Effect of pH on the fractionation of whey proteins with a ceramic ultrafiltration membrane

Journal of Membrane Science 288 (2007) 28–35

Effect of pH on the fractionation of whey proteins with a ceramic ultrafiltration membrane M. Carmen Alm´ecija ∗ , Rub´en Ib´an˜ ez, Antonio Guadix, Emilia M. Guadix Department of Chemical Engineering, University of Granada, 18071 Granada, Spain Received 22 June 2006; received in revised form 25 September 2006; accepted 9 October 2006 Available online 12 October 2006

Abstract The purpose of this work was to investigate the potential of membrane ultrafiltration for the fractionation of clarified whey. Employing a 300 kDa tubular ceramic membrane in a continuous diafiltration mode, the effect of working pH was evaluated by measuring the flux–time profiles and the retentate and permeate yields of ␣-lactalbumin, ␤-lactoglobulin, BSA, IgG and lactoferrin. At pH 3, 9 and 10 permeate fluxes ranged from 68 to 85, 91 to 87 and 89 to 125 L/(m2 h), respectively. On the other hand, around the isoelectric points of the major proteins (at pH 4 and 5), permeate fluxes varied from 40 to 25 and from 51 to 25 L/(m2 h), respectively. For ␣-lactalbumin and ␤-lactoglobulin, the sum of retentate and permeate yields was around 100% in all cases, which indicates that no loss of these proteins occurred. After 4 diavolumes, retentate yield for ␣-lactalbumin ranged from 43% at pH 9 to 100% at pH 4, while for ␤-lactoglobulin, was from 67% at pH 3 to 100% at pH 4. In contrast, BSA, IgG and lactoferrin were mostly retained, with improvements up to 60% in purity at pH 9 with respect to the original whey. © 2006 Elsevier B.V. All rights reserved. Keywords: Whey proteins; Ceramic membranes; Ultrafiltration

1. Introduction Whey proteins are commonly used in the food industry due to their wide range of chemical, physical and functional properties. The most important functional properties of whey proteins are solubility, viscosity, water holding capacity, gelation, emulsification and foaming [1–4]. In addition to their general properties, individual whey proteins have their own unique nutritional, functional and biological characteristics. For instance, ␣-lactalbumin has been claimed as a nutraceutical and a food additive in infant formula owing to its high content in tryptophan [5,6] and as a protective against ethanol and stress-induced gastric mucosal injury [7]. ␤-Lactoglobulin is commonly used to stabilize food emulsions because of its surface-active properties [8]. Bovine serum albumin (BSA) has gelation properties [9] and it is of interest in a number of food and therapeutic applications [10], for instance, because of its antioxidant properties [11]. Bovine immunoglobulins can enhance

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0376-7388/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.memsci.2006.10.021

the immunological properties of infant formula and they can be used therapeutically in the treatment of animal neonates [10] and, in the form of special supplements, they can offer, in many situations, an important reduction of risk to acquire diarrhoea causing infections and other illnesses [12]. Bovine lactoferrin, which exists at relatively low concentrations, has important biological activity. Lactoferrin has been reported to have antimicrobial, immunostimulatory and anti-inflammatory activity [13–15] since it affects growth and development of a wide range of infectious agents. This protein plays a fundamental role in iron metabolism as an iron-transport molecule [13]. Furthermore, recent studies have demonstrated that lactoferrin suppresses tumor growth and metastasis in mice and rats and moreover may inhibit angiogenesis [16–18]. In addition, whey proteins, partially digested, serve as a source of bioactive peptides [19–21]. Therefore, whey fractionation for the recovery and isolation of these proteins has a great scientific and commercial interest. Separation processes to fractionate whey proteins reported in the literature fall into a number of categories, such as selective precipitation, colloidal gas aphrons, selective adsorption and selective elution.

