Investigations on the emulsifying properties of egg white protein : a thesis presented in partial fulfilment of the requirements for the degree of Master of Food Technology at Massey University, Auckland, New Zealand
Loading...
Date
2018
DOI
Open Access Location
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Massey University
Rights
The Author
Abstract
Egg white proteins (EWP) have excellent foaming and gelling functional properties.
However, their emulsifying properties are considered poor when compared to soy proteins
or milk proteins. Some studies have attributed the poor emulsifying properties to the
hydrophobic amino acid groups buried deeply in the interior of the protein conformational
structure which is crucial for emulsification. Several methods, such as heat treatment,
acid/acid-heat treatment, Maillard reaction, phosphorylation and enzymatic hydrolysis,
have been used by some researchers to improve the emulsifying properties of EWP.
Preliminary experiments carried out in this study showed that oil-in-water (O/W) emulsions
prepared with egg white liquid (EWL) generated lots of visible large aggregates, which no
other study has reported. Therefore, it was important to investigate the factors responsible
for the formation of these aggregates. Investigations into improving EWP's emulsifying
properties could offer opportunities in developing unique and well-defined egg white-based
emulsions.
The objective of this research project was to produce egg white emulsions with little or no
aggregates. This thesis comprises three main parts. The first part focused on the effects of
pH and heat treatment on protein aggregation and partial denaturation of proteins in EWL.
The second part investigated the effects of heat treatment, oil concentration and protein
concentration on the reduction of large visible aggregates in emulsions prepared with EWL.
The third part studied the effect of enzymatic hydrolysis on the degree of hydrolysis and
emulsifying properties of EWP hydrolysates. The emulsifying properties of original EWP
and EWP hydrolysates were characterised in terms of size and zeta (ζ)-potential of emulsion
droplets and emulsion stability (e.g. turbidity, microscopic examination and phase
separation).
Firstly, an experimental study was carried out to evaluate the effect of pH on protein
aggregation and precipitation in EWL containing different protein concentrations (0.5, 1,
2, 3, 4, 5 and 10% w/w). It was found that at all the protein concentrations used and at pH
less than around 5, ζ-potential values were all positive but decreased as pH increased from
2 to 5. At pH 5, ζ-potential was close to zero (this is the pI of most egg white proteins),
while, at pH levels above 5, ζ-potential became negative and increased as pH increased
from pH 5 to 11. The spectral absorbance (turbidity) of emulsion samples was also measured at 600 nm which revealed that for all protein concentrations, turbidity was
observed to be higher at acidic pH of 3, 4 and 5, indicating the aggregation of EWP. At
alkaline conditions of pH 7, 8, 9 and 10 the EWL solutions remained to be transparent. The
effect of heat treatment and holding time on the denaturation of EWP in EWL was also
studied at different temperatures (57-62oC) and heating times (0-19 minutes). Higher
turbidity due to protein aggregation was observed as temperature increased from 57 to 62oC
and the heating time increased from 5 to 19 minutes. It is therefore concluded that EWL
can be safely pasteurized with little or no denaturation or aggregation at around 57-58oC
for less than 5 minutes. At 60oC, it was observed that EWL began to thicken and after 5
minutes coagulation and gelation occurred rapidly.
Studies were also carried out to determine the cause of visible large aggregates formed in
emulsions prepared with EWL using various factors, such as heat treatment, oil
concentration and protein concentration. It was found that heat treatment (60oC for 30
minutes) of 1% (w/w) EWP solution prior to homogenisation had no effect on reduction of
aggregates in emulsions containing 5, 10, 15 and 20% (w/w). However, the formation of
aggregates was reduced significantly as oil concentration was reduced to 5%. Therefore,
the effect of lower oil concentrations (1, 3, 5, 6, 7 and 10% w/w) on the formation of
aggregates in emulsions prepared with 1% or 3% EWP concentrations was also
investigated. Little or no visible aggregates were formed when emulsions were prepared
with 1% EWP and ≤ 5% oil or 3% EWP and 1% oil. Therefore, the results indicated that
both protein and oil concentrations played a significant role in the formation of visible
aggregates in emulsions prepared with EWP as an emulsifier.
