UNIVERSITÉ PAUL CÉZANNE, AIX MARSEILLE III - IMEP

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Résultats et discussion. Chapitre 2: Optimisation de la production du spawn Therefore, to our knowledge, there is no report on estimation of quantity and quality of biomass during the production of medicinal Lentinula edodes spawn on wheat grain. In the present study, we report for the first time ergosterol-biomass conversion factor in spawn of L. edodes. Non-alkaline protocol of extraction of ergosterol is developed for estimation of both quantity and quality ways of L. edodes biomass during spawn running. Effect of substrate of culture and age of mycelium were evaluated in model system to determine the quantity of spawn. The correlation between ergosterol levels and CO2 production was determined to estimate the quality of L. edodes spawn. 2. Materiel and methods 2.1. Microorganism Lentinula edodes Le119 was previously selected among collection of sixteen strains for its capability of colonization of olive wastes. The preserved stock culture of strain was maintained in sterilized Potato Dextrose Agar (PDA) (39 g.L -1 , Sigma, France) with 10% of Olive mill wastewater (OMW) (Lakhtar et al., 2009) with periodic transfer of one month. 2.2. Culture Media 2.2.1. Liquid state cultivation Liquid cultures were grown on Malt Extract medium (ME) (30 g.L -1 , Sigma, France) and on diluted OMW (10% of v/v) without any supplements. The culture media (100 mL) were sterilized (121°C, 20 min) and then inoculated with three plugs (1 cm diameter) of earlier uniformly colonized PDA of one week. Medium was incubated at 25°C under shaking (120 rpm). Triplicate flasks were harvested for determination of mycelial biomass dry weight and ergosterol content. Mycelium was harvested from the liquid medium by filtration (Watman filter N°1) and frozen dried. 2.2.2. Solid state Cultivation on model system Three different culture media were used for solid culture: PDA, PDA supplemented with 10% of OMW and Ebly Agar (EbA) consisting of 24 g of wheat grain (Ebly®, Casino, France), which were milled to powder, and 15 g of agar in 1 mL of distillated water. These media were used to determine ergosterol content in mycelial biomass of L. edodes Le119 strain. Biomass and ergosterol content was estimated using sterilized cellophane disc placed on the agar surface from Petri dishes (diam. 90 mm) (De Araujo et al., 2002). After colonization, the mycelium was separated from the substrate and cellophane membrane. It 77
Résultats et discussion. Chapitre 2: Optimisation de la production du spawn was then cut out and frozen dried. Each part of mycelial colony with different ages was tested to quantify ergosterol content (Fig. 1). 1 2 3 4 Figure 1. Mycelium growth of L.edodes Le119 on solid medium with cellophane membrane incubated at 25°C, 1: Inoculum (6 mm), 2: with mycelium 20 days old, 3: with mycelium 12 days old, 4: with mycelium 5 days old. 2.3. Solid state fermentation of L. edodes on wheat grain Tow spawn kinds of L. edodes Le119 strain were prepared. White spawn (WS) was prepared as described by Sobal (2002) with modification. Quantity of 200 g of wheat grain of Ebly® were humidified with distilled water to 50% (w/w). While the brown spawn (BS), was prepared with addition of OMW; 200 g of wheat was wetted with 10% of OMW to get 50% (w/w) of humidity, heated with microwave for 5 minutes to allow absorption of OMW by Ebly®. The substrates were autoclaved for 30 min at 121°C and allowed to cool for 24h. Pre- weighed “Raimbault columns” (De Araujo et al., 1997) were filled with sterilized Ebly® alternated by inoculum (three plugs of 5 mm) cut from PDA previously colonized with Le119 strain of 1 week. Forced aeration of the substrate was regulated to 25 mL.min -1 (which allowed five time of air renewal in column per hour). The CO2 production was recorded using Infra-red detector which was calibrated with air containing 1 and 5% of CO2. 2.4. Extraction of non esterified ergosterol Pure mycelium and spawn (Ebly® fermented) samples were frozen-dried to constant weight and milled to a uniform fine powder using grinder (IKA Labortechnik, Germany). Ergosterol was extracted from 50 mg samples of pure mycelium and spawn by homogenization in 1 mL of methanol. The mixtures were vortexed for 1 minute and subsequently shaken vigorously and then incubated at 4°C for overnight in the dark. Supernatants were recovered by centrifugation for 10 minutes at 14 000 rpm. The supernatant was then filtered through 0.2 µm syringe filter (Iso-Disc TM Filters, USA). 78
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UNIVERSITÉ PAUL CÉZANNE, AIX MARS
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Je remercie le Dr. Antonis PHILIPPO
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Résumé L’objectif du travail a
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2.2.4.2. Culture de champignons sup
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3.7.1. La mesure de l’humidité .
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Liste des Tables Tableau 1. Bilan m
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1. INTRODUCTION Introduction Au Mar
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Introduction L'utilisation de la fe
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Matériel et Méthodes Figure 2. Ev
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Matériel et Méthodes et de l’hu
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Matériel et Méthodes composition
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Matériel et Méthodes Il ressort d
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Matériel et Méthodes catéchol-m
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Matériel et Méthodes Pour une év
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Matériel et Méthodes nombreuses o
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Matériel et Méthodes agricoles (P
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Matériel et Méthodes b) Traitemen
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Matériel et Méthodes pour former
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ABSTRACT Résultats et discussion.
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Résultats et discussion. Chapitre
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3. Results and discussion Résultat
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Synthèse générale 142
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Synthèse générale produits de l
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Synthèse générale Suite à l’o
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Synthèse générale molécules ren
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Conclusions et perspectives 150
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Conclusions et perspectives La mise
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Références bibliographiques Réf
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Références bibliographiques Bing
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Références bibliographiques D'Ann
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Références bibliographiques mill
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Références bibliographiques Jo J.
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Références bibliographiques Match
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Références bibliographiques Pietr
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Références bibliographiques Sebab
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Références bibliographiques Zerva
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UNIVERSITÉ PAUL CÉZANNE, AIX MARSEILLE III - IMEP

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