M.C. Alm´ecija et al. / Journal of Membrane Science 288 (2007) 28–35

Selective precipitation involves adjusting the physical properties to promote insolubility. For example, Outinen et al. [22], Bramaud et al. [23], Hinrichs [24] and Alomirah and Alli [25] precipitated ␣-lactalbumin from whey using heat, acid and/or ultra-high pressure. Da Fonseca and Bradley [26], Tolkach et al. [27] and Alomirah and Alli [25] described processes to separate ␤-lactoglobulin. Colloidal gas aphrons are stabilized microbubbles generated by intense stirring of a surfactant, which is mixed with a given volume of whey. The mixture is allowed to separate into two phases: the aphron (top) phase and the liquid (bottom) phase. Using this technique Fuda et al. separated ␤-lactoglobulin from sweet whey using a cationic surfactant [28] and recovered lactoferrin and lactoperoxidase using an anionic one [29]. In selective adsorption, a single purified protein is produced in conjunction with a treated whey solution depleted in that protein. For ␣-lactalbumin adsorption, Gurgel et al. [30] used an immobilized hexapeptide ligand affinity resin, Turhan and Etzel [31] used cation-exchange chromatography and Noppe et al. [32] used immobilized phenyl groups. For ␤-lactoglobulin adsorption, Wang and Swaisgood [33] used immobilized retinal and Schlatterer et al. [34] used ceramic hydroxyapatite chromatography with sodium fluoride as a displacer. In selective elution, all the whey proteins are trapped simultaneously onto an adsorbent, rinsed free of contaminants, and then eluted one-by-one. Etzel [35], Outinen et al. [22], Ye et al. [36], Mozaffar et al. [37], Couriol et al. [38], Doultani et al. [39] and Heeboll-Nielsen et al. [40] developed ion exchange or affinity chromatography processes to separate whey proteins. Apart from these interesting techniques, membrane filtration has also provided promising results [10] for the fractionation of whey proteins. Membrane filtration has traditionally been based solely on differences in molecular mass [41–44]. Until recently, membranes were thought to achieve separation only between proteins differing in size by at least a factor of 10. Van Reis et al. [45,46] coined the name high performance tangential flow filtration (HPTFF) to describe a very selective process, which allows the separation of proteins with very similar molecular weight. HPTFF is based on the proper choice of pH and ionic strength in order to maximize the differences in the effective hydrodynamic volume of the different proteins [47]. Recent publications have demonstrated the feasibility of HPTFF for the fractionation of some whey proteins. Muller et al. [48] studied the effect of pH and ionic strength in a two-step cascade in order to produce purified ␣-lactalbumin from acid whey. Tubular ceramic membranes were employed (150–300 kDa) and a two-fold increase in protein purity was achieved with a yield of 0.53. Cheang and Zydney [47] designed a two-stage ultrafiltration process for the purification of both ␣-lactalbumin and ␤-lactoglobulin from whey protein isolate. Flat composite regenerated cellulose membranes of 100 and 30 kDa were employed. The purification of ␣-lactalbumin achieved was greater than 10-fold at 90% yield. Bhattacharjee et al. [49] studied the separation of ␤-lactoglobulin from whey protein concentrate using a two-stage process with 30 and 10 kDa flat polyethersulfone membranes followed by ion-exchange membrane chromatography. In each stage, the effect of pH on permeate flux and rejection

29

was investigated. As a result, an 87.6% purity of ␤-lactoglobulin was obtained. The aim of this research work was to study the effect of pH on the fractionation of clarified whey proteins. A 300 kDa tubular ceramic cross-flow ultrafiltration membrane was employed in order to obtain two fractions: (1) a permeate containing a significant percentage of the original ␣-lactalbumin and ␤-lactoglobulin and (2) a retentate enriched in BSA, immunoglobulins and lactoferrin. Following a continuous diafiltration procedure up to 4 diavolumes, the flux–time profiles and yields in both retentate and permeate of the individual proteins were evaluated. The results obtained were explained in terms of membrane–protein and protein–protein interactions. 2. Experimental 2.1. Preparation of clarified acid bovine whey Acid whey was prepared from pasteurized whole milk. First, the milk was centrifuged at 4500 rpm and 4 ◦ C for 30 min in order to eliminate fat. Then, the pH of the defatted milk was reduced to 4.2 by adding 2N HCl for casein coagulation, which was separated from the whey by further centrifugation at 4500 rpm and 4 ◦ C for 30 min. In order to enhance the subsequent permeate flow, the acid whey was pretreated following a similar procedure to those proposed by Rinn et al. [50] and Gesan et al. [51]. First, CaCl2 (1.2 g/L) was added to the whey at 2–5 ◦ C. Then, pH was raised to 7.3 using 6N NaOH and temperature was increased to 55 ◦ C. These conditions were held for 8 min, involving the aggregation of complex lipid–calcium phosphate particles. Finally, the whey was cooled down to 10 ◦ C and centrifuged at 4500 rpm for 30 min. The precipitate contained the insoluble calcium phosphate aggregates and the supernatant was the clarified whey. 2.2. Experimental set-up and procedure The experimental rig consisted of a 2 L feed tank immersed in a thermostatic bath at 30 ◦ C, a precision positive displacement recirculation pump (Procon, Murfreesboro, TN, USA), a membrane housing, one back-pressure valve, two manometers located before and after the membrane, a flowmeter (Badger Meter, Milwaukee, WI, USA), pH and temperature probes, and two tanks for permeate collection and diafiltration water supply. The ceramic membrane employed was a Clover Inside C´eram (Tami, Nyons, France) made of ZrO2 –TiO2 , 1.20 m long, and a filtration area of 0.045 m2 . The membrane module has three channels with a hydraulic diameter of 3.6 mm. Cross-flow ultrafiltration experiments were performed in the continuous diafiltration mode. The clarified acid bovine whey was adjusted to the working pH using HCl or NaOH and vacuum filtered in order to remove any possible aggregates. Afterwards, 2 L of whey were added to the feed tank. While in operation, retentate was recirculated to the feed tank under the following conditions: transmembrane pressure 1 bar, temperature 30 ◦ C, cross-flow velocity 3.5 m/s. The permeate was collected in the

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M.C. Alm´ecija et al. / Journal of Membrane Science 288 (2007) 28–35

cumulated permeate tank and diafiltration water (at the same pH and temperature of the feed) was added to the retentate tank at the same rate of the permeate flow. The system was operated up to 4 diavolumes, i.e. 8 L of permeate were collected. Permeate flux was calculated from mass measurements in an analytical balance. Samples of retentate and permeate were taken at each diavolume for quantification of individual proteins. In order to regenerate the ceramic membrane after whey ultrafiltration, the following cleaning procedure was performed: (1) initial rinse with demineralised water; (2) total recirculation of a solution of 20 g/L NaOH + 0.1 g/L SDS at 50 ◦ C for 60 min; (3) final rinse with demineralised water until neutrality. This cleaning protocol was repeated until the clean membrane resistance was recovered. 2.3. Protein analysis Individual protein concentrations, including ␣-lactalbumin, ␤-lactoglobulin, BSA, lactoferrin and IgG, were determined by reversed-phase high-performance liquid chromatography (RPHPLC) using the method described by Elgar et al. [52] and extended by Palmano and Elgar [53]. The HPLC system (Waters, Milford MA, USA) consisted of an Alliance Separation Module 2690 interfaced with a M-474 absorbance detector and a Millenium data acquisition and manipulation system. A 1 mL Resource RPC column (Amersham Biosciences, Uppsala, Sweden) was operated at room temperature at a flowrate of 1 mL/min. Solvent A was 0.1% (v/v) trifluoroacetic acid (TFA) in Milli-Q water and solvent B was 0.09% (v/v) TFA, 90% (v/v) acetonitrile in Milli-Q water. The column was equilibrated in 80% solvent A. The gradient used was: 0–1 min, 20% B; 1–6 min, 20–40% B; 6–16 min, 40–45% B; 16–19 min, 45–50% B; 19–20 min, 50% B; 20–23 min, 50–70% B; 23–24 min, 70–100% B; 24–25 min, 100% B; 25–27 min, 100–20% B; 27–30 min, 20% B. Detection was by absorbance at 214 nm. Prior to RP-HPLC analysis, all samples were filtered through 0.22 ␮m nylon syringe filters and buffers were filtered through 0.45 ␮m Durapore membrane filters (Millipore, Bedford MA, USA) and degassed. Sample injection volumes were 50 ␮L for initial clarified whey and retentate samples and 100 ␮L for permeate samples. TFA and acetonitrile, both HPLC grade, were from Scharlau Chemie (Barcelona, Spain). Protein standards for calibration (␣-lactalbumin, ␤-lactoglobulin A, ␤-lactoglobulin B, BSA, IgG and lactoferrin) were purchased from Sigma (St. Louis, MO, USA). 2.4. Calculation of the purity improvement of the proteins retained The improvement of purity experienced by the proteins preferably retained was determined as follows. The purity in the original clarified whey for BSA, IgG and lactoferrin was calculated as the ratio between the individual protein concentration and the sum of the concentrations of the five proteins. The purity at the end of the ultrafiltration process was calculated considering the values of protein concentration at this point. Finally,

the purity improvement factor was the ratio between final and initial purity. As a consequence, factor values above 1 involve to some extent a satisfactory process performance since the purity of the protein is increased. On the contrary, values below 1 involve a negative effect of the ultrafiltration since the protein of interest is even less pure than in the original whey. 3. Results and discussion 3.1. Composition of the clarified whey A RP-HPLC chromatogram of the clarified whey is shown in Fig. 1. The major peaks, corresponding to the main whey proteins (␣-lactalbumin and ␤-lactoglobulin, variants A and B), appeared at elution times of 11.4 and 19.6 min, respectively. In order to show significant areas for the other proteins, AU214 nm was limited to 0.60 in the y-axis. The peaks of lactoferrin and BSA appeared between the major peaks at times of 15.5 and 16.9 min, respectively. The last peak eluted corresponded to IgG at 22.8 min. Taking into account the calibration curves, the concentration for each individual whey protein was obtained as follows: ␣-lactalbumin, 1.00 g/L; ␤-lactoglobulin, 2.70 g/L; BSA, 0.1 g/L; IgG, 0.40 g/L; lactoferrin, 0.04 g/L. 3.2. Permeate flux In Fig. 2, it is represented the flux–time profiles for the different pH values. At the lowest pH assayed, pH 3, an initial flux of 68 L/(m2 h) was obtained. After this point, a linear increase was observed, reaching 85 L/(m2 h) after 2.2 h at the end of the operation (4 diavolumes). The slowest filtrations were achieved at pH 4 and 5. Permeate fluxes decayed from an initial value of 40 and 51 L/(m2 h), respectively, to 25 in the first 3 h, remaining practically constant until the end of operation (more than 6 h). Almost identical curves appeared at pH 6 and 7. The main decrease (from 65 to 42 L/(m2 h)) was obtained in the first 2 h and a steady value was maintained up to 3.8 h. A similar behaviour was observed at pH 8, in which flux decayed from 73 to 50 L/(m2 h) with a total time of 3.2 h. An oscillating curve can be seen for pH 9. Flux decreased from 91 to 80 L/(m2 h) in 1 h. Then, a constant flux of 87 was obtained up to 2.1 h. Finally, the fastest operation was achieved at pH 10, in which flux increased almost linearly from 89 to 125 L/(m2 h) in 1.6 h. In order to explain the evolution of permeate flux in this continuous diafiltration, two opposite phenomena have to be considered. First, total protein concentration decreases during the process provided some protein transmission through the membrane occurs. This promotes an increase in permeate flux. Second, membrane fouling as a consequence of protein adsorption on to the membrane involves a decrease in permeate flux. Both mechanisms (dilution and fouling) depend mainly on the electrostatic interactions protein–protein and protein–membrane. Therefore, the isoelectric points of the whey proteins (␣-lactalbumin, 4.5–4.8; ␤-lactoglobulin, 5.2; BSA, 4.7–4.9; IgG, 5.5–8.3; lactoferrin, 9.0) [10] and the point of zero charge of the membrane, should play a crucial role. The point of

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Fig. 1. Reverse phase HPLC chromatogram of clarified bovine whey. The concentration of the individual proteins is shown in the text box.

zero charge of the membrane could be calculated from its chemical composition (64% Al2 O3 , 27% TiO2 , 9% SiO2 [54–55]), which gave a value between 7.3 and 8.0. As an example, at the extreme pH values (3 and 10), all the whey proteins and the membrane have the same charge sign (positive and negative, respectively). Fouling, which is not favoured because of the repulsion, is dominated by the dilution in the retentate tank involving an increase in permeate flux with time. On the other hand, at pH 4 and 5 (around the isoelectric points of the most abundant whey proteins) fouling dominates due to the deposition on to the membrane of aggregates of uncharged ␣lactalbumin, ␤-lactoglobulin and BSA molecules. This involves a sharp permeate flux decrease in the first part of the process until a steady flux is reached, probably due to the sweeping effect of the tangential retentate stream. 3.3. Retentate and permeate yields of individual proteins In order to evaluate the sieving characteristics of the membrane, the retentate and permeate yields of each of the whey

Fig. 2. Flux–time profiles at different pH values for the continuous diafiltration up to 4 diavolumes of clarified bovine whey through a 300 kDa ceramic membrane.

proteins analysed was monitored during the 4 diavolumes of operation for both retentate and cumulated permeate. The yield of retentate and permeate were calculated as the ratio between the mass of protein in the instantaneous retentate and cumulated permeate, respectively, and the mass of protein in the initial feed. 3.3.1. α-Lactalbumin and β-lactoglobulin The yields of ␣-lactalbumin as a function of pH and the number of diavolumes are shown in Table 1. It can be seen that the sum of retentate and permeate yields was around 100%, which means that no loss of this proteins took place. Three different behaviours were observed. For pH 4 and 5, a very low permeate yield was achieved, involving that practically all the initial ␣-lactalbumin remained in the retentate. For pH 3, 6 and 10 retentate yield suffered a significant decrease from 100 to about 60%. Finally, at pH 7, 8 and 9, the highest permeate yields were observed since more than 50% of the original protein passed through the membrane. The retentate and permeate yields of ␤-lactoglobulin are shown in Table 1. Again, the sum of retentate and permeate yields was close to 100%. No significant protein transmission was observed at pH 4 and 5. On the contrary, maximum transmissions were achieved at pH 3, 7, 8 and 9, in which permeate yield increased up to 33% approximately, giving a retentate yield around 67%. Lower permeate yields (20%) occurred at pH 6 and 10. Considering only the molecular weight of the monomer (␣-lactalbumin, 14 kDa; ␤-lactoglobulin, 18 kDa), it could be thought that both proteins should pass through the membrane with ease. However, a variety of permeate yields was obtained at changes of the electrostatic environment since the extent of the aggregation of the protein molecules is pH-dependent. For instance, at pH 4 and 5 both proteins are essentially uncharged, large aggregates of up to 8 molecules are formed [56,57] which results in very low transmissions. At pH in the 7–9 interval, maximum transmissions were achieved since interactions between the proteins and the neutral membrane are minimised. When protein and membrane repel each other (i.e. at pH 3 and 10), permeate yields values were greater than expected, probably due to the importance of convective transport of solute as a consequence of the high permeate flux. A special case occurs for

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Table 1 Retentate (R) and permeate (P) yield of whey proteins at 1 and 4 diavolumes as a function of pH for the continuous diafiltration of clarified bovine whey through a 300 kDa ceramic membrane pH

Diavolumes

R/P

␣-Lactalbumin

␤-Lactoglobulin

BSA

IgG

Lactoferrin

3

1

R P

0.71 0.29

0.75 0.25

0.95 0.05

0.92 0.08

0.94 0.06

4

R P

0.61 0.42

0.66 0.34

1.00 0.00

0.89 0.11

1.00 0.00

1

R P

1.00 0.04

1.00 0.01

1.00 0.00

1.00 0.00

0.95 0.00

4

R P

1.00 0.04

1.00 0.02

1.00 0.00

1.00 0.00

0.74 0.00

1

R P

0.96 0.06

0.99 0.02

1.00 0.00

0.95 0.00

0.70 0.00

4

R P

0.95 0.08

0.94 0.02

0.96 0.00

0.53 0.00

0.27 0.00

1

R P

0.72 0.29

0.88 0.14

0.96 0.00

1.00 0.00

0.69 0.00

4

R P

0.53 0.44

0.80 0.19

0.95 0.00

0.86 0.14

0.51 0.00

1

R P

0.67 0.32

0.81 0.19

0.94 0.00

0.95 0.05

0.64 0.00

4

R P

0.46 0.54

0.70 0.30

0.96 0.00

0.85 0.15

0.26 0.00

1

R P

0.66 0.33

0.79 0.20

0.96 0.00

1.00 0.00

0.68 0.00

4

R P

0.45 0.56

0.69 0.33

0.99 0.00

1.00 0.00

0.60 0.00

1

R P

0.65 0.35

0.77 0.21

1.00 0.00

1.00 0.00

0.85 0.00

4

R P

0.43 0.58

0.65 0.33

0.96 0.00

0.99 0.00

0.91 0.00

1

R P

0.76 0.27

0.85 0.13

0.97 0.00

0.97 0.00

1.00 0.00

4

R P

0.59 0.46

0.79 0.21

0.98 0.00

0.97 0.00

1.00 0.00

4

5

6

7

8

9

10

␤-lactoglobulin at pH 3, where it is present as a monomer and, therefore, shows permeate yields as high as those obtained in the range 7–9. 3.3.2. BSA, immunoglobulins and lactoferrin In Table 1, the yields of BSA are shown. In all cases, no significant transmission of protein took place. Therefore, for all the pH values assayed, retentate yield remained around 100% during the 4 diavolumes. The yields of IgG are shown in Table 1. All the IgG remained in the retentate for pH 4, 8, 9 and 10. Low permeate yields (around 13%) were obtained at pH 3, 6 and 7 with complementary retentate yields up to 100%. A special behaviour was observed at pH 5. Although null permeate yield was obtained, only 53% of the original IgG remained in the retentate.

The yields of lactoferrin are shown in Table 1. Null permeate yields were obtained in all cases. At the extreme pH values (3 and 10) the retentate yield was 100%. In contrast, in the other experiments, retentate yields ranged from 26 to 91%. It can be seen that the retention for these proteins was much higher that the observed for ␣-lactalbumin and ␤-lactoglobulin, owing to their larger size (BSA, 69 kDa; IgG, 150–1000 kDa; lactoferrin, 78 kDa). The loss of significant amounts of IgG at pH 5 and lactoferrin at pH 4–9 could be due to three possible causes: (1) protein adsorption to the membrane; (2) protein denaturation by shear stress caused by the circulation of the retentate stream at high velocities [58]; (3) association with one of the major whey proteins. For example, lactoferrin forms non-covalent complexes with ␤-lactoglobulin or BSA with molar ratios 2:1 and 1:1 [59]

M.C. Alm´ecija et al. / Journal of Membrane Science 288 (2007) 28–35

Fig. 3. Improvement of purity achieved for the retained proteins (bovine serum albumin, immunoglobulin G and lactoferrin) as a function of pH in the continuous diafiltration up to 4 diavolumes of clarified bovine whey through a 300 kDa ceramic membrane.

provided no protein–protein repulsion occurs. This aggregation is favoured during the diafiltration because of the elution of salt ions in solution, which are in equilibrium with ions joined to local charges of the proteins. 3.4. Purity improvement of the proteins retained The results shown above present some general trends. Except for pH 4 and 5, important protein yields for ␣-lactalbumin and ␤-lactoglobulin were obtained in the permeate. On the other hand, for the rest of the proteins studied, there was no significant transmission through the membrane. This suggests that the 300 kDa membrane could be employed to fractionate the original array of whey proteins in to parts: ␣-lactalbumin and ␤-lactoglobulin in the permeate and BSA, IgG and lactoferrin in the retentate. In order to evaluate qualitatively this possibility, the improvement of purity experienced by the proteins retained was calculated. The purities in the original clarified whey were: BSA, 2.4%; IgG, 9.4%; lactoferrin, 0.9%. In Fig. 3, it can be seen that some purity improvement was obtained for BSA in the entire interval of pH assayed. Maximum increments around 50% were achieved at pH 3 and 9. For IgG, the factor decreased from 1.3 at pH 3 to 0.6 at pH 5 and then increased up to 1.6 at an optimum pH of 9. Finally, improvements for lactoferrin were obtained only at the extremes, with two maxima of 1.5 at pH 3 and 9. Therefore, if our objective were to fractionate BSA, IgG and lactoferrin from ␣-lactalbumin and ␤-lactoglobulin with the 300 kDa membrane, working pH should be adjusted to 3 or 9. Moreover, both values are equally appropriate since almost identical filtration times were needed to complete the 4 diavolumes (2.2 and 2.1 h, respectively). 4. Conclusions Regarding the permeate flux, the slowest filtrations occurred at pH 4 and 5, i.e. around the isoelectric point of ␣-lactalbumin,

