Membrane Fractionation of a β-Lactoglobulin Tryptic Digest: Effect of

Article pubs.acs.org/IECR

Membrane Fractionation of a β‑Lactoglobulin Tryptic Digest: Effect of the Hydrolysate Concentration Ayoa Fernández† and Francisco A. Riera*,† †

Department of Chemical Engineering and Environmental Technology, University of Oviedo, C/Julián Clavería 8, 33006 Oviedo, Spain ABSTRACT: The effect of the hydrolysate concentration on the fractionation of a complex peptide mixture through different organic Ultrafiltration/Nanofiltration membranes was evaluated in this paper. The role of changes induced by feed concentration on the selectivity of the membrane received particular attention. The results showed that an increase in feed concentration resulted in an increase in the transmission of anionic peptides, while the transmission of positively charged species decreased. These results tend to work against the selectivity of the process and suggest that the dual size/charge separation mechanism of charged membranes is lost, at least partially. Although in this particular case a slight increase in the separation factor between bioactive and nonbioactive peptides was obtained at high concentration regimes, the presence of an important flux decline may not compensate for it.

■

INTRODUCTION In recent decades, it has been demonstrated that whey protein hydrolysates are a rich source of biologically active peptides: specific protein fractions that have a positive impact on body functions or conditions.1 However, not all the peptides contained in the whole hydrolysate possess beneficial characteristics, and some of them may even show antagonist effects. In order to fractionate protein hydrolysates and obtain enriched products with increased functionality, a separation technology which can recognize small differences in charge, size, and hydrophobicity is needed. Membrane separation techniques, especially tight Ultrafiltration (UF) and Nanofiltration (NF), seem to be well suited for this purpose.2−4 The mechanism responsible for the transmission of each peptide through this kind of membrane is a result of a combination of size and charge effects and is strongly affected by physicochemical conditions such as pH and ionic strength.5 In spite of all the promising perspectives for membrane fractionation of hydrolysates, there are still some unresolved problems that slow down its application on an industrial scale. One of the most important problems to overcome when working with concentrated solutions of peptides is membrane fouling.6 It has been demonstrated that concentration polarization and fouling phenomena are crucial in the case of protein hydrolysates, especially at acid pH values.3,4 Furthermore, most of the UF and NF studies in the literature involving amino acids and peptides have dealt with highly diluted solutions and very few of them have focused on concentrated feeds, which are the most likely to be found in industrial processes, and some of them have reported conflicting results. In the case of NF research studies involving amino acids, Li et al.7 reported that high protein concentration reduces the efficiency of the process and leads to less selective separation. They also observed that while the rejection of Gln increased with increasing feed concentration, the rejection of Glu decreased, especially at lower pH values. Results obtained for Gln were attributed to a polymerization of the amino acid, but this hypothesis has not been proved. Kovacs and Samhaber8 © 2012 American Chemical Society

reported a decrease in both amino acid rejection and permeate flux as a consequence of the increase in the feed concentration when filtrating single amino acid solutions. In addition, rejection became more concentration-dependent at higher pH values because of the increased net charge of the amino acids. Shirley et al.9 studied the behavior of five different amino acids as a function of concentration and compared experimental rejection data with a model derived by combining steric and charge effects. However, experimental data did not show good agreement with the complete model in all the cases. While in the case of amino acid studies, the concentration is increased until the solute reaches its limit of solubility, UF/NF experiments involving protein hydrolysates and complex systems are usually carried out using diluted solutions (1−5 g L−1),3,5 and it is not clear what is the role played by feed concentration on peptide transmission through the membrane and on membrane selectivity. The main objective of the present work was therefore to evaluate the influence of feed concentration on the fractionation of a tryptic hydrolysate of bovine β-lactoglobulin (β-lg), an enzymatic digest that contains several bioactive peptides, through three different UF/NF membranes. Special attention was given to the relation between peptide charge and its transmission through the membrane, as well as its effect on the selectivity of the membrane. In addition, flux decline at different feed concentration was also followed.

