Lau, Shong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 124-129). / Abstracts in English and Chinese. / Abstract of thesis --- p.i / 碩士論文摘要 --- p.iii / Acknowledgement --- p.iv / Declaration --- p.v / Abbreviations --- p.vi / Genetic symbols --- p.viii / Table of content --- p.ix / List of figures --- p.xiii / List of tables --- p.xv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Thesis outline --- p.1 / Chapter 1.2 --- A. chrysogenum --- p.3 / Chapter 1.3 --- Antibiotic industry --- p.4 / Chapter 1.4 --- Cephalosporins --- p.4 / Chapter 1.5 --- CPC biosynthetic pathway --- p.5 / Chapter 1.6 --- DAC --- p.8 / Chapter 1.7 --- Aims of study --- p.10 / Chapter Chapter 2 --- Construction of transformation cassettes --- p.11 / Chapter 2.1 --- Introduction --- p.11 / Chapter 2.2 --- Materials and Methods --- p.13 / Chapter 2.2.1 --- Construction of cassette RTCAH --- p.13 / Chapter 2.2.1.1 --- Cultivation of R. toruloides --- p.13 / Chapter 2.2.1.2 --- Preparation of R. toruloides genomic DNA --- p.13 / Chapter 2.2.1.3 --- PCR of R. toruloides CAH gene --- p.14 / Chapter 2.2.1.4 --- Construction of pRTCAHhyr --- p.16 / Chapter 2.2.2 --- Construction of cassette GHG --- p.17 / Chapter 2.3 --- Results and Discussion --- p.18 / Chapter 2.3.1 --- pGHG construction --- p.18 / Chapter 2.3.2 --- pRTCAHhyr construction --- p.18 / Chapter Chapter 3 --- Optimization of integrative transformation of A. chrysogenum --- p.22 / Chapter 3.1 --- Introduction --- p.22 / Chapter 3.2 --- Materials and Methods --- p.24 / Chapter 3.2.1 --- Strain and culture medium --- p.24 / Chapter 3.2.2 --- Reagents --- p.24 / Chapter 3.2.3 --- Standard transformation procedures --- p.25 / Chapter 3.2.3.1 --- Cell cultivation --- p.25 / Chapter 3.2.3.2 --- Protoplast preparation --- p.25 / Chapter 3.2.3.3 --- PEG mediated protoplast fusion --- p.27 / Chapter 3.2.4 --- Examination of transformation parameters --- p.28 / Chapter 3.3 --- Results and Discussion --- p.29 / Chapter 3.3.1 --- Cell growth period --- p.30 / Chapter 3.3.2 --- DNA concentration --- p.32 / Chapter 3.3.3 --- PEG molecular weight --- p.35 / Chapter 3.3.4 --- Transformation additives --- p.37 / Chapter 3.3.5 --- Modified transformation protocol --- p.39 / Chapter Chapter 4 --- Metabolic engineering of A. chrysogenum --- p.42 / Chapter 4.1 --- Introduction --- p.42 / Chapter 4.2 --- Materials and Methods --- p.43 / Chapter 4.2.1 --- Transformation of A. chrysogenum --- p.43 / Chapter 4.2.2 --- Screening of transformants --- p.43 / Chapter 4.2.3 --- HPLC analysis --- p.43 / Chapter 4.2.4 --- A. chrysogenum genomic DNA preparation --- p.44 / Chapter 4.2.5 --- Genotyping by PCR --- p.45 / Chapter 4.2.5.1 --- GHG transformants --- p.45 / Chapter 4.2.5.2 --- RTCAH transformants --- p.47 / Chapter 4.2.6 --- Genotyping of GHG transformants by Southern hybridization --- p.47 / Chapter 4.2.6.1 --- Preparation of DIG-labeled GHG probe by PCR --- p.48 / Chapter 4.2.6.2 --- PCR preparation of DIG-labeled Cef-EF probe --- p.48 / Chapter 4.2.6.3 --- Restriction digestion of genomic DNA of GHG transformants --- p.49 / Chapter 4.2.6.4 --- Agarose gel electrophoresis and DNA transfer to positively charged nylon membrane --- p.50 / Chapter 4.2.6.5 --- Pre-hybridization and hybridization --- p.52 / Chapter 4.2.6.6 --- Membrane washing and blocking --- p.52 / Chapter 4.2.6.7 --- Membrane detection --- p.53 / Chapter 4.2.7 --- Genotyping of RTCAH transformants by Southern hybridization --- p.53 / Chapter 4.2.7.1 --- PCR preparation of DIG-labeled RTCAH probe --- p.54 / Chapter 4.2.7.2 --- Restriction digestion of genomic DNA of RTCAH transformants --- p.54 / Chapter 4.2.7.3 --- Agarose gel electrophoresis and Southern hybridization of RTCAH transformants --- p.55 / Chapter 4.3 --- Results and Discussion --- p.56 / Chapter 4.3.1 --- Hygromycin B screening and HPLC analysis of GHG transformants --- p.56 / Chapter 4.3.2 --- Genotyping of GHG232 by PCR --- p.56 / Chapter 4.3.3 --- Genotyping of GHG232 by Southern hybridization --- p.58 / Chapter 4.3.4 --- Hygromycin B screening