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Sélection et mise en oeuvre "optimale" de souches microbiennes en bioréacteur, pour la production d'acide hyaluronique

Abstract : This work intended to develop a laboratory scaleproduction process ofhyaluronic acid (HA), a biopolymer of health and cosmetic interest, using a naturally AH producing lactic acid bacterium, Streptococcus zooepidemicus. A literature review allowed to identify the following critical points: firstly, the composition of the culture medium (identification of essential nutrients for microbial growth and synthesis of HA), secondly, the oxygenation level (oxygen transfer and associated redox modifications), and finally, in relation with, the production and metabolism abilities of the considered strains. A preliminary step was dedicated to the developmentorthe improvementof analytical techniques in order todispose of appropriate tools for the monitoring of Streptococcus zooepidemicus metabolism. The quantification of the biomass without considering capsular HA fraction as well as detection of hyaluronidase activity have been developed while other chromatographic and enzymatic methods have been more basically applied to and validated with the substrates and metabolites considered. The laboratory scale cultures of collection (ATCC) as well as “environmental” strains were initially used to formulate a workable cultivation broth and to define suitable culture conditionsfor a use at a larger scale to produce HA. Very positive results were obtained with higher production of HA in comparison with literature assays, while critical influencing factors such as the initial glucose concentration or the oxygenation levelin cultures were highlighted. The influence of these factors was thoroughly studied with bioreactor cultures in both batch and fed-batch modes leading to improved cultivation conditions and culture mode. In parallel, another important step consisted in the highly performance improvement of initially low HA producing "environmental" strains via random mutagenesis. Very promising overproducing mutants have therefore been generated, characterized in their kinetic and metabolic capabilities and long-term stored. At last, one of the best and most reliable mutant has been cultivated with the best previously selected medium composition and operating conditions. Both the HA production level, productivity and size observed validated the findings of this process development work, while helping to identify new improvement domains and strategies
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Pierrick Leblanc. Sélection et mise en oeuvre "optimale" de souches microbiennes en bioréacteur, pour la production d'acide hyaluronique. Alimentation et Nutrition. Université de Lorraine, 2014. Français. ⟨NNT : 2014LORR0228⟩. ⟨tel-01751238⟩

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