Article
Physical and oxidative stability of flaxseed oil-in-water emulsions fabricated from sunflower lecithins: Impact of blending lecithins with different phospholipid profiles Li Liang, Fang Chen, Xing-Guo Wang, Qingzhe Jin, Eric A Decker, and David Julian McClements J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 23 May 2017 Downloaded from http://pubs.acs.org on May 26, 2017
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Journal of Agricultural and Food Chemistry
Physical and oxidative stability of flaxseed oil-in-water emulsions fabricated from sunflower lecithins: Impact of blending lecithins with different phospholipid profiles Li Liang a,b, Fang Chen b,c, Xingguo Wang a, Qingzhe Jin a, *, Eric Andrew Decker b
a
, David Julian McClements b, *
State Key Laboratory of Food Science and Technology, Synergetic Innovation Center of Food
Safety and Nutrition, School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122 China b
Department of Food Science, University of Massachusetts Amherst, Amherst, MA 01003, USA
c
School of Public Health, Nanchang University, Nanchang, Jiangxi 330006, China
* Corresponding authors E-mail addresses:
[email protected] (Q. Jin),
[email protected] (D.J. McClements).
ACS Paragon Plus Environment
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ABSTRACT
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There is great interest in the formulation of plant-based foods enriched with
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nutrients that promote health, such as polyunsaturated fatty acids. This study evaluated
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the impact of sunflower phospholipid type on the formation and stability of flaxseed
5
oil-in-water emulsions. Two sunflower lecithins (Sunlipon 50 and 90) with different
6
phosphatidylcholine (PC) levels (59 and 90%, respectively) were used in varying ratios
7
to form emulsions. Emulsion droplet size, charge, appearance, microstructure, and
8
oxidation were measured during storage at 55 °C in the dark. The physical and chemical
9
stability increased as the PC content of the lecithin blends decreased. The oxidative
10
stability of emulsions formulated using Sunlipon 50 was better than emulsions
11
formulated using synthetic surfactants (SDS or Tween 20). The results are interpreted
12
in terms of the impact of emulsifier type on the colloidal interactions between oil
13
droplets, and the molecular interactions between pro-oxidants and oil droplet surfaces.
14 15
Keywords: sunflower phospholipids; flaxseed oil; emulsion; physical stability;
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oxidative stability; delivery
17 18 19 20 21 22 23 24
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INTRODUCTION
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Epidemiological and animal studies suggest that consuming a sufficiently high
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level of w-3 polyunsaturated fatty acids (ω-3 PUFAs) may prevent the development and
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progression of a number of chronic diseases. As a result, consumers in many developed
29
countries are being advised to increase the level of ω-3 PUFAs consumed. Flaxseed oil,
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which principally contains α-linolenic acid (ALA), and fish oil, which principally
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contains eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are two major
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sources of long-chain ω-3 PUFAs in the human diet. There is strong scientific evidence
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from human trials that ω-3 PUFAs from fish or fish oil supplements have a beneficial
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cardio-protective effect 1, 2. However, the widespread consumption of fish oil sources is
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often limited because of their fishy taste, smell, toxin content, allergies, high cost, and
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tendency to cause eructation (“burping”). Moreover, ω-3 PUFAs obtained from fish are
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unsuitable for consumption by vegetarians, vegans and certain religious groups. ALA is
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a natural precursor of EPA and DHA that may be partially converted into these forms
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within the human body after ingestion 3-5. Consequently, flaxseed oil, which is rich in
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ALA, can be used as a natural source of plant-based ω-3 PUFAs in functional foods and
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supplements 6. Clinical and animal experiments suggest that flaxseed oil has potential
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health benefits, such as reduction in cardiovascular, atherosclerosis, diabetes, cancer,
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arthritis, osteoporosis, autoimmune and neurological disorders 6-9.
