Literatura académica sobre el tema "Saccharomyces cerevisiae – Cultures cellulaires"

This work aimed at the assessment of fermentative capacity of original diploid, monocellular haploid and recuperated diploid cultures of S. cerevisiae in sterilized sugar-cane wort. Twenty eight cultures were analyzed, four being original diploids (URM-4420, Itaiquara Ferment FIT, Lallemand Ferment FLA and Wild Ferment SEL); 12 monocellular haploids from original ones and 12 recuperated diploids from the monocells. The ethanol percentage ranged from 1.7 to 6.2% and the unfermentable reducing sugars from 0.45 to 0.50g/100mL. The highest ethanol percentages were produced by the monocellular cultures URM-MH3 (4.8%) in 12 h and SEL-MH1 (6.2%) in 24 h, corresponding to the productivity values of 3.15 and 2.03 g.L-1.h-1, respectively. The recuperated diploids did not present detectable ethanol content by the method used. The results showed that depending on the physiological state of the yeasts, being in the diploid or monocellular haploid form, different percentages of ethanol could be produced.

В статье представлены результаты скрининга коллекционных культур дрожжей на антагонистическую активность против возбудителя бактериального ожога Erwinia amylovora. Из 36 исследованных штаммов дрожжей 4 культуры обладали ингибирующей активностью. К ним относятся: Torulopsis kefir var kumis №114 3, Kluyveromyces marxianus19, Torulopsis sphaerica№117, Saccharomyces cerevisiae (vini) 2 комплекс №20. Зоны подавления роста патогена у них составляли 22, 23,5, 22,5 и 26 мм, соответственно. Наибольшая антагонистическая активность была у штамма дрожжей Saccharomyces cerevisiae (vini) 2 комплекс №20. Анализ компонентного состава 4 дрожжевых культур (Torulopsis kefyr var.kumis №114 3, Kluyveromyces marxianus19, Torulopsis sphaerica№117, Saccharomyces cerevisiae (vini) 2 комплекс №20) показал, что у культур Saccharomyces cerevisiae (vini) 2 комплекс №20 и Torulopsis sphaerica№117 выявлено 13 химических соединений, из которых основное соединение - фенилэтиловый спирт. Его процентное содержание составляет 43,1%. У культуры Torulopsis kefyr var.kumis №114 3 обнаружено 12 химических веществ, из которых основное соединение - изоамиловый спирт - 15,7%. А у культуры Kluyveromyces marxianus 19 10 соединений, основное из которых этилацетат – 12,24%. Полученные данные свидетельствуют о том, что исследованные дрожжевые культуры представляют интерес для дальнейшего изучения их как агентов биоконтроля возбудителя бактериального ожога E. аmylovorа. The article presents the results of screening of collection yeast cultures for antagonistic activity against the causative agent of bacterial burn Erwinia amylovora. Of the 36 yeast strains studied, only 4 cultures had inhibitory activity. These are Torulopsis kefir var kumis №114 3, Kluyveromyces marxianus 19, Torulopsis sphaerica№117, Saccharomyces cerevisiae (vini)2 complex №20. Their pathogen growth suppression zones were (22, 23.5, 22.5 and 26 mm), respectively. The greatest antagonistic activity was in the yeast strain Saccharomyces cerevisiae (vini)2 complex №20. The analysis of the component composition of 4 yeast cultures (Torulopsis kefyr var.kumis №114 3, Kluyveromyces marxianus 19, Torulopsis sphaerica №117, Saccharomyces cerevisiae (vini) 2 complex №20) showed that 13 chemical compounds were detected in cultures of Saccharomyces cerevisiae (vini) 2 complex №20 and Torulopsis sphaerica №117, of which the main compound is Phenylethyl alcohol. Their percentage is 43.1%, respectively. The culture of Torulopsis kefyr var.kumis №114 has 12 chemical compounds, of which the main compound is 1-Butanol, 3-methyl - 15.7%.And the Kluyveromyces marxianus culture has 19 10 compounds, the main compound Ethyl Acetate is 12.24%. The data obtained indicate that the studied yeast cultures are of interest for further study of them as agents of biocontrol against the causative agent of bacterial burn E. amylovora.

Enhancing the sensory profile of wines by exposing the aromas of the grape variety through the involvement of microorganisms has always been a challenge in winemaking. The aim of our work was to evaluate the impact of different fermentation schemes by using mixed and pure cultures of different Saccharomyces species to Sauvignon blanc wine chemical composition and sensory profile. The Sauvignon blanc must has been inoculated with mixed and pure cultures of S. pastorianus and S. cerevisiae strains. For the mixed fermentation schemes, one strain of S. pastorianus has been inoculated with different proportions of S. cerevisiae (S. pastorianus to S. cerevisiae: 99%–1%, 95%–5%, 90%–10%, 80%–20% and 70%–30% w/w) in co-inoculation with two commercial strains of S. cerevisiae. A total of 13 fermentations trials, three monocultures and 10 mixed cultures were performed in biological triplicate. The fermentation kinetics have been controlled by density measurement and classical oenological analyses were performed based on the International Organisation of Vine and Wine (OIV) analytical methods. The population dynamics were evaluated by the specific interdelta PCR reaction of the Saccharomyces species at the beginning and at the end of the fermentation process. The volatile compounds of the wine aroma, such as the esters, higher alcohols and thiols were analyzed by GC/MS. Sensory assessment by trained panel was carried out for all produced wines. Complete depletion of the sugars was achieved between 10 and 13 days for all the fermentation trials. The population dynamics analysis revealed that the S. cerevisiae strain was the most predominant at the end of the fermentation process in all inoculation ratios that were tested. The wines that were fermented with S. pastorianus, either in pure or mixed cultures, were characterized by significantly lower acetic acid production and higher malic acid degradation when compared to the wines that were fermented only with S. cerevisiae strains. The aroma profile of the produced wines was highly affected by both inoculation ratio and the S. cerevisiae strain that was used. The presence of S. pastorianus strain enhanced the production of the varietal thiols when compared to the samples that were fermented with the S. cerevisiae pure cultures. The mixed inoculation cultures of Saccharomyces species could lead to wines with unique character which can nicely express the varietal character of the grape variety.

The aim of our work was to evaluate the impact of different fermentation schemes by using mixed and pure cultures of S. pastorianus and S. cerevisiae or S. bayanus. For the mixed fermentation schemes, one strain of S. pastorianus has been inoculated under different proportions (99%/1%, 97%/3%, 95%/5%, 90%/10% and 70%/30% w/w) in co-inoculation with two commercial strains of S. cerevisiae and one commercial Saccharomyces bayanus strain. The fermentation kinetics has been controlled by density measurement and classical oenological analyses were performed based on OIV analytical methods. The population dynamics was evaluated by the specific interdelta PCR reaction of the Saccharomyces species in the beginning and in the end of the fermentation process. Volatile compounds of the wine aroma, such as esters, higher alcohols and thiols were analyzed by GC/MS. Sensory assessment by trained panel was carried out for all wines. The wines fermented with S. pastorianus, either in pure or mixed cultures, were characterized by significantly lower acetic acid production and higher malic acid degradation when compared to the wines fermented with S. cerevisiae strains. The presence of S. pastorianus strain enhanced the production of the varietal thiols when compared to the samples fermented with the S. cerevisiae pure cultures. The wines produced by the S. bayanus or cerevisiae monocultures and the ones produced by the co-culture with S. pastorianus at 70%-30% ratio (S. pastorianus to S. bayanus or cerevisiae) were overall better rated from a sensory point of view.

The increasing demand for craft beer is driving the search for novel ale yeast cultures from brewing-related wild environments. The focus of bioprospecting for craft cultures is to identify feral yeasts suitable to imprint unique sensorial attributes onto the final product. Here, we integrated phylogenetic, genotypic, genetic, and metabolomic techniques to demonstrate that sour beer during aging in wooden barrels is a source of suitable craft ale yeast candidates. In contrast to the traditional lambic beer maturation phase, during the aging of sour-matured production-style beer, different biotypes of Saccharomyces cerevisiae dominated the cultivable in-house mycobiota, which were followed by Pichia membranifaciens, Brettanomyces bruxellensis, and Brettanomyces anomalus. In addition, three putative S. cerevisiae × Saccharomyces uvarum hybrids were identified. S. cerevisiae feral strains sporulated, produced viable monosporic progenies, and had the STA1 gene downstream as a full-length promoter. During hopped wort fermentation, four S. cerevisiae strains and the S. cerevisiae × S. uvarum hybrid WY213 exceeded non-Saccharomyces strains in fermentative rate and ethanol production except for P. membranifaciens WY122. This strain consumed maltose after a long lag phase, in contrast to the phenotypic profile described for the species. According to the STA1+ genotype, S. cerevisiae partially consumed dextrin. Among the volatile organic compounds (VOCs) produced by S. cerevisiae and the S. cerevisiae × S. uvarum hybrid, phenylethyl alcohol, which has a fruit-like aroma, was the most prevalent. In conclusion, the strains characterized here have relevant brewing properties and are exploitable as indigenous craft beer starters.

ABSTRACT The so far largely uncharacterized central carbon metabolism of the yeast Pichia stipitis was explored in batch and glucose-limited chemostat cultures using metabolic-flux ratio analysis by nuclear magnetic resonance. The concomitantly characterized network of active metabolic pathways was compared to those identified in Saccharomyces cerevisiae, which led to the following conclusions. (i) There is a remarkably low use of the non-oxidative pentose phosphate (PP) pathway for glucose catabolism in S. cerevisiae when compared to P. stipitis batch cultures. (ii) Metabolism of P. stipitis batch cultures is fully respirative, which contrasts with the predominantly respiro-fermentative metabolic state of S. cerevisiae. (iii) Glucose catabolism in chemostat cultures of both yeasts is primarily oxidative. (iv) In both yeasts there is significant in vivo malic enzyme activity during growth on glucose. (v) The amino acid biosynthesis pathways are identical in both yeasts. The present investigation thus demonstrates the power of metabolic-flux ratio analysis for comparative profiling of central carbon metabolism in lower eukaryotes. Although not used for glucose catabolism in batch culture, we demonstrate that the PP pathway in S. cerevisiae has a generally high catabolic capacity by overexpressing the Escherichia coli transhydrogenase UdhA in phosphoglucose isomerase-deficient S. cerevisiae.

Fresh juices obtained under sterile conditions, including pomegranate juice, cherries, cherries, red grapes, watermelon juice, beetroot juice, sugar cargo, as well as flushes from the surface of juice-containing berries growing in the Turkestan region were used as sources of yeast cultures. Of 180 isolated yeast species, the majority are Saccharomyces - 159, 71 pure cultures are the most typical for the region and suitable for fermentation. A subsequent study of the morphological characteristics of cells, physiological and biochemical properties, clarification of antagonistic activity, and resistance to antibiotics made it possible for further selection of strains. The most highly active and appropriate by technological parameters were selected: Saccharomyces cerevisiae Al-06 (from grapes), Saccharomyces cerevisiae Gl -8 (from sugar sorghum juice) and Saccharomyces cerevisiae-Az-12 (from pomegranate juice). Thus, the analyzes showed the possibility of using plant materials not only as freshly squeezed juice of pomegranate, cherry, grape, watermelon juice, sugar cargo, but also as sources of active yeast.

Stimulation of lactate utilization by Selenomonas ruminantium and Megasphaera elsdenii may help in reducing problems associated with rumen acidosis. The objective of this study was to determine the effect of a Saccharomyces cerevisiae live culture and Saccharomyces cerevisiae fermentation products on in vitro growth and fermentation of lactate-utilizing ruminal bacteria, S. ruminantium (ATCC 19205) and M. elsdenii (ATCC 25940). The cultures were run for 0, 6, 12, 24 and 48 h under anaerobic conditions on a growth medium supplemented with a yeast live culture (SC) or with yeast fermentation products (SCFP) and, as reference, on the same medium without supplementation (CON). Neither SC nor SCFP had a significant effect on the growth of S. ruminantium after 6, 12 and 24 h of incubation, but the live yeast culture significantly (P≤0.05) improved the growth of these bacteria after 48 h of incubation. The yeast fermentation products significantly (P≤0.05) decreased pH and increased lactate synthesis by S. ruminantium. The Saccharomyces cerevisiae live culture significantly improved the growth of M. elsdenii after 12 and 24 h of incubation, and the S. cerevisiae fermentation products increased its growth after 48 h. The After 24 and 48 h of incubation the Saccharomyces cerevisiae live culture reduced the concentration of total volatile fatty acids (VFA), while caproate was the main product of in vitro fermentation of M. elsdenii (P≤0.05). Saccharomyces cerevisiae live cultures may improve microbial fibre fermentation in the rumen by maintaining optimal pH conditions.

