The low electrostatic repulsion between proteins during the heat-induced
assembly process favours the growth of the aggregate into spherical structures
[102]. Then, particulate aggregates are formed at close to protein pI or in the
presence of salts due to the screening of exposed ionised groups. pH, ionic strength,
hydrophilic–hydrophobic balance in the polypeptide chain, protein concentration,
heating temperature ramp and heating time are factors tuning the size of the
particulate aggregates formed [74, 100, 103] and governing their assembly
behaviour and stability in solution [100]. Stable suspensions of particulate
aggregates of β-Lg (absence of sedimentation) are obtained after heating β-Lg
solution at low ionic strength and at pH slightly below (pH 4.5–4.7) or slightly
above (pH 5.7–5.9) the isoelectric point. Under these conditions, the particulate
aggregates are individualised and monodispersed, with an average diameter of
around 200 nm. Their size is slightly higher at pH 4.6, under which conditions
the net charge of the particulate aggregates is lower than at pH 5.8 (+30 mV at pH
4.6 versus À40 mV at pH 5.8) [75]. It was hypothesised that the charge of the
particulate aggregates limits their growth and prevents their assembly into larger
supramolecular structures. A macroscopic phase separation appears rapidly when
the repulsion between the particulate aggregates decreases [100]. In another study,
aggregates with different sizes and morphologies were produced by heating β-Lg at
80
C in the pH range between 6 and 6.8. Large spherical aggregates with an
average hydrodynamic diameter of 96 nm were formed at pH 6 against smaller
and linear aggregates with an average hydrodynamic diameter of 42 nm at pH 6.8
[104]. Particulate aggregates are also formed by heating a mixture of β-Lg and α-La
(ratio 80:20) at pH 5.7. For higher proportion of α-La in the mixture, the yield of
conversion of native proteins into particulate aggregates decreases and the particles
lose their spherical shape and/or aggregation [15]. At pH 5.7, both proteins are
negatively charged but it was suggested that the presence of α-La in the aggregates
reduces the hydrophobic attractive interactions for the formation of round and
dense particles. The yield of conversion of native proteins into particulate
aggregates increases in the presence of salts because salts promote aggregation
[15]. This indicates that mineral composition and protein composition influence the
assembly process. A suspension of spherical aggregates, with core–shell structure
of diameter around 100 nm, is also obtained by heating a mixture of oppositely
charged proteins (Ova/LYS or ovotransferrin/LYS) [105, 106]. Interestingly, these
studies described lysozyme-rich core and ovalbumin-rich shell particles whose
charge depend on the medium pH: at pH 5–10 the core carries positive charges
and the shell carries negative charges; at pH around 5, formed particles are neutral;
below pH 5, particles with homogeneous positive charge are formed; and above pH
10, particles with homogeneous negative charge are formed. The mechanism of
protein assembly is not understood yet, but under the same physicochemical
conditions it is not possible to obtain the same supramolecular structures with
only one protein. This indicates the prevalence of the interactions between proteins
of opposite charge. The procedure for the formation of nanoparticles of Ova and
LYS consists first of the pH adjustment of the solution at 5.3, between the pI of the
two proteins, promoting Ova–LYS interactions (Fig. 3). Then, the pH is increased
and, when the value comes close to the pI of LYS, the LYS molecules aggregate
Spontaneous Assembly and Induced Aggregation of Food Proteins
79
assembly process favours the growth of the aggregate into spherical structures
[102]. Then, particulate aggregates are formed at close to protein pI or in the
presence of salts due to the screening of exposed ionised groups. pH, ionic strength,
hydrophilic–hydrophobic balance in the polypeptide chain, protein concentration,
heating temperature ramp and heating time are factors tuning the size of the
particulate aggregates formed [74, 100, 103] and governing their assembly
behaviour and stability in solution [100]. Stable suspensions of particulate
aggregates of β-Lg (absence of sedimentation) are obtained after heating β-Lg
solution at low ionic strength and at pH slightly below (pH 4.5–4.7) or slightly
above (pH 5.7–5.9) the isoelectric point. Under these conditions, the particulate
aggregates are individualised and monodispersed, with an average diameter of
around 200 nm. Their size is slightly higher at pH 4.6, under which conditions
the net charge of the particulate aggregates is lower than at pH 5.8 (+30 mV at pH
4.6 versus À40 mV at pH 5.8) [75]. It was hypothesised that the charge of the
particulate aggregates limits their growth and prevents their assembly into larger
supramolecular structures. A macroscopic phase separation appears rapidly when
the repulsion between the particulate aggregates decreases [100]. In another study,
aggregates with different sizes and morphologies were produced by heating β-Lg at
80
C in the pH range between 6 and 6.8. Large spherical aggregates with an
average hydrodynamic diameter of 96 nm were formed at pH 6 against smaller
and linear aggregates with an average hydrodynamic diameter of 42 nm at pH 6.8
[104]. Particulate aggregates are also formed by heating a mixture of β-Lg and α-La
(ratio 80:20) at pH 5.7. For higher proportion of α-La in the mixture, the yield of
conversion of native proteins into particulate aggregates decreases and the particles
lose their spherical shape and/or aggregation [15]. At pH 5.7, both proteins are
negatively charged but it was suggested that the presence of α-La in the aggregates
reduces the hydrophobic attractive interactions for the formation of round and
dense particles. The yield of conversion of native proteins into particulate
aggregates increases in the presence of salts because salts promote aggregation
[15]. This indicates that mineral composition and protein composition influence the
assembly process. A suspension of spherical aggregates, with core–shell structure
of diameter around 100 nm, is also obtained by heating a mixture of oppositely
charged proteins (Ova/LYS or ovotransferrin/LYS) [105, 106]. Interestingly, these
studies described lysozyme-rich core and ovalbumin-rich shell particles whose
charge depend on the medium pH: at pH 5–10 the core carries positive charges
and the shell carries negative charges; at pH around 5, formed particles are neutral;
below pH 5, particles with homogeneous positive charge are formed; and above pH
10, particles with homogeneous negative charge are formed. The mechanism of
protein assembly is not understood yet, but under the same physicochemical
conditions it is not possible to obtain the same supramolecular structures with
only one protein. This indicates the prevalence of the interactions between proteins
of opposite charge. The procedure for the formation of nanoparticles of Ova and
LYS consists first of the pH adjustment of the solution at 5.3, between the pI of the
two proteins, promoting Ova–LYS interactions (Fig. 3). Then, the pH is increased
and, when the value comes close to the pI of LYS, the LYS molecules aggregate
Spontaneous Assembly and Induced Aggregation of Food Proteins
79