UNIVERSITÉ PAUL CÉZANNE, AIX MARSEILLE III - IMEP

Résultats et discussion. Chapitre 2: Optimisation de la production du spawn
2.5. Recovery of ergosterol from spawn spiked with ergosterol and mycelium.
Assessment of the recovery efficiency of ergosterol was carried out by artificially
addition of pure ergosterol (from Sigma) or mycelium which its ergosterol content was
already determined [3.55 mg (ergosterol).g -1 (mycelium)]. Recovery of pure ergosterol was
determined after mixing 0.5 mL of methanol containing 100 µg dissolved ergosterol with 10 g
(dry weight basis) of Le119 spawn. After mixing, sub-samples were extracted using the
described procedures and ergosterol was determined by HPLC. For recovery of fungal
ergosterol, portion of 100 mg of pure mycelium were mixed with 10 g (dry weight) of spawn.
After mixing, sub-samples were extracted using the described procedures and the amount of
ergosterol was determined.
2.6. HPLC analysis
Final separation of ergosterol from extract matrix was performed by reversed-phase
high-performance liquid chromatography (RP-HPLC). The chromatographic systems used
were an Agilent Series 2000. Volume injection was 20 µl of sample. The chromatographic
separation was achieved on 5 µm dC18 (4.6x250 mm) reversed-phase column (Atlantis,
Ireland) and detection was set at 282 nm. Elution of ergosterol at column temperature of 40°C
was performed with isocratic run with 100% methanol at a flow rate of 1 mL.min -1 . Peak
identity was checked on the basis of retention times of pure ergosterol. Correlation between
dry biomass and ergosterol content was verified by measuring the peak height and area. UV
spectrum of ergosterol extracted from spawn was recorded using the same apparatus from 265
to 300 nm. All experiments were carried out in triplicate.
2.7. Statistical Analysis
One way analyses of variance were tested for effects of medium composition and
culture age on the ergosterol concentration in mycelium. The significance level was set at P =
0.05. The correlation coefficient was used to determine the relationship two properties
(biomass and ergosterol; ergosterol and CO2 production). The analysis were performed using
Xlstat® software.
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