L'objectif est de quantifier le comportement dynamique de la levure Saccharomyces cerevisiae CBS 8066 en hautes densités cellulaires afin d'intensifier de façon rationnelle les productions microbiennes selon des critères industriels définis (titre, productivité, rendement). Nous avons développé un bioréateur continu bi-étagé avec recyclage cellulaire (BBRC) permettant de maîtriser de façon indépendante la concentration, l'environnement et l'état physiologique de la levure. Son originalité réside dans la gestion de l'activité microbienne via une boucle de recirculation entre les deux étages. L'influence des hautes densités cellulaires (de 30 gms. L-1 à 223 gms. L-1) a été caractérisée en régime permanent d'un point de vue physico-chimique, physico-mécanique et biologique aux échelles macro et microscopique. En focalisant l'interprétation des résultats sur la biomasse viable, nous avons montré qu'il n'y a pas d'impact des hautes densités cellulaires sur les cinétiques microbiennes de croissance et de production d'éthanol dans le domaine de variation considéré. Les effets de la concentration totale en biomasse ont été identifiés et quantifiés au niveau cellulaire (composition élémentaire, eau intracellulaire et volume cellulaire) et au niveau des paramètres physiques (rhéologie). L'application du BBRC à la production intensive d'éthanol a permis d'atteindre une des meilleures performances internationales (41 kg. M-3. H-1) en combinant productivité, titre et rendement<br>The study aims to quantify the dynamic behaviour of the yeast Saccharomyces cerevisiae CBS 8066 in high cell density conditions to improve microbial production taking into account defined industrial criteria (titre, productivity and yield). We developed a Two-stage Bioreactor with Cell Recycling (TBCR) to have independent controls on the cell concentration, the environment and the physiological state of the yeast. The bioprocess originality in the management of the microbial activity by the use of a recycle loop beetween the two stages. The influence of very high cell density (up to 223 gdw. L-1) was quantified on both biological and physical phenomena using macroscopic, microscopic, physico-mechanical and physico-chemical analysis in continuous steady state cultures. Considering the active part of the biomass, no biomass inhibition was shown on both growth and production kinetics. . . Total cell concentration impacts were identified and quantified at a cellular level (elementary composition, intracellular water content and cell volume) and at a physico-mechanical leved (rheology). We experimentally demonstrated the pertinence of the TBCR application to intensive bio-ethanol production according to industrial criteria such as ethanol concentration and yield to perform one of best international high bio-ethanol productivity (41 kg. M-3. H-1)

L’objectif était de caractériser les mécanismes d’interaction entre la bactérie lactique Lactococcus lactis et la levure Saccharomyces cerevisiae dans des écosystèmes modèles. Le comportement macro-cinétique de L. Lactis, analysé en co-culture avec la levure et en culture pure, semble indépendant de la présence de la levure. Par contre, la levure utilise ses propres produits de fermentation (éthanol, acétate), ainsi que le lactate produit par L. Lactis, pour prolonger sa croissance en aérobiose. Une méthode pour réduire les hybridations croisées sur les puces à ADN de L. Lactis lors de l’analyse transcriptomique des co-cultures a été développée. Ainsi, pour la première fois, le transcriptome de L. Lactis a été analysé avec des puces à ADN dans une culture mixte. La comparaison directe des cultures pure et mixte en phase exponentielle de croissance met en évidence de nombreux gènes du métabolisme des pyrimidines sous-exprimés en co-culture. Ce phénomène, confirmé par RT-qPCR, semble lié à la présence d’éthanol produit par la levure. Selon une autre stratégie, l’analyse cinétique comparative des données de transcriptome a été soumise à différents traitements statistiques pour déterminer la robustesse des données. Malgré des différences entre listes de gènes sélectionnés, le profil d’expression global est similaire en cultures pure et mixte, caractéristique de l’adaptation à la carence en glucose. Même si certains gènes de régulateurs semblent différemment exprimés en cultures pure et mixte, L. Lactis semble finalement peu affecté par la présence de la levure

Les systèmes microbiologiques naturels (colonne d’eau), semi-naturels (station d’épuration), mais surtout industriels ou de laboratoire (bioréacteurs) sont communément représentés par des modèles mathématiques destinés à l’étude, à la compréhension des phénomènes ou au contrôle des processus (de production, par exemple).<p><p>Dans l’énorme majorité des cas, lorsque les cellules (procaryotes ou eucaryotes) mises en jeu dans ces systèmes sont en suspension, le formalisme de ces modèles non structurés traite le système comme s’il était homogène. Or, en toute rigueur, il est clair que cette approche n’est qu’une approximation et que nous avons à faire à des phénomènes hétérogènes, formés de plusieurs phases (solide, liquide, gazeuse) intimement mélangées. Nous désignons ces systèmes comme « polyphasiques dispersés » (SPD). Ce sont des systèmes thermodynami-quement instables, (presque) toujours ouverts.<p><p>La démarche que nous avons entreprise consiste à examiner si le fait de considérer des systèmes dits « homogènes » comme des systèmes hétérogènes (ce qu’ils sont en réalité) apporte, malgré une complication du traitement mathématique, un complément d’information significatif et pertinent. <p><p>La démarche s’est faite en deux temps :<p>·\<br>Doctorat en sciences, Spécialisation biologie moléculaire<br>info:eu-repo/semantics/nonPublished

Le trehalose et le glycogene sont les sucres de reserve chez la levure, definis comme source de carbone et d'energie. Les concentrations intracellulaires dependent tres fortement des conditions de croissance (source de carbone, limitation nutritionnelle, stress, etc. ), et du mode de culture (regime respiratoire ou fermentaire, batch ou chemostat). L'objectif principal de cette these est de clarifier le role de ces reserves dans la dynamique de croissance. Dans un premier temps, la caracterisation physiologique de la souche de reference cen. Pk113-7d a mis en avant l'intime connexion entre l'accumulation de ces reserves et l'intensite du flux glycolytique. Une accumulation potentielle de trehalose est demontree par l'action du transporteur d'-glucosides code par le gene agt1. La dynamique cellulaire est abordee par des cultures continues sur glucose dans la zone oxydative des taux de croissance et l'application d'un saut du taux de dilution de 0. 05 a 0. 15 h 1. Dans ce contexte, d'importants bouleversements physiologiques sont observes : en l'occurrence, l'oscillation du taux de croissance, la mobilisation du trehalose et du glycogene et l'induction du bourgeonnement. L'augmentation du flux carbone s'accompagne d'une modification du flux energetique respiro-fermentaire. Cette dynamique macroscopique apparait fortement liee a la dynamique microscopique plus globale : stimulation des voies centrales du metabolisme de la levure et activation des voies dites du by-pass. La mise en uvre de souches deficientes dans la biosynthese du trehalose (tps1) et du glycogene (glg1glg2) a conduit aux memes observations. Toutefois, la presence de glycogene chez le triple mutant tps1glg1glg2 suggere l'existence d'un moyen independant du mode d'action des glycogenines. L'approche analytique de ces souches demontre une etroite correlation entre l'intensite du bourgeonnement et la quantite des reserves mobilisees et le role du trehalose dans le maintien d'un bon rendement energetique.

La paroi cellulaire de la levure est l'organite le plus externe de la cellule de la levure. Elle est composée d'une couche interne de polysaccharides, constituée de β-glucannes majoritairement réticulés à une quantité mineure de chitine, et à laquelle sont liées des mannoprotéines par des liaisons covalentes ou non. Les mannoprotéines forment la couche externe de la paroi cellulaire de la levure et sont considérées comme son deuxième composant le plus abondant. Les mannoprotéines de la paroi cellulaire de la levure sont des protéines fortement mannosylées par des O- glycannes simples courts et par des N-glycannes complexes et longs. Elles présentent des propriétés fonctionnelles particulières et une valeur nutritionnelle exceptionnelle liées à leur structure moléculaire, mais ont été peu étudiées. Ce travail vise à étudier la structure moléculaire des mannoprotéines de la paroi cellulaire de la levure par différentes techniques basées sur la spectrométrie de masse haute résolution.Les mannoprotéines ont été extraites par des procédés physiques ou chimiques, couplés éventuellement à un traitement enzymatique de la souche de référence S288C cultivées selon différents modes dans des bioréacteurs en présence d’un milieu de culture, ou d'échantillons levure industrielle, ou de produits industriels déjà fractionnés de levure. Ensuite, ces mannoprotéines ont été O- et N-déglycosylées chimiquement ou enzymatiquement. Les peptides obtenus ont été analysés par nanoESI-LC-MS/MS. L’analyse bio-informatique de ces données a permis l'identification et la quantification des mannoprotéines grâce à la base de données de la souche de référence de Saccharomyces cerevisiae S288C. La déglycosylation des peptides a également été vérifiée. Une analyse de l'ontologie des gènes a été ensuite réalisée pour déterminer la localisation subcellulaire des protéines identifiées. Les O- et N-glycannes ont été chimiquement dérivées par une réaction d’amination réductrice, puis analysées respectivement par µESI-LC-MS et électrophorèse capillaire.Ce travail nous a permis de comparer différentes méthodes d'extraction des mannoprotéines de la paroi cellulaire de levure. Ces méthodes entraînent un enrichissement qualitatif ou quantitatif de différents types de ces mannoprotéines, mais aussi conduisent à la présence d'autres protéines principalement annotées comme étant liées aux membranes des organelles (en particulier mitochondriales et nucléaires). Une purification supplémentaire des isolats de paroi a été appliquée pour réduire le nombre de ces protéines n’appartenant pas à la paroi. Le développement d’un protocole de N-déglycosylation enzymatique utilisant une méthode utilisant un seul contenant sans transfert de l’échantillon a permis l'augmentation de la couverture des mannoprotéines identifiées. Avec d’autres enzymes le même protocole permet une O-déglycosylation douce mais qui dégrade les O-glycannes en monosaccharides. La N-déglycosylation enzymatique combinée avec une O-déglycosylation chimique permet d’isoler simultanément les O- et N-glycannes des mannoprotéines, permettant leur analyse ultérieure par spectrométrie de masse et électrophorèse capillaire après modification chimique avec des marqueurs appropriés par réaction d'amination réductrice. Les profils de protéines diffèrent qualitativement et quantitativement selon la phase de croissance et le mode de culture. Nous avons identifié certains de leurs marqueurs protéiques, qui sont des marqueurs de la privation de glucose exprimée dans la phase de croissance stationnaire de la culture discontinue en lot et de la culture discontinue alimentée.Cette approche glycoprotéomique a également été appliquée à la caractérisation glycoprotéomique et peptidomique des produits générés par différentes méthodes de traitement industriel, destinés à un usage commercial, permettant de déchiffrer leur nature complexe en termes de composition et de structure<br>Yeast cell wall is the outermost organelle of the yeast cell. It composed of an inner layer of polysaccharides, consisting of β-glucans mostly cross-linked to a minor amount of chitin, and to which are linked mannoproteins by covalent or non-covalent bonds. Mannoproteins form the outer layer of the yeast cell wall and are considered as its second most abundant component. Yeast cell wall mannoproteins are proteins that are highly mannosylated by short simple O-glycans and by large complex N-glycans. They have particular functional properties and exceptional nutritional value related to their molecular structure, but have been little investigated. This work aims to study yeast cell wall mannoproteins at the molecular level by different techniques based on high resolution mass spectrometry.The mannoproteins were extracted by physical, chemical methods eventually coupled to enzymatic methods from the reference strain S288C grown in different modes in bioreactors in the presence of a culture medium, or from industrial yeast samples, or from already fractionated industrial yeast products. These mannoproteins were then O- and N-deglycosylated chemically or enzymatically. The resulting peptides were analyzed by nanoESI-LC-MS/MS. Bioinformatics analysis of these data allowed the identification and quantification of mannoproteins using Saccharomyces cerevisiae S288C reference strain database. The deglycosylation of the peptides was also verified. Gene ontology analysis was then performed to determine the subcellular location of the identified proteins. The O- and N-glycans were chemically derivatized by a reductive amination reaction and then analyzed by µESI-LC-MS and capillary electrophoresis respectively.This work allowed us to compare different methods of extraction of mannoproteins from the yeast cell wall. These different methods result in qualitative or quantitative enrichment of different types of mannoproteins and in the presence of other proteins mainly annotated as being related to organelle membranes (especially mitochondrial and nuclear). Further purification of wall isolates was applied to reduce the number of these non-cell wall proteins. The development of an enzymatic N-deglycosylation protocol using a one-pot method without sample transfer allowed an increase in the coverage of identified mannoproteins. Using other enzymes, the same protocol allows a gentle O-deglycosylation but degrades O-glycans into monosaccharides. Enzymatic N-deglycosylation combined to chemical O-deglycosylation allows the simultaneous isolation of O- and N-glycans from mannoproteins, allowing their subsequent analysis by mass spectrometry and capillary electrophoresis after chemical derivatization with appropriate labels by reductive amination reaction. The protein profiles differ qualitatively and quantitatively according to the growth phase and culture mode. We identified some of their protein markers, which are markers of glucose deprivation expressed in the stationary growth phase of batch and fed batch culture.This glycoproteomic approach was also applied to the glycoproteomic and peptidomic characterization of products generated by different industrial processing methods, intended for commercial use, allowing to decipher their complex nature in terms of composition and structure