and HPLC analysis of RTCAH transformant --- p.61 / Chapter 4.3.5 --- Genotyping of RTCAH transformants by PCR --- p.61 / Chapter 4.3.6 --- Genotyping of RTCAH transformants by Southern hybridization --- p.63 / Chapter Chapter 5 --- Characterization of modified strains and fermentation study --- p.65 / Chapter 5.1 --- Introduction --- p.65 / Chapter 5.2 --- Materials and Methods --- p.67 / Chapter 5.2.1 --- Comparison of DAC production in GHG232 and RTCAH transformants by small-scale fermentation --- p.67 / Chapter 5.2.2 --- CPC conversion assay --- p.67 / Chapter 5.2.3 --- Evaluation of fermentation media --- p.68 / Chapter 5.2.4 --- Factorial design for medium formulation --- p.68 / Chapter 5.3 --- Results and Discussion --- p.71 / Chapter 5.3.1 --- Evaluation of DAC production profiles of GHG232 and RTCAH transformants --- p.71 / Chapter 5.3.2 --- CPC conversion assay --- p.79 / Chapter 5.3.3 --- Medium formulation --- p.81 / Chapter 5.3.4 --- Factorial design for medium formulation --- p.85 / Chapter 5.3.4.1 --- CSL and NH4OAc --- p.85 / Chapter 5.3.4.2 --- CaC03 --- p.91 / Chapter 5.3.4.3 --- Sucrose --- p.94 / Chapter 5.3.4.4 --- Starch and soy oil --- p.97 / Chapter 5.3.4.5 --- Methionine --- p.99 / Chapter Chapter 6 --- Conclusive remarks --- p.101 / Appendix: Study of reusability of commercial plasmid extraction kit --- p.103 / Chapter A1 --- Introduction --- p.103 / Chapter A2 --- Materials and Methods --- p.105 / Chapter A2.1 --- Preparation of competent E. coli DH5a cells --- p.105 / Chapter A2.2 --- Transformation and cultivation ofE. coli DH5a cells --- p.106 / Chapter A2.3 --- Column and solutions for plasmid DNA preparation --- p.107 / Chapter A2.4 --- Plasmid preparation --- p.108 / Chapter A2.5 --- Regeneration and storage of DNA extraction column --- p.109 / Chapter A2.6 --- Yield and quality assessment of the prepared plasmid DNA --- p.109 / Chapter A3 --- Results and Discussion --- p.111 / Chapter A3.1 --- Plasmid DNA yield and purity comparison --- p.111 / Chapter A3.2 --- Column regeneration and EtOH storage --- p.111 / Chapter A3.2.1 --- DNA yield after column regeneration --- p.111 / Chapter A3.2.2 --- Purity of plasmid DNA --- p.112 / Chapter A3.2.3 --- Restriction digestion --- p.117 / Chapter A3.2.4 --- E. coli DH5α cells transformation --- p.121 / Chapter A4 --- Conclusive remarks --- p.123 / Bibliography --- p.124

Microaerobic fermentation (MF) is a process of controlled degradation of organic waste material that occurs in enclosed fermentors under micro-aerobic conditions at near-room temperature. MF processing of vegetal materials progresses to endpoints in about 2-5 weeks. During MF processing, an acidic leachate rich in organic acids and alcohols is produced. The research presented in this thesis focuses on the efficiency of MF pre-processing of feedstock containing fibrous lignocellulosic (FLC) materials; efficiency of microbial and insect larvae-based treatments of MF leachate; tolerance of the Black Soldier fly larvae (BSFL) to various biological inhibitors common in leachate; and effectiveness of using MF and BSFL solid and liquid processing products as agricultural fertilizers. Results indicate that MF is unsuitable for pre-processing of FLC materials. Enhanced MF leachate treatment may increase efficiency of FLC processing though. Leachate can be efficiently treated using BSFL which decrease overall leachate toxicity. BSFL are able to tolerate increased levels of many of the biological inhibitors within the leachate including ethanol, acetate, pH extremes and temperature. MF solid residues increased corn plant growth when amended into soil, but residues resulting from BSFL processing of solid organics stunted corn plant growth. Short-term phytotoxicity of MF leachate was eliminated by diluting it 10 - 10,000 times or through BSFL processing. It can be concluded that MF processing of organics is beneficial for producing solid soil amendments from non-FLC materials and that dilution or BSFL treatment of MF leachate leads to a beneficial liquid fertilizer.

