Fractionation of pasteurized skim milk proteins by dynamic filtration

Food Research International 43 (2010) 1335–1346

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Fractionation of pasteurized skim milk proteins by dynamic filtration Valentina Espina a, Michel Y. Jaffrin b,*, Luhui Ding a, B. Cancino c a

Technological University of Compiègne, EA 4297 TIMR, BP 20529, 60205 Compiègne, France Technological University of Compiègne, UMR 6600, BP 20529, 60205 Compiègne, France c Dept. of Food Technology, Pontificia Universidad Catolica de Valparaiso, Waddington 716, Valparaiso, Chile b

a r t i c l e

i n f o

Article history: Received 6 October 2009 Accepted 23 March 2010

Keywords: Milk fractionation Shear enhanced filtration a-Lactalbumin b-Lactoglobulin

a b s t r a c t This paper describes a two-stage process for separating milk proteins from pasteurized skim milk in three fractions: casein micelles, b-Lactoglobulin (b-Lg) and other large whey proteins, and a-Lactalbumin (aLa). Casein micelles were extracted in the retentate of a microfiltration using rotating ceramic disk membranes. a-La and b-Lg transmissions remained between 0.8 and 0.98. Their yields in permeate reached 81% for a-La and 76.6% for b-Lg at a VRR of 5.4. The separation between b-Lg and a-La was carried out by UF using a rotating disk module equipped with a 50 kDa PES circular membrane. Permeate fluxes were very high, remaining above 340 L h1 m2 at VRR = 5 and 40 °C. a-La transmission remained generally between 0.2 and 0.13 giving yields from 28% to 34%. b-Lg rejection was above 0.94, giving a maximum selectivity of 4.2. These data confirm the potential of dynamic membrane filtration for separating a-La and b-Lg proteins from skim milk. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction The fractionation and extraction of various milk proteins by membrane filtration represents a challenging task as milk is a complex fluid and several of its components induce membrane fouling. For instance casein micelles should be, in principle, easily separated from whey proteins by microfiltration (MF) with 0.1– 0.2 lm pores membranes, but in order to retain good whey protein transmission during the process, it is necessary to combine a high tangential velocity (7–8 ms1) and a low uniform transmembrane pressure (denoted as UTP mode (Daufin et al., 1993). These two conditions can be met with co-current recirculation of permeate in order to obtain the same longitudinal pressure gradient on both sides of the membrane, which regulates TMP, but consumes more energy than normal MF. Le Berre and Daufin (1996) microfiltered pasteurized skim milk with a Membralox ceramic tubular membrane of 0.1 lm pores equipped with co-current permeate recirculation in order to separate casein from whey proteins. They obtained a permeate flux of 110 L h1 m2 at a fluid velocity of 7.2 ms1, 50 °C and a VRR of 2. a-Lactalbumin (a-La) transmission was initially equal to 0.95, but dropped to 0.6 after 140 min of filtration. Corresponding transmissions of b-Lactoglobulin (b-Lg) were 0.82 and 0.5. Gésan-Guiziou, Boyaval, and Daufin (1999) used the same filtration bench in UTP mode with a Kerasep membrane of 0.1 lm pores and reported fluxes of about 80 L h1 m2 at a wall shear stress of * Corresponding author. Tel.: +33 3 44 23 43 98; fax: +33 3 44 23 79 42. E-mail address: [email protected] (M.Y. Jaffrin). 0963-9969/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodres.2010.03.023

100 Pa. Their a-La and b-Lg transmissions were similar to those of Le Berre and Daufin (1996), but permeate turbidity was relatively high, ranging from 100 NTU to 300 NTU at a mean transmembrane pressure (TMP) of 100 kPa, due to incomplete casein rejection. Vadi and Rizvi (2001) used a pilot equipped with a Membralox ceramic multichannel membrane of 0.2 lm pores operated in UTP mode, in MF of skim milk. They found that optimal operating conditions were a velocity of 7.1 ms1 and a TMP of 240 kPa, and obtained, at VRR = 2, a maximum flux of 100 L h1 m2. By contrast, Lawrence, Kentish, Connor, Barber, and Stevens (2008), using PVDF spiral wound membranes of 0.3 lm pores in non UTP mode, reported steady state fluxes of 32–50 L h1 m2 and a b-Lg transmission of only 0.22 at 50 kPa, decreasing to 0.08 at 150 kPa. The next fractionation step is the separation and purification of major whey proteins such as a-La and b-Lg which present various applications for the food and pharmaceutical industries. a-La is used as food additive in infant formula (Muller, Chaufer, Merin, & Daufin, 2003) due to its high tryptophan content and nutritional value and has also medical applications due to its high cytotoxicity (Svensson, Hakansson, Mossberg, Linse, & Svanborg, 2000). b-Lg proteins have important emulsification and gelling properties (Cayot & Lorient, 1997; Dunlap & Coté, 2005). Until recently, whey proteins were separated by selective precipitation, or selective adsorption (Bramaud, Aimar, & Daufin, 1997). Separation by membrane of a-La and b-Lg was difficult as these proteins had close molecular weight, 14 kDa for a-La and 36 kDa for b-Lg in dimer form. Van Reis, Brake, Charkoudian, Burns, and Zydney (1999) proposed a selective crossflow filtration process based on optimisation of pH and ionic strength to enhance their separation.

