Food Research International 74 (2015) 177–184

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Food Research International

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Interactions of caseins with phenolic acids found in chocolate

Shuting Zhou a, Sooyoun Seo a, Inteaz Alli a, Yu-Wei Chang b,⁎
a
Department of Food Science and Agricultural Chemistry, McGill University, Macdonald Campus, 21,111 Lakeshore Road, Ste-Anne-de-Bellevue, Quebec, Canada H9X 3V9
b
Department of Food Science, National Taiwan Ocean University, Keelung 20224, Taiwan

a r t i c l e i n f o a b s t r a c t

Article history: To investigate the interactions between caseins and phenolic acids, such as the ones present in chocolate, casein
Received 29 January 2015 was incubated with protocatechuic acid or p-coumaric acid at 55 °C. In addition, casein was isolated from
Received in revised form 22 April 2015 chocolate and the phenolic compounds within these caseins were quantified. Electrophoresis results revealed
Accepted 2 May 2015
that casein–phenolic interactions were induced by incubation; minor aggregation of casein subunits was
Available online 8 May 2015
observed after incubation of casein with protocatechuic acid. Minor aggregation of casein isolated from
Keywords:
milk chocolate was also observed. In vitro hydrolysis of casein control, casein–protocatechuic acid, casein–p-
Casein–phenolic interactions coumaric acid, caseins isolated from milk chocolate and white chocolate using trypsin showed degree of
Protocatechuic acid hydrolysis of 19.3, 18.6, 17.7, 10.4 and 17.8% respectively. The presence of protocatechuic acid and p-coumaric
p-Coumaric acid acid in the model system and the presence of phenolic compounds in milk chocolate, in addition to the structural
Milk chocolate changes occurring during processing, affected the peptide profiles of casein hydrolysates.
White chocolate © 2015 Elsevier Ltd. All rights reserved.

1. Introduction (2.65%) with antioxidant and antigenotoxic properties (Ferguson, Zhu,

Among minor food components, phenolics or polyphenols have
recently received considerable attention because of their functional
properties such as antioxidant, antimutagenic, anti-inflammatory,
antiatherogenic and antitumour activities (González-Sarrías, Li, &
Seeram, 2012; Nahar, Driscoll, Slitt, & Seeram, 2014; Ortega et al.,
2008; Tanaka, Kojima, Kawamori, Yoshimi, & Mori, 1993; Živković
et al., 2014). Furthermore, it has been demonstrated that many phenolic
compounds play important roles in preventing certain human diseases,
such as osteoporosis, cancers, and cardiovascular diseases (Kaume,
Gbur, DiBrezzo, Howards, & Devareddy, 2014; Morton, Caccetta,
Puddey, & Croft, 2000; Oliveras-López, Berná, Jurado-Ruiz, López-García
de la Serrana, & Martín, 2014). Black tea, green tea, red wine and
cocoa are good sources of phenolics as they are rich in phenolic
phytochemicals (Lee, Kim, Lee, & Lee, 2003). Protocatechuic acid is a
hydroxybenzoic acid that can be found in many foods such as olives,
flaxseed, and wine (Minussi et al., 2003; Van Hoed, 2010). It is also
the most important phenolic acid (69.16%) found in cocoa liquor
(Ortega et al., 2008). It has been reported to have several physiological
functions including antioxidant, antibacterial activity, antimutagenic
activity, antitumour activity, and anticancer effects (Yin, Lin, Wu, Tsao,
& Hsu, 2009). Coumaric acids, hydroxy derivatives of cinnamic acid,
are another important group of phenolic compounds found in cocoa
⁎ Corresponding author. Tel.: +886 2 2462 2192x5152; fax: +886 2 2463 4203.
E-mail address: bweichang@mail.ntou.edu.tw (Y.-W. Chang).
& Harris, 2005; Kikugawa, Hakamada, Hasunuma, & Kurechi, 1983).
