Gel Formation of Peptides Produced by Extensive Enzymatic
Hydrolysis of -Lactoglobulin
Dany Doucet and E. Allen Foegeding*
Department of Food Science, North Carolina State University, Raleigh, North Carolina 27695-7624
Received December 6, 2004; Revised Manuscript Received January 9, 2005
The purpose of the present study was to identify which peptides were responsible for enzyme-induced gelation
of extensively hydrolyzed -lactoglobulin with Alcalase in order to gain insight into the mechanism of
gelation. Dynamic rheology, aggregation measurements, isoelectrofocusing as well as chromatography and
mass spectrometry were used to understand the gel formation. A transparent gel was formed above a critical
concentration of peptides while noncovalently linked aggregates appear with increasing time of hydrolysis.
Extensive hydrolysis was needed for gelation to occur as indicated by the small size of the peptides.
Isoelectrofocusing was successful at separating the complex mixture, and 19 main peptides were identified
with molecular weight ranging from 265 to 1485 Da. Only one fragment came from a -sheet rich region
of the -lactoglobulin molecule, and a high proportion of peptides had proline residues in their sequence.
Introduction
Biopolymer gelation has received much interest lately due
to its application in many fields including tissue engineering
and drug delivery.
1,2
Biopolymer gels can be formed from a
variety of high molecular weight natural polymers such as
gelatin,
3
fibrin,
4
and polysaccharide-derived polymers.
5
An
alternative approach is the use of globular proteins or peptides
to create biopolymer gels.
6
Besides the development of gel-
based systems, interest in peptide self-assembly and gelation
has increased in recent years due to similarities with amyloid
fibrils associated with Alzheimer’s disease, variant Creutzfeldt-
Jakob disease, type II diabetes, and many others.
7
Peptide
gelation seems to result from the assembly of peptides into
large polymeric networks rather than nonspecific entangle-
ments. -sheet forming peptides tend to display this behavior.
Assembly and gelation occurs by hydrogen bonding between
peptides to form intermolecular -sheets that further associate
into a wide variety of nonprotein-like structures, such as
tapes, ribbons, fibrils, and fibers.
8
Whey proteins are the proteins remaining soluble at pH
4.6 and 20 °C after the removal of caseins from milk.
9
Based
on this classification, whey proteins make up about 20% of
the proteins in milk and are comprised mainly of -lacto-
globulin (-LG), R-lactalbumin (R-LA), bovine serum
albumin (BSA), immunoglobulins, and proteose peptones.
-LG, the most abundant whey protein in bovine milk, has
a monomeric molecular weight of 18 362 Da for genetic
variant A and 18 276 for variant B, the two most common
variants.
10
The primary structure is composed of 162 amino
acids with 5 cysteine residues, four of which occurs as
disulfide bonds with the fifth as a free thiol. The secondary
structure of -LG has been elucidated as 43% -sheet, 10%
R-helix, and 47% unordered structure, including -turns.
11
The structure is highlighted by an eight-stranded, antiparallel
-barrel to form the shape of a flattened cone where the
interior is rich in hydrophobic amino acids and the opening
is lined with hydrophilic amino acids. An additional -strand
and the R-helix are attached to this flattened cone.
12
-LG
has a critical role in heat-induced gelation of whey proteins.
Heat-induced gelation of -LG is thought to involve three
main steps. First, there is activation of the molecule to a
reactive structural form followed by the formation of linear
strings of beads polymers via sulfhydryl-disulfide inter-
change reaction. Finally, setting of the string of beads occurs
into a gel network via noncovalent interactions namely
hydrophobic, hydrogen bonding, and van der Waals interac-
tions.
13
In previous work, we have shown that extensive hydrolysis
of a whey protein isolate (WPI) by Alcalase, a commercial
proteolytic enzyme from Subtilisin Carlsberg, led to
gelation.
14-16
The composition of this WPI in -LG, R-LA,
and BSA was 66, 22, and 4% respectively. The first step
toward a better understanding of the self-assembly of whey
protein peptides is to determine which of the two major
proteins, -LG or R-LA, is responsible for gelation. Peptides
responsible for aggregation and gelation of -LG during
limited enzymatic hydrolysis with a protease from Bacillus
licheniformis (BLP) have been identified.
17
Seven major
peptides were identified and the fragment f135-158 was
suspected as the initiator of aggregation. This peptide
contains several basic and acid amino acids alternating with
hydrophobic amino acids. Therefore, aggregation was pro-
posed to occur through electrostatic and hydrophobic interac-
tions. Gelation of R-LA has also been reported when
incubated with the protease BLP.
18
The microstructure of
the gels consisted of nonbranching, apparently hollow
strands, similar in overall structure to microtubules. The
mechanism behind the self-assembly of the partially hydro-
* To whom correspondence should be addressed. E-mail:
allen_foegeding@ncsu.edu. Telephone: (919) 513-2244. Fax: (919) 515-
7124.
1140 Biomacromolecules 2005, 6, 1140-1148
10.1021/bm0492273 CCC: $30.25 © 2005 American Chemical Society
Published on Web 02/16/2005