Aggregation of Whey Protein Hydrolysate Using Alcalase 2.4 L
Citation: Liu C, Liu W, Feng Z, Li D (
Aggregation of Whey Protein Hydrolysate Using Alcalase 2.4 L
Chunhong Liu 0
Wen Liu 0
Zhibiao Feng 0
Dongmei Li 0
Sabato D'Auria, CNR, Italy
0 Department of Applied Chemistry, College of Science, Northeast Agricultural University , Harbin , China
Here, we describe peptide aggregation, which is also known as enzymatic protein resynthesis. Whey protein hydrolysate (WPH) is the starting material for assembling peptides. Analyses of the involved amino acids, intrinsic fluorescence, fluorescence phase diagram, secondary structure, turbidity, and surface hydrophobicity were performed to investigate the reaction process. The aggregation mechanism consists of two parts: 1) formation and 2) aggregation of the building blocks that form the ordered secondary b-sheet structure. Constructing the building blocks requires at least one intermediate state, which is formed after 0.5 hours. Non-synergistic changes in the secondary and tertiary structures then allow the intermediate state to emerge.
Protein hydrolysates can aggregate into high-molecular
substances and/or less soluble substances. Some chemists initially
called this reaction the plastein reaction, which was
characterized by enzymatic protein degradation and resynthesis . Guo
et al. reported that b-conglycinin could form soluble aggregates,
but the association of glycinin with the aggregates led to the
appearance of insoluble materials . Some aggregates
demonstrate amorphous organization , whereas others form highly
ordered amyloid-like fibrils [4-6]. These findings suggest that
protein aggregation may be a common structural property of
proteins and a much more widespread event than previously
In food industry applications, these aggregating protein
hydrolysates can incorporate limited amino acids into
polypeptides, remove the bitter taste of protein hydrolysates, and improve
the nutritional value and functional properties of food proteins
. Therefore, aggregation reactions have enormous potential
applications in industry.
Although peptide aggregation has been researched for a long
time, researchers have not reached a consensus. Yamashita et al.
considered the condensation reaction was the main aggregation
mechanism . Edwards & Shipe reported that aggregates were
held together by noncovalent bonds, rather than covalent peptide
bonds and they concluded that physical forces such as
hydrophobic interactions play a major role . Andrews performed
aggregate reactions on casein hydrolysate and reported that the
reaction was an entropy-driven physical aggregation process .
The results of Yuan et al. indicated that the soluble soy protein
isolate (SPI) - chitosan (CS) aggregate was driven by electrostatic
interaction . Ringgenberg et al. concluded that short range
interactions play a major role in the formation of the protein
network in soymilk curd .
The aim of the present study is to research the factors associated
with peptide aggregation. It is important to determine a unified
and clear process, which will be of great value to further improving
protein hydrolysate applications. Here, we study the mechanisms
of peptide aggregation by investigating the reaction process,
instead of the aggregated product. We believe this protocol may
also prove useful in peptide aggregation.
Materials and Methods
Alcalase 2.4 L was purchased from Novozymes (Bagsvaerd,
Denmark). 1-anilinonaphthalene-8-sulfonate (NAS) was purchased
from Fluka (Switzerland). All other chemicals and reagents used in
this study were analytical grade.
Whey protein hydrolysate
Whey protein was dissolved in water to a protein concentration
of 5% (w/w), and Alcalase 2.4 L was added at an enzyme/
substrate (E/S) ratio of 7% (w/w). Hydrolysis was carried out at
50uC for 5 hours. The pH of the hydrolysis system was kept at
pH 9 6 0.05 by continuously adding 1 mol/L NaOH during
hydrolysis. The enzyme was inactivated by heating the hydrolysis
system in boiling water for 15 minutes. The pH of the hydrolysis
system was adjusted to pH 4.0 by adding 2 mol/L HCl in order to
precipitate the non-hydrolyzed protein. The mixture was
centrifuged at 1800 g for 20 minutes to collect the supernatant, then
lyophilized to obtain whey protein hydrolysate (WPH) as the
substrate of the aggregation reaction.