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The numerous potential health benefits of ω-3 PUFAs, combined with the current
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low levels of consumption 1, 2, have meant that increasing numbers of functional foods
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fortified with ω-3 PUFA are being developed in both the United States and
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Europe 10. Nevertheless, the successful development of functional foods enriched with
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ω-3 PUFAs is challenging due to their high susceptibility to lipid oxidation 11. For this
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reason, encapsulation technologies, such as those based on emulsions, are being
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developed so that they can be successfully incorporated into foods 12, 13. The success of
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this approach is highly dependent on the food system involved, and each encapsulation
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system must be carefully designed for a particular food product. Previous research has
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ACS Paragon shown that encapsulation systems can be Plus usedEnvironment to improve the oxidative stability of ω-
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3 PUFAs in milk 14, yogurt 15, cheese 16, mayonnaise 17, meat products 18, and energy
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bars 19.
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In emulsions, the interface between the oil and the aqueous phase is the place of
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contact between lipids and hydrophilic pro-oxidative components (such as transition
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metal ions, photosensitizers, and enzymes), and therefore it plays a major role in lipid
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oxidation in emulsions 20, 21. For ω-3 PUFAs delivery systems, it is therefore important
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to select an emulsifier that produces an interfacial layer around the oil droplets that
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provides good physical and chemical stability 21, 22. As consumers become more label
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conscious, there is a movement away from synthetic ingredients toward more natural
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ingredients 23. Many kinds of natural emulsifiers are available for utilization in foods,
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including proteins, polysaccharides, and phospholipids 24, 25. Phospholipids act as good
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emulsifiers because they contain a lipophilic part (two fatty acid groups) and
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a hydrophilic part (phosphoric based esters) on the same molecule. Consequently, they
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can adsorb to oil droplet surfaces, reduce the interfacial tension (thereby facilitating
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emulsion formation), and generate repulsive interactions (thereby enhancing emulsion
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stability). For phospholipids, electrostatic repulsion is usually the most important
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repulsive interaction opposing droplet flocculation, but steric repulsion plays an
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important role in inhibiting droplet coalescence 25. Phospholipids from various sources
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have been shown to have good antioxidant properties, such as those derived from milk
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26
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properties of phospholipids have mainly been attributed to their ability to chelate
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transition metal ions, scavenge free radicals, or act synergistically with tocopherols 31,
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32
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natural emulsifiers for the formulation of delivery systems for ω-3 PUFAs.
, egg yolk
27
, marine species
28
, soybean
29
and sunflower
30
. The antioxidant
. These physical and chemical attributes make phospholipids particularly promising
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The main objective of the current study is to investigate the possibility of forming
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both physically and chemically stable flaxseed oil-in-water emulsions using sunflower
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phospholipids. In our previous research, we showed that emulsions could be prepared
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from sunflower lecithins using both low-energy and high-energy homogenization
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methods
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phosphatidylcholine (PC) contents was investigated for their ability to form and
33, 34
. In these studies, a series of sunflower lecithins with different ACS Paragon Plus Environment
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stabilize emulsions, and it was shown that they behaved differently depending on their
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phospholipid composition. In the current study, we investigated the influence of using
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a blend of two sunflower lecithins with different PC contents on the physical and
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chemical stability of flaxseed oil-in-water emulsions. Additionally, emulsions were
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prepared using two other surfactants, Tween 20 (non-ionic) and SDS (anionic), so that
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direct comparisons could be drawn between the performance of natural zwitterionic
90
phospholipids and synthetic nonionic or anionic surfactants.
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MATERIALS AND METHODS
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Materials. Flaxseed oil was purchased from a local grocery store and used
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without further purification (AAK Ltd., England, UK). Two phospholipid ingredients
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derived from sunflower oil were kindly donated by Perimondo (New York, NY, USA).
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Polyoxyethylene-20-sorbitan monolaurate (Tween 20), sodium dodecyl sulfate (SDS)
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and Nile Red were purchased from the Sigma-Aldrich Co. (St. Louis, MO). All other
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reagents were of analytical or chromatographic grade. Double-distilled water (Milli-Q)
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was used for the preparation of all solutions.
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Characterization of the Phospholipids.
Phospholipid, fatty acid
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compositions, peroxide value and tocopherol content were provided by the
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manufacturer (Table 1), and metal ion content were determined according to methods
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reported previously 35.