■

EXPERIMENTAL SECTION Preparation of the Hydrolysate. A 3 L solution containing 30 g L−1 of bovine β-lg (Davisco Food Co, USA) was used for hydrolysate preparation. The protein content of the commercial substrate was 97.9% (w/w), and β-lg Received: Revised: Accepted: Published: 15738

September 4, 2012 November 8, 2012 November 12, 2012 November 12, 2012 dx.doi.org/10.1021/ie302376g | Ind. Eng. Chem. Res. 2012, 51, 15738−15744

Industrial & Engineering Chemistry Research

Article

The membrane cassette was flushed with distilled water prior to and after filtering process solutions as well as prior to cleaning. The membrane was cleaned after each experiment following the manufacturer’s recommendations. The membrane cleaning procedure consisted of the recirculation of a 0.1 N NaOH solution during 30 min at 40 °C. Cleaning efficiency was calculated by the measurement of water permeability. After the cleaning process the membranes were stored in 0.1 M NaOH solution at 4 °C. Analytical Methods. Permeate and retentate samples were analyzed using an RP-HPLC system (Agilent 1200 series, Agilent Technologies, USA) equipped with a binary pump, auto sampler, and a photodiode-array detector. Separations were performed on a Microsorb-MV C18 column (250 × 4.6 mm × 1/4″, Varian, USA) at 25 °C and at a flow rate of 1.0 mL min−1. A gradient of solvent A containing 0.1% trifluoroacetic acid (TFA) (v/v) in water and solvent B consisting of 0.1% TFA (v/v) in acetonitrile was used to elute the peptides as follows: linear gradient from 5 to 45% B in 55 min, linear gradient from 45 to 90% B in 10 min, isocratic elution at 90% B for 10 min, and then return to starting conditions in 2 min. Total analysis time per sample was 77 min. Elution was monitored at 214 nm to enable protein and peptide detection, and sample injection volume was 20 μL. All samples were diluted with ultrapurified water and filtered through cellulose acetate filters of 0.45 μm pore diameter (Teknokroma, Spain). Ultrapure water (MilliQsystem, Millipore, USA), acetonitrile grade HPLC (Prolabo, Belgium), and TFA for UV analysis (Panreac, Spain) were used to prepare the eluents. Peptides present in the chromatographic profile were identified by MS analysis as previously described.11 Figure 1 and Table 2 show the chromatographic profile of a total hydrolysate sample and identified peptides as well as their main physicochemical characteristics, which could be responsible for membrane-peptide interactions. Calculations. The transmission (Tr) of each peptide by the membrane was calculated according to eq 1

represented 91.5% of the total protein. The substrate solution was equilibrated at 37 °C, and the hydrolysis reaction was initiated by adding TPCK-trypsin (T1426-5G, Sigma, USA) at a final enzyme:substrate ratio of 1:450 (w/w). The pH of the reaction mixture was maintained at pH 8.0 by addition of 1 M NaCl. The degree of hydrolysis (DH) was determined from the quantity of base consumed according to the pH-stat method.10 The reaction was stopped after 23 h of hydrolysis when the DH was 6.8. Two hydrolysis batches were performed, and aliquots were stored in a freezer in order to obtain enough stock solution of the hydrolysate to perform all the experiments. Permeation Experiments. The diluted tryptic hydrolysate was filtered using a Pellicon 2 miniholder (Millipore, USA) equipped with three different tangential flow membrane cassettes (membrane characteristics are shown in Table 1), Table 1. Membrane Characteristics membrane

manufacturer

material of separation layer

MWCOa (kDa)

PES-1 HYD-2 PES-5

Sartorius Sartorius Millipore

polyethersulfone stabilized cellulose (Hydrosart) polyethersulfone

1 2 5

a

Molecular weight cut off.