The lyophilized WPH powder was dissolved in water to a
protein concentration of 30% (w/w), and then the solution was
adjusted to pH 7.0. Alcalase 2.4 L was added at an E/S ratio of
2% (w/w) and incubated at 37uC for 0.1, 0.25, 0.5, 1, 2, 3, 4 or
6 hours. The enzyme was inactivated by heating the hydrolysis
system in boiling water for 10 minutes. After the reaction was
completed, the solution was centrifuged at 11,100 g at 4uC for
30 minutes. The synthetic product was then vacuum freeze-dried.
Amino acid analysis
Amino acids were analyzed according to the professional
standards of the Peoples Republic of China (GB/T14965-1994)
. The sample was hydrolyzed for 22 hours at 110uC with
6 mol/L HCl in sealed glass tubes filled with nitrogen. The amino
acid concentration in the hydrolyzed samples was diluted to
50 nmol/L using 0.2 mol/L sodium citrate buffer (pH 2.2). The
pH-adjusted samples were analyzed using a Biochrom 20
Automatic Amino Acid Analyzer (GE, USA).
Intrinsic fluorescence analysis
The synthetic products were dissolved in phosphate buffer
solution (pH 7.0) and 0.5 mmol/L sodium dodecyl sulfate (SDS)
to a protein concentration of 1% (w/w). The liquid was
homogenized at 8000 rpm for 1 minute, and then centrifuged at
1500 g for 10 minutes. The supernatant was collected by filtering
through a 0.45-mm cellulose acetate membrane, then diluted 1006
with phosphate buffer solution for use as a stock solution. The
fluorescence spectrum was measured using the PE LS-55
Fluorescence Spectrophotometer (Perkin Elmer). Each scan was
repeated 56 at a 295 nm excitation wavelength, 10 nm emission
slit width, 10 nm excitation slit width, 1200 nm/minute scanning
speed, and 300450 nm scan range.
Measurement of the infrared spectra and data
Fourier transform infrared (FTIR) spectra were measured using
a Tensor 27 FTIR spectrometer (Bruker, Germany) at a resolution
of 4 cm21, and 64 scans were obtained between 4004000 cm21.
The system was continuously purged with N2. Reference spectra
were recorded under identical conditions, except the KBr media
contained no protein.
Second derivative spectra and Fourier deconvolution spectra
(18 cm21 full width at half maximum [FWHM]; 2.8 enhancement
factor) were determined using OPUS 6.0. By curve-fitting the
deconvolution spectra, multiple iterations were constructed to
ensure that the residual mean square (RMS) was ,0.01.
Secondary structure analysis of the aggregation reaction
Accurately weighed samples of potassium ferrocyanide (3 mg;
internal standard) and the synthetic products (10 mg; obtained at a
different incubation times) were mixed to produce the FTIR
spectra. The integrated areas of the amide I band (1595
1705 cm21) and internal standard band (19442132 cm21) were
determined on the infrared spectra.
The synthetic product solution was diluted 206 with phosphate
buffer solution (pH 7.0), and absorbance at 420 nm was measured
using UV-2500 spectrophotometer (Shimadzu, Japan). All
measurements were performed in triplicate. Distilled water comprised
the blank sample.
Determination of surface hydrophobicity
ANS (8-anilino-1-naphthalenesulfonic acid)-based measurement
of surface hydrophobicity is the most appropriate way to assess
proteins and determine the overall three-dimensional structure in
The synthetic products were gradually diluted to 0.0050.1%
with 0.1 mol/L phosphate buffer (pH 7.0). Aliquots of the solution
(5 mL) were added to 50 mL ANS solution (8 mmol/L ANS and
0.01 mol/L phosphate buffer at pH 7.0) and allowed to stand in
the dark for 3 minutes. The fluorescence spectra were obtained
using a LS-55 spectrofluorophotometer (Perkin Elmer). The
excitation wavelength was 338 nm, and the emission wavelength
was 496 nm. The protein concentration was determined using
Folin phenol reagent according to the Lowry method . Surface
hydrophobicity can be calculated from the initial slope of the
fluorescence intensity curve following protein concentration.
Results and Discussion
Amino acid analysis
The amino acid compositions of WPH and the aggregates were
investigated after incubation for 0.5 or 12 hours, respectively.
Table 1 shows the different amino acid compositions of WPH and
the aggregates, while the aggregates that were incubated for 0.5
and 12 hours were almost the same. If the aggregation reaction
was simply a gradual process, then the amino acid composition of
the products in the initial stages of the reaction should be similar to
WPH and different from the products identified in the later stages.