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Nomenclature C M R0 Tr Y

protein concentration (kg m3) protein mass (kg) apparent rejection transmission yield (percent recovery)

Subscripts i initial f final

Muller et al. (2003) ultrafiltered acid casein whey with a 150 kDa Carbosep M1 membrane in order to separate a-La in permeate from bovine serum albumin (BSA) and immunoglobulin (Ig) in retentate with limited b-Lg transmission. They obtained a a-La transmission of 0.4 at 280 kPa and a permeate flux of 50 L h1 m2. Cheang and Zydney (2003) diafiltered a binary mixture of pure of a-La and b-Lg proteins in a NaCl solution with 1 mM Na2HPO4 prefiltered at 0.2 lm to remove particles, using a small Amicon stirred cell equipped with a 30 kDa cellulosic membrane. a-La transmission was 0.26 at a permeate flux of 14 L h1 m2, against only 0.005 for b-Lg. These transmissions increased with increasing ionic strength to reach 0.6 for a-La at a strength of 150 mM at pH = 5.5, and 0.4 at pH = 7.2. b-Lg transmissions also increased with increasing ionic strength, but were maximum at pH = 7.2. Selectivity (ratio of a-La to b-Lg transmission) was maximum (58) at a pH of 5.5 and an ionic strength of 50 mM, but decreased to 35 when permeate flux was doubled, due to increase in b-Lg transmission at high ionic strength. With a 50 kDa PES Biomax membrane, the maximum selectivity was 10.5 at pH = 5.5 and a strength of 150 mM. The authors attributed this lower selectivity to the higher zeta potential of the Biomax membrane, 15 mV, versus 2.2 mV for the cellulose membrane and to the higher pore size. They concluded that it was possible to separate a-La and b-Lg proteins with a high selectivity and a high yield rate, by optimal choices of pH, ionic strength and membrane cut-off. Bhattacharjee, Bhattacharjee, and Datta (2006) separated b-Lg from whey protein concentrate obtained from raw casein whey by centrifugation followed by a MF at 0.45 lm. They used a dynamic filtration module consisting in a circular polymer membrane of 76 mm diameter rotating inside a cylindrical housing, near a disk stirrer rotating in opposite direction at 500 rpm. The retentate was then ultrafiltered at 30 kDa, after addition of hydrochloric acid to lower the pH to 2.8 in order to obtain monomer b-Lg and a-La, while bovine serum albumin, lactoferrin and immunoglobulins were collected in retentate. With the membrane at rest, the flux decayed from 200 L h1 m2 to 20 L h1 m2 after 20 min of filtration. With the membrane rotating at 300 rpm, the flux stabilized at 100 L h1 m2 and reached 115 L h1 m2 at 600 rpm. The final separation between monomer b-Lg and a-La was obtained by ion-exchange membrane chromatography. This literature survey shows that fractionation of milk protein by membrane and, more specifically, the separation of b-Lg from a-La in milk, is a challenging task, requiring an elaborate process. In most papers that we have quoted, the starting fluid was not from milk, but pre-treated whey or, as in Cheang and Zydney (2003) a binary mixture of pure proteins. In this paper, we propose to use a MF + UF shear enhanced (or dynamic) filtration process to recover separately a-La and b-Lg. The MF step will use rotating ceramic disk membranes in a MSD module to separate whey proteins from caseins. Recently, Espina, Jaffrin, Frappart, and Ding (2008), have shown that this module

p r MF TMP UF UTP VRR

permeate retentate microfiltration transmembrane pressure (kPa) ultrafiltration uniform transmembrane pressure volume reduction ratio

gave the best MF performance, but tests were made on UHT skim milk because of easy access and fluid reproducibility. In the present work, we have used a pasteurized skim milk provided by a dairy plant, which, unlike UHT milk, had the same whey protein composition as fresh milk and is more representative of real industrial conditions. Because ceramic disks with proper cut-off were not available, we have used, for the UF step, a module containing a metal disk with vanes rotating near a circular polymeric membrane. Our module differs from that used by Bhattacharjee et al. (2006) by its capacity to generate higher membrane shear rates due to higher rotation speeds and the presence of vanes on the disk. 2. Materials and methods 2.1. MSD pilot The MSD pilot (Westfalia Separator, Aalen Germany), described by Ding, Jaffrin, Mellal, and He (2006) consists normally in 12 ceramic membrane disks rotating on two parallel hollow shafts, enclosed in a stainless steel housing. Only six ceramic membranes on one shaft for a total membrane area of 0.060 m2 were used in these tests. The permeate was collected through the hollow shaft via radial channels inside the disks. The TMP was calculated from pressure pc of measured at a tap in the housing, close to disk periphery, using a Validyne DP 15 pressure transducer (Validyne Corp., Northridge, CA, USA) as,

TMP ¼ pc 

qx2 ðR21 þ R22 Þ 4

ð1Þ

where R1 = 4.5 cm and R2 = 1.02 cm are, respectively, the outer and inner radius of membrane disks and x is the disk angular velocity. 2.2. Rotating disk module The rotating disk module has been described previously in (Espina et al. (2008)) (Fig. 1). It was equipped with a single-polymeric membrane, of 188 cm2 area (outer radius R1 = 7.75 cm) fixed on the cover of a cylindrical housing in front of the disk. The disk was equipped with eight 6 mm-high vanes in order to increase the core fluid angular velocity kx, between membrane and disk, where x is the disk angular velocity and k is the velocity factor. Its rotation speed can be adjusted between 500 and 2500 rpm. Peripheral pressure (pc) and inlet pressures were measured as described in Bouzerar, Jaffrin, Ding, and Paullier (2000). Values of velocity factor k were obtained from measurements of pc at different speeds and found to be 0.89 for this disk. The pressure was adjusted by a valve on outlet tubing. The TMP was then determined as:

1 2 TMP ¼ pc  qk x2 R2 4 where R is the inner housing radius.

ð2Þ

V. Espina et al. / Food Research International 43 (2010) 1335–1346

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Fig. 1. Schematic of filtration bench with rotating disk module.

2.3. Membranes and cleaning procedure Ceramic membranes (a-Al2O3) used in the MSD pilot were made by Westfalia Separator and had a nominal pore size of 0.2 lm and a skin thickness of 10 lm. Their hydraulic permeability, determined by measuring the permeate flux with deionised water at various TMP and at 20 °C, was found to be 886 ± 227 L h1 m2 bar1. For UF tests in rotating disk module, a new 50 kDa cut-off PES membrane (Microdyn-Nadir, France) was used in each test, since b-Lg was in dimer form. Its hydraulic permeability at 20 °C was 687 ± 231 L h1 m2 bar1. After each test, ceramic membranes were rinsed with deionised water before cleaning, carried out with a P3 Ultrasil 10 (Ecolab) solution at 0.5% and 40 °C for 1 h in closed circuit. Then, the system was drained and membranes were rinsed with demineralised water until a pH of 7.0 was obtained. Initial permeabilities were recovered after cleaning for ceramic membranes. 2.4. Test fluid 2.4.1. MF The test fluid for MF was pasteurized milk collected right after skimming by a centrifuge at Lactalis Company, Clermont 60, France with a pH of 6.8 and a mean total protein concentration for the three tests of 30.0 ± 1.0 g L1, corresponding to 1.34 ± 0.05 g L1 of a-La and 2.07 ± 0.13 g L1 of b-Lg. Individual concentrations of other whey proteins were not measured. 2.4.2. UF Permeates of milk MF tests were used as feed in UF tests. 2.5. Experimental protocol 2.5.1. MF The MSD module was fed from a stirred tank, thermostated at 55 °C in order to obtain a feed temperature of 40 °C, by a volumetric diaphragm pump. The permeate was collected in a beaker placed on an electronic scale (Sartorius B3100 P, Gottingen, Germany) connected to a computer in order to measure the permeate

flux. Tests were conducted without permeate recycling (concentration tests). Retentate and permeate samples were collected every 30 min for analysis. 2.5.2. UF The rotating disk module was fed from the same tank and pump as the MSD. A disk with eight-6 mm-high vanes rotating at 2000 rpm was used in all tests. Tests were also conducted with a feed at a temperature of 40 °C. In order to investigate the effect of TMP, some tests were performed with permeate and retentate recycling. Permeate and retentate samples were collected every 15 min for further analysis, after flux stabilisation in tests at variable TMP. 2.6. Analysis

a-La and b-Lg concentrations in permeate and retentate were measured by HPLC samples collected at the permeate and retentate outlets of the module, according to a procedure described by Espina et al. (2008). A Waters 510 chromatograph, a UV detector at 280 nm and a Vydac-C4 column thermostated at 40 °C, were used according to the method of Jaubert and Martin (1992). A calibration curve was made using pure a-La and b-Lg samples from bovine milk (Sigma Aldrich, Germany) of known concentrations. Solvents used for the HPLC analyses were: Trifluroacetic acid (solvent A) at 0.1% in milli-Q water (Sigma Aldrich), and 80% of methanol with 0.096% TFA and 20% of milli-Q water (solvent B, Prolabo, France). In order to have a better resolution, a change in solvents proportion was made as follows: initial proportions were: 30% of A, 70% of B; then the proportion of B was gradually increased to 100% and decreased back to 70% over a period of 35 min. Before injection into the column, 5 mL of each sample were pre-treated in order to precipitate caseins and denatured proteins. Its pH was lowered to 4.3 with acetic acid (10% w/v) and restored to 4.6 after 5 min with a 1 M sodium acetate solution. After heating the sample for 20 min at 40 °C, the supernatant was filtered using a 0.45 lm Minisart RC 45 (Sartorius) syringe filter, before injection into the HPLC. Proteins concentrations of native (non denatured) proteins in g L1 were calculated from

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C a-La ¼ 6:89  107  A R2 ¼ 0:985

ð3Þ

C b-Lg ¼ 7:86  107  A R2 ¼ 0:980

ð4Þ

2.7. Calculated parameters

a-La and b-Lg transmissions (Tra, Trb) were calculated by Tr ¼

where A denotes the area in mm2 under the corresponding peaks of the chromatogram. Measurement error was estimated to be 5%. Numerical coefficients in Eqs. (3) and (4) are different from those reported in Espina et al. (2008) due to use of methanol, instead of acetonitrile as solvent. Permeate turbidities, which characterize the transmission of casein micelles through the membrane, were measured with a Hach turbidimeter (Colorado, USA).

Table 1 Characteristics of MF (1,2,3) and UF (4,5,6) tests. Test

Vr, L init

Vr, L final

Vp fin

VRR

1 2 3 4 5 6

13.5 20.0 19.5 4.5 15.3 15.5

3.86 4.58 3.60 1.46 1.74 2.88

9.63 15.4 15.8 3.04 13.55 12.61

3.50 4.37 5.41 3.07 8.81 5.37

Table 2 Protein concentrations and masses in retentate and permeate for MF tests. Test protein

1 b-Lg 2 a-La 2 b-Lg 3 a-La 3 b-Lg

Initial Final Initial Final Initial Final Initial Final Initial Final Initial Final

Mr, g

1.334 2.20 2.13 2.73 1.30 1.57 2.19 3.80 1.41 1.42 1.93 2.42

18.0 8.48 28.71 10.54 26.0 7.17 43.74 17.40 27.44 5.10 37.65 8.70

Cp, g L1 0.986

Mp, g 9.44

CF

Yp%

1.66

52.5

1.88

18.10

1.284

63.1

1.18

18.70

1.20

72.0

1.70

26.20

1.74

60.0

1.41

22.28

1.087

81.1

1.826

28.85

1.252

76.6

ð5Þ

where Cp denotes the permeate protein concentration and Cr the retentate one. The yield (percentage of proteins recovered in permeate (Yp) was calculated as

Y p ¼ 100 M p =M ri

ð6Þ

where Mp denotes the protein mass in permeate and Mri the initial feed one. The selectivity (S) was calculated as:

S¼

Tra Trb

ð7Þ

a-La purity (P) in UF permeate was calculated using Eq. (8): P¼

Cp CT

ð8Þ

where Cp is the permeate concentration for a-La and CT is the sum of a-La and b-Lg concentrations as other proteins in permeate had a larger molecular weight than b-Lg and their concentrations were very small. 3. Results 3.1. Separation of caseins from whey proteins by MF on rotating ceramic membranes Three tests were carried out without permeate recycling using the MSD module at same TMP of 60 kPa, same disk rotation speed of 1930 rpm and a temperature of 45 °C, except in test 3 (40 °C). Other characteristics of these tests are listed in Table 1. Table 2 shows that initial a-La concentrations varied from 1.30 to 1.41 g L1among the three tests, while the spread of initial b-Lg concentrations was a little larger, from 1.93 to 2.187 g L1.

100 Test 1 Test 2 Test 3

90 80

JTest1 = -40.8Ln(FRV) + 88.9 R2 = 0.9955 JTest2 = -43.3Ln(FRV) + 94.6 R2 = 0.9925 JTest3 = -37.4Ln(FRV) + 83.5

70

R2 = 0.9938

J (L h-1 m-2)

1 a-La

Cr, g L1

Cp Cr

60 50 40 30 20 MSD. Ceramic membranes 0.2 µm. 1930 rpm. TMP = 60 kPa. Q = 3 L/min.

10 0 1

VRR Fig. 2. Variation of permeate fluxes with VRR in semi-log coordinates for MF tests 1, 2, 3.

10

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Variations of permeate flux with VRR are plotted in Fig. 2 in semilog coordinates for the three tests. The highest flux was obtained for test 2 performed at 45 °C, while the lowest was for test 3 performed at 40 °C. This was expected, as a lower temperature increases the viscosity which decreases the membrane shear rate and the flux in mass transfer-limited regime, as was the case in Fig. 2. These fluxes decayed linearly with Ln(VRR) according to the thin film theory of Blatt, Dravid, Michaels, and Nelson (1970). The maximum theoretical VRR extrapolated to zero flux was equal to 9 for the three tests. Corresponding permeate turbidities, which are caused primarily by the presence of casein micelles in permeate, are plotted versus VRR in Fig. 3. These turbidities were highest

for test 1 and decayed when VRR increased for the three tests, as the thickness of rejected proteins layer on the membrane rises with concentration and decreases micelles transmission. They are higher than those observed with the same pilot and under same operating conditions by Espina et al. (2008) with UHT skim milk which were below 50 NTU, perhaps due to higher whey protein concentration in permeate, but they still indicate a very good micelle rejection by the membrane, as a turbidity of 50 NTU corresponds to a casein rejection of 99% (Gésan-Guiziou, 2008). a-La permeate concentrations for the three tests are plotted in Fig. 4 as function of VRR. They remained between 0.9 and 1.5 g L1, except when VRR exceeds 3.6 in test 3, where it reached 2.1 g L1.

200 Test 1 Test 2 Test 3

180 160

Turbidity (NTU)

140 120 100 80 60 40 20

MSD. Ceramic membranes 0.2 µm. 1930 rpm. TMP = 60 kPa. Q = 3 L/min.

0 1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

VRR Fig. 3. Variation of permeate turbidities for MF tests 1, 2, 3.

α-La permeate concentration (g/L)

2.5

2.0

1.5

1.0

Test 1 Test 2 Test 3

0.5 MSD. Ceramic membranes 0.2 µm. 1930 rpm. TMP = 60 kPa. Q = 3 L/min. 0.0 1.0

1.5

2.0

2.5

3.0

3.5

4.0

VRR Fig. 4. Variation of a-La concentration in permeate with VRR for MF tests 1, 2, 3.

4.5

5.0

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b-Lg permeate concentrations, plotted in Fig. 5, remained between 1.5 and 2 g L1, except again in test 3 at VRR > 3.5 and in test 1 at VRR = 2.7. Corresponding a-La transmissions calculated from Eq. (6) and displayed in Fig. 6, fluctuate between 0.62 and 0.98, due probably to measurement errors, but remain high at all VRR, especially for test 3. These transmissions were highest for test 3, which explains why a-La permeate concentration increased with VRR for this test in Fig. 4. b-Lg transmissions were a little lower than those of a-La, as seen in Fig. 7, which is logical as b-Lg has a larger molecular weight in dimer form, and did not decay at large VRR. Largest transmissions were also observed for test 3, reaching 0.84 at VRR = 4.6.

Table 2 lists final a-La and b-Lg concentrations in retentate (Cr), the mean concentration in collected permeate (Cp), together with initial and final protein masses in retentate (Mr) and the final one in permeate (Mp). The concentration factor of each protein (CF) was calculated as

CF ¼ C rf =C ri

ð9Þ

where subscripts f and i denote respectively final an initial values. Final concentration factors varied little with VRR since transmissions were high, while yields (Yp) increased with increasing VRR. They were largest for a-La in tests 2 and 3, but b-Lg was largest in test 1, with the smallest VRR.

β-Lg permeate concentration (g/L)

3.0

2.5

2.0

1.5

1.0

Test 1 Test 2 Test 3

0.5 MSD. Ceramic membranes 0.2 µm. 1930rpm. TMP = 60 kPa. Q = 3 L/min. 0.0 1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

VRR Fig. 5. Variation of b-Lg concentration in permeate with VRR for MF tests 1, 2, 3.

1 0.9 0.8 0.7

Tr α-La

0.6 0.5 0.4 0.3

Test 1 Test 2 Test 3

0.2 MSD. Ceramic membranes 0.2 µm. 1930rpm. TMP = 60 kPa. Q = 3 L/min.

0.1 0 1

1,5 1.5

2

2,5 2.5

3

3,5 3.5

4

VRR Fig. 6. Variation of a-La transmission versus VRR for MF tests 1, 2, 3.

4,5 4.5

5

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1 0.9 0.8

Tr β-Lg

0.7 0.6 0.5 0.4 0.3 Test 1 Test 2 Test 3

0.2 0.1

MSD. Ceramic membranes 0.2 µm. 1930 rpm. TMP = 60 kPa. Q = 3 L/min.

0 1

1.5 1,5

2

2,5 2.5

3

3.5 3,5

4.5 4,5

4

5

VRR Fig. 7. Variation of b-Lg transmission versus VRR for MF tests 1, 2, 3.

Table 3 Protein concentrations, masses and percent recovery in permeate for a-La and in retentate for b-Lg for UF tests 4, 5, and 6. Test/protein 4 a-La 4 b-Lg 5 a-La 5 b-Lg 6 a-La 6 b-Lg

Init Final Init Final Init Final Init Final Init Final Init Final

Cr, g L1

Mr, g

0.986 2.18 1.88 5.18 1.18 7.19 1.70 13.37 1.41 5.33 1.83 8.80

4.437 3.17 8.46 7.60 18.04 12.48 25.99 23.21 21.85 15.35 28.30 25.35

3.2. UF at 50 kDa of wheys produced by permeate from tests 1 to 3 These tests, numbered 4, 5 and 6, using respectively permeates of test 1, 2 and 3, were carried out without permeate recycling, in order to separate b-Lg in retentate from a-La in permeate. The rotating disk module was used at the same TMP of 400 kPa, same rotation speed of 2000 rpm and same fluid temperature of 40 °C in the three tests. In order to check the effect of membrane cleaning on whey protein transmission, membranes were not chemically cleaned before carrying out tests 5 and 6, while test 4 was started after a period of filtration of 40 min at VRR = 1 with full recycling in order to study the effect of TMP on permeate flux and transmissions. Other characteristics of these tests are listed in Table 1, while initial and final retentate a-La and b-Lg concentrations and masses are listed in Table 3. Variations of permeate flux with VRR are plotted in Fig. 8 in semi-log coordinates for the three tests. The highest flux was obtained for test 6 and this flux decayed relatively little until the maximum VRR of 5.37. Test 4 gave the lowest initial flux; this was due to the preliminary filtration step at VRR = 1, not shown on the graph, which also explains the absence of rapid initial decay at beginning of the test, caused by protein adsorption (Yee, Wiley, & Bao, 2009). The permeate flux of test 5, in which the highest VRR (8.81) was reached, decayed fas-

Cp, g L1

Mp, g

CF

a: Yp, b: Yr%

Purity

0.400

1.214

2.21

27.4

0.58

0.267

0.810

2.76

89.8

0.390

5.28

6.09

29.3

0.200

2.71

7.88

88.9

0.502

6.33

3.78

29.0

0.228

2.875

4.82

89.6

0.72

0.67

ter with increasing VRR than for the other tests, due probably to membrane deterioration as it has been stored for a long time. As seen in Fig. 9, a-La permeate concentrations for the three tests increased almost linearly with VRR, but at a slower rate in test 5 than in tests 4 and 6. This is coherent with our assumption that membrane fouling was more severe in test 5 than in the other two and will be later confirmed by the lower transmission observed for this test. b-Lg permeate concentrations, plotted in Fig. 10, also increase quasi linearly with VRR at a fastest rate in test 4 and a slowest in test 5. Variations of protein transmissions with VRR, which ranged from 0.13 to 0.18 for a-La (Fig. 11) and from 0.03 to 0.1 for b-Lg (Fig. 12) were lowest for test 5 and highest for test 4. The lower transmissions of tests 5 and 6 are probably due to the fact that membranes were not chemically cleaned before these tests, and to the higher initial a-La concentration. They were similar to those obtained with UHT milk by Espina, Jaffrin, and Ding (2009). Variations of selectivity given by Eq. (8) with VRR are displayed in Fig. 13. These selectivities increased with increasing VRR for tests 4 and 5 and reached a maximum of 4.3 at VRR = 4.8 for test 5. In test 6, the selectivity reached a maximum of 3.5 at VRR = 3.5. Table 3 gives the same information as Table 2, but for UF tests 4, 5, and 6, with the addition of concentration factors (CF) and

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700 Test 4 Test 5 Test 6

600

J (L h-1 m-2)

500

400

300

200

100 Rotating disk module. Disk with vannes. 50 kDa PES. 2000 rpm. 40°C. PTM = 400 kPa. Q = 3L/min. 0 1

10

VRR Fig. 8. Variation of permeate flux with VRR in semi-log coordinates for UF tests 4, 5 and 6.

1.2

α-La permeate concentration (g/L)

Rotating disk module. Disk with vannes. 50 kDa PES. 2000 rpm. 40°C. PTM = 400 kPa. Q = 3L/min. 1.0

0.8

0.6

0.4 Test 4 Test 5 Test 6

0.2

0.0 1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

VRR Fig. 9. Variation of a-La concentration in permeate with VRR for UF tests 4, 5, 6.

purities. Initial retentate protein concentrations were equal to those of MF permeates and were a little smaller than initial concentrations of MF tests. Final retentate concentrations were higher than in MF tests, especially for b-Lg, due to its small transmission. Yields factors in permeate for a-La range from 27.4% to 29.3%. For b-Lg, the relevant yield factor is that in the retentate (Yr) calculated from

Y r ¼ 100M rf =Mri

ð10Þ

where Mrf is the final protein mass in retentate. This yield ranged from 88.9% to 89.8% and was thus independent of VRR, due to small b-Lg transmission. a-La purities are given in the last column of Table 3. The highest purity (0.72) was obtained, as expected in test 5 with highest VRR.

In order to check the consistency of our transmission data in UF, we have compared measured final concentration factors CF for each UF test with theoretical values calculated from corresponding VRR. If solute rejection R0 is constant, it is shown in Appendix A, that CF is given by

CF ¼ VRRR

0

ð11Þ

The comparison between final CF values of Table 3 and regression lines of Eq. (11) for a-La and b-Lg is plotted versus VRR in Fig. 14, in log–log coordinates. Slopes of regression lines give theoretical rejections of 0.786 for a-La and 0.935 for b-Lg with squared correlation coefficients R2 close to 1. These theoretical rejections correspond to mean values for the three tests. Mean theoretical

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β-Lg permeate concentration (g/L)

0.60 Test 4 Test 5 Test 6

0.50

0.40

0.30

0.20

0.10 Rotating disk module. Disk with vannes. 50 kDa PES. 2000 rpm. 40°C. PTM = 400 kPa. Q = 3L/min. 0.00 1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

VRR Fig. 10. Variation of b-Lg concentration in permeate with VRR for UF tests 4, 5, 6.

0.35 Test 4 Test 5 Test 6 Mean theoretical transmission

0.30

Tr α-La

0.25

0.20

0.15

0.10

0.05 Rotating disk module. Disk with vannes. 50 kDa PES. 2000 rpm. 40°C. PTM = 400 kPa. Q = 3L/min. 0 1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

VRR Fig. 11. Variation of a-La transmission versus VRR for UF tests 4, 5, 6.

transmissions (given by 1  R0 ) are 0.212 for a-La and 0.064 for bLg. Even though actual transmissions were not constant and Eq. (11) was not strictly valid, it is interesting to observe in Figs. 11 and 12 that these mean theoretical transmissions are reasonable approximations of mean experimental transmissions. 4. Discussion The separation in pasteurized milk of whey proteins from casein by MF with the MSD pilot at 1930 rpm and a low TMP of 60 kPa gave better transmissions than those we obtained by Espina et al. (2008) using the same pilot with UHT skim milk, which were around 0.75 for a-La and less than 0.25 for b-Lg at a speed of

1492 rpm. In the present work, transmissions remained very high, up to 0.98 for a-La and up to 0.81 for b-Lg until at least VRR = 4.6, while casein rejection was excellent. These transmissions were as good as those reported in UTP mode by Gésan-Guiziou et al. (1999). Our permeate fluxes were also high, 90–95 L h1 m2 at VRR = 1 and 45 °C, and 30–35 at VRR = 4, while those of Gésan-Guiziou et al. (1999) were 80 L h1 m2 at 50 °C. In the UF step of whey proteins fractionation at 50 kDa, we obtained very high permeate fluxes, which remained above 320 L h1 m2 until VRR = 5, except in test 5 and a high b-Lg rejection, around 0.94, except in test 4. However, our a-La transmission (about 0.2) was disappointing as it was lower than some data from the literature. For instance, Lucas, Rabiller-Baudry, Millisime,

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0.12 Test 4 Test 5 Test 6 Mean theoretical transmission

0.10

Tr β-Lg

0.08

0.06

0.04

0.02 Rotating disk module. Disk with vannes. 50 kDa PES. 2000 rpm. 40°C. PTM = 400 kPa. Q = 3L/min. 0 1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

VRR Fig. 12. Variation of b-Lg transmission versus VRR for UF tests 4, 5, 6.

6.0

5.0

Selectivity

4.0

3.0

Test 4 Test 5 Test 6

2.0

1.0 Rotating disk module. Disk with vannes. 50 kDa PES. 2000 rpm. 40°C. PTM = 400 kPa. Q = 3L/min. 0.0 1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

VRR Fig. 13. Variation of selectivity versus VRR for UF tests 4, 5, 6.

Chaufer, and Daufin (1998) obtained a-La transmissions ranging from 0.15 to 0.37 and b-Lg ones from 0.05 to 0.1, for a selectivity about 3, with a 50 kDa Carbosep membrane. Cheang and Zydney (2003) obtained, with a 30 kDa membrane, transmissions of 0.26 for a-La and 0.005 for b-Lg, but their permeate flux was much lower at 14 L h1 m2. Our low a-La transmissions may be due to the fact that our membranes have been stored for a few years before use. Other reasons may be that we ultrafiltered a complete whey which may induce more membrane fouling that the binary protein mixture used by Cheang and Zydney (2003) and an electrostatic repulsion between membranes and proteins. At the test pH of 6, a-La proteins and the membrane are negatively charged and repulsion forces may explain the lower transmission. We can compare our data with those of Muller et al. (2003) who used mineral mem-

branes of 150 kDa. They reported a higher a-La transmission of 0.4, but at a lower permeate flux of about 50 L h1 m2. It is legitimate to inquire why decays of permeate flux with increasing VRR were so different between MF tests (Fig. 2) and UF ones (Fig. 8). As said earlier, the linear decay of MF fluxes with ln(VRR) is explained by the law of concentration polarization, which, paradoxally, does not seem to apply to UF tests, at least at low VRR. A possible clue is perhaps given by Yee et al. (2009) who observed in UF of whey at 10 kDa, a 50% drop of flux during the first 15 min of filtration, which they attributed to protein adsorption and the formation of concentration polarization layer. In our case, the initial drop was only 30–35%, but it may be due to the different cut-off. The subsequent rate of flux decay with Ln(VRR) in UF was smaller than those observed in MF, but this

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2.5 a-La b-Lg Ln(CFb-Lg) = 0.936Ln(VRR)

2.0

R2 = 0.995

LnCF

1.5

Ln(CFa-La) = 0.786Ln(VRR)

1.0

R2 = 0.975

0.5

0.0 0.0

0.5

1.0

1.5

2.0

2.5

LnVRR Fig. 14. Variation of Ln(CF) with ln(VRR) for a-La and b-Lg in UF tests 4, 5, 6.

may be explained by the higher shear rates used in UF as the disk radius was 60% larger than in MF. Another possible reason is that retentate viscosity in UF increased less rapidly than in MF, due to the lower protein concentration of whey and the membrane shear rate decayed less rapidly.

Separating variables and integrating A3 assuming constant rejection during the process yields 0

CV R ¼ const ¼ C 0 V R0

0

where subscript 0 denotes initial conditions. Eq. (A4) may be rewritten as

5. Conclusion

0

This work has confirmed the promising potential of rotating ceramic membrane disks for separating whey from casein by MF of skim pasteurized milk. a-La and b-Lg transmissions remained high and unaffected by increasing concentrations. Casein rejection, which was initially high, increased with concentration. The whey protein fractionation step by UF at 50 kDa was satisfactory with regards to permeate flux and b-Lg rejection, which was very high and led to yield factors near 90% in retentate. a-La transmission was moderate at about 0.2 and efforts will be made to improve it by testing new membranes and optimizing pH and ionic strength in order to reduce electrostatic repulsion. Acknowledgements V. Espina has been supported by a scholarship No. E06D101610CL of the Alban European Union Program for Latin America. The authors thank Westfalia Separator for the loan of a MSD pilot, Mrs Dupont, Lactalis Co., Clermont, for donating the milk and Ms. C. Orellana for her help with HPLC analysis Appendix A Volume conservation in a batch system (tank of volume V + filter) gives

dV=dt ¼ Q F

ðA1Þ

where QF is permeate flow rate. Solute conservation yields

dðVCÞ=dt ¼ Q F C F ¼ Q F ð1  R0 ÞC b

ðA2Þ

using CF = (1  R0 )C, where C is the solute tank concentration. Multiplying A1 by C and subtracting it from A2 gives

