Le gari, une semoule de manioc (Manihot esculenta, Crantz) fermentée, gélifiée et déshydratée, représente le produit alimentaire le plus populaire de lAfrique de lOuest. Les bactéries lactiques constituent lun des plus importants groupes de micro-organismes impliqués dans létape de la fermentation du manioc, principalement en raison de leurs rôles établis dans le développement de la saveur et la préservation des aliments obtenus. Dans le but de fournir au consommateur un produit sain, de qualité organoleptique constante et acceptable, des efforts damélioration et de contrôle de la fermentation du manioc ont été entrepris. Seize souches de bactéries lactiques ayant des caractéristiques technologiques appropriées, ont été précédemment isolées et sélectionnées au cours de la fermentation du manioc en vue de leur utilisation comme culture starter pour la production de gari. Lobjectif de ce travail est de contribuer à un meilleur contrôle de la fermentation du manioc à travers la production dun starter de bactéries lactiques lyophilisées pour la production de gari. A lissue de la première partie du travail, les souches Lactobacillus plantarum VE36, G2/25, L. fermentum G2/10 et Weissella paramesenteroides LC11 ont été sélectionnées sur base de : (i) leur capacité à survivre après un test de déshydratation dans une solution de glycérol de concentration croissante suivi dun marquage par des marqueurs fluorescents, (ii) leur taux de survie après la lyophilisation, (iii) leur capacité dutilisation dune source de carbone après la lyophilisation, (iv) leur stabilité au cours du stockage sous forme lyophilisée. Nous avons cependant noté que la plupart des souches lyophilisées perdaient plus de 50% de leur viabilité après 60 jours de stockage à basse température. Dans la seconde partie du travail, nous avons essayé de comprendre lun des mécanismes à lorigine de la perte de viabilité au cours du stockage des souches lyophilisées : loxydation des lipides membranaires. Pour ce faire, limpact de la dégradation des acides gras polyinsaturés sur la survie cellulaire ou le pouvoir dacidification de la souche lyophilisée W. paramesenteroides LC11 a été évalué pendant 90 jours de stockage dans différentes conditions de stockage (différentes températures, présence ou absence dair et dhumidité). Un dosage des produits primaires de dégradation des acides gras polyinsaturés a aussi été effectué pour les échantillons stockés dans des conditions daération. A lissue de cette étude, une corrélation a été établie entre la perte de viabilité cellulaire et/ou du pouvoir dacidification et la dégradation des acides gras polyinsaturés. Dans loptique de confirmer ces résultats, leffet de composés protecteurs tels que les cryoprotecteurs et/ou des antioxydants (acide ascorbique et/ou butyle hydroxytoluene) sur la viabilité cellulaire, lintégrité membranaire et loxydation des lipides de la souche lyophilisée W. paramesenteroides LC11, ont été évalués pendant 90 jours de stockage dans des conditions daération et en présence dhumidité. Après le 90ième jour de stockage, les acides gras membranaires totaux ainsi que ceux de la fraction polaire ont également été analysés. La présence des cryoprotecteurs et du butyle hydroxytoluene avait considérablement réduit la perte de viabilité et dintégrité membranaire, ainsi que la dégradation des lipides de la fraction polaire. Une possible relation entre loxydation des acides gras polyinsaturés, plus précisément ceux de la fraction polaire des lipides, et la perte de viabilité cellulaire au cours du stockage des souches lyophilisées, a été confirmée. Des corrélations entre loxydation des acides gras polyinsaturés de la fraction polaire des lipides et la perte dintégrité cellulaire et, dautre part, entre la perte dintégrité cellulaire et la perte de viabilité cellulaire, ont été établies. Alors que les phénomènes doxydation des lipides se déroulaient au cours du temps, la perte de viabilité cellulaire survenait dès les premières heures du stockage. Les changements dans la perméabilité membranaire de la souche W. paramesenteroides LC11 ont ainsi été évalués 20h après la lyophilisation, à travers les déterminations du nombre de cellules viables et de la conductivité dune solution de réhydratation dont nous avons fait varier la température, la concentration en sels (NaCl), protons et composés protecteurs (glycérol, saccharose, maltodextrine, glutamate monosodique). Une augmentation de la température et de la concentration en sels (NaCl) ou en glutamate monosodique, ainsi quune baisse de la concentration en protons entraînaient une augmentation de la concentration en substances ioniques dissoutes et une baisse du nombre de cellules viables. Cependant, une augmentation de la concentration en glycérol, sucrose ou maltodextrine entraînait une diminution de la concentration en substances ioniques dissoutes et un maintien de la viabilité cellulaire. Ces résultats nous suggèrent quil pourrait se produire une rupture cellulaire durant la déshydratation et que cette situation pourrait influencer la stabilité de la souche lyophilisée dès les premières heures du stockage.

Ibourahema COULIBALY (2010). Contribution à létude de la résistance au séchage des bactéries lactiques (thèse de doctorat en français) ; Université de Liège, Gembloux Agro-Bio Tech, (Belgique), 260 p., 22 tableaux, 39 figures. Résumé Lutilisation de souches lactiques, à léchelle industrielle, nécessite une sélection sur base des propriétés technologiques, physiologiques et biochimiques, ainsi que la mise en oeuvre de procédés biotechnologiques bien maîtrisés pour leur production et leur conservation. A cet effet, le séchage est une étape importante. Sous forme sèche, le produit est plus facilement manipulable. Cependant au cours du séchage, les microorganismes subissent un stress thermique et/ou osmotique qui se traduit par une perte de viabilité pendant le stockage. Lutilisation dagents protecteurs au cours du stockage a permis dobtenir une bonne viabilité. En effet, les bactéries sont soumises pendant la lyophilisation et au cours du stockage à une mortalité liée en grande partie à loxydation des acides gras membranaires. Lanalyse de ces acides au cours des différents procédés de fabrication et de stockage permet de noter la présence des hydroperoxydes. Cette oxydation est engendrée par laction de loxygène sur les acides gras polyinsaturés principalement lacide linoléique (C18:2) et linolénique (C18:3) ce qui engendre la formation des hydroperoxydes : acide hydroperoxyoctadecadienoique (HPOD) et acide hydroperoxyoctadecatrienoique (HPOT) méthylés ou non en position 13- et 9-. Une analyse des composés secondaires émanant des poudres de bactéries lactiques indique la présence de composés volatils. Parmi ceux-ci, apparaissent des molécules volatiles (principalement des aldéhydes), qui modifient la flaveur dorigine des corps gras. Les études menées au niveau des constituants phospholipidiques de la membrane montrent un lien entre le degré doxydation et la mortalité cellulaire au cours du stockage. Les différents phospholipides déterminés au nombre de sept (7) subissent tous cette oxydation à des degrés divers. Lutilisation dantioxydant au cours du stockage permet dinhiber les phénomènes de peroxydation des lipides et permet également de maintenir la valeur nutritionnelle (teneur en AGPI, réduction des dérivés peroxydés nocifs) du produit. Ibourahema COULIBALY (2010). Contribution to the study of resistance of lactic acid bacteria subject to drying. (Dissertation in French) ; University of Liège, Gembloux Agro- Bio Tech , (Belgium), p. 260 p., 22 tables, 39 figures. Summary The use of lactic strains in industrial scale requires selection on the basis of technological, physiological and biochemical properties, as the implementation of biotechnological processes under control for their production and conservation. At this effect drying process is an important step. In dry form, the product is more easily manipulated. However during the drying process, microorganisms undergo heat stress and/or osmotic resulting in a loss of viability during storage. The use of protective agents during storage has resulted in a good viability. In fact, the bacteria are subjected during lyophilization and during storage to a mortality largely oxidation the membrane fatty acids. The analysis of these acids during the process helps to note the presence of hydroperoxydes which are the cause of this mortality. This oxidation is caused by the action of oxygen on polyunsaturated acids linoleic acid (C18:2) and linolenic (C18:3) mostly, causing the formation of hydroperoxydes: hydroperoxyoctadecadienoic acid (HPOD) and hydroperoxyoctadeca-trienoic acid (HPOT) at position 13 - and 9 - methyl or not. An analysis of secondary compounds from the powders indicates the presence of birds. These products appear volatile compounds (mainly aldehydes), hydrocarbons, alcohols, acids etc., which altered the original flavour of fat. Studies in phospholipids constituents of the membrane show a link between the degree of oxidation and cell death during storage. The various phospholipids determined, seven (7) are all undergo this oxidation to various degrees. The use of antioxidants during storage can inhibit the process of lipid peroxydation and helps also to maintain the nutritional value (PUFA content, reduction of harmful peroxide-derived) product.

La present tesi doctoral es centra en l'aplicació dels bacteris de l'àcid lactic (BAL) com a agents bioprotectors davant microorganismes patògens i deteriorants.Es van aïllar i seleccionar BAL de fruites i hortalisses fresques i es van assajar in vitro davant 5 microorganismes fitopatògens i 5 patògens humans.Es van realitzar assajos d'eficàcia en pomes Golden Delicious amb tots els aïllats enfront les infeccions causades pel fong Penicillium expansum. La soca més eficaç era Weissella cibaria TM128, que reduïa el diàmetre de les infeccions en un 50%.Les soques seleccionades es van assajar enfront els patògens Salmonella typhimurium, Escherichia coli i Listeria monocytogenes en enciams Iceberg i pomes Golden Delicious.Els BAL interferien eficientment amb el creixemet de S. typhimurium, and L. monocytogenes, però van mostrar poc efecte enfront E. coli.Finalment, es van realitzar assajos dosi-resposta amb les soques Leuconostoc mesenteroides CM135, CM160 and PM249 enfront L. monocytogenes. De totes les soques assajades, la soca CM160 va ser la més efectiva. / The present thesis focuses on the use of lactic acid bacteria as bioprotective cultures to inhibit pathogenic and spoilage microorganisms.Lactic acid bacteria were isolated and selected from fresh fruit and vegetables and tested in vitro against five plant pathogens and five human pathogen test bacteria.Efficacy trials with all the isolates were performed in Golden Delicious apples against the blue mould rot infections, caused by Penicillium expansum. The highest effectivity found in this assay was of about 50%, with strain Weissella cibaria TM128.Selected lactic acid bacteria were tested against Salmonella typhimurium, Escherichia coli and Listeria monocytogenes in Iceberg lettuce and Golden Delicious apples. Lactic acid bacteria interfered efficiently with the growth of S. typhimurium, and L. monocytogenes, but showed little effectivity over E. coli.Finally, dose-response assays were done with Leuconostoc mesenteroides strains CM135, CM160 and PM249 against L. monocytogenes.Among the three strains tested, strain CM160 showed the highest effectivity.