The effect of EWP concentrations (0.1, 0.3, 0.5, 0.8, 1 and 2% w/w) on the formation and
properties of 5% oil emulsions at ~pH 8 was then investigated. It was discovered that little
or no aggregates were produced in emulsions when prepared at 0.1-1% EWP while large
aggregates were formed at 2% EWP concentration. The size of emulsion droplets was
observed to increase significantly from 242.1 to 703.7 nm as protein concentration
increased from 0.1 to 2%. ζ-potential was however not significantly affected by protein
concentration and ranged from -35.3 to -39.2 mV. The emulsions prepared were also heat
treated at 60-90oC for 30 minutes. No sign of instability with a significant change in the
size of emulsions due to heat treatment was observed from all emulsion samples prepared
at different EWP concentrations (0.1 - 2%). However, phase separation of the emulsions was observed upon freezing at -20oC and thawing at 4 and 20oC, respectively, at all protein
concentrations used. Also, the stability of emulsions was affected by the addition of salts,
such as CaCl2 (5-100 mM) and NaCl (50-600 mM), with an increase in droplet size and
phase separation. However, the emulsions were relatively more stable to salt-induced
flocculation, especially against NaCl, at higher protein concentration (1-2%) than lower
protein concentrations (0.1-0.8%). Lastly, the effect of pH 2-10 was also determined from
the emulsions prepared at 1% EWP and 5% oil. Extensive droplet aggregation was observed
at pH 4 and 5 as expected which is around the pI of most egg white proteins. On the other
hand, it was not observed at extremely acidic pH 2.0 and alkaline pH 9-10 and in the control
emulsion prepared at pH 8.3.
In another part of the study, the effects of enzyme type (bromelain, ficin and papain),
enzyme concentration (0.3, 0.5, 1, 2 and 4% w/w; enzyme/substrate (E/S) ratio) and
hydrolysis time (0, 30, 60 and 120 minutes) on the degree of hydrolysis (DH) of EWP were
investigated by diluting EWL containing 10% EWP to different EWP concentrations
followed by adding enzymes into the EWL solutions. DH was observed to increase
significantly (p < 0.05) with increasing enzyme concentration and hydrolysis time. A
significant difference (p < 0.05) among the different types of enzymes was only observed
from the samples with 4% E/S ratio at 120 minutes of hydrolysis time. Papain yielded the
highest DH of 7.69% while bromelain and ficin yielded similar DH levels of 5.03% and
4.99%, respectively. The results of SDS-PAGE revealed that the protein bands
corresponding to ovalbumin and ovotransferrin disappeared due to their enzymatic
hydrolysis into smaller peptides but it was not significantly different between the samples
treated with different E/S ratios and hydrolysis reaction times.
The effects of enzyme concentration, DH and hydrolysis time on the emulsifying properties
of hydrolysed EWP prepared with bromelain and ficin were investigated. Surprisingly,
enzymatic hydrolysis significantly improved the appearance of emulsions prepared with
EWL containing hydrolysed EWP by producing an emulsion free of aggregates compared
to the control emulsions prepared from original EWP which had lots of large aggregates in
it. For example, emulsions containing 10% oil and various EWP concentrations (1, 5 and
10%) prepared with hydrolysed EWP (4% E/S, DH 5.16%) yielded smaller droplet size
(0.66-0.98 μm) than those of original EWP emulsions (1.22-39.35 μm). However, phase
separation occurred immediately after preparation at all protein concentrations (1, 5 and 10%) used while phase separation occurred in only emulsions stabilised with 5 and 10%
original EWP. When the emulsions were heat treated at 60-90oC for 0-30 minutes, gelation
occurred in the emulsions prepared with 5 and 10% EWP concentrations while the
emulsions prepared with 1% EWP had no gelation but had aggregation and phase
separation after heat treatment. Emulsions prepared with 1% E/S ficin (DH 4.03% and
4.96%, respectively, after 2 and 4 hours of hydrolysis time) yielded smaller droplets size
(0.75-0.87 μm) than droplet size (6.40-7.37 μm) of emulsions prepared with 1% E/S
bromelain (DH 4.10% and 4.87% after 2 and 4 hours of hydrolysis time). Droplet size
decreased as hydrolysis time increased from 2 to 4 hours for both ficin and bromelain
hydrolysates with phase separation occurring the following day after the preparation of
emulsions. Thus, DH and enzyme type had some influence on the emulsifying properties
of EWP hydrolysates.
In conclusion, this study demonstrated that egg white emulsions can be prepared with little
or no aggregates using low oil (≤5%) and low protein (1%) concentrations and by
enzymatic hydrolysis of EWP. Emulsions containing 5% oil prepared with a relatively
higher protein concentration (1-2%) were more stable to destabilization to ionic strength
(salt concentration), especially against NaCl. These could lead to production of egg white
protein based-emulsions with distinct appearance and characteristics.
Description
Figures 2.2 & 2.3 were removed for copyright reasons, but Figure 2.1 remains for ease of access.
Keywords
Eggs, Composition, Egg processing, Emulsions, Research Subject Categories::TECHNOLOGY::Chemical engineering::Food technology