ELS EVI E R Current Opinion in Colloid & Interface Science 5 (2000) 182-187

www.elsevier.nl/locate/cocis

Influence of high pressure processing on protein solutions

and emulsions

Vanda B. Galazkaa, Eric Dickinson”, Dave A. Ledwardb.*

aProcterDepartment of Food Science, University of Leeds, Leeds LS2 9JT, UK
bDepartment of Food Science and Technology, University of Reading, Whiteknights, Reading RG6 U P ,UK

Abstract

Static high-pressure technology is likely to be used increasingly over the next few years in the preservation and processing of
food. This ‘clean’ technology offers an effective and safe method of modifying protein structure, and self-assembly properties.
Pressure processing can lead to globular protein denaturation, and different states of aggregation or gelation depending on
the protein system, the treatment temperature, the solution conditions, and the magnitude and duration of the applied
pressure. 0 2000 Elsevier Science Ltd. All rights reserved.

Keywords: High pressure treatment; Protein-protein interaction; Protein functionality; Casein micelles; P-lactoglobulin; Bovine serum
albumin; Ovalbumin; 11s globulin Vicia faba

1. Introduction hydration properties, dependent upon protein-water

The effect of high pressure processing on food
systems was first reported over 100 years ago by Hite
[l]. However, few studies were published until the
1980s because of technical difficulties and costs asso-
ciated with high pressure processing units and packag-
ing of materials. Nowadays, these limitations are seen
to be surmountable and a great deal of research is
being undertaken to understand the effect of high
pressure on food and food ingredients [2’]. One of
the important aspects of pressure treatment is that
food can be processed with minimal effect on the
natural colour, flavour, taste, and texture with little or
no loss of vitamins [31.
Generally, the functional properties of food pro-
teins may be classified into three main groups: (a)
+
*Corresponding author. Tel.: 44-118-9875123.
E-mail address: d.a.ledward@afnovell.reading.ac.uk
Ledward).
interactions which have an important bearing on wet-
tability, swelling, adhesion, dispersibility, solubility,
viscosity, water absorption, and water holding; (b)
interfacial properties including surface tension, emul-
sification and foaming characteristics; and (c) aggre-
gation and gelation properties, which are related to
protein-protein interactions.
The effect of high pressure (100-1000 MPa) on the
structures of globular proteins in aqueous solution
has received considerable attention over the last few
years [4’,5,6]. These papers have confirmed the view
that, since the formation of hydrophobic bonds and
ion pairs is accompanied by a large volume change [71,
the application of pressure has a disruptive effect on
intramolecular hydrophobic and electrostatic interac-
tions. Conversely, hydrogen bond formation is associ-
ated with a small (usually negative) volume change,
and so hydrogen bonding interactions are relatively
(D.A. insensitive to pressure. This means that high pressure
disrupts the quaternary and tertiary structure of

