The objective of this research was to examine and investigate the stabilization of $\beta$-galactosidase by histidine. Of the four $\beta$-galactosidases tested, histidine stabilized the enzyme from Kluyveromyces marxianus more than the enzyme from Streptococcus thermophilus, Escherichia coli or Aspergillus niger. The enzyme from K. marxianus was purified to electrophoretic homogeneity on a non-denaturing PAGE (pH 8.0) and its kinetic stability determined at 45$\sp\circ$C. All the twenty amino acids (1 mM) tested, except proline, stabilized the enzyme. Histidine was the most effective stabilizer. It enhanced the half-life of the enzyme 58-fold in the presence of 5% lactose. Increasing the lactose concentration up to 15% increased histidine stabilization. Glucose and maltose did not affect the histidine stabilization while galactose and sucrose enhanced it. Histidine also stabilized the enzyme in the absence of sugars but to a lesser extent. The $\alpha$-amino group and the N-1 nitrogen on the imidazole ring of histidine were essential for histidine stabilization while the carboxylic group played a role in the extent of stabilization. Histidine stabilization decreased with increasing ionic strength. The energy of activation for inactivation of this enzyme in the temperature range of 45 to 51$\sp\circ$C was unaffected by histidine. Binding of histidine to the enzyme was not observed by equilibrium dialysis and gel filtration experiments. Further, the melting temperature (51.4$\sp\circ$C) of the enzyme as detected by differential scanning calorimetry was not affected by histidine. Under isothermal conditions a delay in unfolding of the enzyme in the presence of histidine was observed by absorbance spectroscopy. The delay was temperature dependent and was not detected at 47.5$\sp\circ$C. Histidine is probably acting in the inital stages of unfolding on a partially unfolded molecule but the nature of its action is not completely understood. Lactose alone, delayed the unfolding and increased the melting temperature but did not effectively enhance the half-life of the enzyme. The K$\sb{\rm m}$ for lactose and the apparent binding constant for magnesium were unaffected by histidine. Histidine increased the half-life of this enzyme by 44% in milk at 45$\sp\circ$C but was not effective in milk ultrafiltrate.

This work focused on the effect of pH on the solubilization, water-uptake and gelation of myofibrillar proteins at reduced salt concentrations (≤150 mM). Solubilization of myofibrillar proteins in water was affected by certain possible solubility-inhibiting (PSI) polypeptides and postmortem exposure to a low pH. These PSI polypeptides might act like a binder that prevented the rest of the myofibrillar proteins from disorganization, swelling and subsequent solubilization in water. M-protein (166 kDa), a-actinin (95 kDa) and desmin (56 kDa) were tentatively identified as the PSI polypeptides in the mackerel light muscle. Exposure of myofibrillar proteins to the low pH that accompanies postmortem glycolysis could cause protein denaturation and subsequently the loss of extractability in water. However, over 96% of muscle proteins were solubilized after a pre-wash in a solution of physiological ionic strength at neutral pH. The water-uptake of twice water-washed minced chicken breast muscle at physiological ionic strength was governed by the balance between the driving forces for, and the constraint components against, swelling. pH adjustment from 6.4 to 7.0 increased electrostatic repulsive forces and the osmotic potential of myofibrillar proteins. It also solubilized the constraint components associated with myofibrillar structure. Therefore, it increased the water-uptake. pH adjustment from 6.4 to neutrality improved the gel strength and water-holding capacity significantly. After pH adjustment, the net negative charges of muscle proteins increased; the proteins unfolded more extensively during the heating process. Gels formed at physiological ionic strength consisted of mainly myofibrils. These myofibrils, which tended to form a network of localized aggregates at pH 6.4, formed a more evenly distributed network of myofibrils at neutral pH. Cell segments were capable of expansion before gelation (40–50°C) at neutral pH and their final volumes after heating were larger than those at pH 6.4. The thick filaments formed a porous network within the myofibrillar structure at neutral pH. During the cooling process, the gel strength was improved more at neutral pH than at pH 6.4. Structural disorganization imposed by pH adjustment from 6.4 to 7.0 was found not sufficient to improve the gelation significantly. It is suggested that pH adjustment from 6.4 to 7.0 introduced several favorable effects for gelation and water-holding capacity.