In addition to providing essential nutrients, food proteins are
important functional ingredients due to their effect in maintaining the
quality and sensory properties of foods (Belitz, Grosch, & Schieberle,
2009). Casein, which accounts for 80% of milk protein, is one of the
principal functional food proteins (Fox, 2001; Marchesseau et al.,
2002). It is present as large protein complexes incorporating milk salts
(Marchesseau et al., 2002). Milk casein is in the form of colloidally
dispersed particles (calcium caseinate) which are known as micelles
(Marchesseau et al., 2002; McMahon & Brown, 1984). Caseins are
heterogeneous proteins whose main types are αs1-casein (38%), αs2-
casein (10%), β-casein (36%) and κ-casein (13%) (Fox, 2001; Tuckey,
1963).
It has been demonstrated that different phenolic compounds bind
to a variety of proteins, especially to proteins with a high content of
proline, such as β-casein with 15.6 mole percentage of proline (Baxter,
Lilley, Haslam, & Williamson, 1997; Charlton et al., 2002; Siebert,
Troukhanova, & Lynn, 1996). The binding of phenolic compounds to
proteins has been suggested to reduce their antioxidative potential
due to the reduction in their accessibility (Kilmartin & Hsu, 2003;
O'Connell & Fox, 2001; Serafini, Ghiselli, & Ferro-Luzzi, 1996). On the
other hand, these interactions between phenolic compounds with
caseins have been shown to increase the stability to heat denaturation
(O'Connell & Fox, 2001; O'Connell, Fox, Tan-Kinitia, & Fox, 1998), to
oxidative degradation (O'Connell & Fox, 1999), and the foaming ability
of their micelles (Rosenthal, Bernstein, & Nakimbugwe, 1999; Sausse,
Aguie-Beghin, & Douillard, 2003). Although the interaction between

http://dx.doi.org/10.1016/j.foodres.2015.05.006
0963-9969/© 2015 Elsevier Ltd. All rights reserved.
178 S. Zhou et al. / Food Research International 74 (2015) 177–184
caseins and larger phenolic compounds have been investigated
(O'Connell & Fox, 1999, 2001; O'Connell et al., 1998; Sausse et al.,
2003), the interactions between caseins and smaller phenolic acids,
such as protocatechuic acid and p-coumaric acid that are also present
in food matrices such as chocolate, are unknown. The results obtained
from such investigation will be important for the understanding of
the behavior and characteristics of casein within a food matrix and its
effect on the bioavailability of phenolic acids.
Given this, the objective of this research was to study the casein–
phenolic acid interactions in a model system. In addition, caseins from
milk and white chocolate were isolated and compared with the caseins
in the model system. The casein–phenolic interactions were induced by
heat incubation and casein was extracted from both milk chocolate and
white chocolate. Characteristics of the casein–phenolic complexes were
investigated by using a combination of electrophoresis, reversed-phase
high performance liquid chromatography (RP-HPLC) and tryptic
hydrolysis.
2. Materials and methods
2.1. Materials
Commercial whole casein from bovine milk (Technical Grade),
α-casein, β-casein, κ-casein, protocatechuic acid (3,4-dihydroxybenzoic
acid), p-coumaric acid and trypsin type IX-S from porcine pancreas
(EC 3.4.21.4, 13,700 U/mg, 14,800 U/mg protein) were purchased
from Sigma-Aldrich, Co. (St. Louis, MO, USA). Samples of milk chocolate
and white chocolate were obtained as gifts from Barry-Callebaut
(Montreal, Qc, Canada).
2.2. Induction of casein–phenolic interactions
Casein–phenolic interactions were induced using the procedure
reported by Ali (2002) with modifications. Casein–phenolic solution
mixtures were prepared with 10 mM phosphate buffer (pH 7) and by
mixing 1 ml casein solution (5 mg/ml) with 1 ml protocatechuic acid
solution (1 mg/ml) or 1 ml p-coumaric acid solution (1 mg/ml); the
ratio of casein to phenolic acid was 5:1 in order to resemble the natural
concentration of these compounds observed in chocolate formulations.
Casein control solution (2.5 mg/ml casein) was prepared by mixing 1 ml
casein solution with 1 ml phosphate buffer (10 mM; pH 7). The above
solutions were heated in a water bath at 55 °C for 2 h then cooled to
room temperature.
2.3. Casein extraction from chocolate
Milk chocolate and white chocolate were defatted using the
Soxhlet extraction method following the procedure described by the
Association of Official Analytical Chemists (1990) with modifications.
