Article pubs.acs.org/JAFC
Nanoemulsion-Based Delivery Systems for Polyunsaturated (ω-3) Oils: Formation Using a Spontaneous Emulsification Method Alessandro Gulotta,†,§ Amir Hossein Saberi,† Maria Cristina Nicoli,§ and David Julian McClements*,† †
Biopolymers and Colloids Laboratory, Department of Food Science, University of MassachusettsAmherst, Amherst, Massachusetts 01003, United States § Department of Food Science, University of Udine, Via Sondrio 2/A, 33100 Udine, Italy ABSTRACT: Nanoemulsion-based delivery systems are finding increasing utilization to encapsulate lipophilic bioactive components in food, personal care, cosmetic, and pharmaceutical applications. In this study, a spontaneous emulsification method was used to fabricate nanoemulsions from polyunsaturated (ω-3) oils, that is, fish oil. This low-energy method relies on formation of fine oil droplets when an oil/surfactant mixture is added to an aqueous solution. The influence of surfactant-to-oil ratio (SOR), oil composition (lemon oil and MCT), and cosolvent composition (glycerol, ethanol, propylene glycol, and water) on the formation and stability of the systems was determined. Optically transparent nanoemulsions could be formed by controlling SOR, oil composition, and aqueous phase composition. The spontaneous emulsification method therefore has considerable potential for fabricating nanoemulsion-based delivery systems for incorporating polyunsatured oils into clear food, personal care, and pharmaceutical products. KEYWORDS: nanoemulsions, emulsions, spontaneous emulsification, encapsulation, delivery, low-energy homogenization, particle size, nutraceuticals, pharmaceuticals, functional food, ω-3 oils, polyunsaturated oils
■
INTRODUCTION There is considerable interest in incorporating polyunsaturated lipids (such as ω-3 fatty acids) into foods, supplements, and pharmaceuticals due to their potential health benefits.1−4 The three most common types of ω-3 fatty acids in foods and beverages are α-linolenic acid (ALA, 18:3), eicosapentaenoic acid (EPA, 20:5), and docosahexaenoic acid (DHA, 22:6), with the latter two reportedly having the highest bioactivities. Consumption of sufficiently high levels of ω-3 fatty acids has been linked to reduced risk of certain chronic diseases, such as inflammation, cardiovascular disease, immune response disorders, mental disorders, and poor infant development.5,6 The levels of ω-3 fatty acids required to have a beneficial effect are not currently being reached in the general population, and so there is an emphasis on increasing the levels consumed.7,8 Nevertheless, there are a number of challenges associated with fortifying food and beverage products with polyunsaturated fatty acids (PUFAs). First, PUFAs are highly hydrophobic molecules that have a low water-solubility and therefore have to be incorporated into some kind of colloidal delivery system before they can be introduced into most aqueous-based foods and beverages. Second, PUFAs are oxidatively unstable and tend to degrade during storage, leading to undesirable offflavors, which reduces consumer acceptabilty.9 A number of studies have shown that colloidal delivery systems can be designed to incorporate PUFAs into aqueous environments and to improve their oxidative stability, including emulsions, multilayer emulsions, multiple emulsions, and filled hydrogel particles.10−15 Most of these emulsion-based delivery systems contain particles that have dimensions similar to the wavelength of light, and therefore they scatter light strongly, leading to high turbidity or opacity.16 For certain applications it is advantageous to use a delivery system that is transparent so that it can © 2014 American Chemical Society
be incorporated into optically clear food or beverage products, such as some fortified waters, soft drinks, and dressings. An optically clear delivery system should contain particles that do not scatter light strongly. The light-scattering efficiency of particles depends on their refractive index contrast, concentration, and size.16,17 It is difficult to control the refractive index contrast of oil or aqueous phases, because this typically requires the addition of high levels of cosolvents (such as >50% sugar or polyol) to achieve optical clarity.18 The particle concentration in a delivery system must be relatively high to deliver ω-3 fatty acids at levels reported to promote health benefits, that is, >250 mg per day.19 Consequently, there are limits as to how far the particle concentration within a colloidal delivery system can be reduced to achieve optical clarity while maintaining biological activity. The most practical means of incorporating relatively high concentrations of bioactive lipids in optically transparent products is to use delivery systems containing particles that are so small that they do not scatter light strongly, that is,d < 50 nm.20,21 There are also other advantages associated with utilizing delivery systems containing small particles, such as increased stability and bioavailabilty.22,23 The two most common types of colloidal delivery systems with sufficiently small particles to achieve optical transparency are microemulsions and nanoemulsions.20,24 Both systems contain small particles (d < 200 nm) that have a hydrophobic core and a hydrophilic shell, but microemulsions are thermodynamically stable, whereas nanoemulsions are not.25 Received: Revised: Accepted: Published: 1720
December 5, 2013 January 27, 2014 January 29, 2014 January 29, 2014 dx.doi.org/10.1021/jf4054808 | J. Agric. Food Chem. 2014, 62, 1720−1725
Journal of Agricultural and Food Chemistry
Article
Static Light Scattering. The particle size distribution was determined by measuring the angular dependence of the intensity of light scattered from the emulsions. Mean particle diameters (d32) were calculated from the particle size distribution. All samples were diluited with citric buffer prior to making the measurements to avoid multiple scattering effects. This was achieved by adding a few drops of nanoemulsions into approximately 125 mL of citric buffer in the sample chamber of the instrument until an obscuration of 11−15% was obtained. Turbidity Measurements. The influence of temperature on the turbidity of the nanoemulsions was determined using a UV−visible spectrophotometer with temperature-scanning capabilities (Evolution Array, Thermo Scientific, Madison, WI, USA). The turbidity was measured at 600 nm as the temperature was increased form 20 to 90 °C at 1 °C/min and then decreased back to 20 °C. Statistical Analysis. All experiments were carried out twice with two samples being analyzed for each trial. The results are reported as the calculated mean and standard deviation of these measurements.
