| Sweet whey, a byproduct in dairy industry, is a fluid obtained by separating the coagulum from whole milk with rennet-type enzymes. The most useful components in sweet whey are lactose and whey proteins. Among various whey proteins, lactoferrin and immunoglobulin G are very valuable. It has significant economic and social benefits to separate lactoferrin, immunoglobulin G, whey protein concentrate and lactose from the relatively cheaper sweet whey. Expanded bed adsorption (EBA) is a new separation technology which integrates the clarification, concentration and primary purification into a unit operation. EBA allows the adsorption of target proteins directly from an unclarified particle-containing feedstock.Analysis of feedstock:By comparing the contents of protein and whey proteins of six kinds of sweet whey from different sources, we chose one sweet whey named SWP, which had more valuable whey proteins, as the feedstock for the subsequent production. By analyzing the isoelectric points and molecular weights of whey proteins in SWP, it was found that:the isoelectric point of lactoferrin was the highest so that lactoferrin could be captured directly with cation exchange; Immunoglobulin G had not only the second high isoelectric point but also a lot of hydrophobic groups on the molecular surface which facilitated the separation by mixed mode resin with both cation and hydrophobic function groups; the residual whey proteins could be produced to whey protein concentrate altogether; then the residue could be used to extract lactose. According to this conception, various separation methods and mechanisms were studied and the whole separation process was designed as follows: integrating the separation processes of lactoferrin and Immunoglobulin G to a tandem two-stage expanded bed adsorption for less solution dosage and operation time; ultrafiltrating the flow through of this integrated process to produce whey protein concentrate and lactose. The whole separation process was simple, convenient, feasibility and maneuverable through which the sweet whey could be fully utilized.Separation of lactoferrin:Three widely-used cation exchange EBA adsorbents were compared on the physical and chemical properties, expansion characteristic and liquid mixing in the bed and adsorption properties including adsorption isotherms, adsorption kinetics and breakthrough curves. Fastline SP was more suitable for high operation velocity. Meanwhile the bed stability and adsorption property of Fastline SP was satisfactory. It would be taken as the separation media. During the research for adsorption isotherms, we found that the adsorption capacity decreased with the increase of pH or salt concentration. Then we performed molecular simulation to compare the electrostatic potential of lactoferrin in different conditions, and conjectured that pH changed the protonation of amino acid residues on the protein molecular surface and the charge distribution, while salt decreased the electrostatic potential with electrostatic shielding but not obviously affect the charge distribution. Further more, the binding energy between protein and ligand in different conditions was calculated to quantify the adsorption capacity and characterize the effects of pH and salt concentration.A series of experiments were performed with Fastline SP and lactoferrin, the process was confirmed to be loading 1 L 50 g/L sweet whey (pH7.5) to EBA with a expansion factor of 2.0, eluting by phosphate buffer (pH7.0) containing 0.5 M NaCl, cleaning by 0.5 M NaOH. Finally lactoferrin was successfully purified with a high purity of 88.5% and a reasonable recovery of 77.1% in a single step. The purification factor reached 553.Separation of immunoglobulin G:Immunoglobulin G has the second high isoelectric point and many hydrophobic groups on the molecular surface. So we adopted a mix-mode resin Streamline Direct CST-1 (CST-1) as separation media. After a series of experiments on physical and chemical properties, expansion characteristic and liquid mixing in the bed and adsorption properties, it could be concluded that the bed of CST-1 was stable enough; the optimum expansion factor was 2.0. Then on a deeper level, molecular simulation was performed to research the mechanisms of pH and salt influences on the adsorption. The results indicated that with the increase of pH, negative charges increased and positive charges decreased which against the adsorption; while with the increase of salt concentration, the charge distribution was basically constant and electrostatic potential decreased which weakened the adsorption to some extent. These results obtained from theoretical research were consistent with the experimental data and they all indicated that the appropriate pH value of feed solution was 6.0 or 7.0; the appropriate salt concentration was 0 M; Immunoglobulin G could be eluted by high pH and high salt concentration. At last, the elution condition was optimized to be carbonate buffer (pH10.0) containing 0.5 M NaCl in packed bed and the purity of Immunoglobulin G was 91.8% while the recovery was 86.1%.Process integration:In view of the results of lactoferrin and immunoglobulin G purification, these two expanded beds could be combined in series to be an integrated process. Therefore we proposed three integration strategies to connect the two expanded beds effectively as follows: Strategy I, purifying lactoferrin by EBA as the first stage, then the flow through was directly loaded to the second bed without any adjustments; Strategy II, some as Strategy I except loading the flow through to the second bed after adjusting the pH to 6.0; Strategy III, same as Strategy I except using more adsorbents in the second bed. The results generated from different strategies were compared. Since the operation conditions in the first beds were the same in these three strategies, lactoferrin fractions were the same. For immunoglobulin G, the results were totally different, the purities in these three strategies were 91.9%,83.8% and 92.4% corresponding purification factors were 196,179 and 197 and the recoveries were 14.3%, 63.7% and 29.7%, respectively. By comparing the separation effects and feasibilities of these three strategies, Strategy II was adopted to integrate the process. The stream flowed out of the first bed was needed to adjust to pH6.0 before loaded in the second bed for immunoglobulin G purification. With this integrated process, no storage devices were needed, it could reduce the solution dosage, lower invested capital, shorten operation time and enhance production efficiency.Production of whey protein concentrate and lactose:There were whey proteins and lactose in the stream flowed out of the integrated EBA process. They could be totally separated after ultrafiltration in membrane with a molecular cut off of 10 kDa, at a recirculation rate of 600 mL/min, a transmembranous pressure of 0.24 MPa and adding a certain amount of water at a specific time. Whey protein concentrate could be obtained by drying the retention from ultrafiltration while lactose could be extracted from the permeation via alcohol. The products of WPC 70 and WPC 80 were both up to standard.In a word, with a rationally-designed integrated expanded bed adsorption process, lactoferrin and immunoglobulin G could be successfully recovered from crude sweet whey with high purities and reasonable recoveries. In addition, when operating at the optimum conditions, whey protein concentrate and lactose could be prepared from the flow through of this integrated process. So that the sweet whey was fully separated to four products, i.e. lactoferrin, immunoglobulin G, whey protein concentrate and lactose without waste. The whole process was simple, easy to operate, feasible and could be used for industrial production. |
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