Beta-carotene delivery systems stabilised by dairy proteins
Leonardo Cornacchia, Yrjö H. Roos
School of Food and Nutritional Sciences, University College Cork, Ireland
ABSTRACT
Inclusion of liposoluble bioactive compounds, such as carotenoids, in food ingredients and foods is
challenging due to the labile nature of such molecules and the instability of oil-in-water (O/W) emulsionbased delivery systems. The objective of the present work was to evaluate the impact of different
concentrations of protein-emulsifiers (Whey Protein Isolate, WPI, and Sodium Caseinate, NaCas) and lipid
carrier type (Hydrogenated Palm Kernel Oil, HPKO and Sunflower Oil, SO) on the degradation kinetics of ßcarotene included in the lipid phase of O/W emulsions and to evaluate the physical stability of the dispersed
system. O/W systems with 10% (w/w) lipid phase containing 0.05% (w/w) ß-carotene and a water phase
containing 30% (w/w) sucrose and different protein concentrations (0.1, 0.2, 0.4, 0.8, 1.2, 1.6, 2.0% w/w of
emulsion) were prepared. The assessment of the system stability was done visually and measuring the change
in particle size distribution, by Dynamic Light Scattering, during storage at 20°C. The physicochemical
properties of the encapsulating matrix were characterised by Differential Scanning Calorimetry (DSC) and
X-ray diffraction. Surface protein coverage was also calculated. ß-carotene was extracted with n-hexane and
determined spectrophotometrically at 450 nm. WPI formed a multilayer structure at the O/W interface
whereas a monolayer was formed by NaCas. A zero-order reaction model fitted to the experimental data for
the degradation of β-carotene, therefore, the slope of the curves defined the rate of loss of the active
component. Results highlighted a significant impact of the type of lipid carrier and the type of emulsifier on
the kinetics of β-carotene degradation. Above the particle surface saturation (monolayer), a solid lipid carrier
(HPKO) and NaCas as emulsifier were effective for β-carotene stabilisation.
Keywords: β-carotene, O/W emulsion, whey protein isolate, sodium caseinate, hydrogenated palm kernel oil.
INTRODUCTION
The scientific evidences supporting the health benefits of dietary antioxidants [1-3] have led to the
investigation of efficient delivery systems for antioxidant fortification of liquid food formulations [4-6].
Among antioxidants, bioactive lipids, such as ß-carotene, are important model substances for such studies, as
they are chemically well characterised and measurable.
Bioactive molecules need to be protected from the adverse effects of the environment, because any chemical
modification (e.g. cyclization, double bond migration, introduction of oxygen in various forms and chain
shortening) results in losses of bioactive properties [7]. Oxidation is the major cause of carotenoids
degradation and is due to the interaction with oxidative species, such as singlet oxygen; superoxides;
peroxides; hydroxyl radicals; and transition metals [8]. In the present study, a possible technique to improve
the stability of oxygen sensitive compounds in model food matrices was investigated. ß-carotene was
incorporated into dispersed lipid droplets in oil-in-water (O/W) emulsion-based systems, in which the lipid
carrier (Hydrogenated Palm Kernel Oil, HPKO, or Sunflower Oil, SO) was surrounded by an interfacial
membrane of dairy proteins (Whey Protein Isolate, WPI, and Sodium Caseinate, NaCas). The physical state
of the lipid carrier and the type and concentration of proteins were the main factors considered.
MATERIALS & METHODS
Materials
The WPI used was Bipro from Davisco Foods (Le Sueur, MN, USA). NaCas was purchased from Dairygold
Food Ingredients Ltd (Michelstown, Ireland). HPKO (SIL®-CREAM 90) was obtained from Trilby Trading
(Drogheda, Ireland). SO (Flora Pure Sunflower Oil, Unilever) was purchased at the local market. Sucrose
was purchased from Irish Sugar Ltd. (Carlow, Ireland). ß-carotene, reagents for its analysis [sodium azide,
chloramphenicol, n-hexane, butylated hydroxytoluene (BHT), sodium sulfate, methanol, potassium
hydroxide], reagents for protein determination [Folin-Ciocalteau reagent, potassium sodium tartrate, copper
(II) sulfate pentahydrate] and for peroxide value (chloroform, acetic acid, potassium iodide, sodium
thiosulfate, starch) were purchased from Sigma Chemical Co. (Sigma-Aldrich Ireland Ltd, Dublin, Ireland).