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Free Radical Scavenging Assays. The free radical scavenging ability of
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the phospholipids was determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH) as
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described previously with some modifications 36. Eight percent (w/v) solutions of the
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phospholipids in methanol were prepared and then diluted to obtain a range of
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phospholipid concentrations. Then, 0.1 mL of each phospholipid solution was added to
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3.9 mL of methanolic DPPH solution (60 µM). The loss of DPPH was then determined
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by measuring the reduction in the absorption of light at 515 nm using a UV-visible
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spectrophotometer (Ultraspec 3000 pro, Biochrom Ltd., Cambridge, UK) every 15 min ACS Paragon Plus Environment
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until the reaction reached completion. For the blank, 0.1 mL of methanol was used
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instead of the sample. The percentage of remaining DPPH was calculated according to
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a method described previously 37. The concentration of the test compound needed to
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decrease the DPPH concentration by 50% was calculated and expressed as the EC50.
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Emulsion Preparation. Oil-in-water emulsions were prepared by
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homogenizing 10 wt% oil phase (flaxseed oil) with 90 wt% aqueous phase. The
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aqueous phase consisted of emulsifier (2 wt% Sunlipon 50, 2 wt% Sunlipon 90, 1 wt%
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Tween 20, or 1wt% SDS) and phosphate buffer solution (5 mM, pH 7.0). A lower level
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of the synthetic surfactants was utilized than the phospholipids because they are more
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effective emulsifiers. Sodium azide (0.02% w/w) was added as an antimicrobial
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preservative. Aqueous phases with different mass ratios of Sunlipon 50 and 90 were
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prepared (0 to 100%), while keeping the total emulsifier concentrated fixed at 2 wt%.
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Aqueous phases containing the lecithin ingredients had to be sonicated to evenly
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disperse the phospholipids prior to making the emulsions. The sonication conditions
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used were 20 cycles at an amplitude of 70% and pulse length of 5 s on, followed by 3 s
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off. The emulsifier solutions were incubated in an ice bath during sonication to prevent
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large temperature increases. Emulsions were prepared by blending the oil and aqueous
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phases together using a high-speed blender for 2 min (M133/1281-0, Biospec Products,
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Inc., ESGC, Switzerland), and then passing them through a high-pressure homogenizer
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(M110Y, Microfluidics, Newton, MA) with a 75 µm interaction chamber (F20Y) at a
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pressure of 12,000 psi three times.
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Storage Experiment. Immediately after preparation, emulsions samples (2
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mL) were placed in glass tubes and then sealed with plastic caps and parafilm. The test
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tubes were then stored at 55 oC in the dark to determine their long-term physical and
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chemical stability. Periodically, samples were analyzed for particle size, ζ-potential,
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microstructure, and lipid oxidation over a 28-day period. Prior to particle size analysis,
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the test tubes were vortexed for 30 seconds to ensure the samples were homogeneous.
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Separate test tubes containing 10 mL of samples were prepared to observe changes in
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their visual appearance throughout storagePlus (without vortexing). ACS Paragon Environment
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Droplet Size and Charge. The particle size distribution of the emulsions
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was determined using a static light scattering instrument (Mastersizer 2000, Malvern
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Instruments Ltd., Malvern, Worcestershire, UK). Samples were diluted in aqueous
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buffer solutions to avoid multiple scattering effects, and then stirred (1200 rpm) to
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ensure homogeneity. The refractive indices of phosphate buffer solution and flaxseed
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oil were taken to be 1.330 and 1.474, respectively. The droplet diameter of each sample
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was represented as the surface-weighted mean diameter (d32), which was calculated
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from the full particle size distribution.
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The electrical surface potential (ζ-potential) of the particles in these samples was
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measured using a particle electrophoresis instrument based on light scattering (Nano-
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ZS, Malvern Instruments Ltd.). Samples were diluted with buffer solution (5 mM, pH
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7.0) prior to measurements to avoid multiple scattering effects.
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Microstructure Analysis. A confocal scanning laser microscope with a 60
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× objective lens (oil immersion) and 10× eyepiece (Nikon D-Eclipse C1 80i, Nikon,
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Melville, NY, US.) was used to determine the microstructure of the emulsions. Prior to
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analysis, the samples were dyed with a 1 mg Nile red/mL ethanol solution to highlight
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the location of the oil phase. The excitation and emission spectra for Nile red were 543
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nm and 605 nm, respectively. A small aliquot of emulsions was placed on a microscope
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slide and covered with a cover slip prior to visualization. All microstructure images for
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confocal microscopy were taken and processed using image analysis software (EZ-CS1
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version 3.8, Nikon).