each one of them having a filtration area of 0.1 m2. Transmembrane pressure (PM), feed temperature, and pH were set to 7.5 × 104 Pa, 37 °C, and 8.0, respectively. Total feed volume used in each run was 2.5 L. The experiment was carried out at five different concentrations (1.5, 3, 6, 10, and 15 g L−1 of total protein content) with the aim of studying the role of feed concentration on the selective permeation of hydrolysate peptides. Permeate and retentate streams were recycled into the feed tank, where temperature and pH were controlled, to keep the feed concentration constant. The membrane system was run for 1 h, during which time the pH and temperature of the feed solution as well as transmembrane pressure were carefully controlled and corrected if necessary. Measurements of permeate flux were performed every 5 min, and samples of retentate and permeate were taken at 5, 30, and 60 min for HPLC analysis. All experiments were performed in duplicate.

⎛A ⎞ Tr(%) = ⎜ Pi ⎟ × 100 ⎝ ARi ⎠

(1)

Figure 1. Chromatographic profile of the hydrolysate. 15739

dx.doi.org/10.1021/ie302376g | Ind. Eng. Chem. Res. 2012, 51, 15738−15744


Article

Table 2. Peptide Identification According to Fernández and Riera11 peak number

peptide (MS/MS analysis)

a

MW (Da)

sequence

pI

b

A/ B/ Nc

Hφav (kcal/ res)d

charge pH 8

1 2 3

ALK FDK IIAEK

f(139−141) f(136−138) f(71−75)

330.43 408.45 572.7

8.8 5.84 6

B N N

1.55 1.38 1.63

1 0 (-) 0 (-)

4

IDALNENK

f(84−91)

916

4.37

A

0.95

−1

5 6

f(9−14) f(61−69) + f(61−70)

N A

1.14

0 (-)

f(125−135) f(1−8) f(142−148)

672.78 2125.65 − 2183.65 − 2311.76 1245.31 933.17 837.05

5.84

7 8 9

GLDIQK WEND(G)ECAQK + WEND(G)ECAQKK (Ag.) TPEVDDEALEK LIVTQTMK ALPMHIR

3.83 8.75 9.8

A B B

0.85 1.34 1.54

−4 1 1

10

VAGTWY

f(15−20)

695.77

5.49

N

1.46

0 (-)

11 12 13 14 15

IPAVFK TKIPAVFK VLVLDTDYK TPEVDDEALEKFDK WEND(G)ECAQKK + LSFNPTQLEEQCHI WEND(G)ECAQK + LSFNPTQLEEQCHI SLAMASSDISSLLDAQSAPLR VYVEELKPTPEGDLEILLQK

f(79−83) f(76−83) f(92−100) f(125−138) f(61−70) + f(149−162)

673.85 903.13 1065.23 1635.74 2833.14 (2891.18)

8.75 10 4.21 4.02 4.91 (4.57)

B B A A A

2.02 1.76 1.44 0.97 0.85

0 (+) 2 −1 −4 −3 (−4)

f(61−69) + f(149−162)

2704.97 (2763.01)

4.48 (4.25)

A

0.82

−2 (−3)

f(21−40) f(41−60)

2117.4 2313.67

4.21 4.25

A A

1.01 1.37

−1 −3

16 17 18

bioactivity

hypocholesterolemic,12 antihypertensive13 lymphocyte proliferation14 antihypertensive15

lymphocyte proliferation,14 antihypertensive,16 lymphocyte proliferation,14 antihypertensive,15 antimicrobial17 antimicrobial17 antimicrobial17

a

Amino acid interval within the sequence of the parent protein. bIsoelectric points were calculated using the ExPASy Molecular Biology Server (http://www.expasy.org/). cClassified as A: acidic (pI < 5), B: basic (pI > 7), and N: neutral (5 < pI < 7). dCharge of the most abundant ion at pH 8.