A produção de etanol nas destilarias brasileiras é baseada na atividade fermentativa da levedura Saccharomyces cerevisiae que utiliza o caldo da cana-de-açúcar e/ou o melaço como substrato. Bactérias contaminantes da fermentação alcoólica competem com as leveduras pelos açúcares, afetando o rendimento do sistema produtivo e, consequentemente, causando perdas econômicas significativas às usinas. Biofilmes formados nos tanques de fermentação alcoólica agem como reservatórios de bactérias, contribuindo para contaminações persistentes e de difícil controle. Os biofilmes proporcionam aos seus habitantes certo grau de proteção contra diversas ameaças do meio, incluindo a ação dos antibióticos. Desta forma, o conhecimento da comunidade bacteriana dos biofilmes é fundamental para as medidas que visam o controle das contaminações na produção do bioetanol. No primeiro estudo, a composição e dinâmica da comunidade bacteriana foram determinadas pela análise de sequências do gene 16S rRNA de biofilmes com diferentes períodos de crescimento, correspondentes aos estágios iniciais de estabelecimento destes biofilmes dentro dos tanques de fermentação alcóolica Os resultados mostraram que estas comunidades foram compostas predominantemente pelas bactérias ácido-lácticas (LAB), com destaque para o gênero Lactobacillus. A visualização da estrutura dos biofilmes por microscopia eletrônica de varredura evidenciou que estes são formados por bactérias e leveduras (biofilmes mistos). No segundo estudo, a suscetibilidade aos antimicrobianos (monensina, virginiamicina e beta-ácido derivado do lúpulo) e a capacidade de formação de biofilmes em culturas puras foram avaliadas para isolados de Lactobacillus spp. provenientes de biofilmes (células sésseis) e de vinho bruto (células planctônicas) coletados dos tanques de fermentação. A partir dos resultados foi possível observar que as diferenças na suscetibilidade aos antimicrobianos e na habilidade de formar biofilmes foram estirpe-dependentes e que, em alguns casos, o perfil apresentado para algumas espécies mostrou-se relacionado à fonte de isolamento. Este foi o primeiro estudo sobre biofilmes contaminantes da fermentação alcoólica, em escala industrial, para a produção de etanol a partir da cana-de-açúcar / Bioethanol production in Brazilian distilleries is based on fermentative activity of the yeast Saccharomyces cerevisiae which uses sugarcane juice and/or molasses as a substrate. Bacterial contaminants of alcoholic fermentation compete with yeasts for sugars, affecting ethanol yield and consequently causing relevant economic losses to the fuel ethanol industry. Biofilms formed into fermentors act as bacterial reservoirs, contributing to persistent contaminations that are difficult to control. Biofilms provide a certain degree of protection for their inhabitants against some environmental threats, including antibiotics. Thus, understanding bacterial community within biofilms is essential for actions to control contaminations in bioethanol production. In the first study, composition and dynamic of bacterial community were determined by 16S rRNA gene sequences analysis of biofilms with different growth periods, corresponding to initial stages of biofilm establishment in fermentation tanks. Results showed that these communities were dominated by lactic acid bacteria (LAB), mainly of the genus Lactobacillus. Visualization of biofilm structure by scanning electron microscopy revealed a mixed-species biofilm composed by bacteria and yeasts. In the second study, susceptibility to antimicrobials (monensina, virginiamicina and beta-acids from hops) and capacity to form biofilm in pure culture were evaluated for Lactobacillus spp. isolated from biofilms (sessile cells) and wine (planktonic cells) collected from fermentors. The results showed that differences in the susceptibility to antimicrobials and the ability to form biofilms were strain-specific and, in certain cases, the response of some species was related to the isolation source. This was the first investigation of contaminant biofilms from sugarcane-based alcoholic fermentation on an industrial scale

Le vin en tant qu’écosystème complexe est un modèle particulièrement intéressant pour l’étudie des interactions entre les microorganismes. L’interaction sans contact celluaire (interaction indirecte) entre la levure Saccharomyces cerevisae et la bactérie lactique Oenococcus oeni a un effect direct sur l’induction et l'achèvement de la fermentation malolactique (FML), une fermentation très importante pour la qualité du vin. Une souche levurienne peut être classée FML+ si elle stimule la croissance bactérienne et FML- si elle a un effet inhibiteur. Les métabolites connus qui inhibent ou stimulent la FML ne permettent pas toujours d’expliquer cette distinction phénotypique. Dans ce travail de thèse, nous avon développé un workflow multidisciplinaire qui combine l’approche métabolomique non ciblée, l’analyse classique ciblée, les statistiques et les réseaux. L’objectif premier était de dévoiler des métabolites levuriens impliqués dans l’interaction entre levures et bactéries par une comparaison directe des exométabolome des deux phénotypes.À cet effet et pour la première fois dans l’éude d’interactions inter-espèces, la Spectrométrie de Masse à Résonance Cyclotronique des Ions et à Transformée de Fourier (FT-ICR-MS) et la Chromatographie Liquide couplée à la Spectrométrie de Masses (UPLC-Q-TOF-MS) ont été combinées. Pour mieux visualiser les données à haut débit générées par les deux plate-formes, une méthode statistique non supervisée MetICA a été developpée et validée. Par rapport à l’analyse en composantes principales (ACP), cette nouvelle méthode peut réduire la dimension des données d'une façon plus robuste et fiable. Afin d’extraire des métabolites impliquées dans la distinction phénotypique, nous avons comparé différentes methodes de classification et choisi la meilleure pour chaque jeu de données. Les structures putatives de ces biomarqueurs ont été validés par la spectrométrie de masse MS/MS et leurs rôles physiologiques sur la croissance bactérienne ont été confirmées in vitro. La découverte de biomarqueurs a été complétée par l’analyse ciblée réalisées par Chromatographie en Phase Liquide à Haute Performance (HPLC). La complémentarité entre les différentes techniques métabolomiques a conduit à l’identification de nouveaux biomarqueurs de familles distinctes, comme des composés phénoliques, des sucres, des nucléotides, des acides aminés et des peptides. En outre , l'analyse des réseaux métaboliques a révélé des liens entre les biomarqueurs de levure et a suggéré des voies bactériennes influencés par l’exo-métabolome de levure.Notre workflow multidisciplinaire a révélé une réelle capacité à identifier des signatures moléculaires nouvelles et inattendues de l’interaction levure-bactérie. / As a complex microbial ecosystem, wine is a particularly interesting model for studying interactions between microorganisms. Contact-independent interactions (indirect interactions) between the yeast Saccharomyces cerevisae and the lactic acid bacterium Oenococcus oeni have a direct effect on malolactic fermentation (MLF), induction and completion, which is an important factor in wine quality. Yeast strains could be classified as MLF+ phenotype if it usually stimulates the bacterial growth or MLF- in the opposite case. The known metabolites that stimulate or inhibit the MLF cannot always explain the phenotypic distinction. In this work, a multidisciplinary workflow combining non-targeted metabolomics, targeted analysis, statistics and network was developed. The main objective was to unravel diverse yeast metabolites involved in yeast-bacteria interaction via a direct comparison of exo-metabolomes of MLF+ and MLF- phenotypes.To that purpose, and for the first time in the research of interspecies microbial interactions, two metabolomics platforms, Fourier Transform Ion Cyclotron Resonance -Mass Spectrometry (FT-ICR-MS) and Liquid Chromatography coupled with Mass Spectrometry (UPLC-Q-TOF-MS) were used in combination. To better visualize the high-throughput data generated from the two platforms, a novel unsupervised statistical method, the MetICA was developed and validated. Compared to classical principal component analysis (PCA), the new method reduced the data dimension in a more robust and reliable way. To extract metabolic features involved in the phenotypic distinction, we have compared different statistical classifiers and selected the best one for each dataset. Putative structures of these biomarkers were validated via MS/MS fragmentation analysis and their physiological roles to bacteria were confirmed in vitro. The discovery of biomarkers was complemented by targeted HPLC (high performance liquid chromatography) analysis. The complementarities between different analytical techniques led to new biomarkers of distinct chemical families, such as phenolic compounds, carbohydrates, nucleotides, amino acids and peptides. Furthermore, metabolic network analysis has revealed connections between yeast biomarkers and suggested bacterial pathways influenced by yeast exo-metabolome.Our multidisciplinary workflow has shown its ability to find new and unexpected molecular evidence of wine yeast-bacteria interaction.