Etude des interactions entre les levures non-saccharomyces et Saccharomyces cerevisiae. Cette étude a pour objectif de décrire les changements du profil aromatique du vin du vin d'une part et de réduire le pourcentage d'alcool d'autre part. Pour cela, la maîtrise des levures non-Saccharomyces est nécessaire. Ainsi, les principales molécules aromatiques seront dosées en fonction des conditions de cultures (culture pure - culture séquentielle) et leurs conséquences sur les propriétés organoleptiques seront évaluées. En parallèle, leur besoins en sucre, azote, oxygène et vitamines seront déterminés dans la perspective de diminuer le pourcentage d'alcool dans les vins. / Study of the interaction between the non-Saccharomyces yeast and Saccharomyces cerevisiae. This study aims to describe changes in the flavor profile of the wine on one hand and reducing percentage of alcohol on the other hand. For this, control of non-Saccharomyces yeast is required. Thus, the main aromatic molecules must be measured according to the conditions of cultures (pure culture - sequential culture) and their effects on the organoleptic properties will be evaluated. In parallel, their sugar, nitrogen, oxygen and vitamins requirements will be determined to decrease the percentage of alcohol in the wine.

Via mixed-culture fermentation, the MixAlcoTM produces carboxylic acids, which are chemically converted into industrial chemicals and hydrocarbon fuels. Using pilot fermentation data, The Continuum Particle Distribution Model (CPDM) overestimated acid concentration (30–90% error) but more closely estimated conversion (<15% error). Incorporating the effect of air into the model reduced the absolute error of all predictions by >50%. To analyze fermentation data with semi-continuous streams, the Slope method calculates the average flowrate of material from the slope of the moving cumulative sum with respect to time. Although the Slope method does not significantly improve accuracy, it dramatically reduces error compared to traditional techniques (>40% vs. <2%). Nutrients are essential for microbial growth and metabolism. For a four-bottle fermentation train, five nutrient contacting patterns (single-point nutrient addition to Fermentors F1, F2, F3, F4, and multi-point parallel addition) were investigated. Compared to the traditional nutrient contacting method (all nutrients fed to F1), the near-optimal feeding strategies improved exit yield, culture yield, process yield, exit acetate-equivalent yield, conversion, and total acid productivity by approximately 31%, 39%, 46%, 31%, 100%, and 19%, respectively. To estimate nitrogen concentration profiles, a segregated-nitrogen model uses separate mass balances for solid- and liquid-phase nitrogen; the nitrogen reaction flux between phases is assumed to be zero. Using five fermentation trains, each with a different nutrient contacting pattern, the model predictions capture basic behavior; therefore, it is a reasonable tool for estimating and controlling nitrogen profiles. To determine the optimal scenario for mixed-acid fermentations, an array of batch fermentations was performed that independently varied the C/N ratio and the blend of carbohydrate (office paper) and nutrient (wet chicken manure (CM)). Reactant was defined as non-acid volatile solids (NAVS). C/N ratios were based on non-acid carbon (CNA). A blend of 93% paper and 7% wet CM (dry basis) with a C/N ratio of 37 g CNA/g N had the highest culture yield (0.21 g acidproduced/g NAVSinitial), total acid productivity (0.84 g acidproduced/(Lliq·d)), and conversion (0.43 g NAVSconsumed/g NAVSinitial).