However, the actual situation was different. We surmise that some
basic building blocks are formed in the initial stages; in the latter
stages, these building blocks aggregate together to form
Figure 1 shows the intrinsic fluorescence of the aggregates
according to incubation time. Following aggregation, the
maximum emission wavelength (lem, max) of the aggregates shifted from
357.98 nm to 340 nm. This blue-shift can be explained by the
microstructure, which consists of loose WPH peptide chains that
gradually tend toward nonpolar microenvironments.
Chromophore amino acid residues were embedded inside the molecule,
and the loose WPH peptide chains gradually aggregated to form
the hydrophobic regions that constitute the spatial structure.
Figure 2 shows fluorescence intensity and lem, max of the
obtained aggregates according to incubation time. The rapid
increase in fluorescence intensity was observed in the initial stage
(01 hours), and during this stage lem, max quickly blue-shifted to
340.72 nm. Fluorescence intensity slowly increased through
6 hours, and lem, max remained essentially constant at about
340 nm. Some building blocks with hydrophobic cores and stable
conformations formed between 01 hours. Only the building
blocks aggregated together during later stages, and thus no
additional new building blocks were formed.
Fluorescence phase diagram
Figure 3 shows the phase diagram of the aggregation reaction
process. The phase diagram consists of two straight lines with
different slopes that intersect at 0.5 hours. This indicates that the
aggregate formation process agrees with the three-state model
. To conveniently describe the course, the three-state model
can be expressed as H, I, and P (H represents the loose polypeptide
chains in WPH, I represents the intermediates [i.e., building
blocks], and P represents the aggregates). We conclude that there
Amino acid content(%)
Aggregates (0.5 h)
Aggregates (12 h)
is an intermediate state in the aggregation reaction process, and
transitioning from H to I only requires 0.5 hours.
Secondary structure of the aggregates
Figure 4 shows the infrared protein spectra of WPI, WPH, and
the aggregates. Figure 5A shows the second-derivative Fourier
transform infrared spectra of the proteins, and Figure 5B shows
the deconvolved amide I bands. Second-derivative analysis of
infrared spectra (IR-SD) was used to directly and quantitatively
analyze the secondary structural components of proteins, and this
technique is considered reliable [21-23]. The secondary structure
of WPI, WPH, and the aggregates were analyzed using IR-SD.
Figure 5A shows how the b-sheet absorption (1638 cm21,
1630 cm21) of WPI disappeared in comparison with WPH, while
the random coil (1649 cm21) and b-turn absorption bands
(1668 cm21, 1681 cm21) became even more apparent. After
incubation, WPH aggregated together to form proteins, and a
sharp peak with a wide half-width appeared at 1631 cm21 in the
second-derivative spectra. We conclude that WPH barely has an
ordered structure, but incubation rearranges the loose peptide
chains that form the ordered secondary structure, which mainly
consists of b-sheets.
Figure 2. Fluorescence intensity and maximum emission wavelength of aggregates obtained at different incubation times.
The amide I portion of the spectrum, after deconvolution, is
shown in Figure 5B. The peak of the amide I band also initially
demonstrated red-shift and then blue-shift. Curve fitting was
performed and Gaussian-shaped bands for the deconvolved
components were assumed (Figure 6); the curve-fitting results are
shown in Table 2. We conclude that the aggregates formed
ordered secondary structures , indicating that the aggregation
reaction was not a messy irregular aggregation but a relatively
regular polypeptide self-assembly process. This is different from
Otte et als results . Otte et al. studied aggregation formation
in the hydrolysis process, and found that the secondary structure
gradually disappeared with aggregation formation.
Secondary structure analysis of the aggregation reaction
The fate of the secondary structure during aggregation was
investigated using the infrared internal standard method. The
Figure 4. Infrared spectra of the proteins, 4004000 cm21. a) WPI. b) WPH. c) Aggregates. The system was continuously purged with N2.
above discussion gives some justification for the assumption that
aggregation leads to structural rearrangement; this also means the
disordered WPH structure becomes a well-ordered structure of
aggregates that mainly consists of b-sheet structures. b-Sheets
possess considerable hydrogen bonds, particularly ordered
hydrogen bonding and the orderly arrangement of infrared activity in
Figure 5. Second-derivative Fourier transform infrared spectra of the protein (A). Deconvolved amide I bands (B). a) WPI. b) WPH. c)
Aggregates. (FWHM = 18 cm21; enhancement factor = 2.8).