Parmi les différentes méthodes de lutte contre les microorganismes pathogènes et/ou altérants en agroalimentaire, l’utilisation de bactéries lactiques (LAB) bioprotectrices s'avère être un outil prometteur pour la préservation des aliments. Ce travail de thèse collaboratif, entre l'équipe PAPC (AgroSup Dijon, Université de Bourgogne) et le laboratoire BioDyMIA (Université Lyon1-Isara Lyon), concerne l'étude de systèmes bioprotecteurs immobilisant des cellules entières de LAB dans une matrice polymérique d'alginate de sodium et de caséinate de sodium pour une activité ciblée contre Listeria spp. Dans un premier temps, la méthodologie mise en œuvre a consisté à sélectionner des souches de LAB bioprotectrices sur la base de leur activité antimicrobienne évaluée par la méthode de diffusion en milieu gélosé contre trois souches de Listeria spp. Quatre souches sur 19 ont ainsi été sélectionnées. Une caractérisation partielle des métabolites antimicrobiens produits par ces 4 souches a ensuite été réalisée en appliquant des traitements thermiques et enzymatiques aux surnageants de culture correspondants pour évaluer si ces traitements altéraient l’activité des métabolites antimicrobiens présents. Une purification et une identification partielle des actifs antimicrobiens de nature peptidique ont été réalisées uniquement pour la souche d'intérêt principale : Lactococcus lactis LAB3. Dans un second temps, une formulation de la matrice polymérique d’immobilisation des LAB sélectionnées a été choisie en réalisant le diagramme de phases du système aqueux alginate de sodium/caséinate de sodium : 1,5 % (m/m) d'alginate de sodium / 4 % (m/m) de caséinate de sodium / 20 % (m/m) bouillon MRS. Cette formulation a permis d'obtenir une matrice composée d’une phase continue riche en alginate et d’une phase dispersée riche en caséinate dans laquelle les cellules de LAB se localisent préférentiellement d’après les observations en microscopie de fluorescence confocale à balayage laser. Suite à l'inclusion des cellules de LAB dans ces matrices liquides et gélifiées d'alginate seul et d'alginate/caséinate, leur cultivabilité et leur activité anti-Listeria ont été suivies à 30°C pendant 12 jours. Ceci a révélé que la cultivabilité et l’activité antimicrobienne des cellules de LAB se maintiennent à des niveaux plus élevés dans les matrices d'alginate/caséinate que dans celles uniquement à base d’alginate. Ces matrices à base d’alginate et de caséinate apparaissent donc comme un système prometteur pour l'immobilisation de LAB bioprotectrices. Leur intérêt pour l’inclusion de LAB a pu être corrélé à leur viabilité et à la structure composite de cette matrice à base de protéines qui favoriserait la production et la libération des métabolites antimicrobiens / Among the various methods to control foodborne pathogenic and/or food spoilage microorganisms in food chain, bioprotective lactic acid bacteria (LAB) appear to be promising tools for food biopreservation. This collaborative study, between PAPC (Agrosup Dijon, University of Burgundy) and BioDyMIA (University Lyon1-Lyon Isara) laboratories, concerned the development of sodium alginate/sodium caseinate polymeric matrices intended to entrap LAB cells selected for their anti-Listeria spp. activity. First, 4 LAB strains from 19 LAB strains were selected for their anti-Listeria spp. activity: this screening was performed by the method of agar diffusion against three Listeria spp strains. Then, antimicrobial metabolites produced by the selected LAB strains were partially characterized by assessing the effect of various thermal and enzymatic treatments on the anti-Listeria spp. activity of their culture supernatants. A partial purification and identification of antimicrobial active peptides produced by the main strain of interest (Lactococcus lactis LAB3) was also performed. A composition of the polymer matrix has been selected by performing the phase diagram of sodium alginate/sodium caseinate system: 1.5% (w/w) sodium alginate / 4% (w/w) of caseinate sodium / 20% (w/w) MRS broth. This formulation provides a rich alginate continuous phase and a rich caseinate dispersed phase in which LAB cells localize according to the study by confocal microscopy. LAB cells were immobilized in liquid and gelled matrices of alginate and alginate/caseinate. Culturability and anti-Listeria activities were measured during a storage at 30°C for 12 days. The alginate/caseinate matrices were more effective in better maintaining LAB cells cultivability and their antimicrobial activity than alginate matrix. This effectiveness seemed correlated with cell viability and the dispersion-like structure of the protein-based system which enhance production and release of antimicrobial metabolites. Thus, this type of polymeric matrix appeared as a promising immobilization system of bioprotective LAB

Parmi les bactéries lactiques, Lactococcus lactis est la plus utilisée en fabrication fromagère. Actuellement, les ferments lactiques sont majoritairement choisis pour leurs propriétés acidifiantes, protéolytiques et aromatiques. Un autre paramètre majeur est le potentiel redox (Eh). En effet, un Eh réducteur est souvent associé à une bonne qualité aromatique. L’activité réductrice de L. lactis pourrait donc être un nouveau paramètre à prendre en compte dans la maitrise du Eh dans la fabrication des produits laitiers fermentés. Néanmoins, les mécanismes impliqués dans l’activité réductrice de L. lactis demeurent encore inconnus. L’objectif de ce présent travail de thèse a été de les découvrir. Tout d’abord, nous avons développé des milieux de culture gélosé de discrimination redox utilisant des sels de tétrazolium pour cribler une banque de mutants aléatoires de L. lactis. Ceci a permis de démontrer la participation partielle de la chaine de transport d’électrons (Ménaquinones) dans l’activité réductrice de L. lactis. Ensuite, l’approche biochimique nous a permis de déterminer les composés biochimiques principaux contribuant à la diminution du Eh vers des valeurs très réductrices. La présence de groupements thiols exofaciaux est responsable du Eh réducteur atteint par L. lactis. Enfin, l’analyse protéomique utilisant un marquage spécifique des protéines thiols de surface a mis en évidence la présence d’une dizaine de protéines exposant des groupements thiols exofaciaux potentiellement impliquées dans l’activité réductrice de L. lactis. Les thiols sont connus pour être de très puissants antioxydants ce qui confère à L. lactis un intérêt supplémentaire à prendre en considération dans l’élaboration des produits laitiers fermentés. / Among the Lactic Acid Bacteria, Lactococcus lactis is the most used in cheese making. Nowadays, starters are used mainly for their acidifying, proteolytic and flavor properties. Another important parameter is the redox potential (Eh). Indeed, reducing Eh is often related to good flavor properties. The reducing activity of L.lactis should be therefore a new parameter to take into account in the monitoring of Eh during dairy fermented products making. Nevertheless, the mechanisms involved in the reducing activity of L.lactis are still unknown. The aim of this work was to understand them. First of all, we have developed tetrazolium salts agar plate media in order to screen a random bank of mutants of L. lactis on their redox capacities. These media allowed us to demonstrate the partial implication of the electron transport chain (Menaquinone) in the reducing activities of L. lactis. Secondly, we have determined the biochemical compounds involved in the decrease of Eh to very reducing values thanks to a biochemical approach. Exofacial thiol groups are mainly responsible for the reducing Eh reached by L.lactis. Lastly, a proteomical analysis using a specific staining of thiols surface proteins revealed the presence of about ten proteins displaying thiols exofacials groups. These proteins might be involved in the reducing activity of L.lactis. Thiols are known to be very strong antioxidants which confer to L. lactis an additional interest to consider in dairy products making.

Orientador: Marcelo Cristianini / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-27T18:44:38Z (GMT). No. of bitstreams: 1 Oliveira_ThalesLeandroCoutinhode_D.pdf: 2386676 bytes, checksum: bc4655fa6960e3332639a4ca75c75545 (MD5) Previous issue date: 2015 / Resumo: A redução do cloreto de sódio nas formulações de alimentos processados representa um grande desafio para indústria moderna, tendo em vista a relação estabelecida entre o consumo elevado de sódio e a ocorrência da hipertensão arterial, principal fator de risco associado à ocorrência de distúrbios do cérebro e cardiovasculares. Em produtos cárneos, além de atuar sobre aspectos sensoriais e de textura, o sal desempenha papel chave sobre sua estabilidade microbiológica. Uma simples redução pode resultar em produtos instáveis durante sua vida de prateleira. Logo, para que estratégias de redução de sal sejam implementadas de maneira segura, faz-se necessário a aplicação de tecnologias ou agentes de preservação complementares. Tecnologias emergentes não-térmicas de inativação microbiana como o processamento à Alta Pressão (HPP - High Pressure Processing) surgem como opções factíveis para tal propósito, eliminando a dependência de conservantes químicos em face à redução de sal. A adição de agentes antimicrobianos naturais como barreiras adicionais à HPP (Tecnologia de Múltiplas Barreiras), visando potencializar os efeitos conservadores, têm sido apontada como uma promissora tendência na literatura moderna. Diante deste exposto, após determinação da formulação de trabalho com teor reduzido de sódio, a pesquisa objetivou ajustar as variáveis de processo "carga de pressão" e "tempo de residência" (utilizando baixas temperaturas de 25°C) para o processamento de embutido de peito peru fatiado embalado a vácuo (tipo "Blanquet"). Foram avaliadas respostas de inativação microbiana pós-processo (reduções logarítmicas) para populações inoculadas do patógeno Listeria monocytogenes e bactérias ácido láticas deterioradoras incluindo as espécies Leuconostoc mesenteroides e Lactobacillus sakei; também foram avaliados os atributos de qualidade incluindo cor, atividade de água, pH, sinerese, textura e oxidação lipídica. Em adição, visando à utilização de tecnologia de múltiplas barreiras para conservação, foi estudado o efeito da adição do componente bioativo carvacrol em concentrações de 200ppm, na formulação do produto. Empregando estratégia de simples redução, sem a adição sais substitutos, após avaliação de aspectos físico-químicos, sensoriais e microbiológicos, concluiu-se que níveis de 30% de redução de sal (cerca de 25% menos sódio) sobre o controle formulado com 20g/kg, são viáveis; entretanto, foi evidenciada redução da estabilidade microbiológica. Após avaliação de diferentes faixas de pressão e tempo de processo, seguindo os critérios exigidos de desempenho para inativação pós-processo de Listeria monocytogenes (4-5 reduções logarítmicas), um valor de 600MPa/180 segundos a 25°C se apresentou como um tratamento adequado para o produto com baixo teor de sódio estudado, promovendo reduções logarítmicas eficazes contra o patógeno-alvo estudado e a microbiota deteriorante. Alterações instrumentais (p<0.05) em alguns dos atributos de qualidade avaliados puderam ser destacadas principalmente sinerese, oxidação lipídica e textura; porém são necessários estudos mais amplos de análise sensorial. Adicionalmente, dados apresentados demonstraram que os consumidores não foram capazes de diferir o produto low-sodium processado por alta pressão isostática (600MPa/180sec/25°C) de um não pressurizado; de fato, apesar dos efeitos instrumentais significativos, estas alterações parecem não ser identificadas pelos consumidores. A adição de uma barreira natural complementar, o carvacrol, em níveis sensorialmente aceitáveis, foi capaz de potencializar os efeitos de inativação pós-processo (reduções logarítimicas) dos grupos microbianos alvo; efeitos benéficos combinados ao longo da estocagem refrigerada do produto também foram evidenciados por meio de reduzidas taxas de crescimento e fase lag aumentada, maximizando assim o potencial de conservação. A utilização combinada de HPP e carvacrol representou uma alternativa promissora contra fenômenos de injúrias sub-letais e recuperação celular. Sugere-se que a intensidade do processo HPP (em termos de carga de pressão, tempo de processo e temperatura) necessária para