Antioxidant activity of whey protein fractions isolated by gel exclusion chromatography and protease treatment

Available online at www.sciencedirect.com

Talanta 75 (2008) 705–709

Antioxidant activity of whey protein fractions isolated by gel exclusion chromatography and protease treatment Tu˘gba Bayram a , Murat Pekmez b , Nazlı Arda b , A. S¨uha Yalc¸ın a,∗ ˙ Department of Biochemistry, School of Medicine, Marmara University, 34668 Haydarpa¸sa, Istanbul, Turkey ˙ ˙ Department of Molecular Biology and Genetics, Faculty of Science, Istanbul University, 34118 Vezneciler, Istanbul, Turkey a

b

Received 19 June 2007; received in revised form 22 November 2007; accepted 7 December 2007 Available online 23 December 2007

Abstract Whey proteins were isolated from whey powder by a combination of gel exclusion chromatography and protease (pepsin or trypsin) treatment. Whey solution (6 g/dl) was applied to Sephadex G-200 column chromatography and three fractions were obtained. Gel electrophoresis (SDS-PAGE) was used to identify the fractions; the first one contained immunoglobulins and bovine serum albumin, the second contained ␤-lactoglobulin and ␣-lactalbumin whereas the third fraction contained small peptides. We have also subjected the whey filtrate to proteases (pepsin and trypsin). Treatment with proteases showed that ␤-lactoglobulin can be obtained after hydrolysis of the second fraction with pepsin. When the whey filtrate was treated with pepsin and then applied to Sephadex G-200 column chromatography three fractions were obtained; the first one was bovine serum albumin, the second was ␤-lactoglobulin and the third fraction contained small peptides. After trypsin treatment only two fractions were obtained; the first one was serum albumin and the second fraction was an ␣-lactalbumin rich fraction. We have determined the antioxidant activity of the fractions using an assay based on the measurement of superoxide radical scavenging activity. Our results showed that among the three fractions, the first fraction had the highest superoxide radical scavenging activity. Also, protease treatment of the second fraction resulted in an increase in the antioxidant activity. © 2007 Elsevier B.V. All rights reserved. Keywords: Antioxidant activity; Gel chromatography; ␣-Lactalbumin; ␤-Lactoglobulin; Whey

1. Introduction Milk is a complex mixture of proteins, lipids, carbohydrates, vitamins, and minerals structured to provide a complete diet for infants in mammals. Whey protein is a term often used as a synonym for milk-serum proteins. Sweet whey is produced in large amounts worldwide from milk using the enzymatic action of chymosin (also called rennet enzyme) on the casein fraction [1]. Whey represents a rich and heterogeneous mixture of secreted proteins with wide ranging nutritional, biological and functional-food attributes [2]. Whey proteins include ␣-lactalbumin (␣-La), ␤-lactoglobulin (␤-Lg), bovine serum albumin (BSA), immunoglobulins (Ig), and a number of minor proteins and enzymes [3]. Main constituents of whey are ␤lactoglobulin and ␣-lactalbumin, two small globular proteins

∗

Corresponding author. Tel.: +90 216 4144733; fax: +90 216 4181047. E-mail address: [email protected] (A.S. Yalc¸ın).

0039-9140/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2007.12.007

that account for approximately 70–80% of total whey protein. Historically, whey has either been considered a waste product and disposed of in the most cost-effective manner, or processed into relatively low-value commodities such as whey powder and various grades of whey protein concentrate/isolate [4]. ␤-Lg is a small, soluble globular protein with a monomer mass of 18 kDa. Between pH 3 and 7, it exists in solution as a dimer with an effective molecular mass of about 36 kDa [5]. It has a variety of useful nutritional and functional-food characteristics that have made it an ingredient of choice in the formulation of modern foods and beverages. ␤-Lg exhibits a growing number of biological effects including anti-hypertensive, anti-cancer, hypocholesterolemic, opiodergic, and anti-microbial activities [2,4]. ␣-La is another major whey protein that makes up 25% of total bovine whey protein. It is one of the few proteins that remains intact upon pasteurization, and is a calcium binding protein that enhances calcium absorption. It is also a rich source of the amino acids lysine, leucine, threonine, tryptophan and cysteine [6].

