Studies on the antioxidant activity of milk proteins in model oil-in-water emulsions : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology, Riddet Institute, Massey University, Palmerston North, New Zealand
The present study was aimed at extending our knowledge of the antioxidative properties
of the milk protein products, whey protein isolate (WPI) and sodium caseinate (NaCas),
in oil-in-water (O/W) emulsions rich in polyunsaturated fatty acids (PUFAs). In
particular, the objective was to contribute to our understanding of the compositional and
processing factors that influence the oxidative stability of protein-stabilised O/W
emulsions. Linoleic acid (approximately 60 %) was used as the lipid for the oil phase
(10.6 %). The emulsion samples were usually incubated at 50 °C to accelerate lipid
oxidation. Lipid oxidation indicators were lipid hydroperoxides and headspace hexanal,
determined by solid phase microextraction (SPME) combined with gas chromatography
WPI- or NaCas-stabilised emulsions were prepared using a wide range of protein
concentrations (0.5, 1.0, 2.0, 3.0, 4.0, 7.0 or 10.0 %) at two droplet sizes (d32 = 0.31 and
0.65 µm). In general, higher lipid oxidation levels were found for the larger droplet size.
Increasing protein concentration led to a decrease in the lipid oxidation rate. The
greatest decrease in lipid hydroperoxide levels (values after 4 h) occurred at up to 4.0 %
protein concentration. The greatest decrease in hexanal levels (values after 24 h)
occurred at up to 4.0 % protein concentration in WPI emulsions (0.31 µm). The hexanal
levels were more independent of the protein concentration in the other emulsion types.
The hexanal level decreased at protein concentrations > 4.0 % in NaCas emulsions (0.31
and 0.65 µm) and at protein concentrations > 7.0 % in WPI emulsions (0.65 µm). The
difference between lipid hydroperoxide generation in emulsions with small and large
droplet sizes decreased with increasing protein concentration. This effect was more
pronounced in NaCas emulsions. In general, NaCas was a better inhibitor of lipid
oxidation than WPI, but WPI appeared to be the better antioxidant at some droplet
size/protein concentration combinations.
The protein in the continuous phase, i.e. the unadsorbed protein, played an important
role in lipid oxidation. In principal, the lipid hydroperoxide and hexanal levels showed
the same development over the continuous phase protein concentration as over the
protein concentration in WPI and NaCas emulsions (d32 = 0.31 µm). A low NaCas level
in the continuous phase already led to a relatively low hexanal level, whereas a higher
WPI level was required. When NaCas solution was added to a WPI emulsion or WPI
solution was added to a NaCas emulsion, a synergistic antioxidative effect was
The high molecular weight fractions (molecular weight = 12000-14000) of WPI and
NaCas contained pro-oxidative metal ions that contributed to lipid oxidation in the
emulsions. An enrichment of NaCas emulsions with the low molecular weight fraction
of NaCas (with a molecular weight = 12000-14000) notably inhibited lipid oxidation.
An enrichment of WPI emulsions with the low molecular weight fraction of WPI (with
a molecular weight = 12000-14000) also seemed to inhibit lipid oxidation, but the
effect was not significant. The protein solutions were enriched with these fractions
before emulsion preparation.
Pure WPI solution or mixed WPI/NaCas (1:1, weight/weight) solution with 1.12 or 2.24
% protein concentration was heated at 84 °C for up to 40 min, cooled and then used to
prepare emulsions. Lipid oxidation was generally not affected by the heat treatment or
the degree of whey protein denaturation. However, at the lower WPI concentration,
more hexanal was produced for the longer heating times (20, 30 and 40 min) and this
appeared to be connected with the physical instability of the emulsions. Greater
oxidative stability was found at the higher protein concentration and when the proteins
were mixed, pointing to a possible synergistic antioxidative effect of WPI and NaCas.
The addition of the free radical source 2,2’-azobis(2-amidinopropane) dihydrochloride
(AAPH) greatly increased the oxygen uptake and the generation of lipid hydroperoxides
in the emulsions. The oxidative stability increased with increasing protein concentration
(1.0, 4.0 and 7.0 %). NaCas had a greater antioxidative effect than WPI. The inhibition
of oxygen uptake appeared to be largely influenced by the free-radical-scavenging
activity of the system, determined by the protein type and the protein concentration, as
the radicals were produced linearly over time and oxygen was consumed linearly over
time. It can therefore be concluded that free-radical-scavenging activity represents a
major antioxidative mechanism of the milk proteins.
Oxygen was consumed much faster in emulsions than in protein solutions when the
same level of AAPH was incorporated. In a WPI (1.0 % protein) emulsion, much lower
levels of protein hydroperoxides than of lipid hydroperoxides developed. This pointed
to a much greater reactivity of linoleic acid than of the milk proteins with oxygen. In
contrast, the exposure of WPI to oxidising linoleic acid in an emulsion (1.0 % protein)
or to AAPH in aqueous solution led to oxidative damage of the whey proteins, indicated
by the loss of amino acids. The loss of specific amino acids was different for proteins in
the continuous phase or cream phase of an emulsion or in WPI solution.
The present study confirms the antioxidative potential of WPI and NaCas and gives new
insights into their functionality as oxidative stabilisers in O/W emulsions.