Emulsion preparation
Protein systems were prepared by dispersing 5% (w/w) protein powders in deionised water at room
temperature and stirring for ~3 hours to ensure complete hydration and dispersion. HPKO or SO containing
0.05% (w/w) ß-carotene was heated at 50°C and stirred for at least 1 hour to give a homogeneous system. A
solution of 60% (w/w) sucrose at 50°C was also prepared, mixed to the protein systems at the proper ratio,
and, after adjusting the pH to 7 by using 1M NaOH or 1M HCl, blended to form a coarse pre-emulsion by
using a high speed blender (Ultra-Turrax T25 Digital) for 30 seconds. The pre-emulsions (10% w/w oil and
30% w/w sucrose; 0.1-2% w/w proteins in the aqueous phase) were homogenised using a two stage valve
homogeniser (APV-1000 High-Pressure homogeniser, Wilmington, MA) at 850 bars. Emulsions were cooled
to ~5°C, and 0.02% (w/w) sodium azide and 1 mg/Kg chloramphenicol were added before storage at 20°C.
Particle size analysis and determination of surface protein coverage
The particle size distribution of the emulsions was measured by using a laser diffraction particle size analyser
(Malvern, MasterSizer MSE, Malvern Instruments Ltd, Malvern, Worcestershire, UK). The particles surface
protein concentration (mg/m2) was calculated from the difference between the concentration of protein used
to prepare the emulsion and that measured in the subnatant after centrifugation (15000 rpm for 1h at 4°C), by
the Lowry assay [9], using the surface-weighted average particle size to calculate the surface area [10, 11].
Visual observation
Samples stability during storage was evaluated following the method described by Demetriades et al. [12].
Determination of peroxide value (PO value)
PO value was determined by iodometric titration with 0.01N sodium thiosulfate as described by the official
EEC method [13].
Study of the thermal behaviour and physical state of the lipid carrier
The thermal behaviour of the bulk and dispersed lipid was studied by Differential Scanning Calorimetry
(DSC, Mettler Toledo 821e with liquid N2 cooling). Samples were analysed using DSC aluminium pans (40
µl; Mettler Toledo-27331, Schwerzenbach, Switzerland) and the thermograms were analysed using STARe
thermal analysis software, version 6.0 (Mettler Toledo Schwerzenbach, Switzerland). Lipid crystallinity was
also investigated using X-ray diffraction. XRD data were collected on a Phillips Xpert PW3719
diffractometer using Cu Kα radiation (40 kV and 35 mA).
Analysis of β-carotene
A two steps procedure was used for the extraction of β-carotene from emulsified samples. In the first step
aliquots of ~1g of product stored in screw-capped polypropylene test tubes at 20°C, was saponified by adding
1ml of 50% (w/w) potassium hydroxide in methanol mix in order to separate the lipid carrier (saponised
fraction) from the β-carotene (unsaponised). The mix was heated up to 45°C for 30 minutes to increase the
reaction rate. In the second step, the unpolar β-carotene was extracted three times with 2ml n-hexane
containing 0.1% w/v BHT. The extract was dehydrated by adding a small amount (~0.5g) of sodium sulfate
and analysed spectrophotometrically (Varian Cary 300 Bio UV-Visible Spectrophotometer) at the maximum
absorbance wavelength λmax = 450 nm.
Statistics
All the analytical determination were done in three replicates therefore data are reported as mean and
standard deviation.
RESULTS & DISCUSSION
System development
Thermal characterization of the materials was carried out in order to develop two systems with either waterdispersed solid or liquid lipid as carrier for β-carotene. As summarised in Figure 1, the DSC analyses of
emulsified SO and HPKO showed an exothermic peak of crystallization at -23°C and -8°C, respectively.
Figure 1. Crystallization and melting behaviour of emulsified SO (a) and emulsified HPKO (b).
Upon heating, emulsified SO was completely melted at ~-10°C (Figure 1a), whereas, HPKO showed a broad
melting peak, ranging from ~20°C to ~45°C (Figure 1b). Two different delivery systems containing liquid
SO and solid HPKO could be produced by simply cooling the freshly prepared emulsions below the
crystallization temperature of HPKO (~7°C) followed by storage below the onset melting temperature of
HPKO (~20°C).
To verify the effective solid phase of emulsified HPKO at 20°C, X-ray diffraction analysis was done. As
reported in Figure 2, bulk HPKO showed two main peaks at ~21° and ~23°. Emulsified HPKO showed a
good correlation with the XRD patterns of bulk lipid, as diffraction peaks were between ~21° and ~24°, thus
confirming the presence of HPKO crystals in the systems stored at 20°C.
Figure 2. XRD patterns showing peaks of crystallinity of bulk and WPI-stabilised HPKO-in-water emulsion held at 20°C
The differences of XRD peaks could be due to the different crystal structure caused by the effect of the
hydrophobic part of the proteinaceous emulsifier in the lipid crystal network, according to our earlier
hypothesis [14].