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Lipid Oxidation Studies. Each emulsion (2 mL) was placed in a 10-mL glass
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test tube and then sealed to ensure that it was airtight. Sample vials were then incubated
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at 55 oC in the dark. Lipid oxidation was monitored by measuring the formation of
164
hydroperoxides and thiobarbituric acid reactive substances (TBARS) during storage.
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Lipid hydroperoxides were determined according to a method described previously 38.
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Emulsions (0.3 mL) were mixed with 1.5 mL of isooctane/2-propanol solution (3:1 v/v)
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and vortexed (10 s, 3 times). The mixed solution was then centrifuged at 3,400 g for 10
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min (Centrific centrifuge, Thermo Fisher Scientific Inc., Fairlawn, NJ, USA). The upper
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organic layer (200 µL) was mixed with 2.8 mL of methanol/butanol solution (2:1, v/v),
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followed by the addition of 15 µL of 3.94 M ammonium thiocyanate and 15 µL of Fe2+
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solution. The Fe2+ solution was prepared freshly from the supernatant of a mixture of
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equal amounts of 0.132 M BaCl2 in 0.4 M HCl and 0.144 M FeSO4. The solution was
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vortexed and then held for 20 min at room temperature, and the absorbance was
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measured at 510 nm in a UV−visible spectrophotometer (Genesys 20, Thermo Fisher
175
Scientific Inc., Waltham, MA, USA). Hydroperoxide concentrations were determined
176
using a standard curve prepared using cumene hydroperoxide.
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TBARS were measured according to a method described previously
39
. Briefly,
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1.0 mL of emulsion was combined with 2.0 mL of TBA (thiobarbituric acid) solution
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(prepared by mixing 15 g of trichloroacetic acid, 0.375 g of TBA, 1.76 mL of 12N HCl,
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and 82.9 mL of H2O) in test tubes, and then placed in a boiling water bath for 15 min.
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The tubes were then cooled to room temperature for 10 min and then centrifuged (2000
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rpm) for 15 min. The absorbance of the samples was measured at 532 nm, and the
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concentration of TBARS formed were calculated from a standard curve prepared using
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1,1,3,3-tetraethoxypropane.
185 186
The initial oxidation rate was determined from the slope of plots of hydroperoxides and TBARS versus time during storage, using only the data for the first three days.
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Statistical Analysis. All analysis was performed on two samples and repeated
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at least twice per sample. Results are reported as means and standard deviations of these
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measurements. The means among treatments were analyzed by a one-way ANOVA
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followed by post-hoc Duncan test for the initial oxidation rate, and the paired-samples
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t-test was used for evaluation of the change in particle diameter and ζ-potential before
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and after storage. The means values of the EC 50 of the two lecithin ingredients were
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compared by Independent-Samples t Test. Statistical significance was set as p < 0.05
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(SPSS, IBM Corporation, Armonk, NY, USA).
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RESULTS AND DISCUSSION
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In our previous study, ACS it wasParagon shown Plus that both Sunlipon 50 and Sunlipon 90 could Environment
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form emulsions containing small droplets, but that the electrical charge on the droplets
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was appreciably different 33. At neutral pH, the ζ-potential of the oil droplets was highly
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negative when they were coated by Sunlipon 50, but close to zero when they were
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coated by Sunlipon 90. We hypothesized that differences in the interfacial properties
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of the oil droplets would lead to differences in their functional performances. For the
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sake of comparison, we also prepared emulsions stabilized by two synthetic small
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molecule surfactants: SDS (anionic) and Tween 20 (non-ionic).
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Characteristics of Sunflower Phospholipids. The nature of the
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surfactants used to coat the droplets in oil-in-water emulsions is known to have an
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appreciable impact on their physical and oxidative stabilities
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report the phospholipid, fatty acid, and peroxide contents of both the sunflower lecithins
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used as emulsifiers in this study (Table 1). The major phospholipids in sunflower oil
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are phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol
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(PI), and phosphatidic acid (PA)
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phospholipids used in this study are summarized in Fig. 1. The nature of the polar head
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group on phospholipids is known to impact their ability to form and stabilize emulsions
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41, 42
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of PC they contained. Sunlipon 50 contain around 58% PC, 5% PE and minor amounts
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of other phospholipids, while Sunlipon 90 contained around 90% PC.
