lactoglobulin and sodium alginate, were alternately deposited on the surface of S. cerevisiae. Transmission electron microscopy coupled with immune-cytochemistry and scanning electron m…

1H NMR Spectroscopy As Tool To Study Transglutaminase Crosslinking Of Pea Globulin

Skiathos Island, GREECE - 30 May - 02 June 2013; International audience; A new method based on NMR spectroscopy was developed to detect the G-L (GlutamylLysine) isopeptide bonds formed by the enzymatic transglutaminase reaction. Because of thecomplexity of NMR signals of proteins due to their structures, the method was first developedon a model system (glutamine and lysine) to simplify the detection of the G-L residue. Andthen, the results were applied on the real protein matrix (pea globulin). MTG treatment ofmodel system led to the appearance of a new resonance on NMR spectrum which isoriginated probably from the ε-methylene protons of lysine residues covalently linked toglutamine. The co…

Fusarium proliferatum: induction and intracellular location of a lipoxygenase

The fungus Fusarium proliferatum was grown in a soya oil- and glucose-supplemented medium. Induction of lipoxygenase activity (13-fold) in an extract of Fusarium biomass has been observed at day four on soya oil culture medium. A band at 232 kDa was detected using a specific lipoxygenase stain combined with native PAGE. The method of fungal homogenate obtained has been checked via subcellular marker enzymes activities: succinate dehydrogenase (SDH) for mitochondrial fraction and NADH-cytochrome C reductase (NADH-cyt. C) for the microsomal fraction. Protoplasts production and disintegration followed by subcellular fractionation using differential centrifugation was the best method to recover…

Optimization of the Hydrolysis of Safflower Oil for the Production of Linoleic Acid, Used as Flavor Precursor

Commercial lipases, from porcine pancreas (PPL),Candida rugosa(CRL), andThermomyces lanuginosus(Lipozyme TL IM), were investigated in terms of their efficiency for the hydrolysis of safflower oil (SO) for the liberation of free linoleic acid (LA), used as a flavor precursor. Although PPL, under the optimized conditions, showed a high degree of hydrolysis (91.6%), its low tolerance towards higher substrate concentrations could limit its use for SO hydrolysis. In comparison to the other investigated lipases, Lipozyme TL IM required higher amount of enzyme and an additional 3 h of reaction time to achieve its maximum degree of SO hydrolysis (90.2%). On the basis of the experimental findings, C…

Developing sustainable food : the role of consumer liking in optimization of pea yogurt

International audience; In the development of new food consumers’ acceptability is a critical factor. This study aimed at evaluating the possibility to introduce pea proteins as new fermented food. For this purpose, mixtures of cow and pea milks with different ratios of pea (0, 10, 20, 30, 40 and 100%) were fermented with French commercial yogurt-ferment. Consumer acceptability in two European countries: France (Western Europe) and Bulgaria (Southeastern Europe) was investigated. 60 Bulgarian and 70 French panelists were asked to rate: overall acceptability, appearance, odor, taste and texture on a hedonic scales going from 1 (I do not like at all) to 7 (I like very much). The ANOVA showed …

Predicted secondary structure of hydroperoxide lyase from green bell pepper cloned in the yeast Yarrowia lipolytica

International audience; Fatty acid hydroperoxide lyase (HPL) is a member of the cytochrome P450 family acting on fatty acid hydroperoxides in many organisms. The active green bell pepper HPL, cloned and expressed in the yeast Yarrowia lipolytica, was purified by immobilized metal-ion affinity chromatography (IMAC) in the presence of 2% of Triton X-100R. The secondary structure prediction by bioinformatics servers of HPL was realized by ANTHEPROT software, using the GOR, DPM and Predator methods. The theoretical results which are average values obtained from three different calculation methods showed 33% α-helix, 18% β-sheet, 7% turn and 42% coil. On the other hand, the secondary structure a…

Contribution of exofacial thiol groups in the reducing activity of Lactococcus lactis