1359-0294/00/$ - see front matter 0 2000 Elsevier Science Ltd. All rights reserved.
PII: S 13 5 9 - 0 2 9 4 ( 0 0 ) 0 0 0 5 5 - 8
 K B . Galazka et al. /Current Opinion in Colloid & Interface Science 5 (2000) 182-187
globular proteins with relatively little influence on
their secondary structure. It can also dissociate pro-
teinaceous colloidal aggregates (e.g. casein micelles),
which are held together by ionic and hydrophobic
forces. The pressure-induced denaturation of globular
proteins can lead to aggregation and, ultimately, un-
der appropriate conditions and high enough concen-
trations, to gelation or precipitation. Additionally,
several studies have reported that disulfide bonds play
an important role in high pressure-induced aggrega-
tion of proteins due to the increased reactivity of SH
groups.
2. Ovalbumin
Ovalbumin, the main component of egg white, does
not form a gel when pressurised for 30 min at < 400
MPa. This relatively high high-pressure stability may
be due to the presence of four disulfide bonds and
strong non-covalent interactions stabilising the three-
dimensional structure of the protein [8,9]. Gelation
does occur, however, at higher pressures.
Doi et al. [lo] studied the effects of pressure on the
stability of network bonds in heat-induced ovalbumin
gels. They placed a steel ball on top of a heat-set gel
and then pressure-processed. It was found that at
pressures > 400 MPa, the steel ball penetrated deep
into the gels, suggesting that primarily hydrophobic
and electrostatic interactions were destabilised in the
gel, resulting in the partial melting of the gel. In
contrast, cold-set gelatine gels did not melt under
pressure treatment at 600 MPa, which suggested that
hydrogen bonds were not destabilised by pressure
processing. Other researchers [ l l ] demonstrated that
ovalbumin gels produced by high pressure processing
became harder and less adhesive with increasing pres-
sure. It was also found [12’] that ‘protectants’ such as
sucrose and NaCl can inhibit pressure-treatment-
induced modification.
Oil-in-water emulsions prepared with previously
pressure-treated solutions showed very little change
in droplet size at pressures < 400 MPa, with
marginally larger droplets produced at higher pres-
sures (> 600 MPa) [13]. The presence of NaCl (up to
0.1 M) had little effect on the droplet size for emul-
sions prepared with native ovalbumin. Unsurprisingly,
high pressure processing of the protein in the pres-
ence of 0.02 M salt gave emulsions with a larger
droplet size, this being exacerbated by further addi-
tions of NaC1. It was speculated that pressure treat-
ment denatured the protein and that the presence of
salt induced the polymerisation of ovalbumin, which
was further enhanced as the salt content was in-
creased.
Separate experiments involving pressure treatment
183
( > 600 MPa) of model emulsions after homogenisa-
tion showed that pressurisation induced extensive
droplet flocculation [13] which increased with protein
concentration and severity of treatment (Chia, perso-
nal communication). It was also noted that moderate
thermal processing (80°C for 5 min) had a far greater
effect than high-pressure treatment (800 MPa for 30
min) on the state of flocculation of ovalbumin-coated
emulsion droplets. The increase in emulsion viscosity
and gel strength was due to the formation of network
structures from the aggregation of dispersed oil
droplets and denatured polymers in aqueous solution.
The level of pressure-induced flocculation could be
controlled more efficiently by altering the intensity of
the high pressure treatment than controlling the tem-
perature in thermal processing. This principle can be
viewed as a novel way of manipulating the microstruc-
ture and texture of egg white systems with the mainte-
nance of nutritional value and ‘natural’ flavour.
3. Vegetable proteins
Vegetable proteins such as soy, pea, wheat gluten
and broad bean are being widely used in the food
industry for the formulation of new food products.
Matsumoto et al. [14] and Okamoto et al. [15] have
found that a minimum pressure of 300 MPa for 10-30
min is required to induce gelation. It has also been
reported [15-171 that high pressure produces softer
gels with a significantly lower elastic modulus than
those made by heat treatment. Rheological studies
[16] for wheat gluten show that at low pressures
(200-400 MPa, 40°C for 50 min) there is evidence of
liquid-like behaviour, which may indicate some melt-
ing out due to the weakening of hydrophobic or
electrostatic interactions. The cross-link density in-
creased and more solid-like behaviour was observed
with the increasing severity of treatment. It was sug-
gested that these cross-links were due to the forma-
tion of disulfide or hydrogen bonds [171. Other studies
have shown [18] that soy milk changes from liquid to
solid following treatment at 500 MPa for 30 min.
However, at shorter treatment times (10 min) or
lower pressures, the milk was found to remain in the
liquid state, but with improved emulsifying activity
and stability. In the presence of CaCl,, pressure-set
gels (tofu) have been formed at 300 MPa for 10 min;