where APi and ARi are the i peptide corresponding peak area obtained from the UPLC profiles of permeate and retentate, respectively. The separation factor (Sx/y) was calculated as the ratio of mean transmission values between two peptides or groups of peptides according to eq 2 n

Sx / y =

∑i = 1 m

∑j=1

According to Table 2, most of the peptides present in the hydrolysate have pI values lower than 8, so they are negatively charged under the experimental conditions used in this work. Due to the fact that all the membranes employed have a negative net charge at pH 8,19 repulsive electrostatic forces between the peptide and the membrane do not favor peptide approach to the membrane, and this results in lower Tr values, at least in the case of diluted systems.18 In this sense, negatively charged peptides having a molecular weight (MW) higher than 1500 Da are scarcely transmitted throughout the whole concentration working range, even using the PES-5 membrane (results not shown). Figure 2 shows Tr values obtained for negatively charged peptides whose MW is lower than 1500 Da through PES-1, HYD-2, and PES-5 membranes. All the calculations correspond to the samples taken after 60 min of operation. As can be observed, an increase in feed concentration resulted in an increase in peptide Tr probably due to a combination of charge and diffusion considerations, the predominant forms of transport with this kind of membrane. As expected, Tr values obtained when filtrating with the PES-5 membrane were higher than those seen using the PES-1 membrane. However, the lowest Tr values were usually obtained with the HYD-2 membrane, even though its MWCO has an intermediate value. This is probably due to the membrane composition. As previously mentioned, the HYD-2 membrane is made of an extremely hydrophilic material, and hydrophobic membranepeptide interactions, which could lead to an improvement in peptide Tr, are not favored.

Tri n Trj m

(2)

where n and m are the number of peptides included in groups x and y, respectively. This value was used to evaluate the selectivity of each membrane.

■

RESULTS AND DISCUSSION Influence of Feed Concentration on Individual Peptide Transmission (Tr). Tr values of the peptides identified in the HPLC profile were calculated according to eq 1. Permeate and retentate samples were taken at 5, 30, and 60 min throughout each experiment, but Tr does not vary with experimental time. The only exception was for the 5 and 30 min samples in the case of the most concentrated experiments when filtrating with the PES-1 membrane, probably due to the fact that permeate flux under these conditions is so low that the 5 min sample was still diluted with flushing water. As has been shown in previous work,18 Tr is strongly influenced by peptide charge. In view of experimental results obtained in this study it seems that the behavior of the peptides with respect to feed concentration depends on their charge state too. 15740



Article

Figure 2. Transmission coefficient of negatively charged peptides whose MW < 1500 Da through PES-1, HYD-2, and PES-5 membranes as a function of hydrolysate concentration.

Figure 3. Transmission coefficient of positively charged peptides through PES-1, HYD-2, and PES-5 membranes as a function of hydrolysate concentration.

15741



Article

Table 3. Separation Factor between Basic and Acidic Peptides (SB/A), Basic and Neutral Peptides (SB/N), and Neutral and Acidic Peptides (SN/A) as a Function of Hydrolysate Concentration SB/A

SB/N

SN/A

[β-lg]0 (g L‑1)