Lactococcus lactis can decrease the redox potential at pH 7 (E(h7)) from 200 to -200 mV in oxygen free Man-Rogosa-Sharpe media. Neither the consumption of oxidizing compounds or the release of reducing compounds during lactic acid fermentation were involved in the decrease in E(h7) by the bacteria. Thiol groups located on the bacterial cell surface appear to be the main components that are able to establish a greater exchange current between the Pt electrode and the bacteria. After the final E(h7) (-200 mV) was reached, only thiol-reactive reagents could restore the initial E(h7) value. Inhibition of the proton motive force showed no effect on maintaining the final E(h7) value. These result…

Fermentation des protéines végétales : atouts organoleptique et nutritionnel

Référence : 201511122426; Du fait de l’augmentation de la population de la planète, la production de protéines pour l’alimentation des populations et l’évaluation de la qualité de ces protéines pour satisfaire les besoins de l’Homme pourraient devenir des questions majeures dans les décennies à venir pour l’ensemble du monde. Le développement de nouveaux aliments optimisant l’apport protéique d’origine végétale parait également important dans certaines situations (personnes âgées, sportifs…) où les besoins nutritionnels sont modifiés au plan quantitatif et qualitatif. De nombreux problèmes ont été identifiés suite à l’incorporation de protéines végétales dans nos aliments. En particulier, d…

Recombinant hydroperoxide lyase for the production of aroma compounds: Effect of substrate on the yeast Yarrowia lipolytica

International audience; The aim of this study was to investigate the action mechanism of linoleic acid hydroperoxides (HPOD), which are the major substrates of hydroperoxide lyase for the production of flavour compounds, on the yeast Yarrowia lipolytica by evaluating their effect on the oxidative state of the cells. The total antioxidant capacity (TAC) and the activity of the main antioxidant enzymes, such as glutathione reductase, glutathione peroxidase and superoxide dismutase, of cells treated with HPOD were studied. The potential role of intracellular glutathione, including reduced glutathione (GSH) and oxidized glutathione (GSSG), in conferring HPOD resistance was also been examined. T…

Aqueous two-phase system cold-set gelation using natural and recombinant probiotic lactic acid bacteria as a gelling agent

The present study aimed to entrap probiotic lactic acid bacteria (LAB) in a sodium alginate and sodium caseinate aqueous two-phase gel system. The natural acidifying properties of two therapeutic probiotic LAB were exploited to liberate calcium ions progressively from calcium carbonate (CaCO3), which caused the gelation of the co-existing phases. Bi-biopolymeric matrix gelation of GDL/CaCO3 or LAB/CaCO3 was monitored by dynamic rheological measurements, and the final gels were characterized by frequency dependence measurements and confocal laser scanning microscopy. Weak to strong gels were formed with an elastic modulus G' from 10 to 1.000Pa, respectively. After cold-set gelation of our sy…

Screening of lactic acid bacteria for fermentation of a mix of cow and pea protein

Screening of lactic acid bacteria for fermentation of a mix of cow and pea protein. Vitagora - 9. international taste-nutrition-health congress

Fatty acid hydroperoxide lyase of green bell pepper: cloning in Yarrowia lipolytica and biogenesis of volatile aldehydes

Publication Inra prise en compte dans l'analyse bibliométrique des publications scientifiques mondiales sur les Fruits, les Légumes et la Pomme de terre. Période 2000-2012. http://prodinra.inra.fr/record/256699; International audience; Fatty acid hydroperoxide lyase (HPO lyase) is a cytochrome P450 acting on fatty acid hydroperoxides in many organisms. The expression of green bell pepper HPO lyase in the yeast Yarrowia lipolytica is described for the first time. HPO lyase activity from yeast extract and whole yeast cells is measured and aldehydes production from yeast extract and whole yeast cells is compared. 1200 U/L reaction medium were obtained after 96 h of culture on olive oil rich me…

Monitoring of transglutaminase crosslinking reaction by 1H NMR spectroscopy on model substrates

International audience; A new method based on 1H NMR spectroscopy was developed for monitoring transglutaminase crosslinking reaction with model molecules (CBZ-Gln-Gly and N-α-acetyl-lysine). The transglutaminase reaction led to the appearance of new resonances on NMR spectrum as well as significant decrease in others. The new observed resonances, originated from newly formed ɛ-(γ-glutamyl)lysine isopeptide bonds, evidence the enzymatic reaction and allow to quantify the ɛ-(γ-glutamyl)lysine fragment. Moreover, the decrease in resonance intensity, originated from lysine, permit to determine the crosslinking degree. These results obtained by 1H NMR spectroscopy can be used as an alternative …

Environmentally eco-reasoned design process for functional yeast production and stabilization

Environmentally eco-reasoned design process for functional yeast production and stabilization. 3. European Congress of Applied Biotechnology (ECAB 3)

Secondary structure conformation of hydroperoxide lyase from green bell pepper, cloned in Yarrowia lipolytica, and its activity in selected media

International audience; Circular dichroism (CD) spectroscopy of secondary structure conformation of the purified green bell pepper hydroperoxide lyase (HPL), cloned in the yeast Yarrowia lipolytica, was investigated. The CD spectra of HPL in iso-octane, obtained at 60 °C, in the presence of the reducing agent dithiothreitol showed dramatic increase in α-helix content. The enzyme conformation remained unchanged over a range of pH values of 5.0–7.0. Using 13-hydroperoxide of linoleic acid (13-HPOD) as substrate, the biocatalysis of HPL in organic solvent media, including chloroform, dichloromethane, hexane, iso-octane, octane and toluene, was investigated. The results indicated an increase in…

Size measuring techniques as tool to monitor pea proteins intramolecular crosslinking by transglutaminase treatment

International audience; In this work, techniques for monitoring the intramolecular transglutaminase cross-links of pea proteins, based on protein size determination, were developed. Sodium dodecyl sulfate polyacrylamide gel electrophoresis profiles of transglutaminase-treated low concentration (0.01% w/w) pea albumin samples, compared to the untreated one (control), showed a higher electrophoretic migration of the major albumin fraction band (26 kDa), reflecting a decrease in protein size. This protein size decrease was confirmed, after DEAE column purification, by dynamic light scattering (DLS) where the hydrodynamic radius of treated samples appears to be reduced compared to the control o…

From sensory evaluation to food product development: how to fit a new vegetal fermented product to the consumer taste

International audience; We present a series of studies showing the role of sensory evaluation in the optimization of new vegetal fermented products.The first series of studies showed that French consumers did not accept fermented products with vegetal protein ratio over 10%.Moreover, positive information on vegetal protein benefits did not lead to an increase in liking scores. So, a second series ofstudies was carried out in order to optimize the raw material and processes used to prepare the products and improve theirorganoleptic properties. In a first step, the vegetal proteins were purified permitting to obtain firmer products with less offflavors. In a second step, the effect of four fa…

Formulation, process conditions, and biological evaluation of dairy mixed gels containing fava bean and milk proteins: Effect on protein retention in growing young rats

International audience; Food formulation and process conditions can indirectly influence AA digestibility and bioavailability. Here we investigated the effects of formulation and process conditions used in the manufacture of novel blended dairy gels (called "mixed gels" here) containing fava bean (Vicia faba) globular proteins on both protein composition and metabolism when given to young rats. Three mixed dairy gels containing casein micelles and fava bean proteins were produced either by chemical acidification (A) with glucono-δ-lactone (GDL) or by lactic acid fermentation. Fermented gels containing casein and fava bean proteins were produced without (F) or with (FW) whey proteins. The AA…

Formulation et procédés de fabrication des gels laitiers enrichis en globulines de fèverole : Impact sur la rétention protéique chez les rats jeunes en croissance