Le rétrotransposon Tyl de Saccharomyces cerevisiae possède des analogies structurales et fonctionnelles avec les rétrovirus. Ces rétroéléments partagent notamment l'étape d'intégration, qui est catalysée par leur intégrase (IN) et ciblée vers des régions particulières du génome. Cette sélectivité d'intégration dépendrait d'une interaction entre leur IN et une ou plusieurs protéines cellulaires affines de l'ADN ou de la chromatine au site d'insertion. L'intégration de Tyl s'effectue dans une fenêtre d'une kilobase en amont des gènes transcrits par l'AR? polymérase III (Pol III) et dépend de la structure de la chromatine et de la transcription par la Pol II L'objectif de cette thèse est de caractériser les protéines interagissant avec l'IN de Tyl pour étudier leur rôle dans l'intégration. Nous avons identifié par crible double-hybride une interaction entre l'IN et les protéines AC40, Srl2 et Upc2 qui ont des fonctions liées au métabolisme de l'ADN. La protéine AC40 est une sous unité de la Pol III et pourrait donc jouer un rôle dans la sélectivité d'intégration. Nous avons confirmé l'interaction entre les deux protéines et défini le domaine minimal d'interaction de l'IN avec AC40. L fréquence de rétrotransposition de Tyl dans une souche exprimant une protéine AC40 n'interagissant pas avec l'IN n'est pas altérée, suggérant que l'interaction entre les deux protéines serait impliquée dans la sélectivité plutôt que dans l'efficacité d'intégration. A l'inverse, la fréquence de rétrotransposition diminue dans de souches dépourvues de Srl2 ou Upc2 indiquant que ces protéines joueraient un rôle dans l'efficacité de la rétrotransposition<br>Integration is an essential step in the retrovirus life cycle and is catalyzed by the retroviral integrase (IN). It does not occur randomly throughout the host-cell genome but presents a pattern of preferred sites that is specific to each element. A common targeting mechanism has been proposed, based on the interaction of IN with cellular factors bound at preferential insertion sites. The Tyl LTR-retrotransposon of S. Cerevisiae is analogous to retroviruses in its structure and mode of replication. Tyl integrates in a window of one kilobase upstream of RNA polymerase III (Pol III)-transcribed genes. Tyl preference depends both on the chromatin structure and Pol III transcription. The aim of this work was to identify cellular cofactors of Tyl IN and to elucidate their role in Tyl integration. We discovered an interaction between Tyl IN and AC40, a subunit of Pol III in a two-hybrid screen, suggesting that AC40 could be involved in the selectivity of Tyl integration. We confirmed the interaction between the proteins and showed that the C-terminus part of IN is necessary and sufficient. The frequency of integration is not affected by the loss of interaction, suggesting that it may be involved in the selectivity of integration rather than the efficiency. We also identified Upc2 and Srl2 as Tyl IN interacting proteins and showed that the frequency of integration decreases two-fold in their absence, indicating that these proteins may also play a role in Tyl mobility

Chlorella vulgaris (C. vulgaris) est un organisme modèle qui présente un potentiel commercial élevé dans le domaine de l'alimentation et de l'énergie, avec une faisabilité prouvée de cultures sous forme de biofilms et de co-culture de levures/microalgues pour la valorisation in situ du CO2 dans les processus biotechnologiques. Ce travail de doctorat se concentre sur les colonies immobilisées dans des cultures pures ou mixtes. Il vise une meilleure compréhension des interactions au sein des colonies et entre colonies, avec pour objectif ultime de comprendre et d'optimiser les co-cultures.Pour atteindre ces objectifs, un protocole complet et des dispositifs expérimentaux innovants ont été développés, notamment des techniques d'inoculation, des dispositifs de culture immobilisée avec capteurs de gaz, l'imagerie 3-D à l'aide d'un microscope à lumière structurée, le traitement d'images, un bilan gazeux calibré et l'analyse des données. Des précautions ont également été prises concernant les conditions d'incubation, la détermination de la masse sèche, la concentration en glucose, la taille et la densité des cellules.Tout d'abord, le développement de colonies uniques de C. vulgaris a été étudié dans des conditions hétérotrophes. Sur la base du modèle biologique proposé pour la dynamique de croissance en hauteur et en rayon, nous avons conclu que les colonies se développaient à un taux constant dans la direction horizontale et à un taux décroissant dans la direction verticale. Ces tendances sont cohérentes avec les effets cumulés de la disponibilité du glucose et de l'oxygène. Une calotte sphérique décrit le mieux la forme des colonies pendant la période de croissance. L'interaction intraspécifique de C. vulgaris a été étudiée en faisant croître plusieurs colonies sur la même plaque à des distances de initiales différentes : 1,5 mm, 3 mm et 15 mm. Aucun effet significatif de la coalescence des colonies n'a été observé sur les taux de croissance en rayon et en hauteur.Ensuite, l'effet de la lumière a été testé de deux manières : présence de lumière durant toute la culture et exposition à la lumière après une première période hétérotrophe. La forme de la colonie est significativement affectée par le mode de culture : la colonie en croissance hétérotrophe garde une calotte sphérique, tandis que la colonie en croissance mixotrophe atteint une forme cylindrique, en raison d'une croissance radiale presque complètement arrêtée après quelques jours. Grâce au dispositif de mesure des gaz, les données brutes ont été analysées à l'aide d'une équation de bilan gazeux pour obtenir les termes source biologique d'O2 et de CO2. Le rapport de masse du gaz sur la masse sèche de la cellule a été déterminé pour les différentes conditions de croissance. Une synergie est mise en évidence entre la photosynthèse au sommet de la colonie et l’hétérotrophie à la base.L'interaction inter-espèces de C. vulgaris et S. cerevisiae a été étudiée à deux niveaux : au niveau des cellules dans la même colonie et entre colonies. Au niveau de la colonie, des colonies de C. vulgaris et de S. cerevisiae ont été inoculées avec deux distances de séparation initiales différentes (3 mm et 15 mm). Les colonies ont été observées en continu pendant un mois. Même si des recherches supplémentaires sont nécessaires, la croissance et l'interaction observées semblent s'expliquer principalement par le taux de croissance beaucoup plus élevé de la levure. Après avoir fusionné, les colonies de S. cerevisiae finissent par envelopper les colonies de C. vulgaris. Au niveau des cellules, les colonies mélangées de C. vulgaris et de S. cerevisiae ont été observées en 3D. En raison de leur croissance rapide, les cellules de S. cerevisiae finissent par dominer l'ensemble de la colonie, à l'exception de quelques cellules de C. vulgaris présentes au cœur de la colonie et sur le dessus. Les cellules de C. vulgaris cessent presque de croître lorsque les nutriments sont limités<br>Chlorella vulgaris (C. vulgaris) is a model organism that has high commercial potential in the food and energy field, with proved feasibility of cultures as biofilms and yeast/ microalgae co-culture for in situ CO2 mitigation in biotechnological processes. This PhD work focuses on immobilized colonies in pure or mixed cultures. It proposes a better understanding of the interactions within and between colonies, with the ultimate goal of understanding and optimizing co-cultures.To archive these goals, a comprehensive protocol and required innovative experimental devices were developed including inoculation techniques, immobilized culture devices with gas sensors, 3-D imaging using a structured-light microscope, image processing, calibrated gas balance equation and data analysis. Care was also taken regarding incubation conditions, determination of dry mass, glucose concentration, cell size and density.Firstly, the development of single C. vulgaris colonies under heterotrophic conditions was studied. Based on the biological model proposed for the growth dynamics in height and radius, we concluded that the colonies expanded at a constant rate in the horizontal direction and a decreasing rate in the vertical direction. The trends are consistent with the cumulative effects of glucose and oxygen availability. A spherical cap best describes the shape of the colonies during the growth period. The intraspecies interaction of C. vulgaris was investigated by growing several colonies on the same plate with different initial separation distances: 1.5 mm, 3mm, and 15 mm. No significant effects of colony merging were observed on the growth rates in radius and height.Then, the effect of light was tested in two ways: presence of light throughout the culture and exposition to light after a first, purely heterotrophic, period. The shape of colony is significantly affected by the cultivation mode: the heterotrophic growth colony keeps a spherical cap, while the mixotrophic growth colony reaches a cylindrical shape, due to a radial growth almost completely stopped after some days. Thanks to the gas measurement device, the raw data were analyzed using a gas balance equation to obtain the biological source terms of O2 and CO2. Gas yield (mass ratio of gas to dry mass of cell) are proposed for the different growth conditions. A synergy is highlighted between photosynthesis at the top of the colony and heterotrophy at the base.The interspecies interaction of C. vulgaris and S. cerevisiae were studied at two levels: cell-cell level within the same colony and colony-colony level. At the colony-colony level, colonies of C. vulgaris and S. cerevisiae were inoculated with two different initial separation distances (3 mm and 15 mm). Colonies were observed continuously for one month. Even though additional investigation is needed, the observed growth and interaction seems to be mostly explained by the much larger growth rate of yeast. After merging S. cerevisiae colonies eventually envelop C. vulgaris colonies. At the cell-cell level, C. vulgaris and S. cerevisiae intermixed colonies were observed in 3D. Due to its fast grow, S. cerevisiae cells eventually dominate the whole colony, at the exception of some C. vulgaris cells present in the core of the colony and on the top. C. vulgaris cells almost stop growing when the nutrients are limited

Une protéine prion peut adopter deux conformations distinctes, l’une cellulaire et l’autre prion. La conformation prion est le résultat de son agrégation en fibre amyloïde. Cette fibre est le support de l’information prion à partir duquel les isoformes cellulaires sont convertis en forme prion de façon autocatalytique. La transmission de l’information prion repose donc sur la transmission de cette fibre au cours des divisions cellulaires, qui est réalisée par de petits polymères. Ceux-ci sont le résultat d’un équilibre entre la fragmentation et la polymérisation de la fibre. Une perturbation de cet équilibre provoque une agrégation massive de la protéine prion, menant à la perte de l’information prion.L’objectif de ma thèse était de comprendre ce qui définit in vivo la transmission du prion. Mon modèle d’étude est la protéine Ure2p propageant le prion [URE3] dans la levure S. cerevisiae. J’ai montré que la concentration cellulaire d’Ure2p détermine la vitesse d’agrégation de la protéine prion et donc son efficacité de transmission. En effet, de trop fortes concentrations cellulaires sont incompatibles avec la propagation du prion. La concentration cellulaire d’Ure2p définit également la diversité des souches prions. Un crible génétique m’a permit de mettre en évidence que la présence de séquences centromériques surnuméraires dans la cellule interfère avec la transmission du prion [URE3]. Le même phénomène est observé avec une augmentation du niveau de ploïdie de la cellule. Dans les deux cas, la surexpression du chaperon Hsp104 restaure une propagation normale du prion<br>A prion protein can adopt two distinct conformations, one cellular and one prion. Prion conformation is the result of its aggregation into amyloid fibers. This fiber is the support of the prion information from which the cellular isoforms are converted into prion form by autocatalytic manner. The prion information transmission is therefore based on the transmission of this fiber during cell division, which is done by small polymers. These are the result of a balance between fragmentation and polymerization of the fiber. A disturbance of this balance causes a massive aggregation of the prion protein, leading to the prion information loss.The objective of my thesis was to understand what defined in vivo the prion transmission. My studying model was the Ure2p protein propagating the [URE3] prion in S. cerevisiae yeast. I showed that the Ure2p cellular concentration determined the aggregation speed of the prion protein and thus its transmission efficiency. Indeed, too high cellular concentrations are incompatible with the prion propagation. The cellular concentration of Ure2p also defines the prion strains diversity. A genetic screen allowed me to highlight that the presence of centrometric supernumerary sequences in the cell interferes with the [URE3] prion transmission. The same phenomenon is observed with an increase in the cell ploidy. In both cases, overexpression of the Hsp104 chaperone restores normal prion propagation