PES-1

HYD-2

PES-5

PES-1

HYD-2

PES-5

PES-1

HYD-2

PES-5

1.5 3 6 10 15

19 37.6 30.7 15.6 12.4

13.2 10.1 11.1 10.1 11.5

16.3 15.5 14 7.4 7.4

2.5 1.8 1.5 1.1 0.9

2.6 1.8 1.3 0.9 0.6

1.5 0.9 0.8 0.5 0.6

7.6 20.5 20.6 13.9 13.2

5.1 5.6 16.5 11.8 18.3

10.9 16.7 18.2 15.2 11.5

There are probably two main reasons for these results. First, the saturation of some of the charged groups on the membrane surface by the adsorbed peptides would result in a decrease in electrostatic membrane-peptide interactions. In addition, the higher the hydrolysate concentration, the higher the membrane charge saturation. As a consequence, the Tr of negatively charged peptides would be increased, while the opposite phenomenon would occur in the case of positively charged peptides. This effect on the membrane charge has been previously observed in the case of protein UF. For example, Burns and Zydney20 showed that bovine serum albumin absorption on a 100 kDa PES membrane decreased its electronegative surface charge density when both the protein and the membrane are at pH > pI. In the case of tight UF and NF membranes a similar phenomenon has been reported when increasing the ionic strength of the amino acid or peptide mixture by means of salt addition,5,21 but, to our knowledge, this is the first research work that has addressed feed concentration effect on peptide Tr from this point of view. Second, the formation of a polarization layer on the membrane surface could also influence peptide transmission. Influence of Feed Concentration on Membrane Selectivity. As pointed out in Table 2, β-lg tryptic peptides can be classified into three main groups according to their pI values: acidic peptides (pI < 5), basic peptides (pI > 7), and neutral peptides (5 < pI < 7). It has been shown that each group of peptides behaves differently during membrane processing in a charge-dependent manner due to the size/ charge mechanism responsible for peptide separation by means of charged membranes.18 Table 3 summarizes the separation factors obtained for these three groups of peptides during processing through PES-1, HYD-2, and PES-5 membranes. Particularly relevant are the results obtained for SB/A and SB/N. All of the basic peptides are positively charged under experimental conditions, whereas neutral and acidic peptides are anionic species. The different Tr behavior observed for cationic and anionic peptides with regard to feed concentration (see Figure 2 and 3) is responsible of the decrease in SB/A and SB/N. As a consequence, the selectivity of the process of separation of these two groups of peptides is significantly lower in the case of concentrated systems. Poor selectivity separation at high concentration regimens was also observed previously by Li et al.7 when processing a complex ionic fermentation broth in order to separate the amino acids Gln and Glu. Results obtained for SN/A have not shown a clear trend as both groups of peptides are negatively charged under experimental conditions. Thus, values obtained for SN/A depend more on other individual physicochemical characteristics of the peptide, such as MW, than on their charge. As mentioned in the Introduction, some of the β-lg tryptic peptides studied here have previously been shown to possess different biofunctional properties. Peptides IIAEK,12,13 IDAL-

In view of Figure 2 it could be said that, whatever the membrane used, an increase in feed concentration resulted in an increase in peptide Tr. Thus, for example, the Tr value of peptide FDK doubled when the concentration of the feed solution changes from 1.5 g L−1 to 15 g L−1. This effect was less pronounced in the case of peptides whose MW is close to the MWCO of the membrane (IDALNENK and TPEVDDEALEK). The relation between feed concentration and Tr of anionic species was previously observed when filtrating single amino acid solutions.8,9 However, it had not been proved in the case of complex peptide mixtures as used in this research. Figure 3 shows experimental Tr results obtained in the case of positively charged peptides. Similar to what happened with anionic peptides, the highest Tr values were obtained when using the PES-5 membrane, whereas HYD-2 showed the lowest peptide Tr in most cases. Regarding the effect of hydrolysate concentration, the trend is not as clear as in the case of anionic peptides, but, as a general rule, it could be said that the higher the feed concentration, the lower the Tr. These results show that charge effects can overcome the diffusional effects produced by the increase in feed concentration. This behavior is not common, and it has not been described in the literature due to the fact that most of the studies on this subject involve neutral or negatively charged species. To our knowledge, only Lapointe et al.5 mentioned a similar behavior in the total Tr of basic peptides at pH 9 when the hydrolysate concentration was increased from 1 g L−1 to 5 g L−1. However, they did not show the behavior of individual peptides, and the concentration range tested was too short to draw major conclusions about the trend followed by this group of peptides. It is well established that the selectivity of charged membranes is based on a dual size/charge retention mechanism. Thus, the Tr of negatively charged peptides through a membrane having negative net charge is usually lower than the Tr of cationic species, at least when working with diluted hydrolysates.18 However, Figures 2 and 3 suggest that solute charge effect on peptide Tr is partially masked when working with concentrated solutions. As can be observed, the behavior of peptides GLDIQK and IPAVFK agree with this hypothesis. Although both of them have similar sizes (see Table 2), GLDIQK is an anionic peptide at pH 8, whereas IPAVFK is positively charged under the same conditions. Electrostatic membrane-peptide interactions make the Tr of IPAVFK (82.4%) significantly higher than the value obtained for GLDIQK (26.0%) when the feed concentration was set at 1.5 g L−1. However, an increase in feed concentration up to 15 g L−1 led to a decrease in IPAVFK Tr (60.1%), while the opposite was observed in the case of GLDIQK (40.7%). As a result, the separation factor between the two peptides, SIPAVFK/GLDIQK, decreased from 3.2 to 1.5 over the entire concentration range evaluated in this work. 15742