40
20
. For this reason, we
. The molecular structure of the different
. The two sunflower lecithins used in our study mainly differed in the percentage
216
Palmitic acid (16:0), stearic acid (18:0), oleic acid (18:1) and linoleic acid (18:2)
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were the major fatty acids in both Sunlipon 50 and 90 (Table 1), which is consistent
218
with the values reported in the literature 43-45. The major fatty acids in Sunlipon 50 were
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similar to those in Sunlipon 90, with linoleic acid being the most abundant fatty acid,
220
accounting for more than half of the total. Oleic acid was the second most abundant
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fatty acid, followed by palmitic and stearic acids. The fatty acid chain length of
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surfactants influences the hydrophilic–lipophilic balance (HLB), which influences their
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emulsifying ability
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phospholipids was calculated using the following equation:
225
Swi ´ ni L ACS = Paragon Plus Environment w
46
. The average fatty acid chain length of the two sunflower
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(1)
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Here, ni is the number of carbon atoms in the fatty acid chain, w is the total weight
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of the fatty acids, and wi is the weight fraction of the individual fatty acids. According
229
to this equation, the average fatty acid chain length of the Sunlipon 50 and 90 were
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fairly similar, being 17.7 and 17.8 carbons, respectively.
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The rate of lipid oxidation in emulsions is known to be strongly influenced by the
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presence of any pro-oxidant components, such as transition metals (iron and copper)
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and peroxides, which may be contaminants in commercial emulsifiers
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reason, the levels of these pro-oxidants in the two sunflower lecithins used in this study
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were compared (Table 1). The two lecithins had similar peroxide values, and therefore
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the effect of peroxides on lipid oxidation would be expected to be fairly similar. The
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concentrations of iron and copper were higher in Sunlipon 50 than in Sunlipon 90. In
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particular, the copper content of Sunlipon 50 was about four times higher than that of
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Sunlipon 90, which may impact lipid oxidation, since transition metals are known to be
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highly effective pro-oxidants in emulsions.
20
. For this
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The total tocopherol content of the two lecithin products was also compared
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because studies have shown that tocopherol radicals may be regenerated in the presence
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of phospholipids
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reaction products 50. The tocopherol content of Sunlipon 90 was about 2.5 times higher
245
than that of Sunlipon 50. Consequently, it is possible that differences in tocopherol
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levels could impact oxidative stability. It should be noted that the commercial lecithin
247
ingredients used in this study contained a significant fraction of unknown minor
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constituents (Table 1), which could also have impacted their ability to alter the physical
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and chemical stability of emulsions. The ingredient manufacturer suggested that these
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components were probably glycolipids, which can act as effective emulsifiers.
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47-49
and that tocopherols may promote the formation of Maillard
Antiradical Activity of Phospholipids. The free radical scavenging capacity of a 51, 52
252
food component is an important indication of its antioxidant properties
253
decrease in DPPH concentration was therefore measured over time after adding
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different levels of the two sunflower lecithins to the test system (Fig. 2). The DPPH
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levels decreased rapidly during the first few minutes, but then decreased more slowly
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at longer times until they attained a fairly constant value. The magnitude of the decrease
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in DPPH levels increased with increasing lecithin concentration for both Sunlipon 50
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and 90. However, Sunlipon 50 clearly had a higher radical scavenging capacity than
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Sunlipon 90. Indeed, the EC50 values for Sunlipon 50 and 90 were calculated from the
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data shown in Fig. 2 as 1.69 ± 0.24 and 89.6 ± 1.5 mg/mL, respectively, which were
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significantly different (p < 0.05). In principle, the greater antioxidant activity of the
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Sunlipon 50 may have been due to a number of factors, such as differences in tocopherol
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levels or phospholipid head-group type 41, 42. The tocopherol levels of the Sunlipon 50
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were actually lower than those of the Sunlipon 90 (Table 1), which suggests that some
265
other factor must have been important. It is possible that there were other components
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within the commercial sunflower lecithin ingredients that could act as pro- or anti-
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oxidants, but more detailed analysis of the composition of the ingredients would be
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required to establish this.
