A simple and direct isolation of whey components from raw milk by gel filtration chromatography and structural characterization by Fourier transform Raman spectroscopy.

Abstract

A simple and economical method to isolate whey protein from fresh raw milk is developed by serial defatting, casein eliminating, lactose removing, and separating by gel filtration chromatography. Four major whey components, including immunoglobulin (Ig), bovine serum albumin (BSA), beta-lactoglobulin (beta-Lg) and alpha-lactalbumin (alpha-Lac), and a non-protein of low molecular mass ( approximately 1.7 kDa) but strong absorbance at 280 nm, are detected simultaneously. The small non-protein molecule is rich in aromatic amino acids and thiol groups as supported by the structural characterization with near infrared Fourier transform Raman spectroscopy (FT-Raman). FT-Raman results show that the secondary structure of Ig is dominated by anti-parallel beta-pleated sheet; BSA is mainly in alpha-helix; both beta-form and unordered structure are important in beta-Lg; while alpha-Lac is mostly in alpha-helix coupling with random coil. Differences in the Raman profile for each whey component reflect their intrinsic compositional differences and distinct spatial arrangement. The S-S linkages diverging around 510-540 cm(-1) indicate that the conformation of disulfide bonds in each whey components is different, which may be responsible for their diversified behaviors in solubility, rheological and functional properties.

Full text

Page 1

This article was originally published in a journal published by
Elsevier, and the attached copy is provided by Elsevier for the
author’s benefit and for the benefit of the author’s institution, for
non-commercial research and educational use including without
limitation use in instruction at your institution, sending it to specific
colleagues that you know, and providing a copy to your institution’s
administrator.
All other uses, reproduction and distribution, including without
limitation commercial reprints, selling or licensing copies or access,
or posting on open internet sites, your personal or institution’s
website or repository, are prohibited. For exceptions, permission
may be sought for such use through Elsevier’s permissions site at:
http://www.elsevier.com/locate/permissionusematerial

Page 2

Author's personal copy
Talanta 69 (2006) 1269–1277
A simple and direct isolation of whey components from raw milk by gel
filtration chromatography and structural characterization by Fourier
transform Raman spectroscopy
Mong Liang, Vivin Y.T. Chen, Hsiu-Ling Chen, Wenlung Chen∗
Department of Applied Chemistry, National Chiayi University, Taiwan
Received 12 October 2005; received in revised form 4 January 2006; accepted 4 January 2006
Available online 10 February 2006
Abstract
A simple and economical method to isolate whey protein from fresh raw milk is developed by serial defatting, casein eliminating, lactose
removing, and separating by gel filtration chromatography. Four major whey components, including immunoglobulin (Ig), bovine serum albumin
(BSA), ?-lactoglobulin (?-Lg) and ?-lactalbumin (?-Lac), and a non-protein of low molecular mass (∼1.7kDa) but strong absorbance at 280nm,
are detected simultaneously. The small non-protein molecule is rich in aromatic amino acids and thiol groups as supported by the structural
characterization with near infrared Fourier transform Raman spectroscopy (FT-Raman). FT-Raman results show that the secondary structure of Ig
is dominated by anti-parallel ?-pleated sheet; BSA is mainly in ?-helix; both ?-form and unordered structure are important in ?-Lg; while ?-Lac
is mostly in ?-helix coupling with random coil. Differences in the Raman profile for each whey component reflect their intrinsic compositional
differences and distinct spatial arrangement. The S–S linkages diverging around 510–540cm−1indicate that the conformation of disulfide bonds
in each whey components is different, which may be responsible for their diversified behaviors in solubility, rheological and functional properties.
© 2006 Elsevier B.V. All rights reserved.
Keywords: Whey protein; Raw milk; Gel filtration chromatography; FT-Raman; Secondary structure; Disulfide bonds
1. Introduction
Whey protein represents approximately 20% of the original
milk proteins. It exhibits many important functional properties
in the manufacture of food. The nutritional values and physi-
ological benefits of whey protein also receive much attention
[1–8]. Although whey proteins have versatile functional prop-
erties and nutritional values, an estimated 70% of liquid whey
is disposed as waste product. Such large volumes of disposed
protein will seriously impact environmental pollution due to its
high BOD (biological oxygen demand) level. The utilization
of whey proteins is confined because there still exists a large
variability in the composition and functional properties of com-
mercial whey proteins [9–12]. To date, the factors responsible
for the variability remain poorly understood. It is possible that
the incongruence of physico-chemical and functional perfor-
mance of whey protein reported from different groups might
∗Corresponding author. Tel.: + 886 5 271 7965; fax: +886 5 271 7901.
E-mail address: wlchen@mail.ncyu.edu.tw (W. Chen).
result from the compositional variations arising from different
whey resources or processing conditions in commercial whey
products. To clarify this problem, how to obtain whey protein
in its native form, i.e., free from any denaturizing, becomes of
utmostimportance.Manyprocessessuchasultrafiltration,diafil-
tration, polyphosphate complex precipitation, heat coagulation,
and ion-exchange adsorption technology have been developed
for the manufacture of whey protein [13,14]. By these methods,
whey protein is prone to the risk of protein denaturation. For
protein separation, a number of liquid chromatography meth-
ods including reversed-phase [15–18], hydrophobic interaction
[19],andionexchange[20–23]techniqueshavebeenemployed.
For the ion-exchange method, a large amount of mobile phase
is usually prepared with various salts at different concentrations
or at different pH to wash out the compound of interest, and for
reverse-phase HPLC, some organic solvents or an acidic mobile
phase have to be applied, all of which potentially denature pro-
teins. In contrast, size exclusion chromatography is relatively
amiable to protein molecules and has been frequently used in
proteinseparation[24–27].YoshidausedaSephacrylS-200col-
umn to isolated ?-Lg and ?-La from acid whey protein [24];
0039-9140/$ – see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.talanta.2006.01.008