Viren haben als Ursache lebensmittelassoziierter Infektionen eine große Bedeutung. Sie können vor allem über rohe oder unzureichend erhitzte Lebensmittel übertragen werden. In diesem Zusammenhang werden grüner Salat, Erdbeeren, Himbeeren, Frühlingszwiebeln, Muscheln, halbgetrocknete Tomaten, fäkal verunreinigtes Trinkwasser, Backwaren und Rohwürste als häufige Infektionsquellen genannt. Vor allem kurzgereifte Rohwürste gehören aus mikrobiologischer Sicht zu Risikoprodukten. Um eine gleichbleibende Qualität der Produkte zu gewährleisten, ist die Verwendung von Starterkulturen unerlässlich. Als sogenannte Schutzkulturen sollen sie gleichzeitig die Vermehrung unerwünschter bakterieller Pathogene unterbinden. Bisher ist allerdings nicht bekannt, inwieweit diese zur Virusinaktivierung in kurzgereiften Rohwürsten führen bzw. beitragen. Aus diesem Grund war es das Ziel dieser Arbeit, den Einfluss von rohwurstrelevanten Starter- und Schutzkulturen sowie deren Metabolite (Bacteriocine, Milchsäure) auf die Tenazität und Inaktivierungskinetik von Viren zu prüfen. Die Untersuchungen erfolgten mit dem murinen Norovirus (MNV) S 99 sowie dem humanen Influenzavirus H1N1 (A/WSN/33). Antivirale Effekte wurden zum einen anhand von in-vitro-Studien, zum anderen anhand von experimentell mit Viren kontaminierten kurzgereiften Rohwürsten (Mettwurst/Teewurst) geprüft. Die Bacteriocine Sakacin A und Nisin zeigten in phosphatgepufferter Salzlösung (PBS) keine viruzide Wirkung gegenüber MNV S 99 und H1N1 (pH 6,2; 24 °C; Exposition: 3 Tage). Weiterhin wurden anhand von in-vitro-Untersuchungen 29 verschiedene zellfreie Kulturüberstände [Milchsäurebakterien, Staphylococcus spp. (S.), Kocuria (K.) varians] hinsichtlich ihrer antiviralen Wirkung geprüft. Dabei konnte eine signifikante Titerreduktion von MNV S 99 bei Exposition mit dem Kulturüberstand eines Lactobacillus (Lb.) curvatus-Isolates festgestellt werden (p < 0,05). In mit dieser Kultur fermentiertem Tee- und Mettwurstbrät zeigte sich jedoch kein Effekt. Die Virustenazität von H1N1 und MNV S 99 konnte mit D,L-Milchsäure unter rohwurstrelevanten Bedingungen (pH 5,0 bis 6,2) sowohl in-vitro als auch im frischen Mettwurstbrät beeinflusst werden. In-vitro erzielte Titerreduktionen lagen bei 2,5 (H1N1) bzw. 3,25 log-Stufen (MNV S 99) nach drei Tagen (24 °C) Lagerung. Im Gegensatz dazu war MNV S 99 im Vergleich zu H1N1 im Mettwurstbrät stabiler. H1N1 konnte unterhalb von pH 5,5 bereits direkt nach dem Einmischen der Influenzaviren in das Wurstbrät nicht mehr nachgewiesen werden. MNV S 99 wurde hingegen erst nach einem Tag Lagerung (22 °C) maximal um 0,7 log-Stufen reduziert (pH 5,2). Die verwendeten Starter- und Schutzkulturen (Lb. sakei, Lb. curvatus, Lb. paracasei, Lb. plantarum, S. carnosus, S. xylosus, K. varians) zeigten im Mett- und Teewurstbrät im Vergleich zur Kontrolle (ohne Starterkultur) keinen zusätzlichen viruziden Effekt auf MNV S 99. Zunehmende Virustiterreduktionen konnten mit pH-Wert-Erniedrigung beobachtet werden. Nach der Reifung (1 Tag, 22 °C, pH 4,9) von Mettwurst mit Starterkulturen wurde das Virus um maximal 1,65 log-Stufen reduziert. In mit Einzel- beziehungsweise Mehrstamm-Mischkulturen fermentierter Teewurst (7 Tage, 22 °C, pH 4,9) betrug die Titerreduktion maximal 1,10 