Article Cite This: J. Agric. Food Chem. 2019, 67, 671−679
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Study of the Physicochemical Properties of Fish Oil Solid Lipid Nanoparticle in the Presence of Palmitic Acid and Quercetin Morteza Azizi, Yitong Li, Neha Kaul, and Alireza Abbaspourrad*
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Department of Food Science, College of Agriculture and Life Science, Cornell University, Ithaca, New York 14853, United States ABSTRACT: ω-3 polyunsaturated fatty acids, naturally found in fish oil, are highly desirable for their associated health benefits. However, they are highly prone to oxidation and degradation. We examined the feasibility of simultaneously adding a solid lipid (palmitic acid) and an antioxidant (quercetin) into a whey-protein-isolate-stabilized solid lipid nanoparticle emulsion for encapsulating fish oil. The goal was to find a rational and new formulation containing both solid lipid and antioxidant that can encapsulate fish oil and give it the best physicochemical stability. Our results show that adding palmitic acid improved the physical stability of the emulsions by decreasing the size of the oil-in-water droplets. On the basis of the thiobarbituric acid reactive substances assay, we found out that at low concentrations of palmitic acid the addition of quercetin played a dominant role in increasing the oxidation stability of fish oil. On the contrary, at high concentrations of palmitic acid, it was palmitic acid that dominated the oxidation inhibition by the solidification of the encapsulates’ core. KEYWORDS: fish oil, emulsion, solid lipid nanoparticles, palmitic acid, quercetin
1. INTRODUCTION ω-3 polyunsaturated fatty acids (PUFAs), a natural component of many foods, are renowned for their health benefits.1−3 Fish oil contains high levels of ω-3 PUFAs, including eicosapentaenoic acid and docosahexaenoic acid, which are known for their antiinflammatory properties.4 The ingestion of fish oil is associated with the prevention of cardiovascular diseases, a decreased risk of some cancers, reduced inflammation, and improved brain function.5,6 However, the susceptibility of this oil to oxidation limits its application as an additive to fortify foods.7 Oxidation adversely affects oil quality and palatability due to the production of peroxides, dienes, carbonyls, aldehydes, and trienes.8−11 Colloidal systems, specifically oil-in-water (O/W) emulsions, are typically employed to protect PUFAs (lipophilic phase) from oxidation.1,12−15 An emulsifier at the interface of O/W encapsulates added to the encapsulated lipophilic phase can provide protection from harsh environmental conditions, such as extreme temperatures, salt, and mechanical stress during processing and storage, and harsh human gastrointestinal tract conditions, such as enzymatic degradation and acidic pH.1,16−19 Different types of emulsifiers, including macromolecules such as proteins and polysaccharides20−22 or small molecules such as phospholipids,5 have been used to encapsulate fish oil. An appropriate emulsifier in an O/W system to some extent can enhance the protection of the oil and improve the physicochemical stability of the O/W droplets against oxidation, creaming, flocculation, and coalescence effects. The incorporation of other compounds in the O/W droplets has recently been studied to further enhance the physicochemical properties of the emulsion. For example, the inclusion of an antioxidant can potentially improve the oxidation stability of encapsulated oil. Natural antioxidants, such as phenolic compounds, have been employed to enhance the oxidative stability of PUFAs.7 The mechanisms by which antioxidants inhibit or lower oxidation rates include a combination of © 2019 American Chemical Society
chelating pro-oxidative metals, quenching singlet oxygen, scavenging free radicals, and inactivating lipoxygenase.23 In addition, it has been shown that the incorporation of crystalline solid lipids, that is, solid lipid nanoparticles (SLNs), into the dispersed phase can have a significant impact on the emulsion’s physicochemical stability due to the solid lipids’ ability to decrease enthalpy and the Gibbs free energy of the system; the solid phase of materials is thermodynamically more stable.24−26 For this reason, SLNs are being widely studied as delivery systems to encapsulate lipophilic components.27,28 For instance, the influence of different surfactants in SLN emulsions containing encapsulated beta-carotene was evaluated by Helgason et al.29 The authors suggested that high melting point surfactants protect bioactive compounds against degradation because more stable crystal matrices are formed within the SLN particles. The study also found that SLN size plays a more significant role in the oxidation mechanism of susceptible oxidation materials than the crystallization of solid lipids within the droplets’ core. McClements24 also reported that saturated triglyceride carriers, such as tristearin, could be stabilized by adding Quillaja saponaria extract as an emulsifier due to the heterogeneous nucleation and crystallization of the carrier lipid. Another important point of using solid lipids in O/W emulsions is the rate of crystallization and its potential effects on the interior structure of the emulsion system. The rate of crystallization of O/W colloids can be controlled through various means, including the incorporation of impurities or the cooling rate.24,27 Although choosing a good emulsifier can extend the shelf life of fish oil and prevent oxidation, more protection can be incorporated by adding other compounds into the emulsion Received: Revised: Accepted: Published: 671