Page 3

Author's personal copy
from Bio-Rad Lab. (Richmond, CA). Electrophoresis grade
acrylamide, N,N,N,N-tetramethyl ethylenediamine (TEMED),
Trizma base, N,N-methylenebisacrylamide, tris-aminomethane,
ammonium persulfate, 2-mecaptoethanol, bromophenol blue,
coomassie brilliant blue G-250, sodium dodecylsulfate (SDS),
and protein markers were purchased from Bio-Rad Lab. (Rich-
mond, CA). All the chemicals for chromatography were analyt-
1270
M. Liang et al. / Talanta 69 (2006) 1269–1277
Nakai and Al-Mashikhi isolated whey proteins using Sephacryl
S-300 and TSK HW-55 columns [25]. No single method is suit-
able for all whey proteins and the method of choice is usually
based on one or two whey components of interest [17]. More-
over, most previous whey-related studies are sampling from
commercial products such as whey concentrates or acid whey
proteins, of which properties are closely dependent on the man-
ufactureprocesses.Littlehasbeenreportedonfreshrawmilk,to
thebestofourknowledge.Hence,weexpecttodevelopasimple
isolation process of whey proteins directly from fresh raw milk.
FT-Ramanspectroscopyisapowerfultechniqueforthestruc-
tural investigation of protein molecules. Compared to other
techniquesforproteincharacterizationsuchasdifferentialscan-
ning calorimetry (DSC) and polarizing microscopy, viscosity
and turbidity measurements, electron microscopy and chemical
modifications, FT-Raman spectroscopy provides direct, non-
invasive information to the protein structure in either solid, film
or aqueous form. One important factor distinguishing Raman
spectroscopy from many other spectroscopic methods is its
applicability to in situ systems containing high concentration
of proteins, which is critical for the investigation of struc-
tural changes during processes such as foaming, emulsifying,
and gelling [28]. Bands in the Raman spectrum arising from
amide I, amide III, and skeletal stretching modes of peptides
and proteins are useful for characterizing backbone conforma-
tion [29–32]. Valuable information may also be obtained on
SS/SH conversion, CH groups of aliphatic residues, and aro-
matic rings of amino acid residues [33–36]. Thus, changes in
chemicalstructureandmicroenvironmentofproteinside-chains
through either intramolecular or intermolecular interactions can
be easily probed.
In this work, whey protein was first isolated directly from
fresh milk through a simple and economical process includ-
ing defatting, decaseinating, lactose removing and separating
by preparative gel filtration chromatography. It shows that four
major components (immunoglobulin, bovine serum albumin,
?-lactoglobulin, and ?-lactalbumin) and a small non-protein
molecule can be easily collected. The molecular structure of
these whey components is then characterized by FT-Raman.
With its unique spectroscopic advantages, structural informa-
tion on the backbone conformation of whey components and
the microenvironment of important amino acids is elucidated.
2. Experimental
Ammonium sulfate, KCl, NaCl, (NH4)2SO4, NaN3, HCl,
sodiumphosphate,dibasic,12-hydrate,andsodiumsulfatewere
from Sigma Chem. Co. (St. Louis, MO). Protein standards
of ?-lactoalbumin (?-Lac), ?-lactoglobulin (?-Lg), bovine
serum albumin (BSA), immunoglobulin (IgG) were purchased
ical grade, and the rest except for electrophoresis were reagent
grade. All solutions were prepared with ultra pure water, from
an EASYpure RF water system (Barnstead).
2.1. Isolation of whey protein from raw milk
Bovine milk was collected from the Experimental Farm of
the National Chiayi University. Raw milk after milking was
directly refrigerated (at −18◦C) overnight; the fat floated spon-
taneously and then froze on that top layer so that it could be
easilyremoved.Afterremovingthefrozenfat,thelowerportion
(mostly lactose and protein compounds) was thawed under cool
conditions (4◦C) and then centrifuged (4◦C, 15,000rpm) for
30min to isolate protein from other residues. Protein solution
was then treated with diluted HCl solution at pH 4.6, the iso-
electric point of casein, from which it would be precipitated
out. After centrifugation, the pale-greenish transparent solu-
tion (whey protein) was subjected to separation by loading on
an open column (2.6cm×70cm) filled with Sephadex G-200
(Pharmacia, USA) medium, and the elution was collected by a
Gilson FC-205 Fraction Collector (Gilson, France). The flow
rate was controlled at 0.8ml/min, and the volume collected for
each tube was 3ml/tube. The elution buffer was prepared with
Tris–HCl at pH 7.2. Each component was collected, dialyzed,
and lyophilized for further study.
2.2. Molecular mass determination
The molecular weight of each whey protein component was
determined by gel filtration chromatography using a Sephadex
G-200 column (2.6cm×70cm) with molecular mass mark-
ers (Pharmacia). The molecular mass of each whey compo-
nent was determined by the simple linear regression, corre-
lating elution volume with molecular weight, on the calibra-
tion curve. Both high and low molecular weight calibration
curves were quickly constructed by measuring the elution vol-
umes of standard compounds, calculating Kavvalue for each,
and plotting Kavvalue versus the logarithm of each standard
molecular mass. The standard compounds include ribonucle-
ase A (13.7kDa), chymotrypsinogen A (25.0kDa), ovalbu-
min (43.0kDa), bovine serum albumin (67.0kDa), aldolase
(158kDa), catalase (232kDa), ferritin (440kDa), thyroglobu-
lin (669kDa), and blue dextran 2000 (2000kDa):
Kav= (Ve− V0)/(Vt− V0)
where, Veis the elution volume of protein, V0the column void
volume, Vtis the total bed volume.
2.3. SDS-PAGE analysis
Sodium dodecyl sulfate polyacrylamide gel electrophore-
sis (SDS-PAGE) was performed in a vertical mini-gel sys-
tem (Mini-Protean II Dual Slab Cell, Bio-Rad Lab., Rich-
mond, CA) as described by Laemmli [37] with some modifi-
cations. polyacrylamide gels (5% stacking and 14% resolving
gel) were prepared by co-polymerization of acrylamide and

