Oils can become oxidized during processing, distribution and handling, particularly if they are highly unsaturated [1
]. Lipid oxidation in formulated foods is therefore a major concern to food producers. Modern consumers have a preference for ‘clean label’ food products with little to no additives present, e.g., synthetic antioxidants and emulsifiers; thus, it is important to develop new processing technologies that reduce the need of using stabilizing additives.
Oil-in-water (O/W) submicron/nano-emulsions have been studied in order to encapsulate lipophilic molecules in the food, pharmaceutical and cosmetic sectors [3
]. Emulsion stability depends on a range of complex mechanisms, including flocculation, coalescence, Ostwald ripening and phase separation. However, it is well known that one of the key factors determining stability is the average size of fat globules and their size distribution [5
]. Submicron (nano) emulsions are systems containing droplets with diameters below 1 µm and are known to have a high physical stability [6
]. Producing emulsions with small droplet sizes and narrow size distributions can be achieved through the application of a large amount of energy, or the use of surfactants, or the combination of both. Low-energy emulsification processes, such as phase-inversion temperature techniques, require the use of large quantities of surfactants; thus, they cannot be used for large-scale industrial production [7
]. High-energy emulsification utilizes mechanical devices, such as high-pressure homogenizers and microfluidizers or sonication equipment, that are being used in industrial production. These technologies generate intense disruptive forces that break up the oil and water phases and lead to the formation of small oil droplets [8
]. Ultra-high-pressure homogenization (UHPH) is a non-thermal processing technique, which works at significantly higher pressures (≥200 MPa) than high-pressure homogenization (150–200 Mpa) or microfluidization (20–150 MPa) [9
The physical characteristics and emulsion stability of nutritional formulations were evaluated on a pilot-scale HPH unit [11
]. The authors reported that homogenization at pressures from 100–150 MPa yielded more shear-thinning fluids with increased physical stability, representing an opportunity to reduce the concentration of stabilizers in dairy beverages. In another study, UHPH (100–300 MPa) was compared to conventional homogenization at 15 MPa and was found to produce oil-in-water emulsions containing 4.0% (w
) of soy protein isolate (SPI) and soybean oil (10% and 20%, v
) with small d3.2 (µm) values and great physical stability [12
]. Kuhn and Cunha [13
] studied the effects of homogenization pressure and the number of homogenization cycles on the physicochemical characteristics of emulsions containing flaxseed oil and whey protein isolate. The authors reported that these emulsions showed good physical stability without phase separation after nine days of storage, when homogenized at high-pressure (20–100 MPa) with 1–7 homogenization cycles.
Proteins are ingredients widely used in food emulsions as emulsifiers/stabilizers due to their amphiphilic character. Proteins are able to form an interfacial layer that generates repulsive forces (e.g., steric and electrostatic) between oil droplets, which plays an important role in stabilizing the droplets against flocculation and coalescence during long-term storage [14
]. Whey protein isolates (WPI) are widely used as emulsifiers to enhance the formation and stability of (O/W) emulsions [15
]. Whey proteins are also known to inhibit lipid oxidation by preventing pro-oxidants from accessing the droplets [17
The oil volume fraction has a great influence on the physicochemical and viscoelastic properties of emulsions by affecting droplet size distribution, creaming, oxidative stability and rheological properties [18
]. Few data are available concerning the combined effects of varying dynamic high-pressure treatments and the oil volume fraction on emulsion rheology and physical stability. Cortés-Muñoz et al. [19
] studied oil concentrations of 15%, 30% and 45% and pressures up to 300 MPa in O/W emulsions stabilized by whey protein isolate (4%), reporting that optimal droplet breakdown was observed for emulsions with 30% of oil (w
) treated with homogenization pressures ≥200 MPa. Floury et al. [20
] reported that increasing oil concentration resulted in a larger mean droplet diameter at constant homogenizing conditions due to the limitation of the emulsifier. These researchers revealed that the emulsions containing less than 20% of oil followed Newtonian behaviour (n
= 1) regardless of the homogenization pressure applied; however, emulsions containing more than 20% of oil and homogenized at 20 or 150 MPa showed shear-thinning behaviours (n
< 1). Floury et al. [20
] also showed that the application of increasing homogenization pressures resulted in high oil content emulsions (>40%) transitioning from shear-thinning behaviour (at 20 MPa) to Newtonian behaviour (at 300 MPa).