Article

NENK,14 GLDIQK, 15 ALPMHIR,14,16 VAGTWY,14,15,17 IPAVFK,17 and VLVLDTDYK17 are included in this group. One of the main objectives of this research paper was to evaluate the influence of the hydrolysate concentration on the selectivity of bioactive peptide separation in order to produce enriched bioactive peptide preparations with increased biofunctionality potency. The separation factor between bioactive peptides and nonbioactive peptides, SBio/NBio, was calculated for all the experimental conditions (see Table 4). Table 4. Separation Factor between Bioactive and Nonbioactive Peptides (SBio/NBio) as a Function of Hydrolysate Concentration SBio/NBio [β-lg]0 (g L‑1)

PES-1

HYD-2

PES-5

1.5 3 6 10 15

1.7 1.8 2.0 2.0 2.1

1.3 1.2 1.7 1.4 1.8

2.3 2.3 2.3 2.6 2.5

Differences were not as important as in the case of basic and acidic peptides, but a slight increase in SBio/NBio was observed as a consequence of an increase in feed concentration. Thus, for example, SBio/NBio calculated in the case of the PES-1 membrane increased from 1.7 to 2.2 over the concentration range evaluated, from 1.3 to 1.8 in the case of the HYD-2, and from 2.3 to 2.5 in the case of the PES-5 membrane. This observation could be explained by the fact that almost all the bioactive peptides contained in the hydrolysate (except ALPMHIR and IPAVFK) were anionic species under experimental conditions, and their Tr increased with increasing concentration. Influence of Feed Concentration on Permeate Flux Decline. In Figure 4 experimental values of permeate flux are plotted against feed concentration. As would be expected from the MWCO of the membranes employed in this study, the PES-1 membrane showed the lowest permeate fluxes (3.07 L h−1 m−2 when protein concentration was 1.5 g L−1) followed by the HYD-2 (11.09 L h−1 m−2 under the same conditions) and finally the PES-5 (16.36 L h−1 m−2). In addition, an increase in the hydrolysate concentration led to a decrease in the permeate flux. Thus, for example, a flux decline of 54% was seen with PES-5 between the most concentrated and the most diluted experiment. It should be noted that the HYD-2 membrane presented an insignificant flux decline when feed concentration was lower or equal to 6 g L−1. HYD-2 is made of an extremely hydrophilic material which is claimed to prevent membrane fouling and flux decline. However, at a high concentration regime it shows a flux reduction similar to that observed for polyethersulfone membranes (45% between the most concentrated and the most diluted experiment versus 54% in the case of PES-5 and 45% in the case of PES-1). The curves shown in Figure 4 exhibit the same shape whatever the protein concentration and membrane used. The permeate flux decreases with protein concentration, but its value is maintained almost constant during each experiment. The variation of permeate flux with time may simultaneously involve the formation of the polarization layer, membrane fouling, and physical deposition of the insoluble particles. In these experiments, no suspended matter was contained in the hydrolysate or produced during the filtration, so, if flux decline