Page 4

Author's personal copy
M. Liang et al. / Talanta 69 (2006) 1269–1277
1271
bis-acrylamide with the aid of initiator TEMED and ammo-
nium persulfate. Buffer solutions for the stacking and resolv-
ing gels were prepared from 0.125M Tris–HCl (pH 6.8) and
0.375M Tris–HCl (pH 8.9), respectively, incorporating 0.1%
SDS. The running buffer (pH 8.6) consisted of 0.1% SDS,
0.1% 2-mercaptoethanol, 0.19M glycine, 0.025M Tris–HCl,
and 1mM ethylenediamineteraacetic acid (EDTA). The sam-
ple buffer was composed of 0.1% SDS buffer, 10% sucrose,
0.05%bromophenolblueand20mMdithiothreitol.Thesamples
(10?l) were well-mixed with the buffer solution by microcen-
trifugeat1200rpm(HettichGmbH&Co.,Tuttlingen,Germany)
and heated at 100◦C in Dry-Bath (Dubuque, IA, USA) for
3min prior to being loaded to the gels. Electrophoresis was
carried out with a fixed voltage of 180V for 55min. After
electrophoresis, the gel was stained with 0.25% coomassie bril-
liant blue solution containing 12.5% trichloroacetic acid, 20%
methanol, and 7.0% acetic acid for 20min and destained with a
solution of 20% methanol and 7.0% acetic acid overnight. Pro-
tein markers for reduced SDS-PAGE were Myosin (200kDa)
phosphorylase b (97.4kDa), bovine serum albumin (66.2kDa),
ovalbumin (45.0kDa), carbonic anhydrase (31.0kDa), trypsin
inhibitor (21.5kDa), and aprotinin (6.4kDa).
2.4. Identification of whey components by size exclusion
chromatography (SEC)
Identification of whey component was carried out by SEC
using a TSK gel SW guard column (4cm×8mm) and a TSK
G3000 SW (30cm×8mm) (TosoHaas, Japan) loaded on a
HitachiD-7000HPLCsystem(HitachiLtd.,Tokyo,Japan).The
system was equipped with a Model L-7100 pump, a Model L-
7420 UV–vis detector, and a Rheodyne Model 7725 injector.
Peaks were detected at wavelength 280nm, and acquisition and
processing of data were completed by Hitachi B-7000 software
withA/Dinterface.0.1MSodiumphosphatebuffersolution(pH
6.8) with 0.05% sodium azide driven by the pump system was
prepared as mobile phase. Buffer solution was degassed with
Branson 2510 ultrasonic system (Branson Ultransonic Corpo-
ration, Danbury, CT) right before employing. Each whey com-
ponent collected from preparative gel filtration chromatography
described above was dissolved in the buffer solution (1mg/ml),
filtered using 0.45?m sterile units (Millipore Co., Bedford,
MA), and 10?l was injected in the chromatographic system.
A typical analysis could be completed in 30min with the flow
rateof0.6ml/min.Standardproteinsincludingimmunoglobulin
(IgG), bovine serum albumin (BSA), lactoglobulin (?-Lg), and
lactalbumin (?-La) were also run to identify each whey compo-
nent.
2.5. FT-Raman measurement
FT-Ramanspectraofeachwheycomponentwereobtainedby
using a Bruker RFS-100 FT-spectrophotometer (Bruker Optik
GmbH, Lubeck, Germany). Sample was put into the tiny hole
of a stainless steel holder for Raman measurement. A contin-
uous wave Nd-YAG laser (Coherent Lubeck GmbH, Lubeck,
Germany) with wavelength 1064nm, pumped by diode laser,
was used as the near infrared Raman excitation source. An