706

T. Bayram et al. / Talanta 75 (2008) 705–709

In this study, we have isolated whey proteins from whey powder, an industrial by-product. We have also determined the antioxidant activities of the fractions obtained. 2. Experimental 2.1. Sample preparation Whey powder was obtained from a commercial milk and milk products company (S¨utas¸, Bursa-Turkey). Whey solution (6%, w/v) was prepared in ultra pure water and was centrifuged at 4000 × g and 4 ◦ C for 60 min. The supernatant was filtered through a 0.45 ␮m syringe filter. 2.2. Size exclusion chromatography Size exclusion chromatography was performed using a Sephadex G-200 column (1.5 cm × 30 cm). The column was equilibrated and eluted with 0.02 M phosphate buffer, pH 8.6. The flow rate was set as 0.3 ml/min, and fractions of 1–1.5 ml were collected. Absorbances were measured at 280 nm to estimate their protein content. Additionally, quantitative protein analysis was performed according to Lowry et al. [7]. 2.3. Hydrolysis of whey proteins Pepsin (Sigma-P7012; 2500–3500 Units/mg) and trypsin (Fluka-93610; ∼9000 Units/mg) were used for the hydrolysis of whey proteins. For pepsin hydrolysis, the pH of the sample

was adjusted to 1.5 with 1.0 M HCl. The enzymatic reaction was carried out at 37 ◦ C for 30 min with a protein to enzyme ratio of 1:100 (w/w). The reaction was ended by adjusting the pH to 7.8 with 1.0 M NaOH. For trypsin hydrolysis, the pH of the sample was adjusted to 9.0 with 1.0 M NaOH. The enzymatic reaction was carried out at 37 ◦ C for 30 min with a protein to enzyme ratio of 1:50 (w/w). The reaction was stopped by adding 150 mM Na2 CO3 . 2.4. Electrophoretic analysis Gel electrophoresis (SDS-PAGE) was carried out at a constant voltage of 200 mV using 18% separating gel and 4% stacking gel. Samples were loaded to different lanes and protein standards were used to identify individual proteins according to their molecular mass. Gels were stained with 0.05% Coomassie Brilliant Blue R-250. Destaining was carried out with a solution of isopropanol and acetic acid [3]. 2.5. Antioxidant activity Antioxidant activities of different fractions were determined by an assay based on scavenging of superoxide radicals [8]. One ml of the sample (or ultra pure water for control) was added to 3 ml of the reaction mixture made up of 50 mM phosphate buffer (pH 7.8), 13 mM methionine, 2 ␮M riboflavin, 100 ␮M EDTA and 75 ␮M nitro blue tetrazolium (NBT). After incubation under fluorescent light for 10 min, absorbance of the blue colored formazan was measured spectrophotometrically at 560 nm. Per-

Fig. 1. A schematic overview of the isolation procedure used to obtain the fractions.

T. Bayram et al. / Talanta 75 (2008) 705–709

707

centage inhibition of superoxide anion formation was calculated using the following formula.   A0 − A1 %Inhibition = × 100, A0 A0 =Absorbance of the control; A1 =Absorbance of the sample The results were expressed as the protein concentration (mg/ml) leading to 50% inhibition (IC50 ). 3. Results and discussion Preparative chromatographic separation techniques are of importance to the biopharmaceutical industry because they can deliver high-purity products, are relatively easy to develop, and can readily be scaled from the laboratory scale to the desired production level [9]. One reason for the ubiquity of chromatographic steps in preparative protein purification is that they provide a relatively efficient means to meet manufacturing goals of the biopharmaceutical industry [10]. Several processes have been proposed for commercial-scale production of whey protein fractions. These fall into three main categories: selective precipitation induced by adjustment of the solution physical properties, membrane filtration based primarily on differences in size and charge, and selective adsorption [11,12]. A schematic overview of the isolation procedure used in our study is presented in Fig. 1. We have first prepared a whey solution (6%, w/v) which was centrifuged (4000 × g, at 4 ◦ C for 60 min) to remove large particles and coagulated material. The supernatant was filtered through a microfilter (0.45 ␮m) and applied to a Sephadex G-200 column (Fig. 2). The three fractions recovered were identified by gel electrophoresis (SDSPAGE). Immunoglobulins (Ig) and bovine serum albumin (BSA) were eluted in the first peak, ␤-lactoglobulin (␤-Lg) and ␣lactalbumin (␣-La) in the second peak and small peptides in the third peak (Fig. 3). We have also subjected whey filtrate to proteases (pepsin or trypsin). The results are shown in Fig. 4. Three fractions were obtained after pepsin treatment, while trypsin treatment

