Interfacial Concentrations of Hydroxytyrosol Derivatives in Fish Oil-in-Water Emulsions and Nanoemulsions and Its Influence on Their Lipid Oxidation: Droplet Size Effects

Reports on the effect of droplet size on the oxidative stability of emulsions and nanoemulsions are scarce in the literature and frequently contradictory. Here, we have employed a set of hydroxytyrosol (HT) esters of different hydrophobicity and fish oil-in-water emulsified systems containing droplets of different sizes to evaluate the effect of the droplet size, surfactant, (ΦI) and oil (ΦO) volume fractions on their oxidative stability. To quantitatively unravel the observed findings, we employed a well-established pseudophase kinetic model to determine the distribution and interfacial concentrations of the antioxidants (AOs) in the intact emulsions and nanoemulsions. Results show that there is a direct correlation between antioxidant efficiency and the concentration of the AOs in the interfacial region, which is much higher (20–200 fold) than the stoichiometric one. In both emulsified systems, the highest interfacial concentration and the highest antioxidant efficiency was found for hydroxytyrosol octanoate. Results clearly show that the principal parameter controlling the partitioning of antioxidants is the surfactant volume fraction, ΦI, followed by the O/W ratio; meanwhile, the droplet size has no influence on their interfacial concentrations and, therefore, on their antioxidant efficiency. Moreover, no correlation was seen between droplet size and oxidative stability of both emulsions and nanoemulsions.


Determination of interfacial areas in emulsions loaded with HT derivatives by a turbidimetric method
Interfacial areas (IA) of 1:9 (O/W) emulsions obtained by a low energy method loaded with antioxidants prepared with I of 0.005 were evaluated by employing a turbidimetric method as described by Pearce and Kinsella [3]. The interfacial area (IA) of the dispersed oil phase were calculated according to Cameron [4] and expressed as m 2 /mL of emulsion. The calculated interfacial areas (IA) were then use to calculate the average droplet size in these emulsions. Figure S1. Interfacial areas (IA) expressed as m 2 /mL of emulsion of 1:9 (O/W) emulsions obtained by a low energy method loaded with antioxidants prepared with I of 0.005.

2.
Determination of the observed rate constant, kobs, for the reaction between 16-ArN2 + and the AOs in fish oil emulsified systems.
The reaction between 16-ArN2 + and AOs in each region of emulsified systems is the product of the second-order rate constant and the concentration of each reactant in that region in moles per liter of region volume. 16-ArN2 + Has a long hydrophobic alkyl chain and a cationic headgroup and is both water and oil insoluble. Its reactive -N2 + group is located in the interfacial region, where it reacts with AOs, as illustrated in Figure 1, and its concentration in the oil and water regions can be considered negligible. Under pseudo-first order conditions (being [AO] much higher than [16-ArN2 + ]), the observed rate is given by Equation (S1).
In Equation (S1), k2 and kI are the observed second rate constant and the second order rate constant in the interfacial region, respectively; kobs is the observed overall rate; square brackets, [], denote the concentration in mol/L of the total emulsion volume; the subscript T stands for total; parentheses, (), denote the concentration in mol/L of the volume of a particular region; subscript I stands for the interfacial region; and ФI is the emulsifier volume fraction defined as the ratio of the volume of emulsifier divided by the total volume of the emulsion (ФI = Vsurf/VTotal). The reaction between 16-ArN2 + and derivatives of hydroxytyrosol, Scheme S1, was followed spectrometrically by employing the dye derivatization method (azo dye formation) described in detail elsewhere [2]. The methodology exploits the rapid reaction of 16-ArN2 + ions with a suitable coupling agent such as N-(1naphthyl)ethylenediamine dihydrochloride, NED, yielding a stable azo dye whose absorbance can be determined spectrometrically at λ = 572 nm, after dilution with an alcoholic mixture. Solutions of the coupling reagent (NED) were prepared in a 50:50 (v/v) BuOH:EtOH mixture to give [NED] = 0.02 M.
Scheme S1. Values of half-lives, t1/2, for the reactions of 16-ArN2 + with AOs and N-(1-naphthyl)ethylenediamine (NED) obtained under the experimental conditions. Notice that the reaction of 16-ArN2 + with NED is much faster than with AOs, a requirement to get reliable rate constants by using the derivatization method.
kobs Values were obtained by fitting the absorbance-time pairs of data to the integrated first order Eq. S2.In Eq. S2, At, Ao and Ainf are the measured absorbance at any time, at t = 0 and at infinite time, respectively. An illustrative kinetic plot for the reaction between 16-ArN2 + and hydroxytyrosol in fish oil-in-water nanoemulsions is shown in Figure S2. Similar kinetic plots were obtained using fish oil-in-water emulsions.

Determining the partition constants for water-insoluble and oil-insoluble AOs in emulsified systems
For oil-insoluble AOs, i.e., AOs that distribute between the aqueous and interfacial region such as HT, only the constant partition PW I is required to define their distribution and equation 4 of the text can be simplified to give equation S3. For water-insoluble AOs, that is, AOs that distribute between the oil and interfacial regions such as derivatives of hydroxytyrosol with alkyl chain length  6, only the PO I partition constant is required and equation