Dans les produits laitiers, les cultures bioprotectrices et leurs métabolites représentent une alternative d’intérêt aux conservateurs chimiques pour lutter contre les contaminations fongiques. L’objectif de cette thèse était de développer des ingrédients antifongiques, issus de la fermentation d’un substrat laitier par différents microorganismes, bactéries ou champignons, utilisables dans des produits laitiers variés. Pour ce faire, nous avons dans un premier temps caractérisé la diversité des contaminants fongiques des produits laitiers et de leur environnement afin de sélectionner les cibles fongiques les plus pertinentes pour le reste de notre étude. Ensuite, nous avons criblé, in vitro, l’activité antifongique de fermentats issus de la fermentation de 2 substrats laitiers par 698 souches de bactéries lactiques, propioniques et de champignons grâce à une nouvelle méthode de criblage haut-débit, dans une matrice mimant le fromage. Après optimisation des conditions de fermentation pour améliorer l’activité antifongique de ces fermentats, les plus actifs ont été testés à l’échelle du laboratoire sur des fromages blancs et des fromages à pâte pressée à croûte morgée (PPCM) avant d’être testés à l’échelle pilote dans des crèmes fraiches et des fromages à PPCM. Cette étape nous a permis (i) de valider l’activité antifongique des fermentats en produits réels en mettant en place des challengetests et des tests d’usage et (ii) d’évaluer leur impact sur les qualités organoleptiques des produits grâce à des analyses sensorielles. Les molécules impliquées dans l’activité antifongique ont ensuite été identifiées grâce à différentes méthodes (chromatographie en phase gazeuse ou liquide couplée ou non à la spectrométrie de masse) et l’impact de ces composés sur la croissance des cibles fongiques a été évalué. Le criblage in vitro a permis la sélection de 3 ingrédients antifongiques qui se sont tous révélés actifs in situ. Les tests de durabilité ont révélé que le fermentat issu de Lactobacillus rhamnosus CIRM-BIA 1952 avait une activité prometteuse dans les crèmes fraiches. Pour le fromage à PPCM, le fermentat issu de Propionibacterium jensenii CIRM-BIA 1774, qui retarde jusqu’à 16 jours la croissance de Mucor racemosus et Penicillium commune pourrait être utilisé à la place de la natamycine. La caractérisation des composés antifongiques dans les fermentats a mis en évidence certains acides organiques, des acides gras, des composés volatils et un peptide qui, ensemble, jouent très probablement un rôle dans l’activité antifongique de ces fermentats. Nous avons enfin montré l’effet fongistatique du fermentat le plus actif sur Rhodotorula mucilaginosa UBOCC-A-216004 et Mucor racemosus UBOCC-A-109155 ainsi que son impact sur la physiologie des spores de M. racemosus. L’ensemble de ces résultats devrait donc conduire au développement de nouveaux ingrédients laitiers antifongiques qui pourraient remplacer avantageusement les conservateurs dans les produits laitiers, ils apportent en outre des éléments d’information sur les molécules et les mécanismes mis en œuvre dans l’inhibition antifongique. / In dairy products, bioprotective cultures and their metabolites represent an interesting alternative to chemical preservatives. The aim of this PhD thesis was to develop antifungal dairy ingredients, derived from the fermentation of dairy substrates by different microorganisms, bacteria and fungi, which could be used in various dairy products. To do so, we first studied the diversity of spoilage fungi in a large variety of dairy products and their environment in order to select the most appropriate fungal targets. Then, we screened the in vitro antifungal activity of fermentates obtained from the fermentation of 2 dairy substrates by 698 lactic acid bacteria, propionibacteria and fungal strains using a novel high-throughput method in a cheese mimicking model. After optimizing fermentation conditions to enhance antifungal activity, the most active fermentates were tested at a lab scale in semi-hard and fresh cheese before evaluation at a pilot scale in semi-hard cheese and sour cream. This step allowed us (i) to validate the fermentate antifungal activities in real products using challenge- and durability-tests and (ii) to evaluate their impact on the products’ organoleptic properties using sensorial analysis. Antifungal molecules were then identified using different analytical methods (high performance liquid chromatography, gas chromatography coupled or not with mass spectrometry) and impact of these compounds on fungal growth was studied. The in vitro screening allowed selecting 3 antifungal ingredients that were also active in situ. Durability tests revealed that Lactobacillus rhamnosus CIRMBIA 1952 fermentate had a promising activity in sour cream. In semi-hard cheese, Propionibacterium jensenii CIRMBIA 1774 fermentate, which delayed Mucor racemosus and Penicillium commune growth for up to 16 days, could be used instead of natamycin. Antifungal compounds present in fermentates consisted of organic acids, free fatty acids, volatile compounds and peptides which altogether might play a role in the antifungal activity of these fermentates. Finally, the fungistatic effect of P. jensenii CIRM-BIA 1774 fermentate against R. mucilaginosa UBOCC-A-216004 and M. racemosus UBOC-A-109155 was demonstrated, as well as its impact on the physiology of M. racemosus spores. Together, these results should lead to the development of new antifungal dairy ingredients which could replace preservatives in dairy products. Finally, these results gave us new information concerning antifungal molecules and their action mechanisms.

Made available in DSpace on 2014-06-11T19:30:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-12Bitstream added on 2014-06-13T19:00:58Z : No. of bitstreams: 1 murakami_mt_dr_sjrp.pdf: 4603266 bytes, checksum: 354af33ced476bc60fd37bac536c3729 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / This work presents some features of essential biological processes such as the haemostatic system, integrity of biological membranes and thermostability of proteins. Crystallographic, spectroscopic and in silico tools have been used to obtain information at the molecular level of macromolecular complexes, action mechanisms and inhibition pathways. Worms, snakes, ticks, leeches and spiders produce a variety of proteins, which interfere in the regulation of these systems. Different toxins isolated from these organisms were characterized providing necessary information for the development of a new anti-myonecrotic molecule and reveal a new factor Xa exosite that is important for macromolecular substrates recognition and inhibition. The first crystal structure of a member of the sphingomyelinases D family was determined by the quick cryo-soaking technique and the catalytic mechanism was proposed, which involves a magnesium-binding site and two catalytic histidines. An alternative activation of the protein C pathway that does not require thrombomodulin was structurally characterized and revealed the dual role of the elestrotatic surface charge around the active site and the three strategically positioned carbohydrate moieties in the approach, recognition and activation of protein C.

Orientador: Raghuvir Krishnaswamy Arni / Banca: Antônio Carlos Martins Camargo / Banca: Nilson Ivo Tonin Zanchin / Banca: Beatriz Gómez Guimarães / Banca: Roberto da Silva / Abstract: This work presents some features of essential biological processes such as the haemostatic system, integrity of biological membranes and thermostability of proteins. Crystallographic, spectroscopic and in silico tools have been used to obtain information at the molecular level of macromolecular complexes, action mechanisms and inhibition pathways. Worms, snakes, ticks, leeches and spiders produce a variety of proteins, which interfere in the regulation of these systems. Different toxins isolated from these organisms were characterized providing necessary information for the development of a new anti-myonecrotic molecule and reveal a new factor Xa exosite that is important for macromolecular substrates recognition and inhibition. The first crystal structure of a member of the sphingomyelinases D family was determined by the "quick cryo-soaking" technique and the catalytic mechanism was proposed, which involves a magnesium-binding site and two catalytic histidines. An alternative activation of the protein C pathway that does not require thrombomodulin was structurally characterized and revealed the dual role of the elestrotatic surface charge around the active site and the three strategically positioned carbohydrate moieties in the approach, recognition and activation of protein C. / Doutor