April 29, 2018 November 6, 2018 December 3, 2018 January 7, 2019 DOI: 10.1021/acs.jafc.8b02246 J. Agric. Food Chem. 2019, 67, 671−679
Article
Journal of Agricultural and Food Chemistry system. Clearly, there are multiple strategies for stabilizing fish oil emulsions, which can be broadly categorized into the use of antioxidants and solid lipid additives. Quercetin and palmitic acid are two examples of these kinds of materials, respectively, which, to the best of our knowledge, have not been studied in terms of their efficacy in stabilizing fish oil as coencapsulates within an SLN system. To understand how palmitic acid and quercetin simultaneously may improve the physicochemical properties of fish oil encapsulates, we prepared whey-proteinisolate (WPI)-stabilized emulsions using different formulations of palmitic acid and quercetin and at two different cooling regimes (termed slow- and fast-cooling in this study). We then characterized the physicochemical properties of these emulsions, including particle size, ζ-potential, morphology, fish oil encapsulation efficiency, crystallinity, and oxidation stability to demonstrate how effective this system can be in protecting fish oil against physical and chemical instabilities. Our findings show the strong potential of this system for use in functionalized food.
with an atomizer nozzle. The inlet and outlet temperatures were set to 100.0 and 50.0 °C (± 1.0 °C), respectively. The dried powders were collected from both the drying chamber wall and the cyclone and stored at 4 °C until further characterization. The drying process was performed twice for two different batches of each prepared emulsion. 2.3. Characterization. 2.3.1. Encapsulation Efficiency. To investigate the fish oil encapsulation efficiency, a consistent 2:1 ratio of hexane to emulsion was used to wash the surface oil (nonencapsulated oil or free oil) in the continuous phase. The water and oil phases were then separated from each other by centrifuging the sample at 5000 rpm for 10 min. Filter paper (Whatman, No. 4) was used to separate the solid phase (particles) and liquid phase (solvent and extracted oil). However, to ensure that all of the surface oil was fully washed out, the filter paper was again washed using petroleum ether (the same volume as hexane used in the previous step). Then, the washing permeates from the hexane and petroleum ether steps were collected in a beaker, and the solvents were allowed to evaporate at room temperature (20 ± 2 °C) for 3 h. The mass of the remaining material constituted the extracted surface oil.30 The amount of initial and extracted oils allowed us to calculate the fish oil encapsulation efficiency using the following equation
2. MATERIALS AND METHODS fish oil encapsulation efficiency (%) total added fish oil − surface fish oil = × 100 total added fish oil
2.1. Materials. Menhaden fish oil (eicosapentaenoic acid, 10−15%; docosahexaenoic acid, 8−15%) was purchased from Sigma-Aldrich (St. Louis, MO), as was palmitic acid (95 wt %, FCC, FG), quercetin (>95 wt %, HPLC grade), and petroleum ether (anhydrous, 99%). Foodgrade WPI (97.6% protein) was obtained from Davisco Foods International (Le Sueur, MN). Hexane (anhydrous, 95%) was purchased from VWR International (Houston, TX). Milli-Q water was used to prepare all solutions and emulsions. 2.2. Preparation of Fish Oil Encapsulated in Solid Lipid Nanoparticles. The water phase solution was prepared by dissolving WPI (2% w/w) in Milli-Q water (according to the formulations in Table 1) and stirring it at room temperature (20 ± 2 °C) for 1 h. For the