It is well established that emulsions with small droplet sizes and high specific surface areas are very sensitive to lipid oxidation [21
]. Many other factors may affect the oxidative stability of nano-emulsions, such as the physical structure of emulsions, the level and type of emulsifier(s) or the bulk oil-phase [22
]. However, there are few studies that have investigated the oxidative deterioration of these emulsions, including those containing high oil concentrations.
In a previous work of the present authors [23
], submicron emulsions with high physical and oxidative stabilities were obtained using 1 or 2 g WPI /100 g and 100 MPa homogenization pressure or 4 g WPI /100 g and 200 MPa homogenization pressure. Fernandez-Avila and Trujillo [24
] reported that soy protein isolate stabilized emulsions containing 20% of oil (w
), rather than 10% (w
), with homogenization pressures of 100 or 200 MPa from the point of view of oxidation. The researchers attributed this high oxidative stability to the large quantity of oil and the high protein load at the surface. However, to the knowledge of the present authors, no reports on the effect of oil concentration and homogenization pressure on the oxidative stability of whey protein-stabilized emulsions have been referred in the literature. Further research is required to establish the relationship between UHPH treatment of emulsions and the stability to lipid oxidation. The objective of this study was to evaluate the effect of homogenization pressures (100–200 MPa) and oil concentration (10, 30 and 50 g/100 g) on the structure, rheological properties, physical and oxidative stabilities of emulsions containing 4 g whey protein isolate/100 g, in comparison with those produced by colloid mill and conventional homogenization.
2. Materials and Methods
Whey protein isolate (WPI) was obtained from Lactalis (Prolacta 90, Retiers, France). The WPI contained 95.9 g dry solids per 100 g powder and, on a dry basis (w/w), 1.04% non-protein nitrogen (NPN), 89.3% protein ((total N − NPN) × 6.38), 1.1% ash (including 0.27% calcium) and 1.6% lactose, as given by the producer. Protein constituents in the WPI corresponded mainly to β-lactoglobulin (β-Lg) and α-lactalbumin (α-La) (i.e., 68.5% β-Lg and 21.5% α-La per 100 g soluble protein) with small amounts or traces of immune globulins, bovine serum albumin and lactoferrin.
Refined sunflower and olive oils were purchased from Gustav Heess Company (Barcelona, Spain). The characteristics and composition of oils according to the producer were: density (20 °C) = 0.921 and 0.913; acid value = 0.09 (mg KOH/g) and 0.11%; peroxide value (meq O2/kg) = 0.02 and 0.5; absorbance (270 nm) = not determined and 0.29; unsaponifiable (% m/m) = ˂0.05% and ˂1.5%; C16:0 (%) = 6.34 and 11.94; C18:0 (%) = 3.97 and 3.30; C18:1 (%) = 26.65 and 75.23; C18:2 (%) = 61.02 and 6.75; C18:3 (%) = 0 and 0.38; for sunflower and olive oils, respectively.
2.2. Preparation of Emulsions
2.2.1. Experimental Design
A completely randomized factorial design was applied to study the influence of homogenization pressure and oil concentration on the physical and oxidative stability of the emulsions. This design was used with three homogenization methods (colloid mill (CM), conventional homogenization (CH) and ultra-high pressure homogenization (UHPH)). By varying the shear rate (5000 rpm for CM) or homogenization pressure (15 MPa for CH and 100, 200 MPa for UHPH) and oil concentration (10, 30 and 50 g/100 g) according to Table 1
, twelve samples were formulated. Prepared samples were stored in glass bottles under refrigeration (4 °C) until physical analyses. Oxidation analyses were carried out on the first and last day of a 10-day storage at 10 °C in clear glass bottles under light (2000 lux/m2
2.2.2. Preparation of Protein Dispersions
WPI dispersions containing 4 g WPI/100 g were prepared in deionized water using agitation with a high speed mechanical blender (Frigomat, Guardamiglio, Italy) at a rate of 250 rpm at 20 °C. Protein dispersions with pH ≈ 6.5–7 (MicropH 2001, Crison, Alella, Spain) were stored overnight at 4 °C to allow protein hydration.