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Physicochemical Properties and Stability of Emulsions. In the
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absence of oil, a sediment was observed at the bottom of the test tubes when both
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sunflower lecithins were dispersed in aqueous buffer solutions, particularly for the
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solutions containing Sunlipon 90 (data not shown). This result may be due to the fact
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that phospholipids form dense colloidal structures in aqueous solutions, such as vesicles
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and liquid crystals, that sediment due to gravitational forces 53. Previously, it has been
275
reported that the ζ-potential of the colloidal particles in aqueous solutions containing
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Sunlipon 90 and Sunlipon 50 were about +1.7 ± 0.3 mV and -24.0 ± 0.3 mV,
277
respectively, which was attributed to differences in the charge characteristics of the
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phospholipid head groups
279
phospholipids, such as PA, PI, phosphatidylglycerol (PG) and acyl-PE, than Sunlipon
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90 (Fig. 1). Consequently, the greater amount of sediment observed in the Sunlipon 90
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solutions may have been because there was a relatively weak electrostatic repulsion
282
between the colloidal structures in this system, which led to aggregation and
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sedimentation. The presence of these colloidal structures prompted us to sonicate the
284
aqueous phases prior to mixing them with the oil phases so as to evenly distribute the
33
. Sunlipon 50 contains more negatively charged
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lecithin and to avoid blocking the narrow channels in the microfluidizer used to
286
fabricate the emulsions.
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In a previous study, we investigated the possibility of fabricating nanoemulsions
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using sunflower phospholipids 33. The results of this study indicated that phospholipid
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type had a pronounced influence on the electrical characteristics of the droplets.
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Droplets made with Sunlipon 50 had a high negative charge, whereas those made with
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Sunlipon 90 had a small positive charge. It is well known that the electrical charge on
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oil droplets may influence the physicochemical stability and functional performance of
293
emulsions
294
electrostatic repulsion between oil droplets, which can inhibit droplet aggregation. On
295
the other hand, a strong negative surface potential can promote lipid oxidation by
296
attracting cationic transition metals to the droplet surfaces 55. For this reason, the impact
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of using varying ratios of Sunlipon 50 and 90 (0 to 100 wt%) at a constant overall
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emulsifier level (2 wt%) was examined. It was postulated that it may be possible to
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form emulsions with improved physical and chemical stability by using combinations
300
of lecithins, rather than individual lecithins. Two synthetic surfactants (nonionic Tween
301
20 and anionic SDS) were used as controls to highlight the potential advantages or
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disadvantages of using natural surfactants (sunflower lecithins) for this purpose.
54
. A strongly positive or negative surface potential leads to a strong
303
Particle size and electrical charge. The ζ-potential of the initial emulsions
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stabilized with sunflower lecithins became more highly negatively charged as the
305
Sunlipon 50 concentration increased (Fig. 3a), which can be attributed to differences in
306
the phospholipid compositions of the two ingredients (Table 1). PC and PE both have
307
no net charge at neutral pH, whereas PA, PI, PG and acyl-PE are negatively charged 56.
308
Consequently, it is possible that the negative charge on the oil droplets arises from the
309
presence of these anionic phospholipids. There is a higher level of these anionic
310
phospholipids in the Sunlipon 50 (Table 1), which may account for the higher negative
311
charge on the droplets in the emulsions formed using this type of sunflower lecithin.