Fig. 3. Gel electrophoresis (SDS-PAGE) of Sephadex G-200 fractions. Lane 1: whey solution; Lane 2: whey supernatant; Lane 3: whey filtrate; Lane 4: molecular weight markers; Lane 5: F-1; Lane 6: F-2; Lane 7: F-3.

gave two fractions. Further analysis of the fractions obtained by gel electrophoresis following pepsin treatment showed that the first fraction was BSA; the second was ␤-Lg and the third fraction contained small peptides. After trypsin hydrolysis the first fraction was BSA and the second was a fraction rich in ␣-La. The second fraction obtained from Sephadex G-200 column chromatography was hydrolyzed by pepsin in another set of experiments. As shown in Fig. 5, hydrolysis of the second fraction by pepsin yielded ␤-Lg near homogeneity suggesting the possibility of isolating purified ␤-Lg after treatment of the second fraction with pepsin.

Fig. 2. Sephadex G-200 chromatography of whey filtrate.

708

T. Bayram et al. / Talanta 75 (2008) 705–709

Fig. 4. Sephadex G-200 chromatography of whey filtrate before and after protease treatment. (A) Pepsin treatment; (B) trypsin treatment; (C) gel electrophoresis (SDS-PAGE). Lane 1: MW markers; Lane 2: PF-1; Lane 3: PF-2; Lane 4: PF-3; Lane 5: TF-1; Lane 6: TF-2.

Milk contains several antioxidant factors like vitamins and enzymes. Possible antioxidant activity of milk proteins and hydrolysates has also been reported [13]. Accordingly, several investigators have tried to isolate antioxidant proteins and peptides from milk, whey and whey protein hydrolysates [13–15]. Antioxidant activity of the hydrolysates seems be inherent to the characteristic amino acid sequences of peptides derived depending on the protease specificity. Our ultimate aim is to perform a variety of biological activity analyses on whey protein fractions. In this study we have used an antioxidant assay based on superoxide radical scavenging activity. Among the three fractions obtained from Sephadex G-200 chromatography, highest superoxide radical scavenging activity was present in the first fraction (Tables 1 and 2). Also, protease treatment of the fractions resulted in an increase in the antioxidant activity. This was particularly evident for the second fraction which contains ␣-La and

␤-Lg. These results suggest that the superoxide radical scavenging antioxidant activity is inherent in the peptide sequence of ␤-Lg in accordance with recent reports on the antioxidant activity of both ␤-Lg and its hydrolysates [16,17]. The antioxidant activity demonstrated by whey proteins and their hydrolysates suggests that these have potential to enhance product stability by preventing oxidative deterioration. Thus, whey proteins can be readily utilized as functional ingredients in food products. In conclusion, in the present study, we have compared the antioxidant activities of whey proteins and their hydrolysates. We have also observed that ␤-lactoglobulin was resistant to pepsin while ␣-lactalbumin was resistant to trypsin cleavage. Accordingly, ␤-Lg can be easily purified using a combination of Sephadex G-200 size exclusion chromatography and pepsin treatment, whereas an ␣-lactalbumin rich fraction can be obtained after tryptic hydrolysis. Isolating milk-serum

T. Bayram et al. / Talanta 75 (2008) 705–709

709

Table 2 Antioxidant activities of different fractions obtained as described in Fig. 1 Fractiona