the hardness was comparable to that of heat-set gels
prepared at 70°C for 10 min.
Emulsions made with pressure processed 11s have
been shown [19,20] to have poorer emulsifying and
stabilising ability, with respect to initial droplet size,
and creaming behaviour as compared to the native
protein. This is probably mainly due to the enhanced
dissociation of subunits and/or aggregation induced
184 KB. Galazka et al. /Current Opinion in Colloid & Interface Science 5 (2000)182-187
by the formation of intermolecular disulfide bridges
via the -SH/-S-S- interchange. In agreement with
studies on equivalent ovalbumin systems [131, moder-
ate thermal treatments (SOT for 2 min) appear to
have a far greater effect than high-pressure treat-
ments (250 MPa for 20 min) in terms of the associ-
ated changes in droplet-size distribution and the ex-
tent of serum separation. High-pressure processing
was again considered to be a gentler processing oper-
ation in comparison to thermal processing.
4. Milk proteins
4.1. PLactoglobulin
P-Lactoglobulin, the main whey protein compo-
nent, appears to be more sensitive to high-pressure
treatment than ovalbumin or bovine serum albumin.
Solution studies [21,22] of native P-lactoglobulin have
indicated that pressure treatment has a notable effect
on the protein's conformational and aggregation
properties, which are more extensive at higher con-
centrations [21,23']. The formation of aggregates is
most probably due to the generation of intermolecu-
lar disulfide bridges through SH/-S-S- interchange
reactions [22,241. Similar findings have been reported
by Johnston and Murphy [25], who found that the
number of free -SH groups in milk is reduced by
pressure treatment.
High pressure can be used to make whey protein or
P-lactoglobulin gels, which have a sponge-like struc-
ture and are susceptible to exudation [261. Zasypkin et
al. [27] reported that P-lactoglobulin gels can be
stabilised against syneresis by the addition of xanthan,
and confirmed that a more elastic protein gel network
was formed on addition of the polysaccharide. The gel
strength of high-pressure-induced whey protein con-
centrate gels as a function of pH has been studied by
various researchers [28-301. The optimum pH for the
formation of high-pressure-induced gels with the
highest hardness was pH 9, whilst the lowest hardness
occurred in the acidic pH range. Camp and Huyghe-
baert [281 found some significant structural differ-
ences between pressure-induced and temperature-in-
duced whey protein gels made from concentrated
protein solutions. Electron microscopy has revealed
that heat-induced gels (0.1 MPa for 30 rnin at 8OOC)
are stronger and possess a greater number of perma-
nent cross-links between the polypeptide chains. In
contrast, high-pressure treatment (400 MPa for 30
min at 20°C) generates fragile gels with a more porous
network and fewer intermolecular crosslinks [31,26].
The combined use of elevated temperature and
pressure was found to give variable results, which was
highly dependent on the kind of protein under study
[32,33]. For instance, whey protein concentrate formed
gels whose strength increased with an increase in
pressurisation temperature. The improvement was
more noticeable at high protein concentrations ( > 132
g/l) where stronger gels were obtained by pressurisa-
tion (400 MPa at 50°C for 30 min) than by heating
(SOT for 30 rnin).
Emulsions prepared with pressure treated solutions
(up to 800 MPa) with either P-lactoglobulin [34] or
whey protein concentrate [35] as the emulsifier had
substantially larger droplets than those made with the
native protein. The results indicated a modification of
protein structure, leading to the loss of emulsifying
efficiency owing to protein aggregation, despite an
increase in surface hydrophobicity. On the other hand,
when pressure treatment was applied after emulsion
+
formation (0.4 wt.% protein 20 vol.% n-tetrade-
cane) there was less effect on the mean droplet size
and creaming behaviour [34-361. During homogenisa-
tion, the protein probably became partially unfolded
at the interface, and subsequent pressure treatment
caused no further conformational change. However, a
substantial increase in the viscosity, including the
generation of gel-like characteristics of concentrated
P-lactoglobulin-stabilised emulsions (1 wt.% protein,
40 vol.% n-tetradecane) was observed following pres-
sure treatment [371.
As noted previously, high pressure treatment can
be regarded as a milder processing operation than
thermal processing. Strong emulsion gels have been
produced from concentrated emulsion samples (1
+
wt.% protein 40 vol.% n-tetradecane) following
treatment at > 70°C for 5 min [36]. Furthermore, the
increase in viscoelasticity caused by moderate heating
(e.g. 65°C for 5 min) resembles the change in rheolog-
ical behaviour induced by severe pressure treatment
of 800 MPa for 60 min. The rheological properties of
moderately dilute P-lactoglobulin-stabilised emulsions
(0.25 wt.% protein, 10 vol.% n-tetradecane) was shown
[36] to be unaffected by thermal treatment (SOT for 5
min) or pressure processing at 800 MPa for 60 min.
Under conditions of higher ionic strength or lower
