UNIVERSITÉ PAUL CÉZANNE, AIX MARSEILLE III - IMEP

Résultats et discussion. Chapitre 3: Evaluation des facteurs de croissance
studied. Asymmetrical screening design was established and processing the results with the
Nemrodw software (New Efficient Methodology for Research using Optimal Design) enabled
us to quantify the direct effects of the various factors on the responses chosen and to identify
those that will require fine-tuning during the optimization phase.
2. Materials and methods
2.1. Microorganism and spawn preparation
Single strain of L.edodes (Le119) was used in the experiments. The strain was
maintained at 4 °C on potato dextrose agar (PDA, Sigma, France) supplemented with 10% of
OMW (Lakhtar et al., 2009). Wheat grains (Ebly®, Casino, France) were used as substrate
and growth support for spawn preparation. The grains were cooked for 5 min in a water
solution containing 10% (v/v) of OMW at a ratio of 1:1 (Ebly®: Solution; w: v). Once
cooked, the wheat grains were packaged (250 g of wheat grains in jar of 250 ml) and
sterilized in an autoclave at 121 °C for 30 min. After sterilization, the grains were inoculated
with 5 agar disks of 5 mm diameter containing mycelium and then placed in “Raimbault
columns” (De Araujo et al., 1997) and incubated at 25±2 °C in the absence of light for a
periode of 10 and 25 days.
2.2. Preparation of substrates
The substrates were composed of different parts; OC, olive twigs and leaves (OTL)
and OMW as wetting agent. OC was dried in greenhouse retaining 10% of humidity (w/w).
OC and OMW used throughout the study were taken from a three phase system used in the
conventional press type olive oil mill plant (Beni-Mellal, Morocco). The OC and OTL were
dried in solar greenhouse with 10% (w/w) of moisture retained. OMW was stored at -20°C
until it was used. OMW dilutions were performed in distillate water, unless otherwise stated.
The OTL were ground and sieved to desirable particle size. The individual ingredients of the
substrate were mixed thoroughly by mechanical means to achieve a homogenous mixture.
2.3. Formulation of substrate for L. edodes culture
In order to determine the optimal formulation of substrates allowed for high mycelia
biomass, different mixtures of OC and OTL (max. particle size: 5 mm) were soaked with 50%
(v/v) of OMW to obtain 50% of moisture (Table 1). Sterilization was carried out at 121°C for
30 min. After cooling, 5g of grains of wheat spawn (15 days age) added to 45 g of sterile
substrate were gently mixed and placed in sterile 200 mL Erlenmeyer flask. The Erlenmeyer
flasks were then incubated at 25±2 °C for 1 month. Mycelial biomass was estimated using
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