Bovine lactoferrin purification from whey using Yellow HE-4R as the chromatographic affinity ligandJ. Sep. Science


María Fernanda Baieli, Nicolás Urtasun, María Victoria Miranda, Osvaldo Cascone, Federico Javier Wolman
Filtration and Separation / Analytical Chemistry


Putting Women First: Women and Health in a Rural Community

Rahul Goswami (from the Foreword)

The distributions of major whey proteins in acid wheys obtained from caprine/bovine and ovine/bovine milk mixtures

Mirjana B. Pesic, Miroljub B. Barac, Miroslav M. Vrvic, Nikola M. Ristic, Ognjen D. Macej, Sladjana P. Stanojevic, Aleksandar Z. Kostic

Differential protein composition of bovine whey: A comparison of whey from healthy animals and from those with clinical mastitis

Caroline J. Hogarth, Julie L. Fitzpatrick, Andrea M. Nolan, Fiona J. Young, Andrew Pitt, P. David Eckersall


484 J. Sep. Sci. 2014, 37, 484–487

Marı´a Fernanda Baieli∗

Nicola´s Urtasun∗

Marı´a Victoria Miranda

Osvaldo Cascone

Federico Javier Wolman

Ca´tedra de Microbiologı´a

Industrial y Biotecnologı´a,

Facultad de Farmacia y

Bioquı´mica, Universidad de

Buenos Aires, Junı´n 956, 1113

Buenos Aires, Argentina

Received October 3, 2013

Revised November 30, 2013

Accepted December 13, 2013

Short Communication

Bovine lactoferrin purification from whey using Yellow HE-4R as the chromatographic affinity ligand

The worldwide production of whey increases by around 186 million tons each year and it is generally considered as a waste, even when several whey proteins have important economic relevance. For its valorization, inexpensive ligands and integrated chromatographymethods need to be developed for specific and low-cost protein purification. Here, we describe a novel affinity process with the dye YellowHE-4R immobilized on Sepharose for bovine lactoferrin purification. This approach based on a low-cost ligand showed an efficient performance for the recovery and purification of bovine lactoferrin directly from whey, with a yield of 71% and a purification factor of 61.

Keywords: Bovine lactoferrin / Dye affinity chromatography / Sweet whey /

Triazine dyes

DOI 10.1002/jssc.201301086 1 Introduction

Lactoferrin (Lf) is a glycoprotein present in mammalian external secretions with iron-chelating capacity [1]. Its biological properties include regulation of iron adsorption in the gastrointestinal tract, modulation of polymorphonuclear production, and antimicrobial and antifungal activity [2, 3]. All these functional properties imply Lf valorization in different sectors such as the dairy, nutraceutical, pharmaceutical, and cosmetic industries.

Bovine Lf (bLf) has been traditionally purified from natural sources such as milk, whey, and colostrum by different chromatographic methods that include hydrophobic interaction, ion exchange, and affinity with antibodies [4–10].

Particularly, ion-exchange chromatography is mainly used in large-scale processes. However, the concentration of bLf in cheese whey is low, therefore, the starting material requires concentration and conditioning to improve the performance of the chromatographic process. These processes reach variable yields between 50 and 96% and bLf is usually copurified with lactoperoxidase, a whey protein with a similar pI. Using commercial sulfonic adsorptive membranes, Chiu et al. [5] copurified lactoperoxidase and bLf from defatted whey with yields of 73 and 50%, respectively. Plate et al. [11] achieved an 84% yield for bLf purification from a sweet whey concentrate using a similar cation-exchange system, however, the final product showed 10% of the initial lactoperoxidase content.

Correspondence: Dr. Osvaldo Cascone, Campichuelo 103 2A, (1405) Buenos Aires, Argentina


Fax: 54-11-4508-3645

Abbreviations: bLf, bovine lactoferrin; Lf, lactoferrin

On the other hand, the use of affinity chromatography makes possible the direct adsorption of bLf at low concentration, but the purification cost significantly increases according to the ligand cost and stability.

Triazine dyes are pseudobioaffinity ligands widely used for chromatography, which allows the purification of proteins with favorable cost/selectivity ratio [12,13]. Triazine dyes used in the textile industry in tons are low-cost ligands. They are easily immobilized on different supports and are chemically and thermally stable. All these advantages make them suitable for industrial-scale protein purification [14]. Previous research showed that the triazine dye Red HE-3B has an acceptable chromatographic performance for bLf purification from colostrum and sweet whey [15,16]. Recently, we studied the interaction of several triazine dyes, including Red HE3B, toward lactoferricin B (bLfcin), a 25 amino acid peptide present in the N-terminal region of bLf, using surface plasmon resonance and concluded that YellowHE-4R had higher affinity for bLfcin than RedHE-3B [17]. Taking this result into account, in the present study we compared the performance of both dyes (Red HE-3B and Yellow HE-4R) for bLf recovery and purification directly from sweet whey. 2 Materials and methods

Red HE-3B dye (C.I. name: reactive red 120) and Yellow HE4R dye (C.I. name: reactive yellow 84) were from Vilmax S.A. (Buenos Aires, Argentina). Both dyes were immobilized on

Sepharose 4B (Sigma–Aldrich, St. Louis, MO, USA) as previously reported [16,18]. These matrices are named hereafter

S-R and S-Y, respectively. The dye density in S-R and S-Y was ∗These authors contributed equally to this work.

C© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

J. Sep. Sci. 2014, 37, 484–487 Liquid Chromatography 485

Figure 1. Structures of Red HE-3B (A) and Yellow HE-4R (B). determined by the hydrolytic procedure reported by Ruckenstein and Zeng [19].

Adsorption isotherms were performed with commercial bLf fromGlanbiaNutritionals (Fitchburg,WI,USA). To study the interactions, 50 mg matrices (S-R and S-Y) were soaked with 1 mL of bLf solutions (0.063–10 mg/mL) in adsorption buffer (20 mM sodium phosphate buffer, pH 7.0) for 16 h at 25C. The concentration of commercial bLf in the supernatants was determined spectrophotometrically at 280 nm considering an absorption coefficient of 1.51mL/mg·cm [20].

The equilibrium concentration of bLf bound to thematrix per unit of total amount ofmatrix was calculated by difference between the concentration of bLf at the beginning of the experiment and that remaining in the soluble phase after adsorption. Maximum capacities (Qmax) and dissociation constants (Kd) were calculated as described by Chase and analyzed according to the Langmuir model [21]. Five elution solutions were tested: 2 M NaCl, pH 7.0; 0.5 M NaSCN (sodium thiocyanate), pH 7.0; 25% ethylene glycol, pH 7.0; 25% ethylene glycol + 0.5 M NaSCN, pH 7.0; and 25% ethylene glycol + 2

M NaCl, pH 7.0.