Improving probiotic viability and functionality by bioreactors engineering

COMMUNAUTE FRANCAISE DE BELGIQUE ACADEMIE UNIVERSITAIRE WALLONIE-EUROPE UNIVERSITE DE LIEGE – GEMBLOUX AGRO-BIO TECH Improving probiotic viability and functionality by bioreactors engineering Hữu-Thanh NGUYỄN Dissertation presented in total fulfilment of the requirement for the degree of PhD in Agricultural sciences and Biological Engineering Promoteurs: Prof Frank Delvigne Prof Philippe Thonart 2016 COMMUNAUTE FRANCAISE DE BELGIQUE ACADEMIE UNIVERSITAIRE WALLONIE-EUROPE UNIVERSITE DE LIEGE – GEMBLOUX AGRO-BIO TECH Amélioration de la viabilité et fonctionnalité des probiotiques en utilisant les génies des bioréacteurs Hữu-Thanh NGUYỄN Essai présenté en vue de l’obtention du grade de docteur en sciences agronomiques et ingénierie biologique Promoteurs: Prof Frank Delvigne Prof Philippe Thonart 2016 Copyright Aux termes de la loi belge du 30 juin 1994, sur le droit d'auteur et les droits voisins, seul l'auteur a le droit de reproduire partiellement ou complốtement cet ouvrage de quelque faỗon et forme que ce soit ou d'en autoriser la reproduction partielle ou complète de quelque manière et sous quelque forme que ce soit Toute photocopie ou reproduction sous autre forme est donc faite en violation de la dite loi et des modifications ultérieures Huu-Thanh NGUYEN (2016) Improving probiotic viability and functionality by bioreactors engineering (PhD thesis) Gembloux, Belgium, University of LiègeGembloux Agro-Bio Tech, 219 pages, tables, 32 figures Abstract: There is a growing market for probiotic bacteria, but their production is still the subject to technical limitations, i.e susceptibility to the stress conditions found during upstream and downstream operations In this work, we have investigated the effect of cultivation and drying conditions on Bifidobacterium bifidum MG 25628, a probiotic bacteria being particularly sensitive to bioprocessing conditions Previous results have shown that microbial physiology, and the ability of microbes to cope with stress, is dependent of the growth rate and the growth phase It is also known that the preliminary exposure of microbial cells to sub-lethal stress enhance its robustness Accordingly, the effect of the exposure of B bifidum in a two-compartment reactor designed in order to expose the strain to different temperature conditions was investigated We found that the exposure of B bifidum at 42°C for 1h at the onset of the stationary phase enhanced significantly survival after freeze-drying It appeared that the increase in cell survival was attributed to the induction of the synthesis and an exopolysaccharide layer surrounding the cells The method involving the exposure of the strain to sub-lethal temperature stress was further successfully scaled-up to a bioreactor volume of 2000 L EPS synthesis can also be stimulated by sparing the bioreactor with carbon dioxide In this context, we investigated the EPS yield in two specific bioreactor designs for the intensification of the CO2 gas-liquid mass transfer, i.e a trickle bed and a falling-film microreactor Depending of the operating conditions, these two bioreactor configurations led to a significant improvement in EPS synthesis (around 21 g/L) Extensive comparative proteomic analysis confirmed the impact on CO2 mass transfer on cell physiology, notably by enhancing the intracellular concentration of two key enzymes implied in carbonate uptake, i.e phosphoenolpyruvate carboxylase and carbamoyl phosphate synthase Taken altogether, these results point out that biochemical engineering parameters can be used as a very efficient strategy for improving probiotic robustness Additionally, this nonGMO approach is more suited to the consumer expectations Huu-Thanh NGUYEN (2016) Amélioration de la viabilité et fonctionnalité des probiotiques en génie des bioréacteurs (Thèse de doctorat) Gembloux, Belgique, Université de Liège – Gembloux Agro-Bio Tech, 219 pages tables 32 figures Résumé : Malgré des technologies de production limitées (e.g sensibilité aux stress engendrés par les conditions de production), le marché des bactéries probiotiques est en croissance continue Au cours de cette thèse, nous avons évalué l’impact des conditions de culture et de séchage sur la souche probiotique Bifidobacterium bifidum MG 25628 particulièrement sensible ces conditions Les résultats précédents ont mis en évidence l’influence du taux de croissance et de la phase de croissance sur la physiologie et la résistance au stress de ce type de bactérie Il est également accepté que l’exposition préalable de cellules microbiennes un stress non létal augmente leur robustesse Sur base de ces affirmations, un rộacteur bicompartimentộ a ộtộ conỗu afin dexposer B bifidum des conditions de températures oscillantes Les différentes expérimentations ont mis en évidence une augmentation significative de la viabilité post lyophilisation suite une exposition