Separation of Lactoferrin from Whey Concentrate

Transcription

1 Separation of Lactoferrin from Whey Concentrate STELA, Dairy Research Centre Nafissatou Ndiaye, Yves Pouliot and Laurent Bazinet INAF, Institute of Nutraceuticals and Functional Foods Université Laval Québec, Canada, G1V 0A6

2 Introduction (the challenge)

3 The challenge Lactoferrin (LF) Present in whey at a concentration 0.01 to 0.3 mg/ml 80 kda glycoprotein N-lobe and C-lobe: containing 2 Fe 3+ Strong base behaviour associated with N-lobe S. A. Moore, et al, 1997

4 The challenge Conserving the properties of lactoferrin Antibacterial e.g. E. coli O157:H7 Antiviral e.g. Hepatitis C lactoferrin Antifungal e.g. Candida albicans Immunomodulating Anti-inflammatory Anti-tumoral T. G. Kanyshkova, et al., 2001 Wakabayashi et al, 2006

5 The challenge Fractionation Separation of LF is complex because of its low concentration (0.1g/l) in whey and interference from major proteins Major Proteins Concentration (g/l) Molecular weight kda Isoelectric point(pi) β-lactoglobulin α-lactalbumin Immunoglobulins Serum albumin Lactoferrin

6 Fractionation processes Process Advantages Disadvantages Chromatography High purity Low yield, high cost Baromembrane Electrobaromembrane High yield Selectivity Increased Clogging decreased Low selectivity, clogging Production of reactions at electrodes Electrodialysis with membrane filtration (EDMF)???

7 What is EDMF? EDMF is an electro-membrane process Coupling of membrane and electric field Only one driving force: electric field EDMF separates bio-molecules selectively based on their charge and molecular weight by the force of an electric field in an electrodialysis cell

8 How does it work? M + Enriched Solution AEM MF CEM Anode + M + Cathode V Mother Solution Recovery Solution

9 Parameters to control Transfer force: electric field Electric Field (Volt.cm -1 ) Ionic molecule movement at an electrophoretic migration speed (cm.s -1 ) ph of the solution charge migration direction: electrophoretic mobility (cm.s -1 /Volt.cm -1 ) Proportional to charge ph < pi, positively charged molecule: migration towards the cathode ph > pi, negatively charged molecule: migration towards the anode.

10 Hypothesis and goals

11 Hypothesis and goals Hypothesis The ph-dependent electrophoretic properties of lactoferrin influence the selectivity of its separation from whey proteins by electrodialysis with ultrafiltration membrane (EDUF). Goals 1) To study lactoferrin electrophoretic mobility as a function of ph. 2) To study the feasibility of lactoferrin separation by EDUF from a model solution. 3) To separate lactoferrin by EDUF from enriched whey.

12 Results and discussion

13 Measurement of electrophoretic mobility 0.1% LF in water 0.1% LF in water 0.1% LF in KCl 2g/l 0.1% LF in KCl 2g/l Adjusted ph Zetasizer 2000

14 Electrophoretic Mobility 4 Is maximal at ph 3.0: m 2.V 1.s 1 in 2 g/l KCl m 2.V 1.s 1 in distilled water Electrophoretic mobility (x 10-8 m 2.V -1.s -1 ) Isoelectric Point Distilled water KCl solution ph

15 Separation from a Model Solution AMX-SB MUF Cl - K + CMX-SB + Anode LF + - Cathode Voltage: 20 V Treatment time: 4h Model Solution at 0.1% NaCl Model Solution of LF KCl NaCl Protein Measurement (BCA protein assay kit, Pierce)

16 LF Migration Migration 46% after 4h

17 Separation from whey AMX-SB MUF CMX-SB + Anode P ± P - P + Cl - K + - Cathode Voltage : 20 V Treatment time: 4h Whey Solution: 6% ST and 0.1% LF NaCl enriched whey permeate NaCl Protein Measurement (BCA protein assay kit, Pierce) Chromatoraphic Profiles (RP-HPLC)

18 Impact of ph on protein migration The greatest migration of LF was at ph 3.0 (15%) Competition between protein species present 15 % 8% 4%

19 Migration of different proteins ph Time Protein (min) GMP α-la LF BSA β-lg IgG 0 0±0.0 0±0.0 0±0.0 0±0.0 0±0.0 0± ± ±1.2 0± ± ± ± ± ± ± ± ± ± ± ±1 14.6± ± ± ± ±0.0 0±0.0 0±0.0 0±0.0 0±0.0 0± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±0.0 0±0.0 0±0.0 0±0.0 0±0.0 0± ± ± ±2 12.8± ± ± ± ± ± ± ± ± ± ± ± ± ± ±2.3

20 Fraction Composition 17% 15% 12% 16% Composition of enriched fractions differs widely, depending on ph 45% 62% 60% 46% Lactoserum permeate permeate permeate

21 Fraction Composition Comparison of the major protein content of EDUF permeates at ph 3.0 and 4.0 (based on 100% recovery) with WPC and WPI as reported in the literature. Proteins WPC WPI MF EDUF permeate ph 3.0 ph 4.0 WPI IE β-lg α-la BSA IgG GMP Composition is similar

22 Conclusions and prospects EDUF is able to separate lactoferrin from whey. A judicious choice of ph can improve separation selectivity Decreasing treatment time does not affect production yield. Studies to optimize yield by increasing the effective membrane surface should be considered.

23 Particularities of EDMF Can be used to separate any charged molecules: Polyphenols (catechin, anthocyanins, etc ) Oligosaccharides Peptides (anti-hypertensive, anti-cancer )! Depending on configuration, it can separate 2 or several molecules in one step Allows simultaneous concentration of molecules of interest Bazinet et al. Process and system for separation of organic charged compounds. Brevet PCT/CA2005/

24 Potential Advantages of EDMF 1) Preserves the value of the non-bioactive fraction; 2) No organic solvent required; 3) High selectivity (size/charge); 4) Decrease in membrane clogging; 5) The process can be adapted to a large-scale production by stacking membranes; 6) Can be integrated easily into an industrial production line; 7) Can be installed in a small production unit near the source of raw material.

25 Thanks Dr. Linda Saucier Dr. Lucie Beaulieu Monica Araya-Farias (M. Sc.) Fréderic Lehance The entire research team