Lysozyme fractionation from egg white at pilot scale by means of tangential flow membrane adsorbers: Investigation of the flow conditions.

The application of membrane adsorbers instead of classical packed bed columns for protein fractionation is still a growing field. In the case of egg white protein fractionation, the application of classical chromatography is additionally limited due to its high viscosity that impairs filtration. By using tangential flow membrane adsorbers as stationary phase this limiting factor can be left out, as they can be loaded with particle containing substrates. The flow conditions existing in tangential flow membrane adsorbers are not fully understood yet. Thus, the aim of the present study was to gain a deeper understanding of the transport mechanisms in tangential flow membrane adsorbers. It was found that loading in recirculation mode instead of single pass mode increased the binding capacity (0.39 vs. 0.52 mg cm(-2)). Further, it was shown that either higher flow rates (0.39 mg cm(-2) vs. 0.57 mg cm(-2) at 1 CV min(-1) or 20 CV min(-1), respectively) or higher amounts of the target protein in the feed (0.24 mg cm(-2) vs. 0.85 mg cm(-2) for 2.5 or 39.0 g lysozyme, respectively) led to more protein binding. These results show that, in contrast to radial flow or flat sheet membrane adsorbers, the transport in tangential flow membrane adsorbers is not purely based on convection, but on a mix of convection and diffusion. Additionally, investigations concerning the influence of fouling formation were performed that can lead to transport limitations. It was found that this impact is neglectable. It can be concluded that the usage of tangential flow membrane adsorbers is very recommendable for egg white protein fractionations, although the transport is partly diffusion-limited.

Analysis of the effect of temperature changes combined with different alkaline pH on the ß-lactoglobulin trypsin hydrolysis pattern using MALDI-TOF-MS/MS.

Temperature and pH influence the conformation of the whey protein ß-lactoglobulin (ß-Lg) monomer, dimer, and octamer formation, its denaturation, and solubility. Most hydrolyses have been reported at trypsin (EC 3.4.21.4) optimum conditions (pH 7.8 and 37 °C), while the hydrolysate mass spectrometry was largely limited to peptides with <4 kDa. There are few reports on trypsin peptide release patterns away from optimum. This work investigated the influence of alkaline (8.65 and 9.5) and optimum (7.8) pH at different temperatures (25, 37.5, and 50 °C) on ß-Lg (7.5%, w/v) hydrolysis. Sample aliquots were drawn out before the addition of trypsin (blank sample) and at various time intervals (15 s to 10 min) thereafter. Matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS) was used to monitor peptide evolution over time with the use of two matrixes: α-cyano-4-hydroxycinnamic acid (HCCA) and 2.5-dihydroxyacetophenone (DHAP). Mass analysis showed that the N- and C-terminals (Lys(8)-Gly(9), Lys(100)-Lys(101), Arg(124)-Thr(125), Lys(141)-Ala(142), and Arg(148)-Leu(149)) of ß-Lg were cleaved early (15 s) implying the ease of trypsinolysis at the exposed terminals. Hydrolyses at 25 °C and pH 7.8 as well as at 50 °C and pH 9.5 were slowed down and ordered. Nonspecific chymotrypsin-like behavior occurred more at higher temperatures (50 °C) than at lower ones (25 and 37.5 °C). In addition to our earlier work in the acid pH region, it can be concluded that there is potential for controlled hydrolysis outside the trypsin optimum, where different target peptides with predictable biofunctionalities could be produced.