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␤-lactoglobulin and BSA. This could be due to the developing of membrane fouling caused by the deposition of aggregates of uncharged protein molecules, which involves a pronounced flux drop in the first 2 diavolumes of operation. On the other hand, increasing fluxes were observed at extreme pH values (such as 3 and 10) since protein–protein and membrane–protein repulsions avoid aggregation and fouling. With respect to protein transmission, ␣-lactalbumin and ␤lactoglobulin were eluted through the membrane due to their lower molecular weight. At pH 4 and 5, low permeate yields took place owing to the presence of protein aggregates, whereas maxima were achieved between pH 7–9 where the membrane has zero net charge. In contrast, essentially no leakage of the larger BSA, IgG and lactoferrin were detected in the permeate. Unwanted losses of IgG at pH 5 and, more importantly, of lactoferrin in the interval 4–9 occurred, probably due to adsorption to the membrane, denaturation by shear stress and/or association between proteins. According to the diafiltration proposed, the purity of the retained proteins could be improved as follows. The purity of BSA and lactoferrin in the original whey (2.4 and 0.9%, respectively) could be multiplied by a factor 1.5 when operating at pH 3 and 9. Similarly, the starting 9.4% was improved 1.6 times for IgG at pH 9. Acknowledgements This research was supported by the Spanish Plan Nacional I+D+I, under the Projects PPQ-2002-02235 and CTQ-200502653. References [1] K.F. Christiansen, G. Vegarud, T. Langsrud, M.R. Ellekjaer, B. Egelandsdal, Hydrolyzed whey proteins as emulsifiers and stabilizers in high-pressure processed dressings, Food Hydrocolloid 18 (2004) 757. [2] N. Neirynck, P. Van der Meeren, S.B. Gorbe, S. Dierckx, K. Dewettinck, Improved emulsion stabilizing properties of whey protein isolate, Food Hydrocolloid 18 (2004) 949. [3] Z. Herceg, V. Lelas, G. Kresic, Influence of tribomechanical micronization on the physical and functional properties of whey proteins, Int. J. Dairy Technol. 58 (2005) 225. [4] J.D. Firebaugh, C.R. Daubert, Emulsifying and foaming properties of a derivatized whey protein ingredient, Int. J. Food Prop. 8 (2005) 243. [5] E.L. Lien, Infant formulas with increased concentrations of ␣-lactalbumin, Am. J. Clin. Nutr. 77 (2003) 1555. [6] J.W.J. Beulens, J.G. Bindels, C. de Graaf, M.S. Alles, W. WoutersWesseling, Alpha-lactalbumin combined with a regular diet increases plasma Trp–LNAA ratio, Physiol. Behav. 81 (2004) 585. [7] H. Matsumoto, Y. Shimokawa, Y. Ushida, T. Toida, H. Hayasawa, New biological function of bovine ␣-lactalbumin: protective effect against ethanoland stress-induced gastric mucosal injury in rats, Biosci. Biotech. Biochem. 65 (2001) 1104. [8] C.A. Dunlap, G.L. Cˆot´e, ␤-Lactoglobulin–dextran conjugates: effect of polysaccharide size on emulsion stability, J. Agr. Food Chem. 53 (2005) 419. [9] N. Matsudomi, D. Rector, J.E. Kinsella, Gelation of bovine serum albumin and ␤-lactoglobulin; effects of pH, salts and thiol reagents, Food Chem. 40 (1991) 55.

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