Figure 6. Infrared spectra of the amide I band with curve fitting before and after aggregation. a) WPI. b) WPH. C) Aggregates.
specific regions in amide I. The regular and orderly arrangement
of hydrogen bonds could only occur in the ordered secondary
structures (a-helix and b-fold). Therefore, following aggregate
synthesis, an ordered structure gradually forms under the
interacting hydrogen bonds and the corresponding narrow
absorption bands appear in specific regions of amide I. The area
of the absorption peak will also gradually increase.
t (%) is the ratio of the peak area of the amide I band to the
internal standard. Because the band of the internal standard is
fixed, any increase in t (%) represents an increase in the band area
of amide I (i.e., an increase in the ordered secondary structure)
[26,27]. Figure 7 shows the t (%) values of the aggregates that were
obtained at different incubation times. The rapid increase in t (%)
was observed in the initial stage (00.5 hours), and then a very
slow increase in t (%) occurred after 0.5 hours.
Turbidity and surface hydrophobicity
Aggregates were gradually synthesized during incubation with
Alcalase 2.4 L (Figure 8), and turbidity and surface
hydrophobicity also gradually changed. Both trends can be divided into three
parts: 03 hours, 36 hours, and . 6 hours. The reaction rates
were fast, medium, and slow, respectively. This means that the
aggregation processes described are the same.
The changes in surface hydrophobicity also illustrate the
hydrophobic interactions of the reaction processes. At 03 hours,
surface hydrophobicity fell very fast due to the strong hydrophobic
interactions generated by the accumulation of hydrophobic side
chains. Surface hydrophobicity further declined over 36 hours
and the reaction speed slowed, indicating that aggregation
approached equilibrium. Finally, at 612 hours, surface
hydrophobicity slightly increased because after achieving equilibrium
because additional noncovalent bonds formed inside the
aggregate, which led to a more compact aggregation structure as the
hydrophobic regions became embedded.
Speculation regarding a small structures group is based on
the assumption of basic building blocks in the construction of
amino acids . Intrinsic fluorescence spectra analysis shows that
1 hour is the turning point for structural changes and when
structural groups form. Small structures have a stable spatial
structure and hydrophobic core, in addition to the ordered
secondary b-sheet. An intermediate structure was identified at
0.5 hours on the phase diagram. At the same time, the turning
points for lem,max and t (%) occurred at 0.5 hours. The
demarcation point at 0.5 hours indicates non-synergy, and the
endogenous fluorescence spectrum changes in terms of fluorescence
intensity and lem,max. Taking into account the non-synergistic
changes between fluorescence intensity and lem,max, we conclude
that the intermediate appeared during the information of the small
Figure 8. Turbidity and surface hydrophobicity of aggregates obtained at different incubation times.
structures group because of non-synergy between the secondary
and tertiary structures. In other words, the secondary structure
forms prior to the tertiary structure; furthermore, when the
secondary structure is formed, the tertiary structure is still being
built. Therefore, this intermediate has a complete secondary
structure and a fairly complete hydrophobic core.
Aggregation consists of two parts according to the results of the
amino acid analysis, intrinsic fluorescence spectra, and infrared
spectra. First, building blocks with hydrophobic cores and a stable
conformation form between 00.5 hours. After 0.5 hours, the
building blocks aggregate together and no additional new building
blocks form. The small structures group is involved in aggregation,
and this process can be divided into three parts: fast, medium, and
The second-derivative Fourier transform infrared spectra and
deconvolved amide I band infrared spectra of the proteins confirm
that the aggregates demonstrate a stable spatial structure,
hydrophobic core, and ordered secondary b-sheet structure
relative to the reaction substrate. Hydrophobic interactions and
hydrogen bonds play an important role in aggregation.
Conceived and designed the experiments: CL. Performed the experiments:
WL. Analyzed the data: ZF. Contributed reagents/materials/analysis tools:
DL. Wrote the paper: CL ZF.
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