The hypocholesterolemic effect of certain polysaccharides has been attributed to their ability to bind bile acids. The purpose of this study was to better understand bile acid - polysaccharide interactions by systematically characterizing the interactions between selected polysaccharides (chitosan and pectin) and anionic surfactants (sodium dodecyl sulfate (SDS) and sodium taurocholic acid (NaTCA)) using isothermal titration calorimetry (ITC), surfactant selective electrode (SSE) and turbidity measurements. Initially, the influence of environmental conditions (pH, ionic strength and temperature) on the properties of SDS and NaTCA in buffer solutions was characterized. The CMC's (critical micelle concentrations) were largely independent of temperature and pH, but decreased appreciably as the ionic strength increased. In general, the micellization behavior of NaTCA was different from that of SDS because of their different molecular structures. The influence of environmental conditions on the interactions between SDS and NaTCA with pectin and chitosan were then studied. SDS bound strongly to chitosan and formed insoluble complexes, which was attributed to electrostatic attraction. For SDS-chitosan interactions, temperature did not have a large affect on T1 (onset binding), T2 (surfactant concentration at polymer saturation) or CMC* (effective CMC in the presence of polymer). Strong binding only occurred at pH values where the chitosan was cationic (pH 3 and 5), but not when it was uncharged (pH 7). Salt (0 to 200 mM) decreased the CMC* because of the depression of the CMC of free SDS in solution. SDS bound weakly to pectin and formed soluble complexes, which was attributed to hydrophobic interactions. The general characteristics of NaTCA-chitosan interactions were fairly similar to those of SDS-chitosan interactions. The binding interaction was exothermic at all temperatures studied (10 to 50°C), suggesting that it was electrostatic in origin. The T1, T2 and CMC* values were influenced by salt and pH as described for SDS. In addition, only a weak binding interaction was observed between pectin and NaTCA. This study provides information that may lead to the rational design of polysaccharide-based food ingredients with beneficial nutritional and functional characteristics.

The physicochemical effects of pH, temperature, ionic strength and ingredient interaction on the formation of heat-denatured whey proteins and their resulting aggregation and gelation were investigated. Results were interpreted based on the molecular interactions that exist between protein molecules. Aggregation was dependent on protein concentration, pH, heat time and heat temperature. Optimal conditions for production of heat-denatured whey for use as a cold-set gelation ingredient were identified as 10 wt%, pH 7 and 75 to 85°C for 10 to 30 minutes, dependent on desired gel time and rheological properties. Whey protein aggregates were further characterized using Ultrasonic Attenuation Spectroscopy (UAS). UAS proved to be a valuable method for the investigation of molecular relaxation and scattering mechanisms in whey proteins. Electrostatic interactions proved crucial to cold-set gel network formation. Gel texture and optical properties were closely related to mineral content and type, with divalent cations inducing gelation via charge shielding and cross-linking, thereby reducing the amount of added salt necessary. Aggregation of heat-denatured whey proteins exhibited a concentration-dependent sensitivity to sucrose addition. Below 8 wt% sucrose network formation was retarded, as detected by suppression of rheological properties. This was attributed to the viscosity contribution by sucrose to the continuous phase, thereby reducing aggregate collision frequency. Above 8 wt%, the trend was reversed due to preferential dehydration of the protein molecules that encouraged protein-protein interaction. The addition of xanthan to a cold-set gelation system increased its textural properties. This was due to phase separation of the xanthan and heat-denatured whey proteins that resulted due to thermodynamic incompatibility. Excluded volume effects increased the effective concentrations of both biopolymers accounting for their resulting synergism. Finally, heat-denatured whey protein was added to an emulsion stabilized by non-ionic surfactant (Tween 20). Addition of salt caused aggregation of the proteins and was found to be dependent on protein and mineral concentration. A gel network formed around the non-interactive oil droplets to produce a thickened emulsion.