He–Ne laser beam was overlapped with 1064nm beam in order
to visualize the Raman sampling spot. The laser light with
power of 100mW was introduced and focused on the sample.
The scattered radiation was collected at 180◦with an ellip-
soidal mirror and was filtered, modulated and reflected back
into the highly sensitive GaAs detector that was cooled by liq-
uidnitrogen.RamanspectrawereproducedovertheRamanshift
0–3500cm−1. Typically, 500 interferograms were coadded at
4cm−1resolution with a sampling period of about 15min. The
intensity ratio of Raman bands 643–621cm−1(I643/621) as well
as 855–832cm−1(I855/832) was used to evaluate the microenvi-
ronmentpropertyoftyrosine[34],andtheratioof881–758cm−1
(I881/758) was used for the analysis of tryptophan, respectively
[35]. The spectra in the 1560–1720cm−1region were subjected
to numerical curve fitting (Grams/386; Galactic Ind. Co.). The
bandshapeswereapproximatedbyaLorentzfunction.Thebase-
line was approximated by a straight line between two points at
1560 and 1720cm−1, chosen at both sides of the band enve-
lope. Each numerical calculation of the Raman intensity ratio
wasbasedontheaverageoftriplicatemeasurementsatleast.FT-
Raman spectra reported in this study were all-original and were
not smoothed, normalized, and baseline corrected through data
manipulation.
Statisticalanalysiswasconductedwithacommercialstatistic
computing software package (STATISTICA, 1999 ed., StatSoft
Inc., Tulsa, OK, USA) in a personal computer. Results were
considered statistically significant at P<0.05.
3. Results and discussion
3.1. Isolation and identification of whey components
Although several methods including chromatographic meth-
ods,precipitationwithchemicalcontrol,ultrafiltrationwithspe-
cific membrane material, reverse osmosis, microfiltration, etc.
have been developed for isolating of whey protein [13,14], to
develop an easy, direct, and economical way to isolate whey
protein from raw milk remains full of challenge. As described
in Section 2, the method we used is relatively simple, natural,
money saving, and free from chemicals abrasive. For instance,
organic solvent such as hexane or ethyl acetate is usually used
to extract lipid/oil from the system of interest. In contrast, we
demonstrate a natural process (without any chemicals usage) to
remove lipid/fat from it. Based on the intrinsic physical prop-
erty of milk, lipid will be frozen and float spontaneously on
the top layer when it is put in the freezer. Thus, it is easier to
remove it from the remainder. Since all the processes of defat-
ting, delactosing, and casein removing are relatively mild as
well as fewer drastic chemicals being used, the risk of protein
denaturizing can be minimised. Fig. 1 shows the separation of
whey protein by preparative gel filtration chromatography. Five
peaks are observed on the chromatogram, which are attributable
to Ig, BSA, ?-Lg, ?-Lac, and a small non-protein molecule.
The uncommonly strong absorbance at 280nm indicates that
the small molecule has either high extinction coefficient or
many conjugated chromophores. The molecular mass of each