Figure 4. Effect of the hydrolysate concentration on the permeate flux through PES-1, HYD-2, and PES-5 membranes. Experimental conditions: PM 7.5 × 10−5 Pa, 37 °C, and pH 8.0.

had been observed during the operation, it would be controlled by concentration polarization and adsorption onto the membrane material.22 As no flux decline was observed after the initial steps of the operation and the formation of the polarization layer is known to occur at the very beginning of the process, it could be said that it is not fouling but concentration polarization the phenomenon that controls flux decline. However, more research should be done to clarify this point. For example, the effect of different feed velocities and PM on peptide Tr in a high concentration regime has not been studied yet, and, if concentration polarization is actually the phenomenon controlling flux decline, changes in the aforementioned conditions could lead to better selectivity results.

■

CONCLUSIONS The effect of the peptide concentration in the fractionation of a β-lg tryptic digest was studied in this work. Individual peptide Tr was influenced by peptide charge, specially at low concentration regimens. Whereas the Tr of anionic species whose MW is smaller than 1500 Da was increased, and even doubled, over the concentration range evaluated in this work, the Tr of positively charged species was decreased. The results suggest that the dual size/charge separation mechanism of charged membranes was lost, at least partially, when working with concentrated solutions. The saturation of some of the charged groups of the membrane by the adsorbed peptides may explain these results but also the formation of a polarization 15743



Article

(7) Li, S.; Li, Ch.; Liu, Y.; Wang, X.; Cao, Z. Separation of Lglutamine from fermentation broth by nanofiltration. J. Membr. Sci. 2003, 222, 191. (8) Kovacs, Z.; Samhaber, W. Nanofiltration of concentrated amino acid solutions. Desalination. 2009, 240, 78. (9) Shirley, J.; Mandale, S.; Williams, P. M. Amino acid rejection behaviour as a function of concentration. Adv. Colloid Interface Sci. 2011, 164, 118. (10) Adler-Nissen, J. Enzymatic hydrolysis of food proteins; Elsevier Applied Science Publishers: London, 1986. (11) Fernández, A.; Riera, F. β-Lactoglobulin tryptic digestion: a model approach for peptide release. Biochem. Eng. J.doi:10.1016/ j.bej.2012.10.001. (12) Nagaoka, S.; Futamura, Y.; Miwa, K.; Awano, T.; Yamauchi, K.; Kanamaru, Y.; Tadashi, K.; Kuwata, T. Identification of novel hypocholesterolemic peptides derived from bovine milk β-lactoglobulin. Biochem. Biophys. Res. Commun. 2001, 218, 11. (13) Janssen, J. G.; Schalk, J. Peptides having and ACE inhibiting effect. Patent US 2006, 2006/0216330 A1. (14) Jacquot, A.; Gauthier, S. F.; Drouin, R.; Boutin, Y. Proliferative effects of synthetic peptides from β-lactoglobulin and α-lactalbumin on murine splenocytes. Int. Dairy J. 2010, 20, 514. (15) Pihlanto-Leppälä, A.; Rokka, T.; Korhonen, H. Angiotensin Iconverting enzyme inhibitory peptides derived from bovine milk proteins. Int. Dairy J. 1998, 8, 325. (16) Mullally, M. M.; Meisel, H.; FitzGerald, R. J. Identification of a novel angiotensin-I-converting enzyme enhibitory peptide corresponding to a tryptic fragment of bovine β-lactoglobulin. FEBS Lett. 1997, 402, 99. (17) Pellegrini, A.; Dettling, C.; Thomas, U.; Hunziker, P. Isolation and characterization of four bactericidal domains in the bovine βlactoglobulin. Biochim. Biophys. Acta 2001, 1526, 131. (18) Fernández, A.; Suárez, A.; Zhu, Y.; FitzGerald, R. J.; Riera, F. A. Membrane fractionation of a β-lactoglobulin tryptic digest: effect of the pH. J. Food Eng. 2013, 114, 83. (19) Susanto, H.; Ulbricht, M. Influence of ultrafiltration membrane characteristics on adsorptive fouling with dextrans. J. Membr. Sci. 2005, 266, 132. (20) Burns, D. B.; Zydney, A. L. Effect of solution pH on protein transport through ultrafiltration membranes. Biotechnol. Bioeng. 1999, 64, 27. (21) Timmer, J. M. K.; Speelmans, M. P. J.; van der Horst, H. C. Separation of amino acids by nanofiltration and ultrafiltration membranes. Sep. Purif. Technol. 1998, 14, 133. (22) D’Alvise, N.; Lesueur-Lambert, C.; Fertin, B.; Dhulster, P.; Guillochon, D. Hydrolysis and large scale ultrafiltration study of alfalfa protein concentrate enzymatic hydrolysate. Enzyme Microb. Technol. 2000, 27, 286.

