Study of the In Vitro Digestion of Olive Oil Enriched or Not with Antioxidant Phenolic Compounds. Relationships between Bioaccessibility of Main Components of Different Oils and Their Composition

The changes provoked by in vitro digestion in the lipids of olive oil enriched or not with different phenolic compounds were studied by proton nuclear magnetic resonance (1H NMR) and solid phase microextraction followed by gas chromatography/mass spectrometry (SPME-GC/MS). These changes were compared with those provoked in the lipids of corn oil and of virgin flaxseed oil submitted to the same digestive conditions. Lipolysis and oxidation were the two reactions under consideration. The bioaccessibility of main and minor components of olive oil, of phenolic compounds added, and of compounds formed as consequence of the oxidation, if any, were matters of attention. Enrichment of olive oil with antioxidant phenolic compounds does not affect the extent of lipolysis, but reduces the oxidation degree to minimum values or avoids it almost entirely. The in vitro bioaccessibility of nutritional and bioactive compounds was greater in the olive oil digestate than in those of other oils, whereas that of compounds formed in oxidation was minimal, if any. Very close quantitative relationships were found between the composition of the oils in main components and their in vitro bioaccessibility. These relationships, some of which have predictive value, can help to design lipid diets for different nutritional purposes.

**The intensity of some of these signals, together with signal F of Table S3, were used to estimate the molar percentages of different kinds of glyceryl structures using the equations [eq. S1eq. S10].
***The assignment of the 1 H NMR signals of the protons was made as in previous studies (Guillén & Uriarte, 2012a;Nieva-Echevarría et al., 2014). Table S3. Chemical shift assignments and multiplicities of the 1 H NMR signals in CDCl3 of protons of acyl groups and fatty acids. AG: acyl groups; FA: fatty acids.

Main acyl groups (AG) and fatty acids (FA)
A 0.88 t -CH3 saturated and monounsaturated ω-9 in AG and FA 0.89 t -CH3 linoleic in AG and FA B 0 . 9 7 t -CH3 linolenic in AG and FA C 1 . 1 9 -1.42 Abbreviations: d: doublet; t: triplet; m: multiplet. *The intensity of these signals was used to estimate the molar percentage of the main acyl groups plus fatty acids by using equations [eq. S11-eq. S14].
**Overlapping of multiplets of methylenic protons in the different acyl groups either in βposition, or further, in relation to double bonds, or in γ-position, or further, in relation to the carbonyl group.
***Overlapping of multiplets of the α-methylenic protons in relation to a single double bond of the different unsaturated acyl groups.
****The assignment of the 1 H NMR signals of the protons was made as in previous studies (Guillén & Ruiz, 2003;Nieva-Echevarría et al., 2014). Table S4. Chemical shift assignments and multiplicities of the 1 H NMR signals in CDCl3 of protons of some oxidation compounds detected in the digestates and formed during the in vitro digestion.  Table S3, were used to estimate the concentration (mmol/molAG+FA) using the equation [eq. S15].

Signal Chemical shift (ppm) Multiplicity Type of protons Structures Oxidation Compounds (OC) Conjugated dienic systems associated with hydroperoxy groups
**The assignment of the 1 H NMR signals of the protons was made with the aid of standard compounds and with the data taken from literature . Table S5. Chemical shift assignments and multiplicities of the 1 H NMR signals in CDCl3 of protons of cycloartenol and methylencycloartenol, esters of cycloartenol and methylencycloartenol, gammatocopherols, hydroxytyrosol acetate and dodecyl gallate detected in the samples before and after in vitro digestion.
**Assignment was made with the aid of standard compounds and with the data taken from the literature (Baker & Mayers, 1991;Pogliani et al., 1994;Kubo et al., 2002;Kawai et al., 2007).

Operating Conditions for the Acquisition of the 1 H NMR Spectra
The 1 H NMR spectra were acquired in duplicate using a Bruker Avance 400 spectrometer operating at 400 MHz. For this purpose, the samples (approximately 0.16 g) were dissolved in 400 µL of deuterated chloroform, which contained tetramethylsilane (TMS), as internal reference (Cortec, Paris, France). First, a standard 1 H NMR spectrum was acquired and in a second step, a NOESYGPPS experiment consisting of a one-dimensional 1 H NMR pulse sequence with selective suppression of certain strong signals was carried out. This NOESYGPPS experiment allows one to obtain a 1 H NMR spectrum with a greater sensitivity than that of the standard single pulse 1 H NMR experiment (Ruiz-Aracama et al., 2017) in the spectral region from 5.8 to 9.8 ppm, at the cost of suppressing some signals in other regions. The relaxation and acquisition times used allow the complete relaxation of the protons, the signal areas thus being proportional to the number of protons that generate them, except in the suppressed signals, making it possible to use them for quantitative purposes as in previous studies (Guillén & Uriarte, 2012).