durant 1h en début de phase stationnaire 42°C Le bénéfice d’une telle approche a été confirmé dans des volumes plus importants, et notamment en bioréacteur de 2000 L La synthèse d’une couche d’exopolysaccharide semble responsable de cette augmentation La synthèse d’exopolysaccharide peut également être stimulée en enrichissant la phase liquide en dioxyde de carbone Dans ce contexte, nous avons mis au point deux approches permettant d'intensifier le transfert du CO2 de la phase liquide vers la phase gazeuse au sein du réacteur Les rendements d’EPS obtenus au sein d’un microréacteur flux-tombant et d’un réacteur lit ruisselant ont été étudiés Les deux systèmes ont induit une amélioration significative de la biosynthèse d’EPS (environ 21 g/L) De plus, les analyses protéomiques ont confirmé l’impact de l'intensification du transfert de masse du CO2 sur la physiologie cellulaire Une augmentation des concentrations intracellulaires d'enzymes clés impliquées dans les bioréactions de consommation de carbonate chez B bifidum a ainsi été mesurée Il s’agit plus particulièrement des enzymes phosphoenolpyruvate carboxylase et carbamoyl phosphate synthase En conclusion, nos résultats ont souligné que des paramètres couramment utilisés en génie biochimique peuvent être la base de stratégies efficaces visant améliorer la robustesse des bactéries probiotiques En outre, cette approche n'impliquant pas d'OGM est plus adaptée l’attente des consommateurs List of scientific productions The following scientific productions have been achieved during this Ph.D thesis: VII.1 Publications Nguyen HT, Razafindralambo H, Blecker C, N’Yapo C, Thonart P, Delvigne F (2014) Stochastic exposure to sub-lethal high temperature enhances exopolysaccharides (EPS) excretion and improves Bifidobacterium bifidum cell survival to freeze–drying Biochemical Engineering Journal 88:85-94 doi:http://dx.doi.org/10.1016/j.bej.2014.04.005 Nguyen HT, Razafindralambo H, Blecker C, N’Yapo C, Thonart P, Delvigne F (2015) Scalable temperature induced stress for the large-scale production of functionalized Bifidobacteria Journal of industrial microbiology & biotechnology:17 doi:http://dx.doi.org/10.1007/s10295-015-1650-5 Nguyen HT, Ayikpe LH, Delepierre A, Truong DH, Bosquee E, Francis F, Delvigne F (2016) Effect of carbon dioxide intensification on exopolysaccharide production and survival of Bifidobacterium bifidum after freeze-drying in preparation for Applied Microbiology and Biotechnology Journal Nguyen HT, Truong TH, Kouhoundé S, Ly S, Delvigne F (2016) A review of biochemical engineering approaches for increasing viability and functionality of probiotics accepted by International journal of Molecular Science 203 VII.2 Posters Nguyen HT, Delvigne F (2015) Effects of carbon dioxide degassing on the exopolysaccharide secretion and the survival of Bifidobacterium bifidum after freeze-drying The 6th Congress of European Microbiologists from June 21-25th, Maastricht, Netherlands Nguyen HT, Brognaux A,Razafindralambo H, Thonart P, Delvigne F (2014) Sublethal temperature increases exopolysaccharide secretion of Bifidobacterium bifidum and improves their survival to freeze-drying The 10th European Symposium on Biochemical Engineering Sciences (10th ESBES) and the 6th International Forum on Industrial Bioprocesses (6th IFIBiop), from September 7th -10th, Lille, France Nguyen HT, Razafindralambo H, Thonart P, Delvigne F (2014) Role of exopolysaccharide excretion on Bifidobacterium bifidum survival after heat stress 19th National symposium on applied biological sciences, Gembloux, Belgium Nguyen HT, Razafindralambo H, Blecker C, N’Yapo C, Thonart P, Delvigne F (2013) Exopolysascharide formation to improve the viability of Bifidobacterium bifidum LMG 25628 by Heat shock scale-down reactor (HS-SDR) GEPROC November 15th, Liege, Belgium Nguyen HT, Razafindralambo H, Blecker C, Thonart P, Delvigne F (2013) Stochastic exposure to heat shock improves Bifidobacterium bifidum viaiblity and cell recovery during freeze drying The 5th Congress of European Microbiologists from July 21-25th, Leipzig, Germany 204 Nguyen HT, Thiry C, Destain J, Thonart P, Delvigne F (2013).The effect of thermal stresses on the viability of post-centrifugation and post-freeze drying and membrane fatty acids composition of Bifidobacterium bifidum in bioreactors: a scale-down approach Probiotics and Their Applications Conference From May 31th – June 1th, Hanoi, Vietnam Nguyen HT, Razafindralambo H, Thiry C, Destain J, Thonart P, Delvigne F (2013) Effect of thermal stresses on the viability and zeta potential of Bifidobacterium bifidum in bioreactors: a scale-down approach (2013) The 18th Symposium on Applied Biological Sciences February 8th, Ghent, Belgium 205 List of figures 207 Chapter I Figure 1: Diagram of methodology of research use for improving the viability and understanding the alteration of physiological and biochemical characters of B.bifidum on different sub-lethal stress treatments 32 Chapter II Figure Fermentation systems used for improving the viability of probiotics 48 Figure Effect of sub-lethal heat………………….