One of the main advantages of nanoemulsions over microemulsions is that they require considerably less surfactant to form them. Food-grade nanoemulsions can be formed by highenergy methods (such as high-pressure homogenization or sonication) or low-energy methods (such as phase inversion temperature, spontaneous emulsification, or emulsion phase inversion).23,26 In this study, we examined the utilization of spontaneous emulsification for forming food-grade ω-3 fatty acid delivery systems suitable for utilization in transparent foods and beverages. This method involves simply injecting an oily phase (typically consisting of a hydrophilic surfactant, a carrier oil, and a lipophilic bioactive) into an aqueous phase (typically consisting of water and cosolvent) with continuous stirring.27 We have recently shown that this method can be used to successfully encapsulate vitamin E in nanoemulsionbased delivery systems.27−30 An advantage of using low-energy methods to form nanoemulsions is that no expensive processing equipment (such as a high-pressure homogenizer or sonicator) is required.
■
■
RESULTS AND DISCUSSION The overall aim of this study was to identify optimum conditions to prepare stable ω-3-enriched nanoemulsions that were optically transparent. We therefore systematically examined the influence of a number of factors on the formation and stability of the nanoemulsions. Influence of Carrier Oil. Preliminary experiments indicated that nanoemulsions with small droplets could not be formed using fish oil alone as the oil phase. We therefore examined the influence of mixing fish oil with a carrier oil (either MCT or lemon oil). Previous studies with vitamin E have shown that this approach can be used to produce nanoemulsions containing small droplets.27 In this series of experiments, the composition of the oil phase was varied, whereas the total oil (10%), surfactant (10%), and buffer (80%) contents were kept constant. Static light scattering (SLS) was used for these experiments because many of the samples formed after spontaneous emulsification had relatively large particle sizes (d > 1000 nm). The influence of carrier oil type and oil phase composition on the particle size of the systems formed by spontaneous emulsification is shown in Figures 1 and 2. The mean particle diameter was relatively small (d < 200 nm) for both carrier oils when the fish oil concentration in the oil phase remained relatively low (≤40% for MCT and ≤50% for lemon oil). On the other hand, there was a steep increase in the mean particle diameter when the fish oil concentration was higher than these values (Figure 1). At low fish oil concentrations, the nanoemulsions had relatively narrow monomodal particle size distributions for both lemon oil and MCT. Ideally, one would like to have small droplet sizes but also a high loading capacity, that is, a relatively high concentration of fish oil within the oil phase. The nanoemulsions prepared using the lemon oil were more suitable for this purpose, because they still had relatively small droplet diameters at relatively high fish oil contents. For example, when the oil phase contained 50% lemon oil and 50% fish oil, the mean droplet diameter was 112 ± 3 nm when measured by static light scattering. This value was close to the mean droplet diameter of 106.0 ± 0.5 nm determined by dynamic light scattering on the sample. We therefore decided to utilize lemon oil as the carrier oil in the remainder of the study. Another advantage of using lemon oil is that it can mask any off-flavors associated with fish oil. Influence of Surfactant Concentration. Previous studies have shown that the size of the droplets produced by low-
MATERIALS AND METHODS
Materials. Fish oil (Denomega 100) was kindly donated by Denofa (Brighton, CO, USA). Lemon oil (SC 020207, lot 2894308) was kindly donated by International Flavors and Fragrances (Union Beach, NJ, USA). The supplier reported that this oil contained ≈60% limonene,