Introduction et but de l’etude L’impact environnemental et les risques pour la sante associes a la production et la consommation excessive de sources de proteines animales pourraient etre limites par l’augmentation de la part des proteines vegetales dans notre alimentation. Les proteines vegetales sont parfois deficitaires en certains acides amines (AA) essentiels. De ce fait, un melange de proteines animales et vegetales permettrait d’assurer une complementarite en AA repondant aux besoins de l’organisme. L’objectif de cette etude etait d’evaluer l’impact, sur la taille des proteines, de la formulation et des processus de transformation des gels laitiers enrichis en legumineuses et d’etudi…

Formulation et procédés de fabrication des gels laitiers enrichis en globulines de fèverole :Impact sur la rétention protéique chez les rats jeunes en croissance

Introduction et but de l’étude : L’impact environnemental et les risques pour la santé associés à la production et la consommation excessive de sources de protéines animales pourraient être limités par l’augmentation de la part des protéines végétales dans notre alimentation. Les protéines végétales sont parfois déficitaires en certains acides aminés (AA) essentiels. De ce fait, un mélange de protéines animales et végétales permettrait d’assurer une complémentarité en AA répondant aux besoins de l’organisme. L'objectif de cette étude était d'évaluer l'impact, sur la taille des protéines, de la formulation et des processus de transformation des gels laitiers enrichis en légumineuses et d’étu…

MOOC : "ProtéiNNOV : Protéines végétales et Innovation"

La prise de conscience d'une raréfaction des ressources en protéines animales dans les décennies à venir et la nécessité de produire plus durablement ces ressources alimentaires nous conduit à repenser aux autres sources de protéines, notamment les protéines d'origine végétale. Parmi les sources de protéines végétales, la plus employée comme substitut des protéines animales est le soja, mais le soja n'est pas la seule source de protéines végétales. Il est donc opportun de faire le point sur les autres sources de protéines végétales et sur leur utilisation. Le développement de nouveaux produits alimentaires riches en protéines végétales passe par la connaissance des atouts tant technologique…

For me the taste of soy is not a barrier to its consumption. And how about you?

International audience; This research investigates the cultural influence on beliefs about and attitudes towards soy foods (French from France vs. Vietnamese from Vietnam) and possible change of beliefs and attitudes and soy consumption habits with a change in cultural environment (French from France vs. French from Vietnam, Vietnamese from Vietnam vs. Vietnamese from France) of French and Vietnamese participants, two countries with very different soy food consumption. Expressed beliefs and attitudes of soy foods resulting from discussions in focus groups, conducted in both countries, were collected and used to derive a questionnaire. French participants differ mainly from Vietnamese partic…

Biogeneration of 1-octen-3-ol by lipoxygenase and hydroperoxide lyase activities of Agaricus bisporus

Abstract The biogeneration of 1-octen-3-ol by lipoxygenase and associated hydroperoxide lyase activities of a homogenate of the cultivated mushroom Agaricus bisporus was investigated using linoleic acid as a substrate. The optimal substrate and protein concentrations were 1.5 mM and 1.5 mg ml −1 , respectively. The V max value for the enzymic coupled reaction lipoxygenase–hydroperoxide lyase was 6 μg of 1-octen-3-ol ml −1 min −1 , with a K m value of 0.3×10 −3 M. The highest enzymic activity was observed in the presence of pure oxygen (22.9 μg of 1-octen-3-ol ml −1 ), with a bioconversion yield of 36%. The production of 380 μg 1-octen-3-ol per g of mushroom homogenate was demonstrated using…

Différences culturelles en perception, description et appréciation des aliments : comparaison entre les consommateurs français et vietnamiens sur les yaourts aux sojas

Modeling hexanal production in oxido-reducing conditions by the yeast Yarrowia lipolytica

International audience; Hexanal produced by cells of a recombinant Yarrowia lipolytica yeast expressing the hydroperoxide lyase (HPL) from green bell pepper fruit was studied under oxido-reducing conditions using the reducing dithiotreitol and oxidizing potassium ferricyanide compounds. The combined effect of pH, linoleic acid 13-hydroperoxides concentration, temperature and oxido-reducing molecules on the hexanal production was studied. Significant positive effects for the hexanal production were found using high concentrations of hydroperoxides (100 mM, 30 g/L). Adding reducing molecules enhanced significantly hexanal production while the oxidizing molecules had an inhibitory effect. Comb…

A new alternative in vitro method for quantification of Toxoplasma gondii infectivity.

