CONICET | Buscador de Institutos y Recursos Humanos

Esterification reactions for the generation of terpenoids as esters have a particular industrialapplication in the manufacture of aromas and fragrances. Lipases (triacylglycerol acyl hydrolasesEC 3.1.1.3) are hydrolytic enzymes that are capable of catalyzing a wide variety of esterificationreactions of monoterpenes in non-aqueous media. It was determined in previous work that theseenzymes can be induced by d-limonene and olive oil. Although there are a wide variety of fungalstrains with lipolytic capacity, those belonging to the genus Penicillium spp are among the mostprominent in bioconversion synthesis and in the production of substances of nutritional,environmental, cosmetic, and medicinal interest.The objective of this work was to determine the production of aromatic esters and relate them withthe lipase activity in culture media supplemented with d-limonene and olive oil of P. citrinum LBM150.The d-limonene bioconversion assays were carried out using 100 mL Erlenmeyer flasks containing40 mL of liquid culture medium with malt extract (10 g L−1), yeast extract (0.5 g L−1), bacteriologicalpeptone (0.5 g L−1), glucose (10 g L−1), Tween 80 (0.1 g L−1) in sodium acetate buffer 0.1 M, pH 5.4.Fungal inoculum with 1.2 mL of a suspension of 2 x 107 spores per mL-1 was added to eachErlenmeyer flask and incubated under static conditions and the absence of light at 28 ± 1°C. After 5days of incubation, each Erlenmeyer flask was supplemented with 951 µL of d-limonene, andcombined treatment was moreover supplemented with 876 µL of olive oil. Positive controls weremade by adding 876 µL of olive oil and fungal inoculum, while negative controls were made withfungal inoculum without additional supplementation in the culture medium. Assays were performedin triplicate. After 7 days of incubation, the mycelium was separated from the supernatant bycentrifugation at 2500 rpm for 15 min. Subsequently, the supernatant with d-limonene was washedwith 10 mL of n-hexane at 100 rpm for 10 min and the supernatant with d-limonene and olive oil wascentrifuged at 250 rpm for 30 min. The lower density upper fraction was separated into another tubeto which an anhydrous sodium acetate spatula tip was added to remove moisture. The identificationand quantification of terpenes and terpenoids were performed by gas chromatography coupled withmass spectrometry (GC/MS Perkin Elmer, Clarus 500 MS). The relative percentage composition wasdetermined by the proportion of chromatographic areas.It identified 19 terpene compounds by the biotransformation of d-limonene and olive oil, which weregrouped into five chemical groups: monoterpenes and diterpenes, oxides, alcohols, aldehydes, andesters. In the treatment in the presence of d-limonene without olive oil, the most abundantcomponents belonged to the group of alcohols, the majority being cis-carveol, cis-p-menta-2,8-dienol,and α-terpineol. To a lesser extent, aldehydes were identified: citral and dihydrocarvone;monoterpenes and diterpenes: α-pinene, sabinene and myrcene. While in the treatment with thepresence of d-limonene and olive oil, alcohols constituted the majority product with 10 identifiedcompounds; with the greater relative abundance of cis-p-menth-2,8-dienol, trans-p-menth-2,8-dienoland terpineol, and the next group was made up of esters with 3 main components: geranyl acetate,carveol propionate, and geraniol propionate. On the other hand, the presence of limonene oxide wasdetermined in both treatments. The results were correlated with previous studies obtained by the working group in which d-limonene and olive oil increased significantly lipase activity in culturemedia of the strain LBM150. In conclusion, the supplementation with d-limonene and olive oil of P. citrinum LBM150 culture media allows for obtaining aromatic esters such as geranyl acetate, carveol propionate, and geraniol propionate which could be related to the catalytic activity of lipases enzymes.

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