pH, P-lactoglobulin-stabilisedemulsions became more
flocculated after pressure or temperature treatment.
Furthermore, the extent of emulsion flocculation fol-
lowing high pressure processing was found [361 to be
greater when there was a larger proportion of free
protein present in the continuous phase, and, there-
fore, unfolded protein molecules would appear to
aggregate in solution as well as bind to the adsorbed
molecules at the surface of oil droplets. The observed
behaviour can perhaps be best explained in terms of
the cross-linking or flocculation of droplets by unad-
sorbed protein after pressure treatment.
 K B . Galazka et al. /Current Opinion in Colloid & Interface Science 5 (2000) 182-187
4.2. Casein
Calcium-free casein is a disordered heterogeneous
protein with little ordered secondary structure and no
tertiary structure. The functional properties are largely
unaffected by heating to temperatures well above
those at which globular proteins become denatured,
aggregated and coagulated. The same is true for the
effect of high-pressure treatment on solutions of indi-
vidual pure caseins or sodium caseinate. Hence, de-
spite previous suggestions to the contrary [381, it has
been found that the extended application of high
pressure up to 800 MPa has no influence on the
surface activity of bovine p-casein [39]. In addition,
under conditions where p-lactoglobulin-stabilised
emulsions become extensively aggregated, equivalent
emulsions stabilised by sodium caseinate remain un-
changed [40]. However, in milk itself, where the ca-
sein is complexed with colloidal calcium phosphate,
the quaternary structure of the casein micelle is subs-
tantially affected by high pressure.
Generally, it is assumed that pressures higher than
- 200 MPa can cause significant destabilisation of
casein micelles in reconstituted skim milk which
greatly modify the physicochemical and functional
properties of the milk [41-431. The average particle
size of the casein micelles has been shown to decrease
from -300 to - 100 nm prior to processing into
chains or clusters of casein sub-micelles, with a rather
wide size distribution ranging from 50 to 500 nm.
Subsequent heating of the milk at 30°C (0.1 MPa)
restores the original particle size [41]. Pressure in-
creases the dynamic viscosity of bovine milk [41,42],
but decreases milk turbidity [41-461, presumably due
to increased light transmittance of pressurised skim
milk. These changes, coupled with a reduction in
non-sedimentable protein, led Johnston et al. [36] to
conclude that high pressure treatment, rather than
causing the extensive release of individual casein
molecules from micelles, results in the formation of
larger casein fragments possibly containing denatured
whey proteins and/or some re-aggregation. The de-
naturation of the whey protein and/or destabilisation
of casein micelles by the exposure of hydrophobic side
chains occurred when skim milk was treated at 600
MPa for 30 min. The effect was found to persist for
up to 8 days during storage at 5°C [461.
Experiments have been carried out on the proper-
ties of acid-set gels derived from high pressure treated
skim milk [44]. It was found that the gel rigidity and
gel breaking strength increased significantly with in-
creasing applied pressure and pressure pre-treatment
time. These gels also had a reduced tendency to
syneresis with increasing pressure. These properties
seem to suggest that the changes to the milk caused
by pressure were further amplified during the acid-in-
185
duced disintegration and reaggregation process, lead-
ing to a gel with an improved structure [44]. At
various pH values in the range 5.5-7.0, it was found
[45] that high-pressure treatment (200 MPa) signifi-
cantly reduced the rennet coagulation time.
Another area of interest is the combined use of
pressure at sub-zero temperature on protein structure
and function without the complication of freezing
occurring [37].
5. Conclusions
Many studies have reported the usefulness of pres-
sure to provide gels or pastes with unique properties.
The use of high pressure as a possible alternative
processing method to thermal treatment has brought
about the need to study the pressure-temperature
behaviour of macromolecular food ingredients since,
for example, the mechanisms of protein gelation are
far from being fully understood. Experimental re-
search on the use of high pressure as a pre-treatment
method to improve the textural properties of real
food products is widespread. As a pre-treatment tool,
high-pressure processing appears effective in improv-
ing meat, egg or soy protein gelation properties, as
well as improving the coagulating properties of milk.
However, the behaviour of real food is complicated,
especially in the presence of enzymes. Further studies
are required to understand the potential of the tech-
nology for rheology control in food protein systems,
as well as to optimise the operating conditions that
should be used during actual processing. This may
then lead to the development of high-value food
products with a potential future within the market-
place.
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