2.2.3. Homogenization Treatments
After rehydration, protein dispersions of 4 g WPI/100 g and different oil concentrations (10, 30 and 50 g/100 g) were equilibrated at 20 °C before mixing. Pre-emulsions (or coarse emulsions) were prepared by mixing the oily dispersed phase (3 parts sunflower:1 part olive oil) with the aqueous continuous phase containing WPI at room temperature to give a total volume of 40 L. The mixture was stirred for 5 min using a colloid mill homogenizer (E. Bachiller B, S.A, Barcelona, Spain) at maximum power (5000 rpm) to obtain the CM emulsions and further processed into CH and UHPH emulsions as follows.
Conventional homogenization of the CM emulsions was performed using an APV Rannie Copenhagen Series Homogenizer (Model 40.120 H, single-stage hydraulic valve assembly, Copenhagen, Denmark) with Tin of 60 °C at 15 MPa (CH emulsions).
CM emulsions were treated by UHPH using a Stansted high-pressure homogenizer (Model/DRG number FPG 11,300:400 Hygienic Homogenizer, Stansted Fluid Power Ltd., Harlow, UK) with a flow rate of 120 L/h as indicated by the manufacturer. Two spiral-type heat exchangers (Garvía, Barcelona, Spain) located behind the second valve were used to cool the emulsion immediately after the HP-valve to minimize temperature retention in the emulsion after treatment, which may affect emulsion composition. Emulsions were UHPH-treated at pressures of 100 and 200 MPa (single-stage) with an inlet temperature (Tin) of 25 °C (UHPH emulsions). Throughout the experiment, the Tin, the temperature after the homogenization valve (T1) and the temperature of the outlet product (T2) were monitored.
The experiment by each preparation method in each study was repeated three times.
2.3. Emulsion Measurements and Analyses
2.3.1. Droplet Size Distribution
The droplet size distribution of the different emulsions was determined just after sample preparation using a Beckman Coulter laser diffraction particle size analyser (LS 13 320 series, Beckman Coulter, Fullerton, CA, USA), as described by Hebishy et al. [23
]. Emulsion samples were diluted in distilled water until an appropriate obscuration was obtained in the diffractometer cell. An optical model based on the Mie theory of light scattering by spherical droplets was applied by using the following conditions: real refractive index of the oil mixture (sunflower oil:olive oil (3:1)), which was obtained by a refractometric measurement (Spectronic Instruments, Inc., Rochester, NY, USA), 1.471; refractive index of fluid (water), 1.332; the refractive index of the protein was assumed to be 0 [25
]; imaginary refractive index, 0; pump speed, 20%. The volume weighted mean diameter (d4.3, μm), surface-weighted mean diameter (d3.2, μm) and specific surface area (SSA, m2
/mL) were determined. Each diluted sample was analysed at least four times in succession to obtain a mean size distribution curve and the corresponding mean values.
2.3.2. Surface Protein Concentration
The surface protein concentration of oil droplets was determined according to the method of Desrumaux and Marcand [26
], as described by Hebishy et al. [23
]. Briefly, the cream layer was isolated by centrifugation and clarified. The protein content of the isolated purified protein layers was determined in triplicate by the Dumas method with a Leco FP-528 nitrogen/protein instrument (Leco Corp., St. Joseph, MI, USA), estimating crude protein content as N × 6.38.
2.3.3. Rheological Measurements
Rheological measurements were performed at 25 °C using a controlled stress rheometer (Haake Rheo Stress 1, Thermo Electron Corporation, Karlsruhe, Germany) with a parallel plate geometry probe (1°, 60 mm diameter). To avoid any structure destruction, samples were left standing for 5 min at 25 °C in order to reach equilibrium. Flow curves were fitted to the Ostwald de Waele rheological model: τ = K·γn, and the consistency coefficient (K, mPa × s) and flow behaviour index (n) were obtained. Rheological measurements were carried out in triplicate.