Saccharomyces cerevisiae is one of the yeasts with major economic, social, and health significance in human culture. Depending on the growth conditions experienced by the cell, S. cerevisiae growth can proceed via fermentative, respirative, or respirofermentative metabolism. Scar formation, unequal division, a limited replicative lifespan, and increase in cell size commensurate with the cell's replicative age are individual characteristics of this yeast affecting the performance of bioprocesses. These characteristics increase the complexity of predictive models and introducing them with ease into a continuous model is not realistic. Nevertheless, an individual-based model is able to accommodate this complexity in a single computational model. Once an individual model is implemented, it has to be parameterized, calibrated, and its adequacy assessed. All these processes ideally require a high number of both individual and system-level experimental observations. The aim of the present thesis is to advance the development of an individual-based methodology to tackle the study of microbial systems driven by the relevant yeast S. cerevisiae. The adequacy of INDISIM-YEAST, an existing individual-based model of a generic budding yeast, is first assessed. In order to obtain valuable individual-based observations to support the desired individual-based methodology, the diversity of S. cerevisiae in experimental individually-oriented observations under different growth conditions and at different stages of the growth curve is verified and assessed. A quantitative individual-based model focusing on the fermentative (anaerobic) growth of the yeast S. cerevisiae has been designed, implemented in Fortran 90, and termed INDISIM-Saccha. The developed model is parameterized, calibrated, its adequacy evaluated, and used to assess in silico ethanol production by means of virtual experiments. The calibration procedure, and the performance and analysis of the data from the virtual experiments is undertaken using the statistical programming language R. The model adequacy is assessed by testing several model predictions both at a system level (glucose depletion, population growth curves) and single-cell level (fraction of budded cells, genealogical age distribution, and cell diameter distribution evolutions). Individual cell diameter observations obtained within the present thesis play a significant role in this assessment. Results of the virtual experiments suggest that differences in cell size distribution can drastically affect the performance and productivity of fermentations, and encourage routine characterization of the inocula in the biotechnological industry. INDISIM-Saccha is also adapted to take into account the aerobic growth of S. cerevisiae and contrasted with two experimental trials with different oxygen levels in the medium. The preliminary simulated results achieved with the model suggest that the approach also has the potential for reproducing aerobic batch cultures of S. cerevisiae. This represents a further step in obtaining a microbial individual-based model to account for the whole set of metabolic alternatives experienced by S. cerevisiae. In order to communicate efficiently, increase accessibility, and favour usability of the INDISIM-Saccha methodology developed, the present thesis also designs and implements INDISIM-YEAST-NL in the freely available programming environment NetLogo. The implementation of this streamlined model in NetLogo lays the foundations for a deeper understanding of the developed methodology and microbial individual-based models in general, and will facilitate future interactions with potential users of INDISIM-Saccha.<br>El Saccharomyces cerevisiae és un dels llevats que gaudeix de més significació econòmica, social i per a la salut humana. Depenent de les condicions experimentades, el llevat S. cerevisiae pot créixer mitjançant un metabolisme fermentatiu, respiratori o respirofermentatiu. La formació de cicatrius, una divisió desigual, una vida replicativa limitada i un increment de la mida de la cèl.lula amb l’edat replicativa són característiques individuals d’aquest llevat que afecten el comportament dels bioprocessos. Aquestes característiques incrementen la complexitat dels models predictius i dificulten, per tant, la seva inclusió en un model continu de manera realista. No obstant això, un model basat en l’individu sí que és capaç d’acomodar tota aquesta complexitat en un únic model computacional. Una vegada implementat, un model basat en l’individu ha de ser parametritzat, calibrat i la seva adequació ha de ser avaluada. Tots aquests processos requereixen idealment un gran nombre d’observacions experimentals, tant individuals com a nivell del sistema estudiat. L’objectiu general de la tesi present és avançar en el desenvolupament d’una metodologia basada en l’individu per estudiar sistemes microbians conduïts pel llevat S. cerevisiae. Primerament s’avalua l’adequació de INDISIM-YEAST, un model basat en l’individu, ja existent, focalitzat en un llevat genèric. Es verifica i s’avalua la diversitat del S. cerevisiae en observacions experimentals orientades a l’individu en diferents condicions de creixement i en diversos estadis de la corba de creixement de la població. Això permet obtenir observacions basades en l’individu molt valuoses a l’hora de donar suport a la metodologia desitjada. Es desenvolupa i s’implementa en Fortran 90 INDISIM-Saccha, un model quantitatiu basat en l’individu i focalitzat en el creixement fermentatiu (anaerobi) del S. cerevisiae. El model desenvolupat és parametritzat, calibrat, la seva adequació és avaluada i és utilitzat per estudiar in silico la producció d’etanol mitjançant experiments virtuals. El procés de calibratge, l’obtenció i l’anàlisi de les dades dels experiments virtuals s’han realitzat utilitzant el programari estadístic R. L’adequació del model s’avalua testejant diferents prediccions del model a nivell de sistema (corbes de disminució de la glucosa i de creixement de la població) i a nivell de la cèllula individual (evolucions temporals de la fracció de cèl.lules gemades, de la distribució d’edats genealògiques i de la distribució dels diàmetres cel.lulars). Les observacions del diàmetre de les cèl.lules individuals obtingudes a la tesi present juguen un paper significatiu en aquesta avaluació. Els resultats dels experiments virtuals suggereixen que les diferències en la distribució de mides cel.lulars poden afectar dràsticament l’evolució i la productivitat de les fermentacions i suggereixen una caracterització rutinària de l’inòcul a la indústria biotecnològica. L’INDISIM-Saccha també és adaptat per tenir en compte el creixement aeròbic del S. cerevisiae i és contrastat mitjançant dos assajos experimentals amb dos nivells d’oxigen al medi. Els resultats preliminars de la simulació denoten que aquesta aproximació també té el potencial de reproduir cultius discontinus aerobis del S. cerevisiae. Això representa un pas endavant cap a l’obtenció d’un model basat en l’individu que tingui en compte tot el conjunt d’alternatives metabòliques experimentades pel S. cerevisiae. Finalment, aquesta tesi també dissenya i implementa INDISIM-YEAST-NL en l’ambient de programació lliure anomenat NetLogo per tal de comunicar de manera eficient, d’incrementar l’accessibilitat i d’afavorir l’ús de la metodologia INDISIM-Saccha. La implementació d’aquest model simplificat amb NetLogo posa les bases per a una comprensió més alta de la metodologia desenvolupada, i dels models microbians basats en l’individu en general, i facilitarà futures interaccions amb usuaris potencials de l’INDISIM-Saccha.

While Saccharomyces cerevisiae is recognized as the yeast species that completes the process of alcoholic fermentation during winemaking, the use of starter cultures from other species has become popular in recent years. Non-saccharomyces yeast cultures are now widely used for their bio-protective effects and/or the contribution they make to a wine’s sensory profile. Conversely, starters of wine lactic acid bacteria are also commonly utilized around the same time as commercial Saccharomyces cerevisiae, as an alternative to encouraging adventitious strains to proliferate. This could be either for initiating malolactic fermentation during alcoholic fermentation, or more recently for biological protection of musts prior to the fermentation process. The interactions between S. cerevisiae and other species are documented in the following chapter. The areas examined in more details include requirements of nutrients compared to S. cerevisiae, whether complimentary of symbiotic. Active bioprotective agents such as killer factors, the role of cell-to-cell contact, and the resultant effects on final wine composition when co-fermenting with S. cerevisiae is also discussed.

Abstract The basic mechanisms of cell-cycle control seem to be similar in all eukaryotes. The yeasts S. cerevisiae and Schizosaccharomyces pombe are two of the simplest and most powerful model systems for studying eukaryotic cell cycle control. Progress has been rapid, and the lessons learned have often been directly relevant to mammals and other eukaroyotes. Some methods useful for studying the yeast cell cycle are described here. However, these can be applied only in the context of the usual techniques of yeast molecular genetics and cell biology (I, 2). Similar methods for S. pumbe are described in Chapter 5, while methods for preparing synchronous cultures of both yeasts are described in Chapter 2.