inativar microrganismos em níveis requeridos, possa ser reduzida na presença de barreiras antimicrobianas adicionais, garantindo a qualidade total do produto processado em condições mais amenas. Em adição, uma série de vantagens industriais podem ser destacadas tais como a redução de custos para a instalação de equipamento inicial e de manutenção (equipamentos operando em cargas mais baixas); e maximização da produção de processamento por ciclos efetivamente encurtados (ciclos de produtividade mais elevados por hora) / Abstract: The reduction of sodium chloride in processed food formulations represents a great challenge for modern industry, considering the relationship established between high sodium intake and the occurrence of arterial hypertension, the major risk factor associated with the occurrence of cardiovascular diseases and strokes. In meat products, in addition to acting on sensory quality and texture, salt plays a key role on the microbiological stability. A simple reduction can result in unstable products during their shelf-life. Therefore, for the success of salt reduction strategies implemented safely, it is necessary the application of complementary technologies or alternative preservatives. Non-thermal emerging technologies for microbial inactivation, such as High Pressure Processing (HPP), appear as feasible alternative for this purpose, eliminating the dependence of chemical preservatives considering the reduction of salt barrier. The addition of natural antimicrobial agents as additional hurdles (Multi Hurdle Technology) to the HPP, aiming to enhance the preservative effects, has been identified as a promising trend in modern literature. Given this exposed, after determination of low-sodium working formulation, this research aimed adjust the processing variables "pressure load" and "dwell time" (using low-processing temperatures of 25°C) for sliced vacuum-packaged ready-to-eat turkey breast. Post-processing microbial inactivation responses were evaluated (by logarithmic reductions) against inoculated populations of the target pathogen Listeria monocytogenes and spoilage lactic acid bacteria including Leuconostoc mesenteroides and Lactobacillus sakei; were also evaluated quality-attributes including color, water activity, pH, syneresis, texture and lipid oxidation. In addition, in order to use multiple barriers for preservation technology, was studied the effect of adding bioactive component, carvacrol at concentrations of 200ppm in the product formulation. Employing a simple reduction strategy, without adding salt substitutes, after evaluation of physicochemical aspects, sensory and microbiological, it was concluded that levels of 30% NaCl reduction (aproximatelly 25% less sodium) on the control made with 20g/kg, are viable; however, problems with microbiological stability were evidenced. After evaluation of different pressure ranges and processing time, following the performance criteria required for post-inactivation process Listeria monocytogenes (4-5 log reduction), a value of 600MPa/180 seconds at 25°C was presented with an adequate treatment for studied product with low sodium content, promoting effective logarithmic reductions against the studied target pathogen and spoilage LAB. Instrumental changes (p<0.05) in some of the evaluated quality attributes could be revealed mainly in syneresis, lipid oxidation and texture; however, a sensory confirmation needs to be established. Additionally, data presented showed that consumers have not been able to differ low-sodium products processed by HPP (600MPa/180sec/25°C) and unpressurised; in fact, despite significant instrumental effects, these changes seems to be not identified by consumers. The addition of natural barrier, such as carvacrol at acceptable sensory levels (200ppm), was able to potentiate the effects of post-process inactivation (logarithmic reductions) of the target microbial groups; combined benefit effects along the refrigerated product storage were also evidenced by reduced growth rates and increased lag phases, thus maximizing their potential for conservation. The combination of HPP and carvacrol represented a promising weapon against phenomena of sub-lethal injuries and cell recovery. It is suggested that the intensity of the HPP process (in terms of pressure load, process time and temperature) required to inactivate microorganisms in required levels, may be reduced in the presence of additional microbial barriers, ensuring the overall quality of the processed product under mild processing conditions. In addition, a number of industrial advantages can be highlighted such as reduced costs for the initial installation and maintenance equipment (low required pressure loads) and maximization of production by effectively processing cycles shortened (higher productivity cycles per hour) / Doutorado / Tecnologia de Alimentos / Doutor em Tecnologia de Alimentos

Nesta pesquisa, estudaram-se as variações ocorridas na relação entre autólise de culturas lácticas e o desenvolvimento da proteólise de queijo Prato produzido em quatro regiões brasileiras: Santa Catarina (Queijo A), Goiás (Queijo B), São Paulo (Queijo C) e Minas Gerais (Queijo D). A análise quantitativa da população de bactérias lácticas durante a maturação mostrou perfis microbiológicos similares para todas as amostras de queijos examinadas. Após 5 dias de maturação, lactococos e estreptococos estavam presentes em números mais elevados do que lactobacilos mesofílicos e termofílicos, leuconostoc e fermentadores de lactato. Contudo, essas populações aumentaram consideravelmente no final do processo de maturação. Enterococos e fermentadores de citrato permaneceram em números relativamente reduzidos ao longo da maturação. A análise qualitativa mostrou a predominância de \"non starter lactic acid bactéria\" (NSLAB) nos queijos das quatro origens, principalmente de Lactobacillus sp. Outros gêneros foram identificados em menor proporção: Enterococcus sp., Pediococcus sp., Aerococcus sp., Tetragenococcus sp. e Streptococcus sp. O queijo C diferiu dos demais por não apresentar Pediococcus sp. e Streptococcus sp. As culturas lácticas adicionadas Lactococcus lacfis sp. e Leuconostoc sp. estavam presentes em populações menores. A autólise foi estudada pela determinação da atividade de aminopeptidases e detecção de autolisinas por zimogramas e de enzimas intracelulares por \"imunoblotting\". Uma maior liberação de aminopeptidases ocorreu no queijo D, seguido dos queijos C, B e A. Não foram detectadas bandas de atividade lítica nos zimogramas dos queijos A e B em todas as condições avaliadas. Nos zimogramas, detectou-se uma banda de 30 KDa nos queijos C e D a pH 7,4 e 44&#176;C, e uma outra de 40 KDa, exclusiva no queijo D, ambas de fraca intensidade. A pH 6,8 e 42&#176;C, detectou-se bandas de 40KDa de fraca intensidade no queijo C e forte no D, além de mais duas de fraca intensidade de 90KDa e 110KDa no queijo D. Na análise em \"imunnoblotting\" com o antisoro anti-Lc, foi observado apenas sinais fracos de reação positiva e em números inferiores àquelas reveladas com o citoplasma bruto de Lac. Lacfis subsp. lacfis (controle positivo), indicando que a autólise foi praticamente inexistente. Com o antisoro anti-D-LDH, também não se detectou sinais de reação positiva nos queijos A e B, enquanto nos queijos C e D, verificou-se sinais positivos de 37KDa, de forte intensidade e correspondentes à proteína D-LDH, indicando a lise de Lab. helveticus. A evolução da proteólise foi determinada quantitativamente durante a maturação e avaliada com base nos índices: NS-pH4,6/NT% e NNP/NT%, teor de tirosina, eletroforese (Uréia-PAGE) e quantificação de aminoácidos individuais livres. Não foram detectadas diferenças significativas entre os queijos A. B, C e D no início da maturação. Contudo, com a fragmentação das proteínas, ocorreu um aumento gradual desses índices, tendo-se observado valores mais elevados no queijo D, seguido dos queijos C, B e A. Os perfis eletroforéticos de proteínas foram similares para os queijos das quatro origens e mostraram claramente que o coagulante e a plasmina foram os responsáveis pela degradação inicial das caseínas. A taxa de degradação da &#945;s1- e &#946;-caseína apresentou a seguinte ordem: D &gt; C &#8805; B &gt; A. O acúmulo de aminoácidos livres também foi mais rápido no queijo D, seguido dos queijos C, B e A. Portanto, a autólise de Lab. helveticus no queijo D acelerou a proteólise, diminuindo o período de maturação em 45% e não afetando negativamente o desenvolvimento de \"flavour\" e nem a textura. No final da maturação (45 dias), os compostos voláteis foram determinados por meio de cromatografia gasosa com espectrometria de massa (CG-MS). Com raras exceções, os queijos das quatro origens continham os mesmos compostos voláteis, embora em quantidades distintas. Os álcoois e ésters foram os compostos majoritários nos queijos A e B e benzaldeído, 3-metil-butanal-2 e hexanal nos C e D. O perfil de textura instrumental (TPA) e a análise sensorial descritiva e quantitativa foram realizados. Os queijos B, C e D apresentaram características mais típicas de queijo Prato, independentemente do fato de que o aroma de manteiga e o sabor doce serem mais acentuados no queijo D. O queijo A foi classificado como tendo as menores características de queijo Prato e apresentou maior nível de defeitos de \"flavour\", principalmente residual e amargor. Os queijos avaliados não apresentaram diferenças significativas quanto à elasticidade e coesividade. Pequenas alterações na composição físico-química dos queijos, principalmente os teores de umidade e de caseína, influenciaram nos parâmetros como a firmeza e a adesividade. O presente estudo demonstrou pela primeira vez a ausência de autólise de Lc. Lacfis sp. em queijo Prato de quatro origens, bem como a ocorrência de autólise de Lab. helveticus nos queijos de duas origens, C e D. A pronunciada autólise dessa espécie teve um impacto positivo na proteólise e foi a responsável pelo aumento da concentração de aminoácidos livres nesses queijos. As diferenças na evolução da proteólise observada entre os queijos C e D, com taxas mais baixas no queijo C, independentemente da autólise pronunciada de Lab. helveticus, foram atribuídas à falta de uniformidade na composição físico-química dos queijos, principalmente pH e os teores de sal na umidade (S/U). / This paper reports a study aimed at evaluating the variations that occur in the interrelationship between autolysis of lactic starter bacteria and the development of proteolysis in Prato cheese produced in four different regions of Brazil: Santa Catarina (Cheese A), Goiás (Cheese B), São Paulo (Cheese C) and Minas Gerais (Cheese D). Quantitative analysis of microbial population yielded similar microbiological profiles for all the cheese samples investigated. After 5 days ripening, lactococci and streptococci were present in higher numbers than mesophilic and thermophilic lactobacilli, leuconostoc and lactate fermenting bacteria. However, the populations of the latter species had considerably increased by the time the ripening process completed 45 days. Enterococci and citrate fermenting bacteria remained present in relatively low numbers throughout ripening. The findings from qualitative analysis confirmed the predominance of non-starter lactic acid bacteria (NSLAB) in the cheeses from four different origins, especially Lactobacillus sp. Other genera of non-starter lactic acid bacteria (NSLAB) were identified in smaller proportions: Enterococcus sp., Pediococcus sp., Aerococcus sp., Tetragenococcus sp. and Streptococcus sp. Cheese C differed from the cheeses in that it no evidence was found of the presence of Pediococcus sp. and Streptococcus sp. The bacteria of the lactic starter culture Lactococcus lactis sp. and Leuconostoc sp. were also found to be present, although in lower numbers. Autolysis was studied by: (1) determination of aminopeptidase activity; (2) detection of autolysins by zymograms and (3) detection of intracellular enzymes by immunoblotting. The release of aminopeptidase was highest in cheese D, followed by C, B and A. No bands of lytic activity were appeared in the zymograms of Cheeses A and B in all conditions evaluated. At pH 7,4 and 44&#176;C, a low-intensity band of 30 KDa was found in cheeses C e D, whereas another low-intensity band was observed only in cheese D. At pH 6,8 and 42&#176;C, bands of 40KDa were observed in cheese C (low intensity) and cheese D (high