Biotrasformazioni di terpeni e oli essenziali con batteri e funghi isolati da frutti del genere Citrus della foresta amazzonica (Ecuador).

Since few years the “Centro di Ateneo per la Cooperazione allo Sviluppo” of the University of Ferrara is evolving research projects in collaboration with VIS (Volontariato Internazionale Sviluppo) about the valorization of the biodiversity as a source of development of the Achuar and Shuar populations of the Amazon forest (Ecuador). Ecuador is a mosaic of different regions where it is present a great biodiversity. The Achuar territory represents one of the last primary forests that connects the Andes with de Amazon forest of Peru and Brazil. The research project carried out in this field combines the transfer of knowledge and technology to the Chankuap Fundation, partner of the developing project, and on the other hand lays the bases of the beginning of little local companies to recycle the waste materials. In this zone I started my Ph.D research work carrying out the isolation of 61 bacteria and 55 fungi from the epicarp of Citrus limon, C. aurantifolia, C. aurantium and C. paradisiaca fruits, harvested in 3 Achuar communities: Wasakentsa, Pumpuentsa e Sewastian. These strains were screened for their aptitude to biotransform some terpenes as limonene, linalool, citronellal, and sabinene, which are the main components of the Citrus limon, C. latifolia, C. aurantium and C. nobilis essential oils. The active strains were tested in biotransformations of Citrus limon, C. latifolia, C. aurantium and C. nobilis essential oils, extracted from plants of the same region. Materials The essential oils were extracted from the leaves by steam distillation and were characterized by GC-MS. The screening has been performed using liquid cultures: Sabouraud for fungi and PCB for bacteria. DMSO was used for emulsifying the substrates in the aqueous reaction system. To the fungal (5 days growth) and bacterial (48 h growth) cultures the substrates (1g/L) were added at 28-30°C. Samples of the reaction mixtures were analysed at regular intervals by GC-MS. The biotrasformations that gave positive results were repeated on preparative scale and the products were isolated by chromatography and characterized (GC-MS, 1H-NMR, 13C-NMR). On the basis of the data obtained with the terpenes, the selected microrganisms were tested in the biotransformations of the essential oils. Finally the antioxidant activity “radical scavenger” of the essential oils, of the standards and of the terpenical mixtures, with equivalent composition to the starting and biotransformed material, were measured with the DPPH test. This activity was compared with Thymus vulgaris chosen as positive control. Results Three fungal strains (1B14, 1C5, 1D2) were active in biotranformation of (±)-linalool. Ten products were obtained: (cis- and trans-)-linalool furanoid oxide, (cis- and trans-)-linalool pyranoid oxide and 6,7- epoxylinalool as known products, and 1-methyl-1-(4-methylpentyl) oxiran-methanol, 6-methyl-5-hepten- 2-one and 6-methyl-5-hepten-2-ol which have never been reported as linalool biotransformation products. Some fungal strains (1D6, 2D15, 1C22, 1B14, 1C5) were active in the (-)-S-citronellal biotransformations. The products isolated were (-)-S- citronellol, (-)-S-citronellic acid, (cis)-p-menthan-3,8-diol, (trans)-pmenthan- 3,8-diol, isopulegol and neoisopulegol. Only one strain (1C22) has biotransformed sabinene. The main products were 4-terpineol, g-terpinene, g- terpineol, (cis e trans)-sabinene-hydrate, α-terpinene, p-cymene, limonene, terpinolene, (cis e trans)-pmenth- 2-en-1-ol. The biotransformations with limonene didn’t give any products. The biotransformation of the essential oils of C. latifolia, chosen for the high concentration of citronellale, and C. limon, chosen for the high sabinene content, gave essentially the same products that have been obtained in the biotransformation of terpenes. However, limonene-1,2-diol, not found during the screening, was obtained from limonene. This important result could be due to an elicitor function of the terpenes mixture on the fungal enzymes. The antioxidant activity of the essential oils and their biotransformation products didn’t afforded significant results in this preliminary phase, if they are compared with Thymus vulgaris essential oil. Conclusions Strains isolated from the epicarp of genus Citrus fruits have demostrated activity in the biotransformation of the model terpenes and the corresponding essential oils. These results suggest the use of these strains for the conversion of terpenes to the corresponding derivatives, and for the bioconversion of essential oils, whose new composition could modify their organoleptic and bioactive characteristics. The main active strains are going to be characterized at the Centraal bureau voor Schimmelcultures (CBS).