UNIVERSITÉ PAUL CÉZANNE, AIX MARSEILLE III - IMEP

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).
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