*3.2. Analytical Methods and Characterization of the Products Deriving from the Biotransformation Experiments*

The crude biotransformation mixtures obtained according to the procedures described below were then acetylated by treatment with pyridine/acetic anhydride (2 mL of a 2:1 mixture) and catalytic DMAP (10 mg) for 6 hours at rt. The obtained acetylated mixture was analyzed by GC-MS. The compounds whose chemical structure couldn't be assigned only by GC-MS analysis were isolated from the biotransformation mixtures by means of chromatographic separation and then characterized by NMR analysis and GC-MS or ESI-MS analysis.

1H- and 13C-NMR spectra and DEPT experiments were recorded/performed at 400, 100 and 100 MHz, respectively, in CDCl3 solutions at rt using an AC-400 spectrometer (Bruker, Billerica, MA, USA); 13C spectra are proton decoupled; chemical shifts in ppm rel to internal SiMe4 (=0 ppm).

TLC: silica gel *60 F254* plates (Merck, Kenilworth, NJ, USA). Column chromatography: silica gel.

Melting points were measured on a Reichert apparatus, equipped with a Reichert microscope, and are uncorrected.

Mass spectrum were recorded on a ESQUIRE 3000 PLUS spectrometer equipped with an ESI detector (Bruker, Billerica, MA, USA) or by GC-MS analyses.

GC-MS analyses: *HP-6890* gas chromatograph equipped with a 5973 mass detector, using a HP-5MS column (30 m × 0.25 mm, 0.25 μm film thickness; Hewlett Packard, Palo Alto, CA, USA) with the following temp. program: 60◦ (1 min)—6◦/min—150◦ (1 min)—12◦/min—280◦ (5 min); carrier gas, He; constant flow 1 mL/min; split ratio, 1/30; *t*<sup>R</sup> given in min: *t*R(**1**) 16.23, *t*R(**2**) 17.57, *t*R(**3**) 16.53, *t*R(**4**) 17.53, *t*R(**5**) 13.40 and 13.77, *t*R(**6**) 15.44, *t*R(**7**) 15.91, *t*R(**8**) 20.42, *t*R(**9a**) 21.41, *t*R(**9b**) 21.61, *t*R(**10**) 18.63 and 18.72, *t*R(**11**) 17.78, *t*R(**12**) 21.52, *t*R(**13**) 20.61, *t*R(**14**) 21.82, *t*R(**15**) 22.03, *t*R(**16**) 22.56, *t*R(**17**) 21.59, *t*R(**18**) 22.18, *t*R(**19**) 22.23, *t*R(**20**) 18.61, *t*R(**21**) 18.55, *t*R(**22**) 16.51, *t*R(**23a**) 21.51, *t*R(**24a**) 21.32, *t*R(**24b**) 21.60, *t*R(**25**) 21.79, *t*R(**26**) 15.67, *t*R(**27**) 17.47, 17.58, 18.01 and 18.08, *t*R(**28**) 24.10, *t*R(**29**) 23.75, *t*R(**30**) 23.92, *t*R(**31**) 19.02 and 19.19, *t*R(**32**) 20.17, 20.33, 20.50 and 20.61, *t*R(**33**) 15.14 and 15.50, *t*R(**34**) 19.37 and 19.67, *t*R(**35**) 21.78, *t*R(**36**) 21.64, *t*R(**37**) 20.15, *t*R(**38**) 21.17 and 21.30, *t*R(**39**) 17.84, *t*R(**40**) 14.62, *t*R(**41**) 20.91, *t*R(**42**) 20.93, *t*R(7,8-dihydro-α-ionone) 15.95, *t*R(7,8-dihydro-β-damascone) 20.95, *t*R(*cis*-2-acetoxy-α-ionone) 21.42, *t*R(*trans*-2-acetoxy-α-ionone) 21.36.

#### *3.3. Microorganisms and Biotransformation Experiments*

Geotrichum candidum (DSM 10452), Yarrowia lipolytica (DSM 8218), Rhizopus stolonifer (DSM 855), Xanthophyllomyces dendrorhous (DMS 5626), Curvularia lunata (CBS 215.54), Mortierella isabellina (CBS 167.60), Aspergillus niger (CBS 626.26) were purchased from the DSMZ (Braunschweig, Germany) or CBS-KNAW (Utrecht, The Netherlands) collections.

*Penicillium corylophilum* (MUT 5838), *Nigrospora oryzae* (MUT 5844), *Penicillium roqueforti* (MUT 5856) and *Fusarium culmorum* (MUT 5855) were isolated as axenic cultures in our laboratory, then identified by the Mycotheca Universitatis Taurinensis (MUT) of the University of Turin and finally deposited in the same institution under the collection number given in brackets.

All the biotransformations were carried out in triplicate and the presented results are the average of three experimental runs.

#### 3.3.1. Representative Procedures for Biotransformations

The experimental conditions used for the biotransformations are based on the type of microorganism used. Here is described a general procedure depending on the different morphological features regarding the various active grow mycelia. The main ones could be classified in yeast-shape mycelia (*Xanthophyllomyces dendrorhous*, *Geotrichum candidum* and *Yarrowia lipolytica*) and spore-forming mycelia (*Aspergillus niger*, *Rhizopus stolonifer*, *Curvularia lunata*, *Penicillium corylophilum*, *Nigrospora oryzae*, *Penicillium roqueforti*, *Mortierella isabellina* and *Fusarium culmorum*). In the first case, a small amount of the active mycelia grew previously in a petri dish, was suspend in 1 mL of sterile water and then inoculated in a 100 mL conical Pyrex flask containing 40 mL of Malt Extract Medium (MEM) for 2 days at 25 ◦C and 140 rpm (with exception of *Xanthophyllomyces dendrorhous* that was grown at 20 ◦C). After this period, the cells were centrifuged 3 minutes, (rt, 3220·*g*) and collected removing the media. The cells (approx. 600 mg wet-weight) were suspended in 3 mL of sterile water than 350 μL of the same suspension were used for inoculating each biotransformation flask containing 40 mL of MEM. In order to ensure aerobic conditions, the flasks were sealed with cellulose plugs. The microorganism was leave to growth for 2 days and then was treated with a solution of 100 mg of substrate dissolved in 400 μL of DMSO. Generally, after 14 days from the substrate injection using the growing condition described above, the reaction media was filtered under vacuum through a celite pad then was extracted 3 times with ethyl acetate. The organic phase was separated, dried on Na2SO4 and the solvent removed at reduced pressure to give the crude biotransformation mixture.

In the case of the spore-forming mycelia, the spore were collected from a sporulated surface cultures and suspended in 3 mL of sterile water. After that, 350 μL of the same suspension were used for inoculating each biotransformation flask containing 40 mL of MEM. The subsequent steps are the same of that described previously. The unique exception was carried out for *Penicillium corylophilum*, in the case of γ-ionone. The toxicity of the compound forced us to keep its concentration lower than the others (7.5 mM) and to block the biotransformation earlier (8 days). After this period, the most important products are degraded. In the case of *Fusarium culmorum* the biotransformation was blocked after 20 days instead of 14 days because the activity of the fungus did not stop in the prefixed time.