layer on the membrane surface could contribute to these results. In the case of negatively charged peptides with a MW larger than 1500 Da, their Tr values were negligible, even when the PES-5 membrane was used. The combination between size and negative charge for this last group of peptides prevents their transmission through the membranes employed in these experiments. According to the results obtained for SB/A and SB/N, an increase in feed concentration works against the selectivity of tight UF and NF processes due to the fact that the charge exclusion mechanism of the process loses significance. A slight increase in the separation factor SBio/NBio over the concentration range tested was observed. In the particular case of β-lg tryptic hydrolysate this is due to the fact that most of the bioactive peptides present in the mixture are negatively charged under experimental conditions (pH 8) and their Tr increased with feed concentration. However, the presence of an important flux decline may not compensate for the slight improvement concerning the separation of bioactive peptides from the hydrolysate. As expected, the membrane having the narrowest MWCO (PES-1) shows the lowest permeate flux values. There is an important flux decline with increased feed concentration over the concentration range tested in this study, reaching 54% in the case of the PES-5 membrane. However, permeate flux is maintained almost constant during the course of each individual process. Experimental results suggest that concentration polarization instead of fouling is the phenomenon that controls flux decline. If so, changes in hydrodynamic conditions such as feed velocity or transmembrane pressure could minimize it. However, more research should be done in order to test these hypotheses.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS Author Ayoa Fernández acknowledges a Ph.D. fellowship from the Severo Ochoa Programme (Principado de Asturias Government).

■

REFERENCES

(1) Kitts, D. D.; Weiler, K. Bioactive proteins and peptides from food sources. Application of bioprocesses used in isolation and recovery. Curr. Pharm. 2003, 9, 1309. (2) Korhonen, H.; Pihlanto, A. Bioactive peptides: production and functionality. Int. Dairy J. 2006, 16, 945. (3) Garem, A.; Daufin, G.; Maubois, J. L.; Chaufer, B.; Leónil, J. Ionic interactions in nanofiltration of beta casein peptides. Biotechnol. Bioeng. 1998, 57, 109. (4) Pouliot, Y.; Wijers, M. C.; Gauthier, S. F.; Nadeau, L. Fractionation of whey protein hydrolysates using charged UF/NF membranes. J. Membr. Sci. 1999, 158, 105. (5) Lapointe, J.-F.; Gauthier, S. F.; Pouliot, Y.; Bouchard, C. Fouling of a nanofiltration membrane by a β-lactoglobulin tryptic hydrolysate: impact on the membrane sieving and electrostatic properties. J. Membr. Sci. 2005, 253, 89. (6) Van der Bruggen, B.; Mänttäri, M.; Nyström, M. Drawbacks of applying nanofiltration and how to avoid them: a review. Sep. Purif. Technol. 2008, 63, 251. 15744


Membrane Fractionation of a β-Lactoglobulin Tryptic Digest: Effect of

Recommend Documents