Antioxidant activityb

Whey solution Whey precipitate Whey supernatant Whey filtrate F-1 F-2 F-3 Pepsin Trypsin F-2-P PF-1 PF-2 PF-3 TF-1 TF-2

n.d. n.d. 33 ± 1 43 ± 3 9.3 ± 0.9 176 ± 7 91 ± 2 101 ± 8 54 ± 1 16 ± 3 21 ± 4 88 ± 3 143 ± 10 n.d.c 60 ± 13

a

The fractions were obtained and designated as described in Fig. 1. Antioxidant activity was assayed as superoxide scavenging activity and expressed as the protein concentration (IC50 ) leading to 50% inhibition of superoxide radical formation under the conditions described in the methods. Results are given as the mean ± S.D. of three different determinations. c n.d. = not determined. b

to determine their biological activities and to identify different bioactive proteins/peptides. Acknowledgements Fig. 5. Gel electrophoresis (SDS-PAGE) of the second fraction before (F-2) and after (F-2P) pepsin treatment. Lane 1: F-2; Lane 2: F-2-P Lane 3: MW markers.

Table 1 Protein concentration of different fractions obtained as described in Fig. 1 Fraction

Protein (mg/ml)

Volume (ml)

Total protein (mg)

Whey solution Whey precipitate Whey supernatant Whey filtrate F-1 F-2 F-3 Pepsin Trypsin F-2-P PF-1 PF-2 PF-3 TF-1 TF-2

9.32 16.42 7.82 7.48 0.38 1.65 2.00 6.48 6.73 1.42 0.13 0.43 1.82 0.13 1.68

52.0 2.7 49.0 44.1 6.0 7.2 9.6 10.0 10.0 4.0 8.4 7.0 14.0 5.0 21.0

484.64 44.33 383.18 329.87 2.28 11.88 19.20 64.80 67.30 5.68 1.09 3.01 25.48 0.65 35.28

The fractions used were obtained as described in Fig. 1.

proteins by a procedure combining gel exclusion chromatography and proteases seems to be an easy method of obtaining whey proteins for further characterization and functional analysis. We are presently carrying out experiments on whey protein fractions

This work was supported by Marmara University Research Fund (SAG-YLS-290506-0092 and SAG-YYP-290906-0208) and Turkish Scientific and Technological Research Council (SBAG-2972/104S507). References [1] A. Tolkach, U. Kulozik, J. Food Eng. 67 (2005) 13. [2] A.S. Yalc¸ın, Curr. Pharm. Des. 12 (2006) 1637. [3] R. Burr, in: S. Roe (Ed.), Protein Purification Applications, Oxford University Press, USA, 2001, pp. 87–115. [4] D.E.W. Chatterton, G. Smithers, P. Roupas, A. Brodkorb, Int. Dairy J. 16 (2006) 1229. [5] H. Roginski, J.W. Fuquay, P.F. Fox (Eds.), Encyclopedia of Dairy Sciences, Academic Press, New York, 2003. [6] E.A. Permyakov, L.J. Berliner, FEBS Lett. 473 (2000) 269. [7] O. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, J. Biol. Chem. 193 (1951) 265. [8] C.A. Martinez, M.E. Loureiro, M. Oliva, M. Maestri, Plant Sci. 160 (2001) 505. [9] E.N. Lightfoot, Ind. Eng. Chem. Res. 38 (1999) 3628. [10] L. Pedersen, J. Mollerup, E. Hansen, A. Jungbauer, J. Chromatogr. B 790 (2003) 161. [11] S. Doultani, K.N. Turhan, M.R. Etzel, Process Biochem. 39 (2004) 1737. [12] A.L. Zydney, Int. Dairy J. 8 (1998) 243. [13] A. Pihlanto, Int. Dairy J. 16 (2006) 1306. ¨ [14] G. Unal, A.S. Akalın, Agro Food Ind. Hi-Tech. 17 (2006) 4. [15] S.C. Cheison, Z. Wang, S.-Y. Xu, J. Agric. Food Chem. 55 (2007) 3896. [16] H.C. Liu, W.L. Chen, S.J.T. Mao, J. Dairy Sci. 90 (2007) 547. [17] R.J. Elias, J.D. Bridgewater, R.W. Vachet, T. Waraho, D.J. McClements, E.E. Decker, J. Agric. Food Chem. 54 (2006) 9565.