V dC=dt ¼ R0 Q F C b ¼ R0 C dV=dt

ðA4Þ

ðA3Þ

C=C 0 ¼ ðV 0 =VÞR

ðA5Þ

which is equivalent to Eq. (11). References Bhattacharjee, S., Bhattacharjee, C., & Datta, S. (2006). Studies on the fractionation of b-lactoglobulin from casein whey using ultrafiltration and ion-exchange membrane chromatography. Journal of Membrane Science, 275, 150–151. Blatt, W. F., Dravid, A., Michaels, A. S., & Nelson, L. (1970). Solute polarization and cake formation in membrane ultrafiltration: Causes, consequences and control techniques. In J. E. Flinn (Ed.), Membrane science and technology (pp. 47–97). New York, NY: Plenum Press. Bouzerar, R., Jaffrin, M. Y., Ding, L. H., & Paullier, P. (2000). Influence of geometry and angular velocity on performance of a rotating disk filter. AIChE Journal, 46, 257–265. Bramaud, C., Aimar, P., & Daufin, G. (1997). Optimization of a whey protein fractionation process based on the selective precipitation of a-lactalbumin. Lait, 77, 411–423. Cayot, P., & Lorient, D. (1997). Structure-function relationships of whey proteins. In S. Damodaran & A. Paraf (Eds.), Food proteins and their applications (pp. 225–256). New York: Marcel Dekker. Cheang, B., & Zydney, A. L. (2003). Separation of a-lactalbumin and blactoglobulin using membrane ultrafiltration. Biotechnology & Bioenergy, 83, 201–209. Daufin, G., Radenac, J.-F., Gésan, G., Kerherve, F. L., Le Berre, O., & Michel, F. (1993). A novel rig for ultra- and microfiltration for separating milk and whey components. Separation Science and Technology, 28, 26–35. Ding, L. H., Jaffrin, M. Y., Mellal, M., & He, G. (2006). Investigation of performances of a multishaft disk (MSD) system with overlapping membranes in microfiltration of mineral suspensions. Journal of Membrane Science, 276, 232–240. Dunlap, C. A., & Coté, G. L. (2005). b-Lactoglobulin–dextran conjugates: Effect of polysaccharide size on emulsion viability. Journal of Agricultural and Food Chemistry, 53, 419–426. Espina, V. S., Jaffrin, M. Y., Ding, L. H. (2009). Separation of a-lactalbumin and blactoglobulin from skim UHT milk by dynamic filtration. In Proc. euromembrane conference (p. 72). Montpellier, France. Espina, V. S., Jaffrin, M. Y., Frappart, M., & Ding, L. H. (2008). Separation of casein micelles from whey proteins by high shear microfiltration of skim milk using rotating ceramic membranes and organic membranes in a rotating disk module. Journal of Membrane Science, 325, 872–879.

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Gésan-Guiziou, G. (2008). Personal communication. Gésan-Guiziou, G., Boyaval, E., & Daufin, G. (1999). Critical stability conditions in cross-flow microfiltration of skimmed milk: Transition to irreversible deposition. Journal of Membrane Science, 158, 211–232. Jaubert, A., & Martin, P. (1992). Reverse phase analysis of goat caseins: Identification of as1 and as2 genetic variant. Lait, 72, 235–247. Lawrence, N. D., Kentish, S. E., Connor, A. J., Barber, A. R., & Stevens, G. W. (2008). Microfiltration of skim milk using polymeric membranes for casein concentrate manufacture. Separation and Purification Technology, 60, 237–244. Le Berre, O., & Daufin, G. (1996). Skim milk crossflow microfiltration performance versus permeation flux to wall shear stress ratio. Journal of Membrane Science, 117, 261–270. Lucas, D., Rabiller-Baudry, M., Millisime, L., Chaufer, B., & Daufin, G. (1998). Extraction of a-lactalbumin from whey protein concentrate with modified inorganic membranes. Journal of Membrane Science, 148, 1–12.

Muller, A., Chaufer, B., Merin, U., & Daufin, G. (2003). Prepurification of alactalbumin with ultrafiltration ceramic membranes from casein whey: Study of operating conditions. Lait, 83, 111–129. Svensson, M., Hakansson, A., Mossberg, A. K., Linse, S., & Svanborg, C. (2000). Conversion of a-lactalbumin to a protein inducing apoptosis. Proceedings of the National Academy of Science, 97, 4221–4226. Vadi, P. K., & Rizvi, S. S. H. (2001). Experimental evaluation of a uniform transmembrane pressure crossflow microfiltration unit for the concentration of micellar casein from skim milk. Journal of Membrane Science, 189, 69–82. Van Reis, R., Brake, J. M., Charkoudian, J., Burns, D. B., & Zydney, A. L. (1999). High performance tangential flow filtration using charged membranes. Journal of Membrane Science, 159, 133–140. Yee, K. W. K., Wiley, D. E., & Bao, J. (2009). A unified model of the time dependence of flux decline for the long term ultrafiltration of whey. Journal of Membrane Science, 332, 69–80.