The fat from 10 g of chocolate sample melted using a water bath at
50 °C was extracted with 100 ml of petroleum ether for 8 h. The defatted
chocolate samples were air dried in a fume hood. Casein was isolated
from milk chocolate and white chocolate using the procedure described
by Veloso, Terxeira, and Ferreira (2002) and Molina (2006) with
modifications. Defatted chocolate powder (10 g) was ground using a
mortar and pestle and was reconstituted with 100 ml of distilled
water and adjusted to pH 4.6 (1 M HCl) to precipitate the casein. The
mixture was allowed to stand for 1 h with continuous stirring then
centrifuged (8000 × g, 25 min), then the supernatant was discarded.
The residue was washed with acetone followed by centrifugation
(8000 ×g, 25 min). The casein was air dried and stored at 4 °C.
2.4. Polyacrylamide gel electrophoresis (PAGE)
Native-PAGE was carried out according to the method reported by
Ornstein (1964) and Davis (1964) with some modifications. Acrylamide
staking gel (4%) and resolving gel (8%) were selected to perform with
a Mini-PROTEAN 3 Cell unit (Bio-Rad, Hercules, CA, USA). Sample
solutions were prepared by dissolving 600 μl casein–phenolic mixtures
or 15–20 mg casein isolated from chocolate in 300 μl sample buffer
(distilled water, 0.5 M Tris–HCl pH 6.8, glycerol and 0.5% bromophenol
blue) separately. Sample solutions (10–15 μl) were loaded into each
individual sample well. The commercially available high molecular
weight calibration kit (Amersham Bioscience, Piscataway, NJ, USA)
was used as standard protein markers. The marker proteins were
thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), lactate
dehydroxygenase (140 kDa) and albumin (67 kDa). Gels were run
at constant current (7.5 mA/gel) for approximately 1.5 h. After electro-
phoresis, gels were fixed for about 10 min in fixing solution (distilled
water: methanol: acetic acid/7:2:1) followed by staining for 1 h with
Coomassie Brilliant Blue R-350. Destaining of gels was done by
frequently replacing the fixing solution until the protein bands were
clearly visible.
SDS-PAGE was performed following the method reported by
Laemmli (1970) with some modifications. A Mini-PROTEAN 3 Cell unit
(Bio-Rad) was used with a stacking gel of 4% as well as a resolving
gel of 12.5%. Sample solutions were prepared by dissolving 250 μl
casein–phenolic mixtures or 15–20 mg casein isolated from chocolate
in 250 μl sample buffer (distilled water, 0.5 M Tris–HCl pH 6.8, glycerol,
10% SDS, 0.5% bromophenol blue and β–mercaptoethanol), respective-
ly, followed by heat treatment at 95 °C for 5 min. Sample solutions
(10–20 μl) were loaded into each individual sample well. Broad molec-
ular weight standard kit (Bio-Rad) was loaded into the first sample well.
The standard protein markers were myosin (200 kDa), β-galactosidase
(116.25 kDa), phosphorylase b (97.4 kDa), serum albumin (66.2 kDa),
ovalbumin (45 kDa), carbonic anhydrase (31 kDa), trypsin inhibitor
(21.5 kDa), lysozyme (14.4 kDa) and aprotinin (6.5 kDa). The molecular
weight of the standard proteins and their migration distance were
used to make a standard curve to estimate the molecular weight of
the protein samples. The electrophoresis of gels was carried out at a
constant voltage (120 V) for approximately 1.5 h. A mixture of methanol
(20% v/v) and acetic acid (10% v/v) was used to fix gels for 10 min. The
gels were stained by Coomassie Brilliant Blue R-350 followed by
destaining with the fixing solution.
2.5. RP-HPLC
RP-HPLC was performed by following the protocol reported by
Alli, Gibbs, Okoniewska, Konishi, and Dumas (1993) with a few
modifications to monitor the profiles of casein–phenolic complexes.
