A Global Study by 1H NMR Spectroscopy and SPME-GC/MS of the in Vitro Digestion of Virgin Flaxseed Oil Enriched or not with Mono-, Di- or Tri-Phenolic Derivatives. Antioxidant Efficiency of These Compounds.

The effect of enriching virgin flaxseed oil with dodecyl gallate, hydroxytyrosol acetate or gamma-tocopherol on its in vitro digestion is studied by means of proton nuclear magnetic resonance and solid phase microextraction followed by gas chromatography/mass spectrometry. The extent and pattern of the lipolysis reached in each sample is analyzed, as is the bioaccessibility of the main oil components. None of the phenolic compounds provokes inhibition of the lipase activity and all of them reduce the lipid oxidation degree caused by the in vitro digestion and the bioaccessibility of oxidation compounds. The antioxidant efficiency of the three tested phenols is in line with the number of phenolic groups in its molecule, and is dose-dependent. The concentration of some minor oil components such as terpenes, sesquiterpenes, cycloartenol and 24-methylenecycloartenol is not modified by in vitro digestion. Contrarily, gamma-tocopherol shows very low in vitro bioaccessibility, probably due to its antioxidant behavior, although this increases with enrichment of the phenolic compounds. Oxidation is produced during in vitro digestion even in the presence of a high concentration of gamma-tocopherol, which remains bioaccessible after digestion in the enriched samples of this compound.

*This signal shows different multiplicity if the spectrum, is acquired from the pure compound or taking part in the mixture. **The intensity of some of these signals, also shown in Figure 1, together with signal F of Table S3, were used to estimate the molar percentages of different kinds of glyceryl structures using the equations [eq. S1-eq. S10]. ***The assignment of the 1 H NMR signals of the protons was made as in previous studies (Guillén & Uriarte, 2012;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. The signal letters agree with those given in Figure 1.
Signal Chemical shift (ppm)

Main acyl groups (AG) and fatty acids (FA)
A 0.88 t -CH3 saturated and monounsaturated ω-9 in AG and FA 0.89 linolenic in AG and FA Abbreviations: d: doublet; t: triplet; m: multiplet. *The intensity of these signals, also shown in Figure 1, was used to estimate the molar percentage of linolenic acyl groups plus fatty acids by using equations [eq. S11]. **Overlapping of multiplets of methylenic protons in the different acyl groups either in β-position, 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 S3, were used to estimate the concentration (mmol/molAG+FA) using the equation [eq. S12]. **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 (Guillén & Ruiz, 2005). 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, gamma-tocopherols, hydroxytyrosol acetate and dodecyl gallate detected in the samples before and after in vitro digestion. Some of the signal letters agree with those given in Figure 2.  Table S3, were used to estimate the concentration (mmol/molAG+FA) using the equation [eq. S12]. **Assignment was made with the aid of standard compounds and with the data taken from the literature (Baker & Mayers, 1991;Kubo et al., 2002;Kawai et al., 2007)

Quantification from 1 H NMR spectral data of several compounds present in the starting samples and/or in the lipid extracts of the digestates.
Bearing in mind that the area of each 1 H NMR spectral signal is proportional to the number of protons that generate it, and that the proportionality constant is the same for all kinds of protons, the area of some spectral signals can be employed to quantify a wide variety of compounds, as detailed below.

A. Equations used to estimate the molar percentage (%) of the several glyceride structures present in the lipid extract of digestates and the glycerol.
In these equations, the number of moles (N) of fatty acids and all the glycerides were expressed as follows: N2-MG=Pc*AK/4 [eq. S1] N1-MG=Pc*AL [eq. S2] N1,2-DG=Pc*(AI+J-2AL)/2 [eq. S3] NTG=Pc*(2A4.26-4.38-AI+J+2AL)/4 [eq. S4] N1,3-DG=Pc*(A4. 04-4.38-2A4.26-4.38 where Pc is the proportionality constant existing between the area of the 1 H NMR signals and the number of protons that generate them, AK, AL, AI+J and AF are the areas of the corresponding signals indicated in Table S2, and A4.26-4.38 and A4.04-4.38 represent the areas of the signals between 4.26 and 4.38 ppm, and between 4.04 and 4.38 ppm, respectively. Using these equations, the molar percentages of the different kinds of glycerides in relation to the total number of moles of glyceryl structures present (NTGS) were determined as follows: where G is each kind of glyceride (TG, 1,2-DG, 1,3-DG, 2-MG and 1-MG) and NG the respective number of moles.
Gol%=100NGol/NTGS [eq. S10] B. Estimation of the molar percentages of linolenic fatty acids (FA) plus acyl groups (AG) (FA+AG). The molar percentages of linolenic (Ln%) FA plus AG, in relation to the total number of moles of AG plus FA (NTAG+FA) present in the starting oils and in the lipid extracts of the corresponding digestates were estimated as follows: Ln%=100*(AH/2*AF) [eq. S11] where AH and AF are the areas of signals H and F indicated in Table S3.
C. Estimation of the concentration of specific compounds in oil samples and in the lipids extract from digestates.
The concentration of the several kinds of specific compounds (SC), expressed as micromoles per mole of the sum of AG+FA present, was estimated by using the following equations: [SC] = [(ASC/n)/(AD/2)]*1000 [eq. S12] where ASC, is the areas of the signals selected for the quantification of each specific compound (SC), present in the oil samples and in the lipid extract from digestates and n the number of protons that generate each signal given in Tables S4 and S5 and Figure 1 and AD is the area of the signal D in Table S3.  Figure S2. Region between 4-30 min of the total ion chromatogram obtained by SPME-GC/MS of the FDJ sample and of the digestate of the virgin flaxseed oil DF.