To study the changes in secondary, tertiary and quaternary structures and the alteration in protein–protein interactions during the protein aggregation upon heating, bovine serum albumin (BSA) was incubated over a range of temperature, and its structure was monitored by various biophysical techniques including circular dichroism (CD), second-derivative UV absorption spectroscopy, dynamic light scattering (DLS) and size exclusion chromatography coupled with multiangle laser light scattering (SEC-MALLS). The experimental data demonstrated that the size of BSA was relatively stable up to 60 1C. Above 65 1C, however, BSA underwent the obvious heat-induced aggregation (a dramatic growth of hydrodynamic radius Rh), accompanied with great unfolding of its secondary structure (a sigmoidal reduction of a-helix with the transition midpoint at 67.6 1C), with significant expansion of its tertiary structure (an increased solvent exposure of some aromatic residues with the transition midpoint at 65.5 1C), with distinct screening of electrostatic repulsion (a great reduction of second virial coefficient A2 above 65 1C) and with remarkable formation of aggregates (an evident increase of molecular weight above 65 1C).

The effect of pancreatic hydrolysis on the aggregate size and molecular weight distribution (MWD) of bovine casein was investigated by size exclusion chromatography (SEC), coupled with multiangle laser light scattering (MALLS) and dynamic light scattering (DLS). The concentrations of both casein aggregates and monomers decreased as the reaction proceeded and almost disappeared after a 10min hydrolysis. The ratio of aggregates to monomers was maintained at 1:(2-2.5) during the initial 10min hydrolysis, which shows that the caseins in the solution are in a dynamic equilibrium system, between aggregates and monomers. Upon limited hydrolysis, casein aggregates displayed a continuous growth in their sizes and molecular weights, together with a decrease in the intermolecular repulsion. This result was further supported by on-line DLS measurements, which showed the increase in the hydrodynamic radii and the decrease in light scattering intensities of casein hydrolysates. Moreover, the release kinetics of peptide fractions with different molecular weights were also followed. It was suggested that the increase in the hydrophobic attraction and the reduction in the protein repulsion might promote casein aggregation, during the limited pancreatic hydrolysis.

Bovine casein was digested with pancreatin at pH 8.0 in a batched stirred tank reactor. In total, 52 casein phosphopeptides (CPPs) in the time-course samples were separated and identified using liquid chromatography-tandem mass spectrometry by means of neutral loss scanning and database searching. Of these peptides, 23, 7, 8, 10 and 4 are singly, doubly, triply, quadruply and quintuply phosphorylated, respectively. Furthermore, 14 peptides contained the cluster sequence, S(P)S(P)S(P)EE, providing the mineral binding sites; these peptides were all formed after 10 min of hydrolysis, but none of them survived after 6 h of pancreatic digestion. A study of the release kinetics of CPPs allowed determination of the degrees of hydrolysis for the preparation of target peptides with high yields. The discrimination of pancreatic attack on different phosphorylated regions of bovine casein was also analyzed in terms of the release of CPPs.-2006 Elsevier Ltd. All rights reserved.

Bovine serum albumin was hydrolyzed with trypsin in a batch stirred tank reactor. The influence of operating parameters on the degree of hydrolysis was investigated by pH-stat method. Molecular weight distribution of hydrolysates was analyzed by high performance sizeexclusive chromatography. The kinetic model of hydrolysis reaction was studied by a lumping technique. The hydrolysates were combined into several lumps according to their molecular weights. Reaction of the lumps was described with intrinsic kinetic equations, and the lumping kinetic model for bovine serum albumin tryptic hydrolysis reaction was established. The rate constant of each reaction was estimated by Marquardt method in two steps from lower level to upper level. The reliability of the model was tested by comparing the computed values with the experimental values.

In the enzymatic membrane reactor for separating casein hydrolysatem backflushing technology has been used to decrease the fouling of the membrance. Predication of the backflushing efficiency poses a complex non-linear problem as the system integrates enzymatic hydrolysis, membrane separation and periodic backflushing together. In this paper an alternative artificaial neural network approach is developed to predict the vackflushing efficiency as a function of duration and interval. A contour plot of backflushing performance is presented to model these effects, and the backflushin conditions have been optimized as duration of 10s and interval of 10 min using this neural network, Also, simpleneural networks are established to predict the time evolution of flux before and after backflushing. The results predicted by the modelass are in good agreement with the experimental data, and the average deviations for all te cases are well within +-5% The neural network approach is found to be capable of modeling the backflushing with confidence.