The system used was equipped with a programmable solvent module
(Model 126, Beckman Coulter, Indianapolis, IN, USA) for completing
the delivery of solvents and a programmable detector module (Model
166, Beckman Coulter) for detecting the absorbance of the eluted
fractions. Samples were prepared by mixing 200 μl casein standard,
casein control, phenolic controls, casein–phenolic mixtures with
800 μl sample buffer (0.1% trifluoroacetic acid (TFA) in 10% acetonitrile
solution) and then filtered through an acetate membrane filter
(0.45 μm, GE, Piscataway, NJ, USA). Solvent A and B were used to
generate a gradient elution system with solvent A composed of 0.1%
trifluoroacetic acid (TFA) in deionized water and solvent B composed
of 0.1% trifluoroacetic acid (TFA) in acetonitrile/deionized water (7:3).
Samples (100 μl) were injected into the system by a manual injector
through a 100 μl loop. Separation of fractions was done through an
Eclipse XDB C18 reversed phase column (5 μm, 4.6 × 250 mm; Agilent,
Santa Clara, CA, USA). The fractions of samples were eluted at a constant
flow rate of 0.5 ml/min with the following gradient: starting at 10%
solvent B and increasing to 70% in 30 min, followed by holding at 70%
solvent B for 20 min, and returning to 10% solvent B in 10 min.
Chromatographic data were analyzed by the Gold System (Beckman
Coulter).
 S. Zhou et al. / Food Research International 74 (2015) 177–184
2.6. Tryptic hydrolysis of casein–phenolic complexes
Tryptic hydrolysis of casein–phenolic complexes was performed to
simulate the conditions of human digestive system using the methods
described by Adebiyi, Adebiyi, Ogawa, and Muramoto (2008) and
Antila et al. (1991) with modifications. Trypsin solution was made
immediately before use by dissolving 2.5 mg of trypsin in 1 ml
phosphate buffer (10 mM; pH 8.0). Sample solutions (2.5 mg casein/
ml) were adjusted to pH 8.0 with 1N NaOH solution. Trypsin solution
was added into each sample solution (trypsin to casein ratio of 1:20)
followed by mixing. The mixtures were incubated (water bath at
37 °C, 3 h) and the reaction was stopped by heating in boiling water
for 10 min. A portion (50 μl) was taken from each enzyme–substrate
mixture at 30 min intervals during the reaction for the measurement
of the degree of hydrolysis.
3. Results and discussion
3.1. Total phenolic content
The total phenolic contents of chocolate samples and of isolated
caseins are summarized in Supplementary Table S1. The total
phenolic contents of milk chocolate and white chocolate were
1.91 ± 0.03 mg/g of chocolate and 1.68 ± 0.06 mg/g of chocolate,
respectively, which are similar to those reported by Meng, Jalil,
and Ismail (2009) (total phenolic contents of 1.61 ± 0.07 mg/g of
chocolate and 1.26 ± 0.08 mg/g of chocolate, respectively)
and their results for total phenolic content varied for different
commercial chocolate samples. Total phenolic contents of milk
chocolate and white chocolate were higher than the defatted
chocolate and the caseins isolated from chocolate suggesting
that some fat soluble phenolic compounds were removed
after the defatting process. The total phenolic content of white
chocolate was much lower than that of milk chocolate which is
similar to previously reported results (Grassi, Lippi, Necozione,
Desideri, & Ferri, 2004; Meng et al., 2009). The lower phenolic
content of white chocolate is due to the fact that white chocolate
is made from cocoa butter, an extract from cocoa liquor with
less phenolic compounds, whereas milk chocolate is made from
cocoa liquor (Meng et al., 2009). No phenolic acid was detected in
the casein isolated from white chocolate; this suggests that
the phenolic compounds in white chocolate are mainly associated
with the cocoa butter and with other ingredients rather than
with casein.
Fig. 1. a) Native-PAGE of caseins incubated at 55 °C (pH 7, 2 h). STD: Standard protein markers; (1) casein control (incubated without phenolic acid); (2) casein–protocatechuic acid
complex; (3) casein–p-coumaric acid complex. b) Native-PAGE of caseins isolated from milk chocolate and white chocolate. STD: Standard protein markers; (1) casein standard (no
incubation); (2) casein control (incubated without phenolic acid); (3) casein isolated from milk chocolate; (4) casein isolated from white chocolate.
179
3.2. Identification of casein–phenolic complexes
Three major bands (I, II, III) were observed from casein and casein
incubated with protocatechuic acid and p-coumaric acid on the
native-PAGE (Fig. 1a). The bands of casein–protocatechuic acid
showed relatively lower migration distance than the casein control
demonstrating a slight increase in weight of casein incubated
with protocatechuic acid. On the other hand, no difference in the
migration distance of the bands between casein–p-coumaric acid
and the casein control could be observed (Supplementary Table S2).
Two bands with migration distances of 0.08 cm and 0.50 cm
were obtained from casein isolated from milk chocolate (Fig. 1b, lane
3 A1, A2, respectively) and two bands with migration distances of
0.08 cm and 0.60 cm were obtained from casein isolate isolated from
white chocolate (Fig. 1b, lane 4 A1, A2, respectively); these bands
were different from the casein standard and the casein control and
suggest that other compounds in milk chocolate and white chocolate
are bound to casein fractions. In addition, the structural changes
occurring in caseins during chocolate processing steps might have
caused the difference in migration distance as compared to the casein
control. The migration distances of the other major bands of casein
isolated from white chocolate (Fig. 1b, lane 4 B and C) are identical to
that of the casein standard and the casein control. The two major
casein bands isolated from milk chocolate (Fig. 1b2, lane 3 B and
C) demonstrated shorter migration distance (3.20 cm and 5.70 cm,
respectively) than those of the casein standard and the casein control.
This difference in migration distance between casein isolated from
milk chocolate and the casein control may be due to the effect of
binding interactions between casein and phenolic compounds in
chocolate (Natsume et al., 2000). Since the total phenolic content of
casein isolated from milk chocolate is higher than that of casein
isolated from white chocolate, the casein–phenolic interactions are
greater in casein isolated from milk chocolate.
The molecular weights of α-, β-, and κ-caseins calculated from
the SDS-PAGE electrophoretogram (Supplementary Fig. S1) are
32.0 kDa, 30.1 kDa and 28.2 kDa respectively, which are close to
those mentioned in the literature (Molina, 2006; Stewart, Willis, &
Mackinlay, 1984). Three major bands (A, B, C) for α-, β-, and κ-caseins
were observed from casein and casein incubated with protocatechuic
acid and p-coumaric acid on the SDS-PAGE electrophoretogram
(Fig. 2a). Casein–protocatechuic acid complex showed losses in band
intensities of α-, β- and κ-casein bands (Fig. 2a, lane 2 A, B, C) and the
appearance of three slower migration bands (Fig. 2a, lane 2 A1, A2, A3),
which are aggregation bands of casein. The presence of aggregation
bands was less significant in the casein control and the casein-p-
180 S. Zhou et al. / Food Research International 74 (2015) 177–184
Fig. 2. a) SDS-PAGE of caseins incubated at 55 °C (pH 7, 2 h); STD: Standard protein markers, (1) casein control (incubated without phenolic acid), (2) casein–protocatechuic acid
complex, (3) casein–p-coumaric acid complex. b) SDS-PAGE of caseins isolated from milk chocolate and white chocolate; STD: Standard protein markers, (1) casein standard (no
incubation), (2) casein control (incubated without phenolic acid), (3) casein isolated from white chocolate, (4) casein isolated from milk chocolate.
coumaric acid (Fig. 2a, lanes 1 and 3, respectively) as compared to
the casein–protocatechuic acid which indicates that protocatechuic
acid favored the aggregation of casein subunits by protein–phenolic
interactions. Although larger and more hydrophobic phenolic com-
pounds have demonstrated stronger binding to proteins (Baxter
et al., 1997), the higher ability of the smaller protocatechuic acid to
aggregate caseins as compared to p-coumaric acid could be due to
the higher molar concentration of the protocatechuic acid added
(6.4 mM) as compared to p-coumaric acid (6.1 mM). It has been
demonstrated that as the concentration of phenolic compounds
increases, the phenolic compounds complexed onto the protein
surface cross-link different molecules leading to aggregation
(Charlton et al., 2002). In addition, the extra hydroxyl group found
on protocatechuic acid may have provided stronger phenolic acid-
protein interactions. Among the five potential types of interactions
between phenolic compounds and proteins including hydrogen
bonding, π-bonding, and hydrophobic, ionic, and covalent linkages,
Scheme 1. Possible hydrophobic interactions and hydrogen bonding between proteins
and (A) p-coumaric acid or (B) protocatechuic acid adapted from Santos-Buelga and de
Freitas (2009).
hydrophobic interactions and hydrogen bonding (Scheme 1) have
been identified as the most important ones (Bianco, Chiacchio,
Rescifina, Romeo, & Uccella, 1997; Hagerman, 1992; Murray &
Williamson, 1994; Santos-Buelga & de Freitas, 2009).
The migration of α-, β- and κ-casein bands was similar between
the casein standard, the casein control and the caseins isolated
from milk chocolate and white chocolate (Fig. 2b). The protein
profile of the casein isolated from white chocolate (Fig. 2b, lane
3) is similar to those of the casein standard (Fig. 2b, lane 1) and the
casein control (Fig. 2b, lane 2), while one band with relatively large
molecular weight was obtained in the casein extracted from milk
chocolate (Fig. 2b, lane 4); this suggests that the interactions
between caseins and phenolic compounds in milk chocolate
(Supplementary Table S1) and the structural changes of caseins
that occur during the additional processing steps required for milk
chocolate, such as refining, conching, and tempering, favored the
aggregation of caseins as compared to white chocolate.
The three major peaks from the RP-HPLC chromatograms of the
casein standard and the casein control (Supplementary Fig. S2 A
and B, peaks 1, 2, 3) were assigned as the κ-, α- and β-casein
fractions respectively according to the literature (Molina, 2006).
Although the retention times of the peaks between the casein
standard and the casein control did not change, there was a slight
thickening of the peaks and the appearance of a new peak at
35 min which demonstrates a slight structural change/aggregation
of incubated caseins. The first peak on the chromatograms of
casein–protocatechuic acid complex (Fig. 3a1) and casein–p-
coumaric acid complex (Fig. 3b1) was the protocatechuic acid
fraction and the p-coumaric acid fraction, respectively. Peak 2 of
casein–protocatechuic acid complex (Fig. 3a2) was the casein
fraction, and peaks 2 and 3 of casein–p-coumaric acid complex
(Fig. 3b2, 3) were α-casein and β-casein. As compared to the
RP-HPLC chromatogram of the casein control (Supplementary
Fig. S2B), the casein profile of casein–protocatechuic acid (Fig. 3a2)
did not show the separation of α- and β-caseins which may be due
to the interactions between protocatechuic acid and β-caseins
leading to a decrease and shift of the β-casein peak as compared to
the control. Gallo, Vinci, Graziani, De Simone, and Ferranti (2013)
showed that caseins incubated with polyphenols did not show any
stable adduct through MALDI-TOF-MS which demonstrates the tran-
sient and weaker nature of these interactions.
 S. Zhou et al. / Food Research International 74 (2015) 177–184 181

Fig. 3. a) RP-HPLC chromatograms of casein–protocatechuic acid complex; (1) protocatechuic acid, (2) casein. b) RP-HPLC chromatograms of casein–p-coumaric acid complex; (1) p-
coumaric acid, (2) α-casein, (3) β-casein. c) RP-HPLC chromatograms of casein isolated from milk chocolate; (1) κ-casein, (2) α-casein, (3) β-casein. d) RP-HPLC chromatograms of casein
isolated from white chocolate; (1) κ-casein, (2) β-casein.

Three peaks obtained from the chromatogram of casein isolated
from milk chocolate (Fig. 3c1, 2, 3) are comparable to the ones
from the casein control (Supplementary Fig. S2B) with similar
retention times and they were assigned as the κ-, α- and β-casein
fractions, respectively. The less defined shape of the peak 1 and
peak 3 of the caseins isolated from milk chocolate as compared to
the casein controls demonstrate the structural changes of κ- and β-
casein that occurred during processing. Only two main peaks were
observed from the chromatogram of casein isolated from white
chocolate (Fig. 3d1, 2). The co-elution of α- and β-casein fractions
isolated from white chocolate also demonstrate the structural
changes of these proteins that occurred during processing.
3.3. Effect of casein–phenolic interactions on the degree of hydrolysis
Tryptic hydrolysis was carried out to study the effect of casein–
phenolic complexes on the digestion by trypsin. As compared to
the unhydrolyzed samples (Fig. 4a, lanes 2, 4, 6 and b lanes 2, 4, 6),
the α-, β- and κ-casein bands of the hydrolyzed casein, hydrolyzed
casein–phenolic complexes (Fig. 4a, lanes 3, 5 and 7) and hydrolyzed

Fig. 4. a) SDS-PAGE of caseins incubated at 55 °C (pH 7, 2 h) and casein hydrolysates; STD: Standard protein markers, (1) casein standard (no incubation), (2) casein control
(incubated without phenolic acid), (3) hydrolysate of casein control, (4) casein–protocatechuic acid, (5) hydrolysate of casein–protocatechuic acid complex, (6) casein–p-coumaric
acid, (7) hydrolysate of casein–p-coumaric acid complex. b) SDS-PAGE of casein isolated from chocolate and its hydrolysate; STD: Standard protein markers, (1) casein standard (no
incubation), (2) casein control (incubated without phenolic acid), (3) hydrolysate of casein control, (4) casein isolated from milk chocolate, (5) hydrolysate of casein isolated from
milk chocolate, (6) casein isolated from white chocolate, (7) hydrolysate of casein isolated from white chocolate.
182 S. Zhou et al. / Food Research International 74 (2015) 177–184
caseins from milk chocolate and white chocolate (Fig. 4b, lanes 5 and
7) disappeared and were converted into subunits of lower molecular
weight on the SDS-PAGE electrophoretograms, which confirmed
the tryptic hydrolysis of caseins. Native casein has demonstrated
resistance against complete digestion without decomposition
(Bican, 1983) and β-casein has demonstrated to be the most
digestible among caseins due to its particularly open conformation
(Swaisgood, 1993).
The highest degree of hydrolysis of casein control within 3 h was
19.3 ± 0.8% (Supplementary Fig. S3A) which is similar to the results
reported by Adamson and Reynolds (1997) where a degree of
hydrolysis of 21% after 130 min was obtained. The highest degree
of hydrolysis of casein–protocatechuic acid, casein–p-coumaric
acid, caseins isolated from milk chocolate and white chocolate
were 18.6 ± 0.2%, 17.7 ± 0.4%, 10.4 ± 0.4% and 17.8 ± 0.6%
respectively. The degree of hydrolysis profiles of casein control
(Supplementary Fig. S3A) and casein–protocatechuic acid (Supple-
mentary Fig. S3B) was similar; however, a lower maximum degree
of hydrolysis was obtained for casein–protocatechuic acid complex.
The degree of hydrolysis profile of casein–p-coumaric acid (Supple-
mentary Fig. S3C) was significantly different from that of casein
control where an initial lowering effect of p-coumaric acid on tryptic
hydrolysis of casein was observed; this may due to the change in
conformation of caseins upon complex formation with p-coumaric
acid (Hasni et al., 2011) leading to a decrease in the accessibilty of
the cleavage sites. These results are comparable to the results
reported by Wehr (1973) where the tryptic digestibility of bovine
serum albumin decreased significantly after incubating with
polyphenol oxidase and catechol. As compared to the degree of
Fig. 5. a) RP-HPLC chromatograms of hydrolysate of casein control (A), hydrolysate of casein–protocatechuic acid complex (B) and hydrolysate of casein–p-coumaric acid complex (C).
b) RP-HPLC chromatogram of (A) hydrolysate of casein control, (B) hydrolysate of casein isolated from milk chocolate and (C) hydrolysate of casein isolated from white chocolate.
hydrolysis profile of the casein control (Supplementary Fig. S3A),
the degree of hydrolysis of the casein isolated from milk chocolate
was lower (Supplementary Fig. S3D), which suggests an inhibitory
effect of phenolic compounds in milk chocolate and also of casein
aggregation occurring during processing on the tryptic hydrolysis
of casein. On the other hand, the degree of hydrolysis of casein iso-
lated from white chocolate was similar (Supplementary Fig. S3E) to
the casein control (Supplementary Fig. S3A), which may be due to
the absence of phenolic compounds in the caseins isolated from
white chocolate (Supplementary Table S1) and also due to the
lower number of processing steps involved in its production as
compared to milk chocolate.
As compared to the chromatogram of the casein control (Supple-
mentary Fig. S2B), several new peaks, which are the peptides
obtained after tryptic hydrolysis, appeared on the chromatogram of
the casein control hydrolysate (Fig. 5a A) in the retention time
range of 20 min to 35 min. The retention times of peaks 1 to 12
(Fig. 5a II) of the hydrolysates of casein–protocatechuic acid
(Fig. 5a B) and casein–p-coumaric acid (Fig. 5a C) showed no
difference as compared to the peptide profiles of the casein control
(Fig. 5a A); however, the retention times of peaks in the range of
15 min to 24 min (Fig. 5a I) were different. Two peaks with retention
times of 6.8 min and 9.9 min, which were absent from the chromato-
gram of the hydrolysate of the casein control (Fig. 5a A), appeared on
the chromatogram of casein–protocatechuic acid hydrolysate
(Fig. 5a B). Similarly, three peaks with retention times of 20.0 min,
22.0 min and 23.2 min that were absent from the chromatogram of
the casein control hydrolysate (Fig. 5a A), were observed on the
chromatogram of casein–p-coumaric acid hydrolysate (Fig. 5a C).
 S. Zhou et al. / Food Research International 74 (2015) 177–184
Phenolic compounds have been reported to decrease the digesti-
bility of proteins by acting as an enzyme inhibitor (Suman,
Monteiro, Ramachandra, & Sudharshana, 1992), in addition to con-
tributing towards the complex formation between phenolic com-
pounds and proteins which can sterically hinder the access of the
digestive sites on the proteins by trypsin (Bican, 1983). This can
lead to differences in digestibility thus leading to the changes in
the elution profile of phenolic acid–casein hydrolysates. The poten-
tial peptides that can be generated from the tryptic digestion of β-
casein are presented in Supplementary Table S3. Proline, the major
amino acid involved in the protein–phenolic compounds interaction
(Charlton et al., 2002), constitute 15% of all amino acids found on β-
casein; however, 60% of all prolines on β-casein are found on two po-
tential peptides that can be generated from tryptic digestion (pep-
tide #8 and 13). This suggests that the phenolic acid–protein
interaction found around the potential cleavage sites leading to
these peptides are stronger which may lead to protein aggregation
resulting in a decrease in the accessibility of these cleavage sites to
trypsin.
The retention times of the peptides obtained after tryptic hydro-
lysis of the casein control were in the 20 to 35 min range (Fig. 5b A),
and those of the casein isolated from milk chocolate and white
chocolate were located in the 24 to 35 min range (Fig. 5b B) and in
the 22 to 37 min range (Fig. 5b C), respectively. This change in
retention time may be due to the decrease in the degree hydrolysis
caused by the structural changes/aggregation that occurred during
processing resulting in larger peptides as compared to the casein
control hydrolysate. In particular, the significant increase in reten-
tion time of the peptides from casein isolated from milk chocolate
(Fig. 4b B) may suggest an effect of casein–phenolic interactions
and of extra processing steps on the structural changes of the protein
(Hasni et al., 2011) and the decrease in the digestibility of casein
isolated from milk chocolate.
4. Conclusions
Casein–phenolic interactions were induced by heat incubation
and protocatechuic acid showed greater interaction with casein
than p-coumaric acid leading to protein aggregation. Protocatechuic
acid and p-coumaric acid affected the degree of hydrolysis of caseins
and the peptide profiles of the hydrolysates. The caseins isolated
from milk chocolate demonstrated more aggregation and resistance
to digestion as compared to white chocolate suggesting the effect of
casein–phenolic interactions and extra processing steps on the
structure of caseins. These results will contribute towards a better
understanding of the behavior and characteristics of caseins within
a food matrix and the effect of casein–phenolic interactions on the
digestibility of caseins and the bioavailability of phenolic acids.
Additionally, in future research, appropriate mass spectrometric
methods would be helpful to unravel structural information on the
enhancement of caseins–phenolic interactions in a complex food
matrix system.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.foodres.2015.05.006.
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