A two-stage ultrafiltration process for fractionation of whey protein isolate
Introduction
A number of recent studies have demonstrated the feasibility of using membrane systems for the separation of proteins with very similar molecular size. van Reis et al. [1], [2], [3] have coined the name high performance tangential flow filtration (HPTFF) to describe these very high selectivity membrane processes. HPTFF exploits a number of different strategies to achieve high resolution separations, including: (1) proper choice of pH and ionic strength to maximize differences in the hydrodynamic volume of the product and impurity, (2) use of electrically charged membranes to enhance the retention of like charged proteins, (3) operation in the pressure-dependent regime to maximize the selectivity, and (4) use of a diafiltration mode to wash impurities through the membrane [3].
van Reis et al. [2] used HPTFF to separate bovine serum albumin (BSA) from an antigen binding fragment of a monoclonal antibody (Fab), achieving more than 900-fold purification and 90% yield of the BSA. van Eijndhoven et al. [4] developed a membrane system for the separation of bovine serum albumin from hemoglobin, two proteins with essentially identical molecular weight, with more than 100-fold purification and nearly 70% yield. More recently, Cheang and Zydney [5] were able to obtain 100-fold purification and greater than 90% recovery of β-lactoglobulin (β-LG) from a binary mixture with α-lactalbumin (α-LA).
Although these studies clearly demonstrate the feasibility of using high performance tangential flow filtration for protein separations, the data were all obtained with model systems consisting of an artificial mixture of two previously purified proteins. Experimental studies with complex multi-component feed streams are much more limited, and the overall performance of these systems is much less impressive. For example, Bottomley [6] described a two-stage membrane process for the purification of α-LA from cheddar whey, but the final product still contained nearly 25% β-LG. Muller et al. [7] used a combined ultrafiltration–diafiltration process for the purification of α-LA from acid casein whey, with the final permeate having an α-LA purity of only 50%. The purification and yield obtained in these studies are significantly less than those obtained with model binary mixtures.
The objectives of this study are to examine the performance of two-stage membrane systems for the purification of α-lactalbumin and β-lactoglobulin from whey protein isolate, a complex protein mixture that is readily available as a by-product from dairy processing. Experimental data were first obtained with different buffer concentration and filtration velocity to determine the optimal filtration performance of two different molecular weight cut-off composite regenerated cellulose membranes. Two purification strategies, both consisting of two separate diafiltration steps, were designed to obtain purified α-LA and β-LG fractions from whey protein isolate. The results obtained using each strategy were compared in terms of the yield and purification factor for each protein product.
Section snippets
Materials and methods
Experiments were performed with whey protein isolate (Bi-Pro) provided by Davisco Foods International, Inc. (Eden Prairie, MN). The whey protein isolate was spiked with additional bovine serum albumin, obtained from Sigma Chemical (Catalog #A 7906, St. Louis, MO), to facilitate the analysis of the overall purification factor of α-lactalbumin and β-lactoglobulin. Pre-weighed quantities of the whey protein isolate and BSA powder were dissolved in a pH 7 phosphate buffer solution (prepared from Na2
Results and analysis
Typical experimental data for filtration of a solution containing 10 g/l of whey protein isolate spiked with 1 g/l BSA are shown in Fig. 1. This solution contained 1.5 g/l of α-LA (MW=14 kDa, pI=4.5–4.8 [5]), 8 g/l of β-LG (MW=18 kDa, pI=5.2 [5]), 1.3 g/l of BSA (MW=67 kDa, pI=4.7–4.9 [5]), and small amounts of other trace whey proteins, including both small molecular weight species and large immunoglobulins, lactoferrin, and lactoperoxidase. The BSA concentration accounts for both the spiked protein
Discussion
Although a number of recent studies have demonstrated the feasibility of using membrane systems for protein separations, these investigations have almost always focused on the behavior of simple model systems consisting of only a single product and impurity. The experimental results obtained in this study provide the most complete analysis to date for the separation of two protein products from a complex mixture: the purification of α-LA and β-LG from a commercial whey protein isolate. The
Acknowledgements
The authors thank Millipore Corp. for donation of the Pellicon XL modules and Davisco Foods International, Inc. for providing the whey protein isolate.
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