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Interestingly, the droplets coated by Sunlipon 90 had a reasonably high negative charge
313
in this study (-26.3 mV), whereas they had a slight positive charge (+1.7 mV) in our
314
previous study
33
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concentration, emulsifier levels, and aqueous phase composition in the two studies. For
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example, flaxseed oil was used in the current study, whereas a fish oil was used in the
317
previous study. As expected, the droplets coated with SDS had the highest negative
318
surface potential (-89.3 mV), which can be attributed to the presence of the sulfate (-
319
SO4-) head group on this anionic surfactant 30. The droplets coated with Tween 20 also
320
had a relatively high negative surface potential (-39.1 mV), despite the fact that it is
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supposed to be a non-ionic surfactant. This phenomenon has also been reported by
322
other researchers 57, 58, and has been attributed to anionic impurities in the surfactants
323
or oils used (such as fatty acids) or the preferential adsorption of hydroxyl ions from
324
water
325
negative after the emulsions were stored for 28-days (Fig. 3a), which suggests that there
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was some change in interfacial composition during storage. This effect could have
327
occurred due to chemical degradation of the oils or surfactants during storage leading
328
to the generation of anionic reaction species, such as short chain organic acids. The
329
chemical degradation of either the surfactant during storage would be undesirable for
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commercial applications due to the generation of off-flavors, or the loss of ingredient
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functionality. In future studies, it would therefore be interesting to monitor the change
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in the chemical structure of the surfactants over time in more detail.
59
. With the exception of the SDS system, the surface potential became more
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The mean particle diameter of the emulsions decreased appreciably as the
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proportion of Sunlipon 50 in the emulsifier phase (Sunlipon 50 and 90) increased from
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0 to 25%, e.g., d32 was 1940 nm and 246 nm at 0% and 25% Sunlipon 50, respectively
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(Fig. 3b). The most appreciable decrease in droplet diameter occurred from 0 to 25%
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Sunlipon 50, with no significant change occurring at higher levels. This result shows
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that relatively small oil droplets can be formed with a range of different interfacial
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compositions by using the mixed sunflower lecithin ingredients. Nevertheless, the
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mean droplet diameter produced using the sunflower lecithin was appreciably higher
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than that obtained using SDS (129 nm). This effect can probably be attributed to the
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fact that the SDS facilitates droplet disruption and inhibits droplet coalescence more
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effectively than the phospholipids.
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There were appreciable changes in the mean particle diameter and particle size
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distribution of some of the emulsions after 28-days storage (Fig. 3), which suggested
346
that they were unstable to droplet aggregation. In particular, the size of the particles in
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emulsions containing low levels of Sunlipon 50 (0, 25 or 50%) increased appreciably
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after storage (Figs. 3c and 3d). This effect may have been due to the relatively low z-
349
potential on these droplets leading to droplet aggregation because of the relatively weak
350
electrostatic repulsion between the droplets. Conversely, the size of the particles in the
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emulsions containing high levels of Sunlipon 50 (75 and 100%) did not change
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appreciably after storage (Figs. 3c and 3d). Presumably, the strong electrostatic
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repulsion in these systems prevented the oil droplets from coming into close proximity
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60
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emulsions containing Tween 20 or SDS (Fig. 3). The Tween 20 emulsion was highly
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unstable to droplet aggregation, whereas the SDS emulsion was highly stable.
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Emulsions stabilized by Tweens are known to be unstable to aggregation when stored
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at elevated temperatures because dehydration of their hydrophilic head groups leads to
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a change in the optimum curvature of the surfactant monolayer and a reduction in the
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steric repulsion
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increase as the temperature approaches the phase inversion temperature (PIT) of the
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surfactant-oil-water system used.
. There was also an appreciable difference between the storage stabilities of the
61
. The rate of droplet coalescence due to this mechanism tends to
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Visual Appearance and Microstructure. The visual appearance and microstructure
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of the emulsions was recorded initially and after 28-days storage (Fig. 4a). Initially, a
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thin cream layer was observed on top of some of the sunflower lecithin stabilized
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emulsions, with the thickness of the cream layer decreasing with increasing Sunlipon
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50 ratio. This effect can be attributed to the relatively large initial droplet sizes of the
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emulsions containing high levels of Sunlipon 90 (Fig. 3). No creaming was observed
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in the initial emulsions stabilized by either Tween 20 or SDS, which is due to the
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relatively small size of the droplets they contained. After 28-days storage, an oil layer
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was visible on top of the emulsions stabilized by sunflower lecithins when they
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contained less than 50% Sunlipon 50, which suggested that droplet coalescence and
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oiling-off had occurred within these systems. At higher Sunlipon 50 levels, only a
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cream layer was observed, which suggested that some droplet creaming had occurred,
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but that the droplets were relatively stable to oiling-off. Creaming may have occurred
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in these emulsions because some of the oil droplets were relatively large 62. In contrast,
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the emulsions stabilized with Tween 20 or SDS retained a whitish color throughout
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storage, although a thin layer of oil was observed on top of the emulsions after 28-days
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storage suggesting that a limited amount of coalescence and oiling-off had occurred.