intensity), in addition to two more low-intensity bands of 90KDa and 110KDa in cheese D. Immunoblotting with antiserum anti-Lc produced only minor signs of positive reaction, evidenced by the formation of low-intensity bands of 100 Kda in cheeses A and B and two high-intensity bands of 75 Kda and 100 Kda in cheeses C and D. Since these were present in smaller numbers to those revealed with crude cytoplasm of Lac. lactis subsp. lactis, it was concluded that autolysis did practically non occur. Immunoblotting with antiserum anti-D-LDH also detected sings of positive reaction in cheeses A and B, whereas in cheeses C e D positive high-intensity signs of 37Kda were found relative to D-LDH protein, indicating lysis of Lab. helveticus. The evolution of proteolysis was determined quantitatively during the ripening process and evaluated on the basis of the following parameters: NS-pH4,6/NT% and NNP/NT% indexes, tyrosine content, electrophoresis (Urea-PAGE) and quantification of free amino acids. No significant differences were found between cheeses A, B, C and D in the ear1y stages of ripening. However, with the on-going fragmentation of proteins during ripening, a gradual increase of the ripening indexes occurred, with the highest values being observed in cheese D, followed by C, B e A. The electrophoretic profiles were similar for the four cheeses investigated and clear1y showed that the clotting agent or milk coagulant and plasmin were responsible for the initial breakdown of the caseins. The degradation rate of Q.sl- and p-casein followed the following order: D &gt; C &#8805; B &gt; A. The buildup of free amino acids was also faster in cheese D, followed by cheeses C, B e A. At the end of the ripening process studied (45 days), the volatile compounds were identified using gas chromatography and mass spectrometry (GC-MS), whereas the instrumental texture profile was measured and evaluated by Texture Profile Analysis (TPA). Cheese samples were evaluated by descriptive and quantitative sensory analysis. With rare exceptions, the cheeses of four different origins contained the same volatile compounds, although in different quantities. Alcohols and esters were the predominant volatile compounds in cheeses A and B and benzaldehyde, 3-methyl-butanal-2 and hexanal in cheeses C and D. Autolysis of Lb. helveticus accelerated proteolysis in cheese D, thereby reducing ripening time by 45% without any negative effect on either flavor or texture development. Cheeses B, C and D exhibited the most typical Prato cheese characteristics, in spite of the fact that the buttery aroma and sweet taste were more pronounced in cheese D. Cheese A was rated as the cheese with the less typical overall Prato cheese profile and was also the one that exhibited the highest degree and number of flavor defects, notably aftertaste and bitterness. The cheeses investigated did not present any significant differences as to elasticity and cohesiveness. Minor changes in the physical-chemical composition of the cheeses - mainly related to the moisture and casein levels - influenced parameters such as firmness and adhesiveness. The present study demonstrates for the very first time the absence of autolysis of Lc. Lactis sp. in Prato cheese from four different origins, as well as the occurrence of autolysis of Lb. helveticus in two of the cheeses analyzed (cheeses C and D). The pronounced autolysis of this species had a positive impact on proteolysis and was responsible for the release of increased quantities of free amino acids in these cheeses. The differences in the evolution of proteolysis observed between cheeses C and D - lower rate of proteolysis in cheese C, in spite of pronounced autolysis of Lb. helveticus - were attributed to poor uniformity of the physical-chemical composition of this cheese, particularly as related to pH and the salt and moisture levels (S/M).

A link between the gut and the brain has been proposed to influence psychiatric disorders. Probiotics have been suggested to modify the gut microbiota and thereby improve autism symptoms in children. Attention deficit hyperactivity disorder (ADHD) has high comorbidity with other neuropsychiatric diagnoses, including autism. This is a follow-up of the first study examining a synbiotic intervention in patients with ADHD (Skott et al., 2019). In the original study, 114 adults participated. In this study, 38 adults were evaluated. The aim was to examine if suggested improvements remained 18 months post treatment. Specifically, if reductions were detected in comorbid autism symptoms, emotional dysregulation or functional impairment. The endpoints were measured using questionnaires: Autism-Spectrum Quotient (AQ), Difficulties in Emotion Regulation Scale (DERS-16) and Weiss Functional Impairment Rating Scale (WFIRS). No Synbiotic2000-specific effect was detected. Synbiotic2000 and placebo improved emotion regulation and life skill-functioning equally well. More research is needed to draw reliable conclusions. / En koppling mellan magen och hjärnan har i studier antytts påverka psykiatriska tillstånd. Probiotika har föreslagits förändra mag- och tarmkanalens bakterieflora och därigenom förbättra psykiatriska symtom hos barn med autism. ADHD har hög komorbiditet med andra neuropsykiatriska diagnoser, däribland autism. Detta är en långtidsuppföljning av RCT-studien som var först med att undersöka en synbiotika-intervention hos patienter med ADHD (Skott et al., 2019). I uppföljningen undersöktes 38 av de 114 vuxna som deltagit i originalstudien. Syftet var att undersöka om indikationerna till förbättring höll i sig 18 månader efter studieavslutet. Frågeställningen var om reduktion i komorbida autismsymtom, svårigheter med emotionsreglering eller funktionsnedsättning kunde identifieras. Detta undersöktes genom självskattningsskalor: Autism-Spectrum Quotient (AQ), Difficulties in Emotion Regulation Scale (DERS-16) och Weiss Functional Impairment Rating Scale (WFIRS). Ingen behandlingsspecifik effekt detekterades. Förbättringar av samma grad identifierades av Synbiotic2000 och placebo, utifrån emotionsreglering samt delskalan färdigheter. Mer forskning på området krävs för att möjliggöra tillförlitliga slutsatser. / BAMBA (Behandla Adhd med MagBakterier)