log-Stufen. Das Influenzavirus H1N1 konnte im Rohwurstbrät mit Starterkulturen auch nach Verwendung hoher Ausgangstiter bereits zu Beginn der Untersuchungen nicht mehr nachgewiesen werden. Aus den erzielten Daten kann geschlussfolgert werden, dass die Bacteriocine Sakacin A und Nisin nicht als antivirale Zusatzstoffe in Lebensmitteln (z. B. Rohwürste) geeignet sind. Das antivirale Potential von zellfreien Kulturüberständen war Bakterienstamm-spezifisch und nur in-vitro ersichtlich. Daher muss die Nutzung des Lb. curvatus 1-Stammes nicht anderen rohwurstrelevanten Starterkulturen vorgezogen werden. Die Verwendung von Milchsäure als Zusatzstoff im Rohwurstbrät eignet sich nur zum Ausschluss einer viralen Exposition im Zusammenhang mit H1N1. Frische Mettwurst muss allerdings hierzu adäquat gesäuert (pH < 5,5) werden. Neben dem antiviralen Effekt durch gebildete Säure, konnte keine weitere spezies-spezifische antivirale Wirkung verwendeter Starter- und Schutzkulturen auf MNV S 99 festgestellt werden. Die Säureleistung einzelner Kulturen ist demzufolge für eine Virusinaktivierung entscheidend. Das antivirale Potential verwendeter Starter- und Schutzkulturen in Rohwürsten ist im Zusammenhang mit MNV S 99 als gering einzuschätzen. Unter der Annahme, dass murine und humane Noroviren eine ähnliche Tenazität in kurzgereiften Rohwürsten aufweisen, sollten diese Produkte im Zusammenhang mit Noroviren als Risikoprodukte eingestuft werden.

Liquid biofuels produced from biomass have the potential to partly replace gasoline. One of the most promising biofuels is butanol which is produced in acetone-butanol-ethanol (ABE) fermentation. The ABE fermentation is characterized by its low butanol concentration in the final fermentation broth. In this research, the simulation of three in situ recovery methods, namely, vacuum fermentation, gas stripping and pervaporation, were performed in order to increase the efficiency of the continuous ABE fermentation by decreasing the effect of butanol toxicity. The non-integrated and integrated butanol production systems were simulated and optimized based on a number of objectives such as maximizing the butanol productivity, butanol concentration, and butanol yield. In the optimization of complex industrial processes, where objectives are often conflicting, there exist numerous potentially-optimal solutions which are best obtained using multi-objective optimization (MOO). In this investigation, MOO was used to generate a set of alternative solutions, known as the Pareto domain. The Pareto domain allows to view very clearly the trade-offs existing between the various objective functions. In general, an increase in the butanol productivity resulted in a decrease of butanol yield and sugar conversion. To find the best solution within the Pareto domain, a ranking algorithm (Net Flow Method) was used to rank the solutions based on a set of relative weights and three preference thresholds. Comparing the best optimal solutions in each case study, it was clearly shown that integrating a recovery method with the ABE fermentation significantly increases the overall butanol concentration, butanol productivity, and sugar conversion, whereas butanol yield being microorganism-dependent, remains relatively constant.