The impact of WPI and NaCas concentration on particle size was studied to produce the minimum mean
droplet size, at the homogenisation conditions used, in order to minimise gravitational separation. The
dependence of the surface weighted mean diameter of the dispersed droplets, D[3,2], from the protein
concentration is shown in Figure 3.
Figure 3. Sauter-average particle diameter, D[3,2], after 1 homogenization cycle at 850 bar, as a function of the protein
concentration in emulsion.
For all the four different combinations of protein and lipid, the protein concentration was limiting stability
below 0.8 % (w/w), as the average droplet diameter was reduced by increasing protein concentration. The
mean droplet size produced by homogenization was determined by the maximum surface area covered by
protein [15]. Above the limit of surface saturation, the droplet size was independent on the protein
concentration, but it was dependent on the energy input of the homogenizer.
Protein assembly at the O/W interface of the disordered flexible NaCas and the compact globular WPI was
studied to understand the particle-surface load and its impact on system and β-carotene stability. As reported
in Figure 4, NaCas and WPI showed different behaviour.
Figure 4. Particles surface protein coverage (mg/m2) of NaCas- (a) and WPI- (b) stabilised emulsions plotted as a
function of the protein concentration in emulsions.
Most likely, WPI formed a multilayer structure at the O/W interface, as the surface coverage progressively
increased above the surface saturation (0.8% w/w) (Figure 4a). On the other hand, a monolayer was formed
by NaCas as, above the saturation, the surface coverage remained constant (Figure 4b). Above the saturation
threshold concentration, excess NaCas remained dispersed in the water phase, whereas, WPI unfolded at the
O/W interface allowing multiple layering of WPI via non-polar, thiol and disulfide groups. These are
normally buried in the interior of WPI native structure, but exposed at the interface, could increase the
hydrophobic interactions and favour intermolecular disulfide bonding of protein molecules through a thioldisulfide exchange reaction or oxidation [16,17].
System stability and β-carotene degradation kinetics
The stability of systems with different formulations were followed over 32 days by particle size distribution
and gravitational separation. Below the saturation concentration of the proteins, extensive instability
occurred, whereas, above this threshold, emulsions appeared stable against gravitational separation and
change in particle size (data not shown).
The decrease in β-carotene concentration at 20°C followed zero-order kinetics (data not shown), so that the
rates were derived from the slopes of concentration against time (Table 1).
Table 1. Slopes of β-carotene loss with solid (HPKO) and liquid (SO) lipid carrier.
[Protein] (% w/w)
NaCas + HPKO
NaCas + SO
WPI + HPKO
0.1
-0.8162
-0.8761
-0.6854
0.2
-0.4479
-0.7275
-0.6750
0.4
-0.2440
-0.6961
-0.4541
0.8
-0.1791
-0.5136
-0.4489
1.2
-0.2480
-0.4815
-0.5478
1.6
-0.2211
-0.4290
-0.5538
2.0
-0.2190
-0.4931
-0.5471
WPI + SO
-1.0821
-0.9249
-0.7777
-0.7299
-0.7068
-0.7148
-0.6981
System stability on β-carotene degradation was crucial. Unstable systems ([Protein] ≤ 0.4) resulted in a high
rate of degradation. The impact of the lipid carrier type on β-carotene stability was significant. The solid lipid
carrier, HPKO, greatly enhanced the stability of the bioactive compound. The rate of β-carotene loss of
emulsion containing HPKO was lower than that of emulsions containing SO, when both NaCas and WPI
were used. As the peroxide value of SO was found fairly constant (~7 mEq/Kg) over the period of
observation because most likely buffered by the presence of natural antioxidants (60mg/100ml as claimed by
the producer), the enhanced protection was ascribed to the solid state of the carrier which provided an
effective barrier against degradation. Despite the fact that WPI formed a multilayer structure at the O/W
interface, the NaCas monolayer was more effective in protecting β-carotene from degradation. This was
presumably due to the different amino acid composition, which resulted in a different radical scavenging
property, and the different thickness of the interfacial membrane, as postulated by Hu et al. [9].
CONCLUSION
Protein-stabilized dispersions may be used for improved food ingredients and foods containing liposoluble
bioactive compounds, such as carotenoids and vitamins. In the present study, the physical state of the lipid
carrier had a leading role in the bioactive protection efficacy of the delivery system. The solid lipid
significantly enhanced stability by reducing the degradation rate of -carotene. The emulsifier type also
influenced protection efficacy. NaCas showed superior barrier properties compared to WPI because of the
different scavenging properties due to the different interfacial structure and amino acid profile.
ACKNOWLEDGEMENTS
This study was financially supported by the Irish Research Council for Science, Engineering and Technology
(IRCSET) “Embark” initiative, funded by the National Development Plan.
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