2.3.2. Particle Sizes and ζ-Potential Measurements. Droplet size, droplet size distribution, and ζ-potential of the resulting dispersions were analyzed in triplicate by a commercial dynamic light scattering device (Nano-ZS, Malvern Instruments, Worcestershire, U.K.). For these measurements, the original samples were diluted 100-fold with Milli-Q water. Three mL of the diluted sample was retained for particlesize and ζ-potential analysis. 2.3.3. Scanning Electron Microscopy of Spray-Dried Powders. The microstructural properties of the spray-dried emulsion samples were investigated using a scanning electron microscope (SEM). Using double-sided carbon tape, samples were loaded onto an aluminum platform. A gold sputter coater was used to coat the samples under argon for 30 s. The images were obtained on a JCM-6000 NeoScope (JEOL, Japan) at 15 kV. 2.3.4. X-ray Diffraction. X-ray diffraction (XRD) analysis of the prepared powder was performed using a Bruker D8 Advance ECO powder diffractometer (Billerica, MA) by running the powder samples from 5 to 45° under continuous scan at a step size of 0.026 with 2θ min−1. 2.3.5. Oxidative Properties of Prepared Fish Oil Emulsions. The oxidative stability of the encapsulates was evaluated using thiobarbituric acid reactive substances (TBARS) assay. TBARS values were determined by mixing 500 μL of the prepared emulsion with 3.0 mL of the TBA reagent (15% w/v trichloroacetic acid and 0.375% w/v thiobarbituric acid in 0.25 M HCl) in test tubes and then heated in a water bath to 90 °C for 15 min. The tubes were cooled at room temperature for 10 min and then centrifuged (1000g) for 15 min to let the hydrophobic phase (mainly chemical reaction products) separate from the aqueous phase. Then, 3 mL of butanol was added and the tubes were vortexed. The supernatant was then carefully separated using a 1 mL pipet, and the absorbance at 532 nm was measured with UV−vis absorption spectroscopy (UV-2600 spectrophotometer, Shimadzu, Japan). The concentration of TBARS was determined using a standard curve prepared using 1,1,3,3-tetraethoxypropane.3 2.3.6. Interfacial Tension. The interfacial tension between the 2% w/w WPI water-phase solution and the oil phase containing 2.50% w/w palmitic acid was measured using the pendant drop method and a standard Ramé-Hart contact-angle goniometer (model 190 CA, RaméHart Instrument, Netcong, NJ). The measurement of the interfacial tension was carried out by filling a standard quartz cell with the preheated oil phase at 70 °C and using a microsyringe with a stainlesssteel 22G needle filled with 2% preheated WPI solution at 70 °C to produce a submerged pendant droplet. The Young−Laplace equation was used to analyze the droplet images and to measure the interfacial
Table 1. Formulations of Emulsions Prepared Using Different Amounts of WPI, Palmitic Acid (shown as P), and Quercetin (shown as Q) sample name 1.25% P, 200 ppm Q 1.25% P, 500 ppm Q 1.25% P, 800 ppm Q 2.50% P, 200 ppm Q 2.50% P, 500 ppm Q 2.50% P, 800 ppm Q 3.75% P, 200 ppm Q 3.75% P, 500 ppm Q 3.75% P, 800 ppm Q
WPI (wt %)
palmitic acid (wt %)
quercetin (ppm)
2
1.25
200 500 800 200 500 800 200 500 800
2.50
3.75
(1)
preparation of the oil phase, fish oil with different amounts of palmitic acid and quercetin was added to a beaker and stirred at 70 °C for 10 min, according to Table 1, to ensure that a homogeneous mixture was obtained. Emulsions with 10 wt % total oil phase were prepared by adding the oil into the water phase solution (90 wt %, preheated to 70 °C for ∼3 min prior) and homogenizing the mixture with a high-speed blender at 15 000 rpm for 3 min. This preheating process prevents palmitic acid crystal formation outside the O/W droplets, which can interrupt the emulsion preparation process before the formation of any fine colloidal particles.24 After preparing the emulsion, it was aliquoted into two falcon tubes (∼40 mL, each). One tube was processed by a “fast-cooling” procedure, involving storing it in an ice bath (∼4 °C), and the other tube was processed by a “slow-cooling” procedure, which involved allowing the solution to cool to room temperature (20 ± 2 °C) on its own. We spraydried the oil-in-water emulsion using a laboratory-scale spray dryer (FT30MkIII-G Spray Dryer; Armfield, Hampshire, England) equipped 672
DOI: 10.1021/acs.jafc.8b02246 J. Agric. Food Chem. 2019, 67, 671−679
Article
Journal of Agricultural and Food Chemistry
Figure 1. (A) Schematic structure of an emulsion and the cross-section of a fish oil SLN encapsulate consisting of a crystalline structure of palmitic acid and quercetin as an antioxidant incorporated into the core. WPI is at the interface to stabilize the emulsion droplets. (C) Particle size and (D) PDI of the prepared WPI-stabilized fish oil emulsions at different concentrations of palmitic acid and quercetin added to the oil phase (core). Peak analysis attributed to SLN formation at different amounts of palmitic acid and quercetin added to the oil phase (core) for two different cooling regimes: (E) slow-cooling and (F) fast-cooling. Results are provided as mean ± SD of three replicate samples. Bars with different letters are significantly different from each other (p < 0.05). tensions. Ten measurements were taken, spaced by 1 s intervals. All reported values are an average of triplicate measurements. 2.4. Statistical Analysis. All experiments were performed in at least triplicate, and the measurements are presented as mean ± standard deviation (SD). A two-way ANOVA analysis with replication test followed by a Student’s two-tailed t test was used to determine statistical differences (p values