380
Surprisingly, the visual appearance of the emulsions after 28-days storage depended on
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the amount of emulsion placed in the test tubes (Fig. 4). The emulsions appeared to
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undergo more phase separation when a smaller volume was placed in the test tube,
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particularly for the Tween 20 system. This surprising result may have been due to
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differences in the amount of oxygen that diffused into the emulsions, which impacted
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their physicochemical stability 20. The volume ratio of headspace oxygen to emulsion
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volume was calculated to be 0.32 and 1.18 for the large and small test tubes,
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respectively. One would therefore expect more oxygen to diffuse into the emulsions in
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the smaller test tubes, and therefore they would be more susceptible to this effect. This
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interesting phenomenon certainly deserves more attention in future studies.
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Emulsion microstructures were determined by confocal laser scanning microscopy
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before and after they were stored at 55 °C for 28-days (Fig. 4b). Initially, all of the
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emulsions contained relatively small oil droplets (stained red) dispersed throughout the
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aqueous phase (black). After 28-days storage, there were appreciable changes in the
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microstructure of the emulsions, which depended strongly on the ratio of Sunlipon 50
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to 90 in the systems. For the emulsions containing 0 or 25% Sunlipon 50, phase
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inversion had occurred, with evidence of some water droplets (black) dispersed in an
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oily continuous phase (stained red). For the emulsions containing 50% and 75%
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Sunlipon 50, the system was still an oil-in-water emulsion but the oil droplets were
399
larger than the initial ones, indicating that some coalescence had occurred. There were
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no appreciable changes in the microstructures of the emulsions containing only
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Sunlipon 50 or SDS, which suggested that these systems were relatively stable to
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droplet coalescence during storage. On the other hand, marked phase separation was
403
observed in the emulsions stabilized with Tween 20, suggesting they were highly
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unstable to droplet coalescence and oiling-off. As mentioned earlier, this effect can be
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attributed to the dehydration of the surfactant head-groups that occurs when the
406
temperature is increased towards the PIT of the system 61.
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The fact that the water-in-oil emulsions and very large oil particles observed by
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confocal microscopy in many of the samples (Fig. 4b) were not observed by static light
409
scattering (Fig. 3) may be due to differences in the sampling procedures used for these
410
two analytical techniques. A highly diluted and stirred emulsion was analyzed for the
411
light scattering measurements, but a non-diluted emulsion was directly placed on the
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microscope slide for the microscopy measurements. Consequently, some of the free oil
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or large oil droplets in the emulsions may have been broken down during the sample
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preparation procedure used for the light scattering measurements, and some of the very
415
large droplets may have creamed to the top of the sample within the instrument and
416
therefore not been analyzed
417
light scattering measurements and microscopy measurements for this type of complex
418
colloidal system.
63
. These results highlight the importance of combining
419
Oxidative Stability
420
Influence of single emulsifier type. Initially, we examined the influence of the
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different types of individual emulsifier on lipid oxidation. Flaxseed oil emulsions were
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prepared using the four different emulsifiers studied: Sunlipon 50 (2%), Sunlipon 90
423
(2%), Tween 20 (1%), and SDS (1%). The formation of lipid oxidation primary
424
products (hydroperoxides) and secondary products (TBARS) was then monitored
425
during storage at 55 oC. In all samples, there was an increase in the concentration of
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hydroperoxides and TBARS detected during storage (Figs. 5a, b). The relative impact
427
of emulsifier type on the lipid oxidation rate was compared by plotting the initial
428
oxidation rate (linear slope of the curve) based on the first 3-days of storage (R2 > 0.96)
429
(Fig. 5c). The rate of lipid oxidation clearly depended on emulsifier type, decreasing in
430
the following order: Sunlipon 90 > Tween 20≈SDS > Sunlipon 50 (for
431
hydroperoxides) and Sunlipon 90 > Tween 20>SDS > Sunlipon 50 (for TBARS)
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(p