…………………………………… 59 Figure Diagram showing the biosynthetic pathways involved for the production of exopolysaccharides from lactose, fructose, galactose and glucose by lactic acid bacteria …………………………………………………………………………………… 63 Chapter III Graphic abstract 84 Figure 1: evolution of the amount of colony forming units (cfu) during cultures performed in the different bioreactor setups 98 Figure 2: evolution of the glucose concentration and the main metabolites during the culture of B bifidum in the three bioreactor setups 99 Figure 3: A: real time flow cytometry detection of fluorescent microbeads at the outlet of the recycle loop of the TCB 101 Figure 4: stochastic simulations of the temperature profile experienced at the single cell level… ………………………………………………………………………………… 103 Figure 5: evolution of the different subpopulations of microbial cells labeled with PI and cFDA……………… 106 Figure 6: evolution of the cell size and the zeta potential determined by dynamic light scatter analysis 108 Figure 7: evolution of the survival rate, i.e the percentage of cfu recovery before and after treatment 110 Figure 8: comparative analysis of EPS produced by B.bifidum LMG 25628 grown either in HS-TCB (solid bars) or TCB (open bars) 113 209 Chapter IV Figure : evolution of the time required to shift from 37°C to 42°C in stirred tank bioreactor according to the operating scale 135 Figure Evolution of cell viability (cfu counting expressed in log10 scale) according to the different bioreactor operating scales 137 Figure 3: excretion of EPS according to the different bioreactor operating scales investigated and operated with (Stressed S) or without (Non stressed NS) sub-lethal heat stress 138 Figure 4: evolution of the S/V ratio during scale-ups, the surface S representing the section of the bioreactor and the volume V being the working volume 140 Figure 5: Effect of carbon dioxide on A : cell viability and B : EPS production 141 Figure 6: Composition of EPS collected in 200L and 2000L bioreactors operating with (S) and without heat stress (NS) 142 Chapter V Figure.1 The evolution of potential redox by using the different level of intensive gases…………………………………………………………………………………………………………………………………….167 Figure 2: Cell concentration of B bifidum LMG 25628 as a function of N2 and CO2 intensification in various bioreactors 168 Figure.3 Evolution of survival rates compared between the CO2 intensification bioreactors/reference bioreactors 169 Figure.4 Evolution of cells concentration according to time of CO intensification ………………………………………………………………………………………………………………………………………….170 Fig.5 Exopolysaccharides secretion in the difference CO2 intensification modes 171 Figure 6A Upregulated and downregulated protein present in cells with CO2 intensification and reference 174 Figure 6B Upregulated and downregulated protein present in supernatants of culture with CO2 intensification and reference 175 Figure Comparative proteomic expression in cells under intensified CO2 and reference 176 Figure.8 Comparative proteomic expression in supernatant under intensified carbon dioxide vs Reference 177 210 Chapter VI Figure.1 Influence of environmental stress factors to EPS production from B.bifidum ………………………………………………………………………………………………………………………………………….191 Fig Alteration of EPS components under temperature sub lethal stresses 192 Fig 3A Relationship between viability and EPS total 193 Fig 3B Relationship between the viability and attached EPS…….…… ………… 194 211 List of Tables 213 List of Tables Chapter II Table 1: Effect of sub-lethal temperature conditions on survival and heat tolerance to lethal temperature in Lactobacillus strains during the exponential growth phase………………………………………………… ……………………………………….52 Chapter V Table 1: Geometry, operating conditions and effectiveness parameters for the different bioreactor set up used in this work…………………………………………………… ……131 Table 2: comparative analysis of cell viability obtained for all the operating scales investigated during this work………………………………… ……….……………………144 Chapter VI Table 1: Technical features of carbon dioxide flow rate for intensifying the CO in bioreactors………… ……………………………………………………………………….161 215 Annex 217 ... dite loi et des modifications ultérieures Huu-Thanh NGUYEN (2016) Improving probiotic viability and functionality by bioreactors engineering (PhD thesis) Gembloux, Belgium, University of LiègeGembloux... biochemical engineering approaches for increasing viability and functionality of probiotics” Nguyen HT, Truong DH, Kouhoundé S, Ly S, Razafindralambo H and Delvigne F (2016) accepted by international... of probiotics on health Probiotic consumption levels are rising quickly in the world despite the fluctuation of their viability and functionality Technological methods aiming at improving probiotic