A Two-Step Bioconversion Process for Canolol Production from Rapeseed Meal Combining an Aspergillus niger Feruloyl Esterase and the Fungus Neolentinus lepideus

Abstract

Rapeseed meal is a cheap and abundant raw material, particularly rich in phenolic compounds of biotechnological interest. In this study, we developed a two-step bioconversion process of naturally occurring sinapic acid (4-hydroxy-3,5-dimethoxycinnamic acid) from rapeseed meal into canolol by combining the complementary potentialities of two filamentous fungi, the micromycete Aspergillus niger and the basidiomycete Neolentinus lepideus. Canolol could display numerous industrial applications because of its high antioxidant, antimutagenic and anticarcinogenic properties. In the first step of the process, the use of the enzyme feruloyl esterase type-A (named AnFaeA) produced with the recombinant strain A. niger BRFM451 made it possible to release free sinapic acid from the raw meal by hydrolysing the conjugated forms of sinapic acid in the meal (mainly sinapine and glucopyranosyl sinapate). An amount of 39 nkat AnFaeA per gram of raw meal, at 55 °C and pH 5, led to the recovery of 6.6 to 7.4 mg of free sinapic acid per gram raw meal, which corresponded to a global hydrolysis yield of 68 to 76% and a 100% hydrolysis of sinapine. Then, the XAD2 adsorbent (a styrene and divinylbenzene copolymer resin), used at pH 4, enabled the efficient recovery of the released sinapic acid, and its concentration after elution with ethanol. In the second step, 3-day-old submerged cultures of the strain N. lepideus BRFM15 were supplied with the recovered sinapic acid as the substrate of bioconversion into canolol by a non-oxidative decarboxylation pathway. Canolol production reached 1.3 g/L with a molar yield of bioconversion of 80% and a productivity of 100 mg/L day. The same XAD2 resin, when used at pH 7, allowed the recovery and purification of canolol from the culture broth of N. lepideus. The two-step process used mild conditions compatible with green chemistry.

Keywords: Aspergillus niger; Neolentinus lepideus; canolol; feruloyl esterase; rapeseed meal; sinapic acid.

Figure 1

Canolol synthesis from the main esterified forms of SA from rapeseed meal.

Figure 2

Time course of sinapine disappearance (A) and SA increase (B) in the reaction mixture of RSM hydrolysis as a function of the cinnamoyl esterase tested.

Figure 3

Time course of sinapine disappearance and SA increase in the reaction mixture of hydrolysis as a function of the amount of AnFaeA and temperature. (A,B): 37 °C, (C,D): 45 °C, (E,F): 55 °C.

Figure 4

SA and FA metabolism by N. lepideus BRFM 15 (A), S. commune BRFM823 (B) and S. hirsutum BRFM889 (C): consumption of SA or FA in relation to the production of canolol and syringic acid, and VG and vanillic acid, respectively.

Figure 5

Comparison of SA metabolism by N. lepideus BRFM15 supplemented with different sources of SA. Cultivations were carried out in the presence of aqueous commercial SA (green symbols), ethanolic commercial SA (blue symbols), and natural ethanolic SA from RSM (red symbols). Consumption of SA in relation to the production of canolol and syringic acid.