Page 5

Author's personal copy
1272
M. Liang et al. / Talanta 69 (2006) 1269–1277
Fig. 1. Gel filtration chromatography pattern of whey protein isolated directly
from raw milk after defatting and decaseinating. Peak 1, Ig; peak 2, BSA; peak
3, ?-Lg; peak 4, ?-Lac; peak 5, small non-protein molecule. Column, Sephadex
G-200; flow rate, 0.8ml/min; absorbance, UV-280nm; buffer solution, 0.05M
Tris–HCl (pH 7.2).
component was determined as: ?-lactoalbumin (∼14kDa), ?-
lactoglobulin (∼18kDa), bovine serum albumin (∼66kDa),
immunoglobulin (∼150kDa), and small non-protein molecule
(∼1700Da). The resolution between ?-Lac and ?-Lg is not
good enough; however, it is acceptable to collect each sample
at the highest absorption for further usage. Fig. 2 shows the
electrophoretic profile of whey components by SDS-PAGE. Six
bands (Lane 6) clearly shown on the SDS-PAGE gel indicate
the distribution of Ig, BSA, ?-Lg, and ?-Lac in whey proteins.
The molecular mass of non-protein molecule is too small to be
detected as evidenced by the absence of any bands (Lane 1) on
the SDS-PAGE. Lane 2 and Lane 3 represent the SDS-PAGE of
?-Lacand?-Lg,respectively.Sincethetwocomponentsarenot
well-resolved by preparative gel filtration chromatography and
due to the large amount of ?-Lg, the band with molecular mass
of about 18.5kDa in Lane 2 is a residue of ?-Lg. The molecular
mass of ?-Lac is further confirmed to be ∼14.0kDa by SDS-
PAGE. Based on SDS-PAGE, the molecular mass of BSA (Lane
Fig. 2. The SDS-PAGE of whey protein and whey constituents collected from
preparative gel chromatography. Lane S: standard protein markers includ-
ing myosin (200kDa) phosphorylase b (97.4kDa), bovine serum albumin
(66.2kDa), ovalbumin (45.0kDa), carbonic anhydrase (31.0kDa), trypsin
inhibitor (21.5kDa), and aprotinin (6.4kDa). Lane 1, non-protein molecule;
Lane 2, ?-La; Lane 3, ?-Lg; Lane 4, BSA; Lane 5, Ig; Lane 6, whey protein.
Electrophoresis was carried out at a constant voltage of 180V for 55min.
Fig. 3. Size exclusion chromatography patterns of each whey component col-
lected from preparative gel chromatography. (a) Immunoglobulin, (b) bovine
serum albumin, (c) ?-lactoglobulin, (d) ?-lactoalbumin, and (e) small non-
proteinmolecule.SeparationwasconductedonaHitachiD-7000HPLCsystem.
Column, TSK G3000; flow rate, 0.6ml/min; absorbance, UV-280nm; buffer
solution, 0.1M sodium phosphate (pH 6.8).
4) is easily obtained as ∼66kDa. The two light bands of higher
molecularmassareattributedtotheresidueofIgG.Threebands
with molecular mass larger than 97kDa appeared for IgG (Lane
5) on SDS-PAGE indicate three different classes of Ig in the raw
milk.
In addition to electrophoretic analysis of whey proteins, each
whey component was further analysed by gel exclusive chro-
matography using a TSK G-3000 SW column and identified
by running protein standards. As shown in Fig. 3, each whey
component (peaks 1–5) has a different elution time. The peak
intensity of each component reflects the relative amount present
in whey protein with ?-Lg and ?-La being present in the largest
quantities. Ig shown single peak in preparative gel filtration
(Fig. 1) can be further separated into three peaks (Figs. 2 and 3)
bySDS-PAGEandSEC.ThisindicatesthatIgconsistsofdiffer-
ent classes of immunoglobulin such as IgG, IgA, and IgM. The
shouldersshownontheprofilesof?-Lgand?-Laclearlyexplain
their incompletely resolved by the preparative gel filtration with