Abstract During the last 25 years a great deal of information has come from cell-cycle studies of yeast, and one of the essential tools has been the production of synchronous cultures. These cultures have been produced by a variety of techniques and the purpose of this chapter is to describe their use in the most frequently studied yeasts, Saccharomyces cerevisiae and Schizosaccharomyces pombe. The choice of method is usually determined by the available apparatus, by the cycle event or cellular component one is investigating, by the degree and duration of synchrony required, and, particularly in S. cerevisiae, by the compatibility of the methods available with particular strains.

Probiotic microorganisms are defined as living microorganisms that provide health benefits on the host when administered in adequate amounts. The benefits include improvement of microbial balance immune system and oral health, provision of cholesterol-lowering effect, and antimicrobial activity against a wide variety of bacteria and some fungi. Kefir microbiota contains active living microorganisms. Many researches were carried out that potential probiotic bacteria such as Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus kefir, Lactobacillus kefiranofaciens, Leuconostoc mesenteroides, or yeasts like microorganisms such as Saccharomyces cerevisiae, Kluyveromyces lactis, and Kluyveromyces marxianus were isolated from kefir grains. This chapter presents the data both on the probiotic bacteria isolated from kefir grains or kefir and the probiotic properties of kefir produced with these microorganisms.

Microprinting has been demonstrated effective in the patterning of surface regions for trapping cells used within microfluidic devices. In this study a polydimethylsiloxane (PDMS) silicone elastomer stamp was microfabricated and used to microstamp concanavalin-A (con-A; protein that binds to yeast) on a glass surface. Yeast cells, Saccharomyces cerevisiae, were brought into contact with patterned con-A producing an array of yeast microscale culture in an ordered array identical to the printed pattern.

Studies on the bioconversion of whey water formed from chickpea and pea grains in the preparation of protein concentrates have been performed. The serum remaining after precipitation of the main part of the protein was subjected to a symbiotic transformation of Saccharomyces cerevisiae 121 and Geotrichum candidum 977 yeast cultures with the formation of protein-containing products with a mass fraction of protein (52.27-57.90% of DS) and a complementary amino acid composition. A microbial-plant concentrate was used as an additive in the feeding of Wistar laboratory rats. After 25 days of feeding, there was no negative effect on the physiological parameters and behavior of animals, which indicates the high quality of the protein product and the prospects of its inclusion in the composition of animal feed and diets.

Investigations were carried out to optimize the growth parameters of the symbiosis of cultures of the yeast Saccharomyces cerevisiae 121 and the fungus Geotrichum candidum 977 on whey waters formed from pea flour as a secondary product in the production of protein concentrates after precipitation of proteins at the isoelectric point. The whey remaining after protein precipitation is bioconverted at optimal parameters of crop growth (pH of the medium, amount of inoculum, temperature) with the formation of microbial plant concentrate (MPC) for feed purposes. Serum cultures assimilated stachyose, glucose, maltose, arabinose, and other pentoses. The mass fraction of protein in the concentrate was 57.90-61.68 % of DS. The composition of MPC obtained from biomass is balanced in essential amino acids with a speed of 107-226 %. The fatty acid composition is represented by 97 % fatty acids and 3 % - esters, aldehydes, ketones with the properties of fragrances, photo stabilizers, odor fixers, preservatives and other compounds. The ratio of the sum of saturated and unsaturated acids is 1:3, the content of cis-isomers is 91.1 %, trans-isomers are 5.1 %, omega-6 fatty acids are 19.73 %. The quality and safety indicators indicated that it is promising for use in the diet of animals.

Rapid detection of pathogens and hazardous elements in fresh fruits and vegetables after harvest requires the use of advanced sensor technology at each step in the farm-to-consumer or farm-to-processing sequence. Fourier-transform infrared (FTIR) spectroscopy and the complementary Raman spectroscopy, an advanced optical technique based on light scattering will be investigated for rapid and on-site assessment of produce safety. Paving the way toward the development of this innovative methodology, specific original objectives were to (1) identify and distinguish different serotypes of Escherichia coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus cereus by FTIR and Raman spectroscopy, (2) develop spectroscopic fingerprint patterns and detection methodology for fungi such as Aspergillus, Rhizopus, Fusarium, and Penicillium (3) to validate a universal spectroscopic procedure to detect foodborne pathogens and non-pathogens in food systems. The original objectives proposed were very ambitious hence modifications were necessary to fit with the funding. Elaborate experiments were conducted for sensitivity, additionally, testing a wide range of pathogens (more than selected list proposed) was also necessary to demonstrate the robustness of the instruments, most crucially, algorithms for differentiating a specific organism of interest in mixed cultures was conceptualized and validated, and finally neural network and chemometric models were tested on a variety of applications. Food systems tested were apple juice and buffer systems. Pathogens tested include Enterococcus faecium, Salmonella enteritidis, Salmonella typhimurium, Bacillus cereus, Yersinia enterocolitis, Shigella boydii, Staphylococus aureus, Serratiamarcescens, Pseudomonas vulgaris, Vibrio cholerae, Hafniaalvei, Enterobacter cloacae, Enterobacter aerogenes, E. coli (O103, O55, O121, O30 and O26), Aspergillus niger (NRRL 326) and Fusarium verticilliodes (NRRL 13586), Saccharomyces cerevisiae (ATCC 24859), Lactobacillus casei (ATCC 11443), Erwinia carotovora pv. carotovora and Clavibacter michiganense. Sensitivity of the FTIR detection was 103CFU/ml and a clear differentiation was obtained between the different organisms both at the species as well as at the strain level for the tested pathogens. A very crucial step in the direction of analyzing mixed cultures was taken. The vector based algorithm was able to identify a target pathogen of interest in a mixture of up to three organisms. Efforts will be made to extend this to 10-12 key pathogens. The experience gained was very helpful in laying the foundations for extracting the true fingerprint of a specific pathogen irrespective of the background substrate. This is very crucial especially when experimenting with solid samples as well as complex food matrices. Spectroscopic techniques, especially FTIR and Raman methods are being pursued by agencies such as DARPA and Department of Defense to combat homeland security. Through the BARD US-3296-02 feasibility grant, the foundations for detection, sample handling, and the needed algorithms and models were developed. Successive efforts will be made in transferring the methodology to fruit surfaces and to other complex food matrices which can be accomplished with creative sampling methods and experimentation. Even a marginal success in this direction will result in a very significant breakthrough because FTIR and Raman methods, in spite of their limitations are still one of most rapid and nondestructive methods available. Continued interest and efforts in improving the components as well as the refinement of the procedures is bound to result in a significant breakthrough in sensor technology for food safety and biosecurity.