Made available in DSpace on 2014-06-11T19:32:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-06-05Bitstream added on 2014-06-13T19:22:45Z : No. of bitstreams: 1 martins_es_dr_rcla.pdf: 4883436 bytes, checksum: 90e9eddebe5cd615b809d1494e86d994 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Pectinases termoestáveis apresentam características interessantes do ponto de vista da sua aplicação industrial, como alta estabilidade ao pH e à temperatura. Além disso, o tipo de processo fermentativo pode influenciar a produção e propriedades físico-químicas destas enzimas. A produção de poligalacturonase (PG) pelo fungo Thermoascus aurantiacus foi realizada em fermentação submersa (FSM) e em estado sólido (FES), usando substratos contendo pectina comercial ou subprodutos agro- industriais como fonte de carbono. A PG bruta obtida em FES apresentou atividade ótima a 65ºC e pH 5,0, com estabilidade na faixa de pH entre 4,0 e 5,0 e entre 7,5 e 8,5 e manteve 85% da atividade original quando incubada a 60ºC, por 1 hora. Em FSM, o melhor meio de cultivo foi a água amarela, com pH inicial de 5,5, após 5 dias de cultivo a 45ºC. A enzima em sua forma bruta apresentou temperatura ótima de 60ºC e pH ótimo de 5,0, maior estabilidade em pH ácido (3,0 a 4,5) e menor termoestabilidade, quando comparada com a obtida em FES, mantendo apenas 13% da atividade original quando incubada a 60ºC, por 1 hora. As enzimas foram purificadas utilizando-se cromatografias de filtração em gel e troca iônica. A PG purificada proveniente da FSM apresentou pH e temperatura ótimos de 5,5 e 60-65ºC, estabilidade em pH 5,0-5,5 e manteve, após 1 hora de incubação, 100% da atividade original até 50ºC. Resultados similares foram obtidos para a PG proveniente da FES. A PG de FES apresentou massa molar de 29,3 kDa, Km de 1,58 mg/mL e Vmáx de 1553,1 ? mol/min/mg, enquanto que a da FSM apresentou massa molar de 30,1 kDa, km de 1,46 mg/mL e Vmáx de 2433,3 ? mol/min/mg. Íons como Fe+3, Ca+2, e K+ praticamente não afetaram a atividade da enzima... / Thermostable pectinases present important characteristics under the view of their industrial application, as their high stability to pH and temperature. Besides, the type of fermentative process used can affect the ir production and physical-chemical properties. The polygalacturonase (PG) production by the thermophilic fungus Thermoascus aurantiacus was carried out by submerged fermentation (SMF) and solid state fermentation (SSF) using substrates containing commercial pectin or agro- industrial residues as carbon sources. The crude PG from SSF presented optimum activity at 65ºC and pH 5.0, with stability at pH 4.0-5.0 and 7.5-8.5 and maintained 85% of its original activity at 60º C for 1 hour. In SMF the best cultivation medium was the liquid waste from juice extraction, with initial pH of 5.5, after 5 days of cultivation at 45ºC. The crude enzyme showed an optimum activity at 60ºC and pH 5.0, higher stability in acid ic pH (3.0 to 4.5) and was less thermostable when compared to that obtained in SSF, wich maintained only 13% of its original activity at 60ºC, for 1 hour. Purification of enzymes was carried out using filtration and ion-exchange chromatographies. The purified PG, from SMF, showed optimum pH and temperature of 5.5 and 60-65ºC, stability at pH 5.0-5.5 and preserved, after 1 hour incubation, 100% of its original activity at 50ºC. Similar results were obtained to PG from SSF. The PG obtained by SSF presented molar mass of 29.3 kDa, Km of 1.58 mg/ml and Vmáx of 1553.1 ? mol/min/mg, while that the enzyme from SMF presented molar mass of 30.1 kDa, km of 1.46 mg/ml and Vmáx of 2433.3 ? mol/min/mg. Ions such as Fe3+, Ca2+ and K+ practically did not affect the enzyme activity, while Mg2+, Mn2+ and Zn2+ decreased 7%, 75% and 50%... (Complete abstract, click electronic address below)

De nombreux problèmes ont été identifiés lors des tentatives d’incorporation de protéines végétales dans nos aliments. En particulier, les faux goûts, le goût et la texture ont été mis en évidence comme de véritables obstacles à l’acceptabilité des produits végétaux par les consommateurs. Le consommateur lui-même est aussi un déterminant important en ce qui concerne le terme « acceptabilité ». Donc, dans le but de développer un nouveau produit fermenté à base de protéines de pois deux volets ont été étudié : le produit et le consommateur. Afin de comprendre la physico-chimie et l’acceptabilité de produits fermentés à base de protéines de pois, plusieurs facteurs ont été étudiés dans des étapes successives tels que la culture, les allégations positives sur la santé et l’environnement, les cocktails de souches lactiques et les procédés de préparation. Dès les premiers tests