2.3.4. Physical Stability
The physical stability of emulsions was assessed by measuring the d4.3 (µm) value at the top or at the bottom of the emulsion tubes stored for 9 days. Measurements were performed in triplicate using the laser diffraction particle size analyser (LS 13 320 series, Beckman Coulter, Fullerton, CA, USA), as detailed before (Section 2.3.1
The stability of emulsions was also determined with a vertical scan analyser Turbiscan MA 2000 (Formulaction, Toulouse, France), as reported by Hebishy et al. [23
]. This equipment allows the optical characterization of any type of dispersion. The light source is an electro-luminescent diode in the near-infrared (λair = 850 nm). Any change due to a variation of the droplet size (flocculation, coalescence) or a local variation of the volume fraction (migration phenomena: creaming, sedimentation) is detected. Under backscattering mode, Turbiscan measures the light backscattered by the sample, which is directly dependent on the droplet mean diameter, at pre-set intervals (30 min for CM emulsions, 3 days for CH and UHPH emulsions) over a selected period of time (5 h for CM emulsions and 17 days for CH and UHPH emulsions). The migration rate or velocity (V
; μm/min) of the clarification front was also calculated using Turbisoft software in order to follow the kinetics of the creaming phenomenon.
2.3.5. Emulsion Microstructure
In order to assess the microstructure of emulsions, emulsion samples were observed under a transmission electron microscope with a Jeol 1400 (Jeol Ltd., Tokyo, Japan) equipped with a Gatan Ultrascan ES1000 CCD Camera. Samples were prepared according to Cruz et al. [27
], as described by Hebishy et al. [23
2.3.6. Stability of Emulsions to Photo-Oxidation
For the determination of primary oxidation products, lipid hydroperoxides were measured by mixing 0.3 mL of emulsion with 1.5 mL of isooctane/2-propanol (3:1, v
) by vortexing (10 s, three times) and isolating the organic solvent phase by centrifugation at 1000× g
for 2 min. The organic solvent phase (200 μL) was added to 2.8 mL of methanol/1-butanol (2:1, v
), followed by 15 μL of 3.97 M ammonium thiocyanate and 15 μL of ferrous iron solution (prepared by mixing 0.132 M BaCl2
and 0.144 M FeSO4
). The absorbance of the solution was measured at 510 nm, 20 min after the addition of the iron [28
]. Hydroperoxide content was expressed as absorbance (A510).
For the determination of secondary oxidation products, thiobarbituric acid-reactive substances (TBARS) were determined according to an adapted method of McDonald and Hultin [29
]. The emulsion (1.0 mL) was combined with 2.0 mL of TBA solution (prepared by mixing 15 g of trichloroacetic acid, 0.375 g of thiobarbituric acid, 1.76 mL of 12 N HCl and 82.9 mL of H2
O) in test tubes and placed in a boiling water bath for 15 min. The tubes were allowed to cool to room temperature for 10 min, and then, the coloured solution was separated by filtration through glass wool. The absorbance was measured at 532 nm. Concentrations of TBARS were calculated from a standard curve prepared using 1,1,3,3-tetraethoxypropane.
2.4. Statistical Analyses
Statistical analyses were performed using SAS System® v9.2 (SAS Institute Inc., Cary, NC, USA), with a nominal significance level of 5% (p ˂ 0.05) and Tukey adjustment for multiple comparisons of means. In order to evaluate the physical and oxidative stabilities depending on the type of emulsion (CM, CH or UHPH) and the concentration of oil (10%, 30% and 50%), a general linear model with repeated measures was performed. The rheological index, consistency coefficient, hydroperoxides and TBARS values were compared between the CM, CH and UHPH emulsions. The other parameters (d3.2, SSA and SPC) were compared only between CH and UHPH emulsions, excluding CM emulsions due to the wide variation of data; these three parameters were compared among the three oil levels in CM emulsions.