An in vitro method to determine the infectious potency of an unknown suspension of the protozoan parasite Toxoplasma gondii based on kinetics of host cells lysis was developed. Mic1-3KO a mutant strain of T. gondii RH tachyzoites was inoculated in 25-cm² flasks containing a 90% confluent monolayer of human foreskin fibroblasts. Lysis kinetics was monitored for infection ratios ranging from 1∶10⁶ to 1∶10; we defined 10⁶ tachyzoites/ml⁻¹ as the threshold value for parasite egress. Results allowed us to build a calibration curve relating the initial infection ratios to the time needed to reach 10⁶ tachyzoites/ml⁻¹. Finally, we validated the method using a known mixture of dead and live parasit…

Design of new sensitive α,β-unsaturated carbonyl 1,8-naphtalimide fluorescent probes for thiol bioimaging

International audience; We report herein an efficient synthesis of news naphtalimide-based fluorescent derivatives functionalized with an a,(3-unsaturated carbonyl group. Probes were synthesized from reaction of an amino-naphtalimide precursor with maleic anhydride. Photophysical study of fluorescent probes and cells labelling were performed, and showed that the reactive derivatives exhibit rich turn on fluorescence properties in presence of different biological thiol (Glutathione and Cysteine) making these systems a very promising way for thiol bioimaging.

Characterization of Purified Green Bell Pepper Hydroperoxide Lyase Expressed by Yarrowia Lipolytica: Radicals Detection during Catalysis

International audience; The optimization of the expression of recombinant 6-His-tagged HPO lyase in Yarrowia lipolytica is described: 1800U/L of culture was detected at 24h of culture on a medium containing olive oil as the sole carbon source. The enzyme was purified by IMAC and showed an optimal pH at 5.5, an optimal temperature at 20^\circC and a Km value of 9μM with 13-HPOD substrate. The participation of radicals during the catalysis of purified bell pepper fruit hydroperoxide lyase has been observed by electron paramagnetic resonance spectroscopy and the yet unidentified radical species might be an alkyl or alkoxyl radical linked to the enzyme.

Toxicity of fatty acid hydroperoxides towards Yarrowia lipolytica: Implication of their membrane fluidizing action

Abstract Linoleic acid hydroperoxide (HPOD), substrate of hydroperoxide lyase, an enzyme of the lipoxygenase pathway, can be transformed into many aromatic compounds, the so-called “green notes”. The presence of linoleic acid hydroperoxide in the culture medium of Yarrowia lipolytica, the yeast expressing the cloned hydroperoxide lyase of green bell pepper, undoubtedly exerted an inhibition on the growth and a toxic effect with 90% of yeast cells died after 120 min of exposition in 100 mM HPOD solution. The increase in cell membrane fluidity evaluated by measuring fluorescence generalized polarization with the increasing concentration of HPOD in the medium confirmed the fluidizing action of…

CCDC 1054357: Experimental Crystal Structure Determination

Related Article: Nicolas Sok, Maria Nikolantonaki, Stephane Guyot, Thanh Dat Nguyen, Anne-Sophie Viaux, Franck Bagala, Yoann Rousselin, Florence Husson, Régis Gougeon, Rémi Saurel|2017|Sens.Actuators,B|242|865|doi:10.1016/j.snb.2016.09.171

CCDC 1054356: Experimental Crystal Structure Determination

Related Article: Nicolas Sok, Maria Nikolantonaki, Stephane Guyot, Thanh Dat Nguyen, Anne-Sophie Viaux, Franck Bagala, Yoann Rousselin, Florence Husson, Régis Gougeon, Rémi Saurel|2017|Sens.Actuators,B|242|865|doi:10.1016/j.snb.2016.09.171