sensoriels, il était clair qu’il ne serait pas facile de convaincre les consommateurs de consommer des produits fermentés à base de pois : les consommateurs français n’acceptent pas les produits même à une faible concentration de pois (10 %). Ni la familiarité pour un produit proche de nos produits étudiés, ni l’encouragement des consommateurs à accepter ce type d’aliment en passant des messages positifs sur les protéines végétales n’étaient assez efficaces. Ainsi, une deuxième série d’études a été réalisée afin d’optimiser les propriétés rhéologiques et organoleptiques de ces produits. La meilleure combinaison, 1- cocktail bactérien (S. thermophilus + Lb. bulgaricus) 2- matière première (globuline de pois isolée dans notre laboratoire) 3- paramètres de préparation (mélange des deux laits avant le traitement thermique à 90 °C) a été sélectionnée afin d’optimiser les produits fermentés en ce qui concerne l’acidité, la fermeté, les profils volatils et peptidiques. Enfin, du point de vue sensoriel, une légère amélioration de l’acceptabilité a été remarquée. 20 % de protéines de pois a donné un produit accepté par la plupart des consommateurs, et 40 % de protéines de pois a été évaluée positivement par certains consommateurs et négativement par d'autres. / Many problems have been identified following the attempts to incorporate vegetal proteins in our food. In particular, off-flavor, taste or texture have been highlighted as real barriers to the acceptability of plant products by consumers. The consumer himself is also an important determinant regarding the term "acceptability." So, in order to develop a new fermented product based on pea, two issues were studied: the product and the consumer. To understand the physico-chemical properties and the acceptability of pea protein-based fermented products, several factors have been investigated in successive stages such as culture, positive health and environmental claims, lactic acid bacteria strains and preparation processes. From the first sensory tests, it was clear that it will not be easy to convince consumers to buy the pea-based fermented product: French consumers did not accept products even in a lower concentration of pea (10%). Neither the familiarity to close products nor encouraging consumers to accept this type of food by transmitting positive messages about vegetal protein were efficient enough. Thus, a second series of studies was carried out in order to optimize the rheologic and organoleptic properties of these products. Best combination of 1- starter culture (S. thermophilus + Lb. bulgaricus) 2- raw material (pea globulin isolated in our laboratory) 3- preparation parameters (mixing both milk before heat treatment at 90 ° C) were selected to optimize the fermented products in terms of volatile compounds and peptide profiles, acidity and firmness. Finally, from the sensory point of view, a slight improvement in the acceptability was noticed. 20% of pea protein gave a product accepted by most of the consumers, and 40% of pea protein was assessed positively by some consumers and negatively by others

Les bactéries lactiques sont utilisées pour la fermentation d'aliments dans le but d'augmenter leur durée de conservation et d'améliorer leurs propriétés organoleptiques et nutritionnelles. Dans le but de diversifier l'offre d'aliments fonctionnels et de limiter les pertes en produit frais, nous proposons des produits fermentés à base de fruits ou légumes, riches en antioxydants et plaisants pour le consommateur. Pour cela, mes travaux de thèse ont été menés en deux étapes : caractériser la flore bactérienne lactique présente à la surface de fruits et de légumes cultivés à La Réunion, puis sélectionner des bactéries autochtones possédant certaines propriétés fonctionnelles. Ainsi, 77 bactéries lactiques isolées de papayes, de tomates et d'achards de la Réunion, appartenant aux genres Leuconostoc, Lactococcus, Weissella, Lactobacillus et Fructobacillus ont été caractérisées génétiquement et phénotypiquement. Parmi la grande diversité observée, certains isolats ont présenté des caractéristiques technologiques (conditions et vitesse de croissance, résistance aux stress environnementaux) et fonctionnelles (production d'exo-polysaccharides) intéressantes pour l'élaboration d'aliments fermentés, en particulier les isolats Weissellla cibaria 64 et 30, Leuconostoc pseudomesenteroides 60 et Lactobacillus plantarum 75. A partir de ces données, des étapes complémentaires de criblage ont été réalisées sur différents substrats (mangue, papaye, ananas, thé vert, thé noir) fermentés, en examinant leurs propriétés organoleptiques et leurs activités antioxydantes. Deux isolats, Lc. pseudomesenteroides 12b et W. cibaria 64, ont augmenté significativement la teneur en composés phénoliques et l'activité antioxydante de jus d'ananas Victoria au cours d'une fermentation de 48h. Le produit fermenté se conserve 16 jours au froid sans aucune altération et conserve l'ensemble de ses bénéfices nutritionnels. Une odeur et des goûts typiques ont été détectés dans les boissons fermentées obtenues. L'augmentation de l'activité antioxydante observée au cours de la fermentation résulte probablement du métabolisme des composés phénoliques par les bactéries. L'identification des molécules produites et des enzymes impliquées est nécessaire afin de comprendre les mécanismes mis en jeu. / Lactic acid bacteria are used for the production of large variety of fermented foods in order to enhance shelf-life together with improved organoleptic and nutritional properties. In order to create new functional foods and to reduce fresh product waste, the objectives of my PhD were to develop and characterize fruit or vegetables fermented foods, rich in antioxidants and pleasant for consumers. To reach these goals, my PhD work was split in two steps: 1- characterization of lactic bacteria present on fruits and vegetables grown at Reunion Island and 2- selection of autochthonous bacteria with functional properties. Thus, 77 lactic bacteria from genera Leuconostoc, Lactococcus, Weissella, Lactobacillus and Fructobacillus were isolated from papaya, tomato and sliced cabbage from Reunion Island. They were genetically and phenotypically characterized. A huge diversity in term of genetic and phenotypic characteristics was determined. Furthermore, several isolates exhibiting specific technological and functional properties (growth rate, resistance to environmental stress, production of exopolysaccharides) were identified. These isolates, potentially useful for the production of fermented foods, were Weissella cibaria 64 and 30, Leuconostoc pseudomesenteroides 60 and Lactobacillus plantarum 75. Then, further screening steps were performed on different food substrates (pineapple, mango, papaya, tea infusions) in order to select isolates able to improve antioxidant and organoleptic properties. Two isolates, W. cibaria 64 and Lc. pseudomesenteroides 12b were shown to significantly enhance the phenolic content and the antioxidant activity of Victoria pineapple juice after a 48h-fermentation. The fermented products exhibited a good shelf-life of 16 days without alteration and preserved nutritional benefits. Characteristic odour and tastes were detected in the fermented drinks. The increase in antioxidant activity observed during fermentation was probably the consequence of a depolymerization of phenolic compounds. Further works are required to identify the composition changes over fermentation and to reach a better understanding of the mechanisms involved.

La réduction de la consommation des carburants fossiles est l’un des enjeux majeurs du XXIème siècle. Le bioéthanol représente une alternative durable à l’essence, mais sa production est limitée puisqu’il est produit à partir de ressources alimentaires. L’éthanol de seconde génération offre une alternative pour relancer son développement. Encore au stade expérimental, il serait produit à partir de biomasse lignocellulosique (bois, paille, plantes annuelles..) et donc ne concurrencerait pas l’alimentation humaine. Cette thèse s’inscrit dans le cadre de production d’éthanol de 2nde génération dans une usine de pâte kraft. Dans ces usines, les hémicelluloses, qui représentent 20 à 30% du bois, ne sont pas valorisées. Cette étude porte sur l’extraction de ces hémicelluloses en amont du procédé et de leur fermentation en éthanol. Elle consiste à la mise au point et l’optimisation d’un procédé global allant du copeau de bois à l’éthanol. / Decreasing the consumption of crude oil derivatives has become one of the main world issues of the 21st century. The only green substitute for gasoline potentially available in large quantities today is bioethanol, but its production has to be limited as it is produced from crops. It is therefore important to develop the production of second generation ethanol, which consists in using lignocellulosic biomass as raw material to avoid to compete with food resources. Second generation ethanol is still at experimental scale nowadays. This thesis deals with the specific case of the production of ethanol from hemicelluloses in a kraft pulp mill. In pulp mills, hemicelluloses, which make up 20 to 30% of the wood, are not value added. It is proposed to extract them from wood prior to the kraft treatment and to ferment them into ethanol. The study talks more specifically of the optimisation of the global process from the woodchip to the ethanol.