*2.7. Statistics*

The experiments were run in triplicate (NMR) and in duplicate (ISO 3657:2013). The results are expressed as the mean values ± standard deviation (sd). Tuckey's test was applied for the significantly different means (*p* < 0.05).

#### **3. Results and Discussions**

#### *3.1. 1H-NMR Spectral Characterization of Fats and Oils*

A typical 1H-NMR spectrum of an oil is illustrated for a rapeseed oil (RO) in Figure 1. The corresponding peak assignment is explained in Table 1. Figure 1 also shows a comparison of the 1H-NMR spectra of tributyrin (TB) and two rapeseed oil–tributyrin binary mixture: RO (30%) + TB (70%) and RO (60%) + TB (40%).

**Figure 1.** Comparative 1H-NMR spectral characterization of tributyrin (TB —), rapeseed oil (RO —) and rapeseed oil–tributyrin binary mixtures: RO (30%) + TB (70%) — and RO (60%) + TB (40%) —. Letters A–J were assigned to resonances according to letters in Table 1.


**Table 1.** Chemical shifts and peak assignment of 1H-NMR spectra of milk fats. Adapted with permission from Refs. [12,19]. Copyright 2004, *Eur. J. Lipid Sci. Technol*.; Copyright 2021, *J. Dairy Sci*.

\* Letters from A–J correspond to specific resonances according to Figure 1.

As reflected from Figure 1, certain signals (i.e., A, C, E and J) cannot be found in the spectrum of tributyrin, because butyric acid is a short chain saturated fatty acid, lacking allylic, bis-allylic and unsaturated protons. The butyric moiety displays the triplet B' characteristic of the terminal methyl group in the structure of fatty acids, the signal D of the protons in position β relative to the ester group, the triplet F generated by the methylene groups adjacent to the ester group and the signals in the specific area of the glycerol backbone (H and I). We have previously shown the assignment of NMR signals in methyl esters of fatty acids as standards for vegetable oil characterization [20]. We have also shown [19] that the resonance characteristic to the terminal methyl group of the fatty acyl chains appears shifted downfield (0.96 ppm) only in the case of linolenic and butyric acyl moieties (B and B', respectively), compared to the rest of the fatty acyl chains (triplet A, 0.85 ppm). It is therefore evident that as the amount of TB added to the vegetable oil increases, all the resonances related to unsaturated specific groups (J) and those in the vicinity of allylic and bis-allylic groups, (E and G) will decrease. The amplitude of signal C also decreases with the addition of TB, as this resonance is dependent on the length of the fatty acyl chains, being absent for TB.

The only signal that increases in intensity is the triplet B from 0.96 ppm, characteristic for the terminal methyl group in butyric acid or linolenic acid. In rapeseed oil, the 0.96 ppm resonance is due to the linolenic acyl moiety (signal B); as the percentage of added TB increases, this resonance also increases in intensity due to the overlapping signal B'. As expected, the unspecific signals present in all fats and oils, regardless of their specific fatty acid profile (such as H and I from the glycerol moiety, as well as D and F adjacent to the ester group), did not show modifications.

#### *3.2. Algorithm for the SV Calculation from 1H-NMR Data*

The general pattern of triacylglycerols (TAGs), as depicted in Figure 2, consists of a glycerol ester backbone and three fatty acyl chains, each with a terminal methyl group and various amounts of methylene and CH=CH double bonds.

**Figure 2.** General representation of a triacylglycerol structure.

As reflected from Figure 2, triacylglycerols consist of a glycerol triple ester backbone, common to all TAGs, the differences occurring in the hydrocarbon residues from fatty acyl chains. Apart from the terminal methyl groups (-CH3), the hydrocarbon chains consist only of methylene groups (-CH2-) and double bonds (-CH=CH-), the number of which differs depending on the length of the chain and on the degree of unsaturation, being characteristic for each individual fatty acid. For example, oleic acid contains fourteen methylene groups (-CH2-) and a single double bond (-CH=CH-), and linoleic acid contains twelve methylene groups (-CH2-) and two double bonds (-CH=CH-). Therefore, the average molecular formula of a triglyceride can be rendered as:
