Food Chemistry 141 (2013) 38373845

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Biological activities and physicochemical properties of Maillard reaction

products in sugarbovine casein peptide model systems
Zhanmei Jiang a,, Lizhe Wang b, Wei Wu a, Yu Wang a
a
b

Key Laboratory of Dairy Science, Ministry of Education, Northeast Agricultural University, Harbin 150030, PR China
Department Environmental and Agro-Biotechnologies, Centre de Rechereche Public Gabriel Lippmann, 41 rue du Brill, 4422 Belvaux, Luxembourg

a r t i c l e

i n f o

Article history:
Received 23 November 2012
Received in revised form 3 May 2013
Accepted 11 June 2013
Available online 20 June 2013
Keywords:
Maillard reaction products
Bovine casein peptide
ACE inhibitory activity
Antioxidant activity
Antiproliferative activity

a b s t r a c t
The purpose of this study was to evaluate the biological activities and physicochemical properties of
Maillard reaction products (MRPs), derived from aqueous reducing sugar (ribose, galactose and lactose)
and bovine casein peptide (BCP) model systems. The uorescence intensity of ribose-BCP MRPs reached
the maximum value within 1 h, while it took 3 h for galactose-BCP MRPs. Size exclusion chromatography
of all the MRPs indicated molecular rearrangements and production of new smaller molecules, as a function of the heating time. The consumption of ribose and amino groups was the highest in the ribose-BCP
MRPs. BCP lost its known angiotensin-I-converting enzyme (ACE) inhibitory activity by the Maillard reaction with reducing sugars. RiboseBCP MRPs had the lowest ACE inhibitory activity, but they showed the
highest 2,2-diphenyl-1-picryl-hydrazyl (DPPH) radical scavenging activity and ferrous reducing power
among all the MRPs. Galactose-BCP MRPs inhibited, slightly the growth of Caco-2 cells, while riboseBCPand lactose-BCP MRPs had no cytotoxicity.
Crown Copyright 2013 Published by Elsevier Ltd. All rights reserved.

1. Introduction
The Maillard reaction is a complex chemical reaction between
carbonyl groups of reducing sugars and free amino groups, mainly
from amino acids, peptides and proteins (Hodge, 1953). The Maillard reaction usually occurs during processing and storage of various foods and forms Maillard reaction products (MRPs), which are
largely responsible for the physicochemical properties and health
effects of foodstuffs. Many scientic studies have focused on the
benecial health effects of the MRPs present in foods. For instance,
indigestible melanoidins produced by the Maillard reaction were
benecial to human health, displaying in vivo antioxidant, antimicrobial and prebiotic activity in the intestine (Wang, Qian, & Yao,
2011). It was also shown that thermally processed foods rich in
MRPs could have a benecial impact on ex-vivo low-density lipoproteins (LDL) oxidation (Dittrich et al., 2009). In vitro studies indicated that MRPs might offer health-promoting activities such as
metal chelating (Wijewickreme & Kitts, 1998) and radical scavenging properties (Hwang, Kim, Woo, Lee, & Jeong, 2011). However,
the Maillard reaction leads to the formation of toxic MRPs and an
overall decrease in the nutritional value of foods (Vagnarelli, Desario, Cuzzoni, Mazza, & Decarli, 1991). Some endogenous MRPs
formed in biological systems, have been associated with diabetes,
cardiovascular diseases and renal diseases (Tessier & Birlouez Corresponding author. Tel.: +86 451 55190459; fax: +86 451 55190577.
E-mail address: zhanmei.jiang@gmail.com (Z. Jiang).

Aragon, 2012). Therefore, the evaluation of MRPs with their benecial or harmful biological properties is fundamental to the production of safe foods and to the development of novel functional
food ingredients.
Although the Maillard reaction involving amino acids has been
extensively explored (Ajandouz & Puigserver, 1999; Brueckner,
Justus, & Kirschbaum, 2001; Zeng, Zhang, Guan, & Sun, 2011), only
a few studies have investigated the MRPs from peptide-sugar model systems and most of these works were focused on the kinetics,
functional properties and antioxidant activities. A temperature effect on peptide degradation and peptide cross-linking often occurs
during the Maillard reaction (Lan et al., 2010). Decourcelle, Sabourin, Dauer, and Guerard (2010) reported that the Maillard reaction
with xylose improved the emulsifying properties of a shrimp
hydrolysate (Pandalus borealis). Functional properties of soy protein hydrolysate were also enhanced by conjugation with curdlan
(Fan et al., 2006). Antioxidative activity of hydrolysates from casein
and sh was improved by 2030% when reacted with glucose
(Guerard & Sumaya-Martinez, 2003). Radical scavenging activity
of a porcine haemoglobin hydrolysatesugar model system was
about four times higher than that of porcine haemoglobin hydrolysate (Sun & Luo, 2011). All the above investigations showed positive effects of the Maillard reaction on bioactive peptides.
Although the functional properties and antioxidant activities of
bioactive peptides were improved by the Maillard reaction, there
is no evidence that the Maillard reaction caused certain functional
peptides to enhance or keep other biological activities.

0308-8146/$ - see front matter Crown Copyright 2013 Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2013.06.041

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Z. Jiang et al. / Food Chemistry 141 (2013) 38373845

Bovine casein peptides are shown to have biological properties,

such as antihypertensive activity (Jiang, Tian, Brodkorb, & Huo,
2010), antibacterial activity (Lopez-Exposito, Minervini, Amigo, &
Recio, 2006), antioxidative activity (Suetsuna, Ukeda, & Ochi,
2000) and opioid antagonist activity (Pihlanto-Leppala, Antila,
Mantsala, & Hellman, 1994).These biological properties make them
widely applied as functional ingredients in many processed foods.
In this study, bovine casein peptides were validated with strong
antihypertensive activity (Jiang et al., 2010). As food products are
generally complex, including proteins or peptides and carbohydrates, the Maillard reaction is one of the most frequent reactions
occurring during heat treatment (Jaeger, Janositz & Knorr, 2010).
Though functional foods contain bioactive ingredients with desired
health benets, their stability and bioactivity throughout processing, storage and gastrointestinal transit are of utmost importance.
Nevertheless, it is not certain whether the Maillard reaction has
negative or positive effects on the biological activity of bovine
casein peptides in food production, processing, storage and distribution. Furthermore, sugar type is one of the numerous factors
controlling the Maillard reaction rate, and its choice is critical because it will remarkably impact the biological activities or functional properties of the MRPs. Therefore, the biological activity,
structure and characteristics of bovine casein peptides and their
possible MRPs with three types of reducing sugars, were examined
as model systems in this study. The results could provide indicative
data for the food industry on how the sugar type undergoing the
Maillard reaction inuences bioactivities and characteristics of bovine casein peptides.
2. Materials and methods
2.1. Materials
Bovine casein was prepared from bovine milk using isoelectric
precipitation developed by the Key Laboratory of Dairy Science,
Ministry of Education, Northeast Agricultural University (Harbin,
Heilongjiang province, China). AS1.398 neutral protease (from
Bacillus subtilis; activity 50,000 U/g) was purchased from Wuxi Enzyme Preparation Company (Wuxi, China). Caco-2 cells (TCHu146),
a human colon adenocarcinoma cell line, was purchased from
Cell Center of Institutes for Biological Sciences of Chinese
Academy of Sciences, Shanghai, China. Hippuryl-L-hisstidyl-L-leucine (Hip-His-Leu), angiotensin-I-converting enzyme (ACE, EC
3.4.15.1), hippuric acid (HA), D-ribose, D-galactose, a-lactose, OPA
(o-phthaldialdehyde), L-leucine, potassium ferricyanide (III)
powder, 2,2-diphenyl-1-picryl-hydrazyl (DPPH), sodium dodecyl
sulphate (SDS), ribonuclease, bacitracin, MSH tetrapeptide,
Leu-Try-Met-Arg, bradykimin, Tyr-Glu, Leu-Phe, Asp-Glu, L-leucine
and triuoroacetic acid (TFA) were purchased from SigmaAldrich
Co. (St. Louis, MO, USA). Other reagents were of analytical grade.
2.2. Preparation of bovine casein peptides
Bovine casein peptides were prepared by the method of Jiang,
Tian, Brodkorb, and Huo (2010). Casein (75 g/L), was dissolved in
pH 7.0, 0.04 mol/L phosphate buffer in a batch reactor. Hydrolysis
was initiated by addition of the AS1.398 neutral protease at 45 C
and the enzyme concentration was xed to 5% (w/w protein).
The pH values of the hydrolysis system were kept at 7.0 by continuous addition of 1 mol/L NaOH using a pH-stat (Mettler Toledo Co.,
Ltd., Shanghai, China). After 6 h of the hydrolysis reaction, the degree of hydrolysis (DH) was 24.93%. Then the hydrolysate samples
were heated at 95 C for 20 min to inactivate the protease. The protein hydrolysate was centrifuged at 4000g for 20 min at 4 C. Subsequently, the supernatant was ultraltrated through a 3 kDa

molecular weight cut-off membrane (Millipore Co., Billerica, MA),

at 35 C under a pressure of 30 bar. The obtained permeates were
collected and then lyophilised to obtain the bovine casein peptides.
2.3. Preparation of Maillard reaction products (MRPs)
Maillard reaction products (MRPs) were prepared by dissolving
casein peptides (30 g/L) and one of three selected sugars (ribose,
galactose or lactose, 60 g/L) in Milli-Q water. The initial pH values
of the solutions were adjusted to 8.0 using 6 mol/L NaOH. The solutions were then transferred to sealed screw-top glass tubes and
heated in a water bath at 95 C. The samples were quenched in
ice water at intervals of 0, 0.5, 1, 2, 3, 4 and 5 h, respectively. Control samples were prepared with casein peptides (30 g/L) heated
without any sugar, or with added ribose, galactose or lactose.
2.4. Determination of browning and UV-absorbance
Browning intensities and UV-absorbance of sugarBCP MRPs
and casein peptide samples were assessed by absorbance readings
at 420 and 294 nm, against water, using a UVVisible spectrophotometer (UV-2401PC, Shimadzu Co., Ltd., Japan). Samples were diluted to 12-fold and 120-fold with Milli-Q water for browning
intensities and UV-absorbance analysis, respectively.
2.5. Determination of uorescence intensity
The uorescence intensity of sugarBCP MRPs and casein peptide samples was determined at the excitation wavelength of
347 nm and emission wavelength of 420 nm using a F-4500 uorescence spectrometer (Hitachi Co., Ltd., Japan). Both excitation
and emission slits were set to 10 nm. The scan rate was 120 nm/
min.
2.6. Determination of free amino group and reducing sugar
Free amino groups were quantied by the OPA method. 100 ll
sample (12-fold dilution) was mixed with 3 ml of OPA reagent.
After vortexing and a minimal 5 min delay in darkness at room
temperature, the absorbance was recorded at 340 nm against
OPA reagent. Calibration curves were obtained by using 00.5 g/L
leucine as a standard and absorbance readings were converted into
free amino group content. A blank reading was determined in the
same manner, except that Milli-Q water was used instead of the
samples. The changes in free amino groups were expressed as relative concentrations (%) in comparison with the content of nonheated samples.
The free ribose, galactose and lactose of the samples were measured with a HPLC system (Waters 2695 Alliance, Waters, USA)
equipped with a high performance carbohydrate column
(4.6  250 mm, 4 lm particle size, Shiseido Co., Ltd., Japan). The
column temperature was 30 C and 30 ll of sample were injected
into the HPLC system. The mobile phase was 70% acetonitrile and
eluted at a ow rate of 1 ml/min. A 2414 refractive index detector
(Waters, USA) was used to detect samples eluting from the column.
The data analysis was performed using Empower Chemstation
software.
2.7. Molecular weight measurement of MRPs
The Molecular weight distribution proles of sugarBCP MRPs
and casein peptide samples were estimated by size exclusion chromatography (SEC) using a series connection column with TSK G
2000 SW (7.5 mm  60 cm, 10 lm, Japan) tted to a TSK guard column (7.5 mm  7.5 cm). 20 ll aliquot samples were injected at a
concentration of 2.5 g/L. The ow rate of the eluent (30%

Z. Jiang et al. / Food Chemistry 141 (2013) 38373845

acetonitrile in 0.1%TFA Milli-Q water) was set at 0.5 ml/min. The

absorbances were monitored at a wavelength of 214 nm. The ten
standards used were bovine serum albumin (67 000 Da),
b-lactoglobulin (36 000 Da), ribonuclease (13 700 Da), bacitraci
(1423 Da), MSH tetrapeptide (764.84 Da), Leu-Try-Met-Arg
(604.77 Da), bradykimin (368.44 Da), Tyr-Glu (310.31 Da),
Leu-Phe (278.35 Da) and Asp-Glu (262.22 Da) as MW calibration
standards (log MW = 0.1347X + 7.9742, with retention time, expressed in minutes, R2 = 0.9851).
2.8. ACE inhibitory activity
The ACE inhibitory activity assay was carried out by HPLC, by a
modication of the method of Jiang et al. (2010). Hip-His-Leu
(5.0 mmol/L) was dissolved in 50 mM sodium borate buffer (pH
8.3) containing 0.5 mol/L NaCl. A mixture containing 120 ll HHL
solution and 20 ll of sample solution was pre-incubated at 37 C
for 5 min, 10 ll of ACE solution (100 Units/L) was then added
and the mixture was further incubated at 37 C for 60 min. The
reaction was interrupted by the addition of 150 ll of 1 mol/L
HCl. Hippuric acid liberated by ACE was determined by RP-HPLC
onto a Yilite SinoChrom ODS-BP C18 column (4.6 mm  250 mm,
5 lm, Dalian, China) at 30 C. Samples were ltered through
0.45 lm lters and 50 ll was injected. A linear gradient from 20
to 70% acetonitrile containing 0.1% TFA was applied over 10 min.
The concentrations of 700% acetonitrile containing 0.1% TFA were
reached within 2 min. The detection wavelength was set at 228 nm
and the ow rate was held at 1 ml/min. The percentage of ACE
inhibitory activity was calculated as (A0  A1)/A0  100%, where
A0 represents the hippuric acid without sample and A1 represents
the hippuric acid with sample.
2.9. Determination of ferrous reducing power
The ferrous reducing power of sugarBCP MRPs and casein
peptide samples was determined according to the method of
Jiang and Brodkorb (2012), with a slight modication. Samples
of 200 ll aliquot were diluted to 12-fold, and then mixed with
500 ll of 0.2 mol/L sodium phosphate buffer (pH 6.6) and
500 ll of 1% potassium ferricyanide. The mixtures were incubated in a water bath at 50 C for 20 min, followed by the addition of 500 ll of 10% trichloroacetic acid. The mixtures were
then centrifuged at 5,000 g for 10 min, and 500 ll of supernatant
was blended with 500 ll of Milli-Q water and 100 ll of 0.1%
FeCl3. After the sample rested for 10 min, its absorbance was
measured at 700 nm. A higher absorbance of the reaction mixture indicated greater reduction power. The results were expressed as absorbance units.
2.10. 2,2-Diphenyl-1-picryl-hydrazyl (DPPH) radical scavenging
activity
The DPPH radical scavenging activity of sugarBCP MRPs and
casein peptide samples was estimated according to the method
of Yen and Hsieh (1995) with a slight modication. The MRPs of
250 ll aliquot were added to a 1 ml solution of DPPH, prepared
fresh daily, at a concentration of 0.1 mmol/L in ethanol. The reaction solution was then shaken vigorously and allowed to stand in
darkness at room temperature for 30 min. The mixture was centrifuged at 4000g for 10 min. The absorbance of the supernatant of
mixtures was recorded at 517 nm. The percentage of DPPH radical
scavenging activity was calculated as follows:

DPPH radical scavenging activity %

Ablank  Asample  Acontrol =Ablank  100%

3839

where Ablank is the absorbance of ethanol added instead of samples,

Asample is the absorbance of samples and Acontrol is the absorbance of
ethanol added instead of 0.1 mmol/L DPPH.
2.11. Cell viability assays
The effects of sugarBCP MRPs and casein peptide samples on
cell death and cell proliferation were determined using an MTT assay. Caco-2 cells were cultured in Dulbeccos Modied Eagles
Medium (DMEM) containing 100 IU/ml penicillin, 100 lg/ml streptomycin, and supplemented with 10% fetal bovine serum (FBS).
Cells were plated at a density of 104 cells in 96-well plates per
100 ll medium and maintained in a humidied incubator at an
atmosphere of 5% CO2 in air at 37 C for 24 h. When the conuence
of cells was up to 6070%, serial dilutions of the samples (200 ll)
were added into each of the 96-well plates, and the cells were incubated for 48 h. After incubation, 20 ll of MTT solution (5 g/L) were
added to each well for 4 h. Again the supernatants were carefully
removed and 150 ll of dimethylsulphoxide was added into each
well. The absorbance was detected at 490 nm with a microplate
reader (Model 680, Bio-Rad Laboratories, Hercules, CA, USA). The
inhibition of cell relative growth rate by the samples tested was
calculated using the following formula: Asample/Acontrol  100%.
2.12. Statistical analysis
Three independent experiments were performed with preparation of Maillard reaction products in sugarbovine casein peptide
model systems. Analyses, including the determinations of browning, UV-absorbance, uorescence intensity, free amino group,
reducing sugar, ACE inhibitory activity, reducing power, DPPH radical scavenging activity and cell viability assays, were run in triplicate. SEC measurements were carried out in duplicate. One-way
analysis of variance (ANOVA) and independent samples t-test were
conducted to determine the signicance of the main effects using
SPSS (SPSS 13.0 for windows, SPSS Inc, Chicago, IL, USA). Experimental results were expressed as means with standard deviation.
Signicant differences (P < 0.05) among means were identied
using the Duncans multiple range tests and independent samples
t-test.
3. Results and discussion
3.1. Changes in browning and UV-absorbance
Development of a brown colour provides a visual evaluation for
the occurrence of Maillard reaction. The absorbance at 420 nm is
used as an indicator for the browning development in an advanced
stage of the Maillard reaction (Morales & Jimnez-Prez, 2001).
An increase in the browning of sugarBCP MRPs, as measured
by absorbance at 420 nm, was observed as the heating time increased (P < 0.05), as shown in Fig. 1A. When the BCP was heated
alone, the change in absorbance, at 420 nm, was negligible. The
MRPs derived from the Ribose-BCP showed the highest increase
in absorbance at 420 nm, followed by those of the galactose-BCP
and lactose-BCP. Ribose yielded MRPs with a 12-fold higher
browning intensity after 5 h of heating time, when compared to
lactose. Browning intensities of galactose-BCP MRPs were about
4-fold higher than those of lactose-BCP MRPs. Lactose-BCP MRPs
showed the lowest browning intensities (P < 0.05). This result validated that the reactivity of sugars in the Maillard reaction declined in the order of pentoses, hexoses, and disaccharides.
Browning intensity of MRPs derived from procine plasma protein
and reducing sugars increased as the heating time increased, and
MRPs derived from 2% galactose showed a greater increase in

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Z. Jiang et al. / Food Chemistry 141 (2013) 38373845

(A)

that the increase in absorbance at 294 nm in sugarBCP MRPs

formed colourless compounds, which might contribute to the
brown pigment formation in Maillard reactions.

Absorbance (420 nm)

0.9
0.8
0.7

3.2. Changes in uorescence intensity

0.6
0.5
0.4
0.3
0.2
0.1
0
0

Heating time (hours)

(B)

Absorbance (294 nm)

0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0

Heating time (hours)

Fluorescence Intensity (A.U.)

(C)

700
600
500
400

Fluorescence compounds, formed prior to the generation of

brown pigments, have been proposed as early indicators of the
Maillard reaction. The uorescence intensity of the ribose-BCP
MRPs reached a maximum within 1 h, compared to 3 and 5 h for
galacose-BCP and lactose-BCP MRPs, respectively (Fig. 1C). Thereafter, uorescence intensity of ribose-BCP and galactose-BCP MRPs
decreased dramatically during 5 h of heat treatment, while lactoseBCP MRPs showed increasing uorescence after 5 h. However, uorescence intensities of BCP heated alone didnt change during 5 h of
heating time of (P > 0.05). The development of uorescent compounds was found to increase with prolonged heat treatment of
rice protein and glucose (Li et al., 2009). Fluorescence intensity of
glucose-casein and fructose-casein were found also to increase
with prolonged heat treatment, while uorescence intensity of
ribose-casein quickly reached a maximum and then decreased to
a plateau phase (Jing & Kitts, 2002). Fluorescence intensity of the
b-lactoglobulin-ribose and a-lactalbumin-ribose MRPs reached
the maximum within 12 h, followed by a rapid decline (Jiang &
Brodkorb, 2012). These reports suggest that uorescence compounds may increase continuously, reach a plateau value, or show
a maximum with heating time during Maillard reaction. In the
present study, results indicated that the source of the reducing sugar inuenced the uorescent development patterns in the sugarBCP model system. These ndings also conrmed that the type of
reducing sugar determined the Maillard reaction rate of bovine
casein peptides. For instance, ribose was the most active among
three sugars, and ribose-BCP MRPs quickly reached the early stage
of the Maillard reaction.
3.3. Changes in free amino group contents and reducing sugar

300
200
100
0
0

Heating time (hours)

Fig. 1. Changes in browning intensity (A), absorbance at 294 nm (B) and
uorescence (C) of ribose-BCP MRPs (j), galactose-BCP MRPs (), lactose-BCP
MRPs (N) and bovine casein peptides (4) during heat treatment at 95 C for up to
5 h. Error bars represent the mean standard deviation of triplicate experiments.

browning intensity than those from 2% fructose and 2% glucose

(Benjakul, Lertittikul, & Bauer, 2005). Laroque et al. (2008) reported
that the browning development was favoured with pentoses compared to hexoses. Our results suggested that the browning rate was
remarkably inuenced by the type of reducing sugar involved in
the Maillard reaction. Based on browning development, ribose
was the most reactive in the sugarBCP MRPs. In addition to Maillard reaction, caramelisation of reducing sugars might occur, leading to browning of the sugarBCP MRPs.
Absorbance at 294 nm was used to determine the intermediates
of the Maillard reaction (Lerici, Barbanti, Manzano, & Cherubin,
1990). Fig. 1B showed that absorbance at 294 nm increased in
the sugarBCP MRPs as the heating time increased up to 5 h
(P < 0.05). MRPs derived from ribose showed the highest increase
in absorbance at 294 nm, followed by galactose and lactose,
respectively. However, no changes in absorbance at 294 nm were
found in BCP samples heated alone (P > 0.05). The result indicated

To assay amino group availability, free amino group contents of

MRPs during heat treatment were measured using the OPA modied method, as shown in Fig. 2A. While heating a bovine casein
peptides system on its own, 8.5% of free amino groups were lost
after 5 h of heating time as compared to initial peptides
(P < 0.05), due to thermal degradation. A continuous decrease
was observed for amino groups of galactose-BCP and lactose-BCP
MRPs when heated for up to 5 h (P < 0.05), in which about 40.1%
and 27.9% of amino groups were modied by galactose and lactose,
respectively.Chevalier, Chobert, Popineau, Nicolas, and Haertl
(2001a)also validated that the shorter the carbonic chain of the
reducing sugar, the more reactive was the sugar with the amino
groups of b-lactoglobulin. However, free amino groups of
ribose-BCP MRPs decreased when the heating time was at 1 h
and thereafter increased up to 5 h (P < 0.05). For instance, 49.8%
of free amino groups in ribose-BCP MRPs were lost within 1 h of
heating time, and free amino groups signicantly increased from
50.2% to 66.1%, corresponding to 1 h and 5 h of heat treatment,
respectively. This observation was in accordance with Zeng et al.
(2011), who demonstrated that free amino groups in the piscoselysine and fructose-lysine system decreased sharply at rst and
then slightly increased. During the Maillard reaction, the loss of
free amino group content was ascribed to the formation of a
Schiff-base, while the release of free amino groups was probably
attributed to the decomposition of Heyns compounds at the
advanced stage of Maillard reaction (Baisier & Labuza, 1992). This
result also indicates that ribose may possess a higher reactivity
than galactose and lactose in the sugar-BCP systems, during the
Maillard reaction.

Z. Jiang et al. / Food Chemistry 141 (2013) 38373845

Free amino group (%)

(A) 100
80
60
40

Ribose
Glactose

20

Lactose
Control

0
0

Heating time (hours)

Reducing sugar (%)

(B)

100
80
60
40
20
0
0

Heating time (hours)

Fig. 2. Changes in free amino groups (A) of ribose-BCP MRPs (j), galactose-BCP
MRPs (), lactose-BCP MRPs (N) and bovine casein peptides alone (4) during heat
treatment at 95 C for up to 5 h. (B) Reducing sugar consumption in heated
solutions of ribose-BCP MRPs (j), galactose-BCP MRPs (), lactose-BCP MRPs (N),
ribose alone (h), galactose (}) and lactose (4) during heat treatment at 95 C for up
to 5 h. Error bars represent the mean standard deviation of triplicate experiments.

The monitoring of sugar consumption is another means to evaluate the degree of reactivity of sugars during Maillard reaction
(Laroque et al., 2008). The loss of sugars in the ribose-BCP, galactose-BCP and lactose-BCP MRPs and sugars heated alone, as a function of the heating time, is shown in Fig. 2B. No obvious changes
were observed in the concentration of any sugar heated alone
within 5 h of heat treatment (P > 0.05). However, ribose in ribose-BCP MRPs underwent noticeable sugar consumption during
the heat treatment (P < 0.05). The consumption of ribose in ribose-BCP MRPs was the highest, followed by that of galactose in
galactose-BCP and lactose in lactose-BCP MRPs (P < 0.05), respectively. Results showed a maximal 50.5% of remaining ribose when
reacted with bovine casein peptides for 5 h at 95 C, while remaining galactose and lactose were 80.1% and 87.9%, respectively. A
sharp decrease of glucose in all MRPs was observed at 100 C for
up to 240 min of heat treatment (Kim & Lee, 2009). A signicant
ribose consumption of 39.3% could be due to the highest sugar
reactivity, when ribose is involved in Maillard reaction with
b-lactoglobulin (Jiang & Brodkorb, 2012). Sugar reactivity can be
related to the length of the carbon backbone. A shorter carbon skeleton may contribute to the open-chain form of sugar, known as the
active form of sugar in Maillard reaction (Bunn & Higgins, 1981). In
this study, consumption of three different reducing sugars also
indicated different degrees of reactivity during Maillard reaction.

3.4. Evaluation of molecular rearrangements in the sugar-BCP MRPs

Ribose-BCP MRPs (Fig. 3A), galactose-BCP MRPs (Fig. 3B), lactose-BCP MRPs (Fig. 3C) and BCP (Fig. 3D) were monitored at

3841

220 nm by size exclusion chromatography. The molecular weight

distribution of the sugar-BCP MRPs and BCP system could be changed as a function of the heating time during the Maillard reaction.
Similar shapes were conserved and the same peaks were eluted for
the chromatograms of the MRPs or BCP systems. As the heating
time increased, the molecular weight of the ribose-BCP, galactose-BCP and lactose-BCP MRPs showed increasing intensities. This
means that the complex mixes produced during the Maillard reaction, mainly differed in quantitative terms. Furthermore, new compounds detected at 220 nm can also be produced in the progress of
the Maillard reaction with increasing heating time, due to molecular rearrangements of the peptide bonds. In the ribose-BCP MRPs,
molecular rearrangements occurred for large fractions between
3500 and 11,000 Da, estimated by molecular weight calibration.
In comparison, corresponding large fractions between 3500 and
8300 Da and between 3500 and 4900 Da were also produced for
galactose-BCP MRPs and lactose-BCP MRPs, respectively. Conversely, those rearrangements of peptide bonds also affected compounds smaller than 250 Da in all MRPs system. This result
indicated molecular rearrangements and production of new smaller molecules in sugar-BCP MRPs as a function of the heating time.
As is shown in Fig. 3E, ribose-BCP MRPs, galactose-BCP MRPs
and lactose-BCP MRPs exhibited proles, similar to those of the
control samples (bovine casein peptides heated alone) after 5 h
of heat treatment at 95 C. Meanwhile, chromatograms corresponding to the ribose-BCP MRPs were characterised by the strongest absorbance at 220 nm, followed by those of the galactose-BCP
and lactose-BCP MRPs, respectively. Among all of the sugars tested,
ribose showed the highest activity, as most of ribose disappearance
occurred within 3 h of the Maillard reaction (Fig. 2B). The order of
reactivity between all sugars tested was also conrmed by the
chromatographic method.
3.5. ACE inhibitory activity
An ACE inhibition assay was performed to separate and identify
antihypertensive peptides. A modied HPLC method was used to
analyse the ACE inhibitory activities of all sugar-BCP MRPs and
BCP systems. ACE inhibitory activities (Fig. 4) of ribose-BCP, galactose-BCP and lactose-BCP MRPs showed signicant differences during heat treatment (P < 0.05). The ACE inhibitory activity of BCP
alone remained relatively high with a small loss of approx. 5.5%
during heat treatment, probably due to the thermal degradation
of bovine casein peptides. In contrast to this, the ACE inhibitory
activity of ribose-BCP MRPs was lower than that of the other two
MRPs. In particular, 37.9% of ACE inhibitory activity of RiboseBCP MRPs was lost after 1 h of heat treatment (P < 0.05), and thereafter ACE inhibitory activity of Ribose-BCP MRPs was almost
invariable. The ACE inhibitory activities of galactose-BCP and lactose-BCP MRPs were lost with increasing heating time, and galactose-BCP MRPs lost more ACE inhibitory activity than Lactose-BCP
MRPs (P < 0.05). For instance, 28.1% and 10.0% of ACE inhibitory
activity loss was observed for the galactose-BCP and lactose-BCP
MRPs, respectively. This indicated that the lost amounts of ACE
inhibitory activities of sugar-BCP MRPs depended on the sugar
reactivity of the Maillard reaction. Potential antihypertensive
activity of food melanoidins derived from coffee, beer and sweet
wine (Ruan-Henares & Morales, 2007) have been validated by
investigating the in vitro ACE-inhibitory activity. Hwang et al.
(2011) reported that fructosemethionine and glucosemethionine MRPs showed a slightly higher ACE inhibitory activity than
an untreated mixture. However, in the present study, the reactant
bovine casein peptides showed high ACE inhibitory activities. Glycosylation of bovine casein peptides by the Maillard reaction with
a reducing sugar diminished their ACE inhibitory activities. Results
showed that consumption of bovine casein peptides affected the

262.22Da

4.5

0.29

0.24

0.24

Heating
Time

0.19

3.5

0.14
3

Heating
Time

0.09

20

25

30

35

40

45

50

55

60

13700Da

4.5

0.19
3.5
0.14

Heating
Time

0.09

2.5

-0.01

2
20

25

30

0.34

4.5

Absorbance (220 nm )

0.24

0.19
3.5

Heating
Time

Heating
Time

0.09

-0.01
30

35

40

45

50

55

13700Da

60

Heating
Time

3.5

0.14
3

0.09

2.5

-0.01

20

60

25

30

35

40

45

50

55

60

Retention Time (min)

0.34

13700Da

262.22Da

4.5

Heating
Time

0.24
0.19

Ribose-BCP

0.14

3.5

Galactose-BCP

Heating
Time

0.09

20

25

30

35

Lactose-BCP
BCP

0.04
-0.01

262.22Da

0.19

0.29

Absorbance (220 nm)

55

4.5

Retention time (min)

(E)

50

0.04

25

45

0.24

2.5

0.04
20

40

0.29

Log(MW)

Absorbance (220 nm)

(D)

0.29

0.14

35

Retention time (min)

262.22Da

13700Da

0.04

Retention time (min)

(B) 0.34

262.22Da

2.5

0.04
-0.01

0.34

Absorbance (220 nm)

Absorbance (220 nm)

0.29

(C)

Log(MW)

13700Da

40

45

50

55

Log(MW)

0.34

Log(MW)

(A)

Z. Jiang et al. / Food Chemistry 141 (2013) 38373845

Log(MW)

3842

2.5

60

Retention time (min)

Fig. 3. Size exclusion chromatograms of ribose-BCP MRPs (A), galactose-BCP MRPs (B), lactose-BCP MRPs (C) and bovine casein peptides (D) at 220 nm during heat treatment
at 95 C for up to 5 h and corresponding size exclusion chromatograms (E) of ribose-BCP MRPs, galactose-BCP MRPs, lactose-BCP MRPs and bovine casein peptides at 220 nm
after 5 h of heating.

ACE inhibitory activities more signicantly than sugar-BCP MRPs.

Consumption of bovine casein peptides by the Maillard reaction
was assumed to cause the main decrease of ACE inhibitory
activities, although some of the sugar-BCP MRPs might potentially
improve the ACE inhibitory activities. Wang, Li, Cheng, Yin, and Li
(2013) also reported that the consumption of peptides with
ACE-inhibitory activity by the Maillard reaction might cause the
decrease of ACE inhibitory activity in douchi (fermented soy product) ripening. Therefore, in order to remain or avoid losing ACE
inhibitory activities of bovine casein peptides, all possible factors

of reaction condition required by the Maillard reaction with bovine

casein peptides should be removed or controlled during processing, storage and distribution of food products.
3.6. Antioxidant activity
3.6.1. Changes in the DPPH radical scavenging activity
DPPH radicals are scavenged by MRPs through the donation of
hydrogen to form a stable DPPH-H molecule. During the Maillard
reaction, intermediates, the nal brown polymer or sugar

Z. Jiang et al. / Food Chemistry 141 (2013) 38373845

the DPPH radical-scavenging activity of its corresponding MRPs.

These results were similar to those previously reported, where
MRPs from sugar-amino acid, -peptide and -protein model systems
showed DPPH radical scavenging activity (Jiang & Brodkorb, 2012;
Zeng et al., 2011).

ACE inhibitory activity (%)

70
60
50
40
30
20
10
0
0

Heating time (hours)

Fig. 4. ACE inhibitory activity during heating of ribose-BCP MRPs (j), galactoseBCP MRPs (), lactose-BCP MRPs (N) and bovine casein peptides alone (4) at 95 C
for up to 5 h. Error bars represent the mean standard deviation of triplicate
experiments.

caramelisation products can be used as hydrogen donors and contribute to the DPPH radical scavenging activity (Matthaus, 2002).
According to Fig. 5A, the DPPH radical-scavenging activity of ribose-BCP MRPs increased signicantly as the heating time increased (P < 0.05) for the rst 2 h but leveled off at 35 h. The
DPPH radical-scavenging activity of galactose-BCP MRPs and Lactose-BCP MRPs increased markedly with increased heating time
up to 5 h (P < 0.05). Ribose-BCP MRPs had the strongest DPPH radical-scavenging activity in relation to galactose-BCPs and lactoseBCP MRPs within the 5 h of heat treatment (P < 0.05). Meanwhile,
galactose-BCP MRPs showed a higher DPPH radical-scavenging
activity than lactose-BCP MRPs. In contrast, heated BCP lacked

DPPH scavenge ability (%)

(A)

90
70
60
50
40
30
20
10
0
0

Absorbance (700 nm)

Heating time (hours)

(B)

0.8

3.6.2. Changes in ferrous reducing power

The ferrous reducing power of ribose-BCP MRPs increased signicantly within 1 h (P < 0.05) and leveled off thereafter, up to
5 h of heating time (Fig. 5B). Ribose-BCP MRPs showed the highest
ferrous reducing power (P < 0.05), compared to galactose-BCP and
lactose-BCP MRPs. The ferrous reducing power of galactose-BCP
and lactose-BCP MRPs increased quickly up to 5 h of heating time,
but galactose-BCP MRPs showed a higher ferrous reducing power
than lactose-BCP MRPs (P < 0.05). However, it was also shown that
there was no ferrous reducing power of the heated bovine casein
peptides. The reducing power of tuna hydrolysates continued to increase in the Maillard reaction with four ketohexoses (Zeng, Zhang,
Guan, & Sun, 2012). Benjakul et al. (2005) also reported that MRPs
of porcine plasma proteinglucose models possessed reducing
power. Those results suggested that MRPs might function as electron donors. Hydroxyl and pyrrole groups of MRPs may play an
important role in the reducing ferrous activity (Yoshimura, Iijima,
Watanabe, & Nakazawa, 1997). In the present study, ribose-BCP
MRPs showed the highest ferrous reducing power, followed by galactose-BCP and lactose-BCP MRPs up to 5 h of heating time, which
revealed that sugar reactivity might be one of the main factors that
affected the reducing power of sugar-BCP MRPs.
From the results of the DPPH radical scavenging activity and
ferrous reducing power in sugar-BCP MRPs and BCP system, BCP
showed strong antioxidant activities after undergoing the Maillard
reaction with a reducing sugar. This suggests that sugar-BCPs may
have a great potential as effective antioxidants in the food
industry.
3.7. Evaluation of potential cytotoxic effects

80

3843

0.6
0.4
0.2
0
0

Heating time (hours)

Fig. 5. DPPH radical scavenging activity (A) and ferrous reducing power (B) of
ribose-BCP MRPs (j), galactose-BCP MRPs (), lactose-BCP MRPs (N) and bovine
casein peptides alone (4) during heat treatment at 95 C for up to 5 h. Error bars
represent the mean standard deviation of triplicate experiments.

It is important to verify whether reaction products are of significant toxicity, when the Maillard reaction occurred. Fig. 6 represents the viability of Caco-2 cells incubated in the presence of
increasing concentrations of sugar-BCP MRPs and BCP with an
MTT assay.
The addition of galactose-BCP MRPs heated for 5 h to cultured
Caco-2 cells resulted in a slight decrease of cell viability
(P < 0.05) within the concentrations ranging from 0.01 to 0.2 mg/
ml, compared with addition of unheated galactose-BCP MRPs
(Fig. 6B). For example, at the concentration of 0.01 mg/ml, the
heated galactose-BCP MRPs showed a 68% relative growth rate
compared with the unheated ones. The formed galactose-BCP
MRPs probably contained certain components with antiproliferative effects of Caco-2 cells. Obrien and Morrissey (1989) reported
that MRPs generated from glucose-glycine and -glutamate models
have potentially toxic effects. Jing and Kitts (2000) further conrmed the toxicity of fructose-lysine MRPs on Caco-2 cells of human origin. Following 48 h incubation, the cytotoxicity for
fructose-lysine MRPs was obtained at both 0.2 and 2 mg/ml concentrations, while glucose-lysine MRPs produced cytotoxicity at
only 2 mg/ml. In our tests, bovine casein peptides were used to
established reaction models, instead of amino acids. This result
also showed that galactose-BCP MRPs after 5 h of heat treatment,
to some extent, had slight toxicity effects on Caco-2 cells after
incubation for 48 h.
However, ribose-BCP MRPs (Fig. 6A), lactose-BCP MRPs (Fig. 6C)
and BCP (Fig. 6D), which were unheated or heated for 5 h, displayed no decrease of cell viability at the concentration range of
0.010.2 mg/ml. Meanwhile, the heated Ribose-BCP MRPs and

3844

Relative growth rate (%)

(A)

Z. Jiang et al. / Food Chemistry 141 (2013) 38373845

120

bw cw

cd
ab

cd

cd
ab

100

system models were reported by Chibber, Molinatti, Rosatto, Lambourne, and Kohner (1997), who found that bovine retinal capillary
endothelial cell (BREC) growth was signicantly increased with
glucose-BSA, while the bovine retinal capillary pericytes (BRP) cell
growth was inhibited. In this study, galactose-BCP MRPs showed a
slight inhibition to Caco-2 cells, while ribose-BCP and lactose-BCP
MRPs had no cytotoxicity on Caco-2 cells, which indicates that sugar type may notably impact the cytotoxicity of MRPs.

ab

80
60
40
20
0

0.01

0.02

0.05

0.1

0.2

Ribose-BCP concentration (mg/mL)

Relative growth rate (%)

(B)

120

100

a
cd

80

4. Conclusion
a
d

60
40
20
0
0.01

0.02

0.05

0.1

0.2

Galactose-BCP concentration (mg/mL)

Relative growth rate (%)

(C)

120

d
a

Relative growth rate (%)

abm cm

abn cdn

bq cdq

0.02

0.05

0.1

0.2

80
60
40
20
0
0.01

(D)

abw cdw

100

Lactose-BCP concentration (mg/mL)

140
120
100
80
60
40
20
0

c
aw

bw

am bm

an bn

MRPs of ribose, galactose and lactose with bovine casein peptides were prepared under high-temperature treatment in alkaline
pH conditions. During the Maillard reaction, free amino groups and
free reducing sugars of the MRPs decreased considerably. Size
exclusion chromatography of sugar-BCP MRPs indicated molecular
rearrangements and production of new smaller molecules during
heat treatment. In comparison with BCP, a considerable loss of
ACE inhibitory activity was observed with the sugarBCP MRPs,
as well as a remarkable increase in the ferrous reducing power
and DPPH radical scavenging activity. Ribose-BCP and lactoseBCP MRPs had no cytotoxicity on Caco-2 cells, while galactoseBCP MRPs resulted in a slight decrease in viable Caco-2 cells.
Therefore, biological activities and physicochemical properties of
bovine casein peptides were greatly affected by the type of reducing sugars involved in the Maillard reaction. SugarBCP MRPs may
be used as potentially effective antioxidants in the food additives
market.
In this study, sugarBCP model systems are of great importance
in elucidating the physicochemical characteristics of the Maillard
reaction and investigating the effects of sugar sources of the Maillard reaction on the bioactivities of BCP. Future work on more comprehensive model studies that closely reect the situation during
the heating stages of food processing, is worth carrying out.
Acknowledgements

0.01

0.02

0.05

0.1

0.2

BCP concentration (mg/mL)

Fig. 6. Antiproliferative activity of ribose-BCP MRPs (A), galactose-BCP MRPs (B),
lactose-BCP MRPs (C) and BCP alone (D) produced by heating at the initial hour
(white) and fth hour (black stripe) on Caco-2 cells. Error bars represent the
standard deviation of the mean of triplicate experiments. The different letters bars
are signicantly different (P < 0.05).

BCP, slightly stimulated the proliferation of Caco-2 cells at concentrations ranging from 0.02 to 0.2 mg/ml and at both concentrations
of 0.1 and 0.2 mg/ml (P < 0.05), respectively, compared with unheated ones. Lactose-BCP MRPs heated for 5 h, exhibited no significant difference (P > 0.05) in cell viability at the concentration
range from 0.02 to 0.2 mg/ml, compared with unheated ones.
Vagnarelli et al. (1991) reported that a heated Ribose-Lysine MRPs
showed some inhibitory tendency to the growth of FT cells (HeLa
clone). It was also shown that there was no enhancement of cytotoxicity by glycated proteins and peptides against monkey kidney
broblast COS-7 and human promyeloblast HL-60 cells (Chevalier,
Chobert, Genot, & Haertl, 2001b). However, our data demonstrated that ribose-BCP and lactose-BCP MRPs had no toxicity effect on Caco-2 cells. Especially, when ribose-BCP MRPs were at
the concentration of 0.02 mg/ml, the cell viability was almost
117%. The same results were obtained by Jing and Kitts (2002),
who reported that Ribose-casein heated mixtures also showed no
signicant toxicity to cultured Caco-2 cells. The cytotoxic effect
of MRPs derived from glucose and bovine serum albumin (BSA)

This study was supported by the Innovative Research Team of

Higher Education of Heilongjiang Province (2010td11), Project for
National Natural Science Foundation of China (31000801) and
the Innovation Group Program of the Northeast Agriculture University of China (CXT007-4-1). Teagasc Food Research Centre,
Moorepark was acknowledged for analysis of size exclusion
chromatography.
References
Ajandouz, E. H., & Puigserver, A. (1999). Nonenzymatic browning reaction of
essential amino acids: Effect of pH on caramelization and Maillard reaction
kinetics. Journal of Agricultural and Food Chemistry, 47(5), 17861793.
Baisier, W. M., & Labuza, T. P. (1992). Maillard browning kinetics in a liquid model
system. Journal of Agricultural and Food Chemistry, 40(5), 707713.
Benjakul, S., Lertittikul, W., & Bauer, F. (2005). Antioxidant activity of Maillard
reaction products from a porcine plasma proteinsugar model system. Food
Chemistry, 93(2), 189196.
Brueckner, H., Justus, J., & Kirschbaum, J. (2001). Saccharide induced racemization of
amino acids in the course of the Maillard reaction. Amino Acids (Vienna), 21(4),
429433.
Bunn, H. F., & Higgins, P. J. (1981). Reaction of monosaccharides with proteinspossible evolutionary signicance. Science, 213(4504), 222224.
Chevalier, F., Chobert, J. M., Genot, C., & Haertl, T. (2001b). Scavenging of free
radicals, antimicrobial, and cytotoxic activities of the Maillard reaction products
of beta-lactoglobulin glycated with several sugars. Journal of Agricultural and
Food Chemistry, 49(10), 50315038.
Chevalier, F., Chobert, J. M., Popineau, Y., Nicolas, M. G., & Haertl, T. (2001a).
Improvement of functional properties of b-lactoglobulin glycated through the
Maillard reaction is related to the nature of the sugar. International Dairy
Journal, 11(3), 145152.
Chibber, R., Molinatti, P. A., Rosatto, N., Lambourne, B., & Kohner, E. M. (1997). Toxic
action of advanced glycation end products on cultured retinal capillary

Z. Jiang et al. / Food Chemistry 141 (2013) 38373845

pericytes and endothelial cells: Relevance to diabetic retinopathy. Diabetologia,
40(2), 156164.
Decourcelle, N., Sabourin, C., Dauer, G., & Guerard, F. (2010). Effect of the Maillard
reaction with xylose on the emulsifying properties of a shrimp hydrolysate
(Pandalus borealis). Food Research International, 43(8), 21552160.
Dittrich, R., Dragonas, C., Kannenkeril, D., Hoffmann, I., Mueller, A., Beckmann, M.
W., et al. (2009). A diet rich in Maillard reaction products protects LDL against
copper induced oxidation ex vivo, a human intervention trial. Food Research
International, 42(9), 13151322.
Fan, J. F., Zhang, Y. Y., Szesze, T., Li, F. J., Zhou, M. Y., Saito, M., et al. (2006). Improving
functional properties of soy protein hydrolysate by conjugation with curdlan.
Journal of Food Science, 71(5), C285C291.
Guerard, F., & Sumaya-Martinez, M. T. (2003). Antioxidant effects of protein
hydrolysates in the reaction with glucose. Journal of the American Oil Chemists
Society, 80(5), 467470.
Hodge, J. E. (1953). Dehydrated foods-chemistry of browing reactions in model
systems. Journal of Agricultural and Food Chemistry, 1(15), 928943.
Hwang, I. G., Kim, H. Y., Woo, K. S., Lee, J., & Jeong, H. S. (2011). Biological activities
of Maillard reaction products (MRPs) in a sugar-amino acid model system. Food
Chemistry, 126(1), 221227.
Jaeger, H., Janositz, A, & Knorr, D (2010). The Maillard reaction and its control during
food processing. The potential of emerging technologies. Pathologie Biologie,
58(3), 207213.
Jiang, Z., & Brodkorb, A. (2012). Structure and antioxidant activity of Maillard
reaction products from a-lactalbumin and b-lactoglobulin with ribose in an
aqueous model system. Food Chemistry, 133(3), 960968.
Jiang, Z., Tian, B., Brodkorb, A., & Huo, G. (2010). Production, analysis and in vivo
evaluation of novel angiotensin-I-converting enzyme inhibitory peptides from
bovine casein. Food Chemistry, 123(3), 779786.
Jing, H., & Kitts, D. D. (2000). Comparison of the antioxidative and cytotoxic
properties of glucoselysine and fructoselysine Maillard reaction products.
Food Research International, 33(6), 509516.
Jing, H., & Kitts, D. D. (2002). Chemical and biochemical properties of caseinsugar
Maillard reaction products. Food and Chemical Toxicology, 40(7), 10071015.
Kim, J. S., & Lee, Y. S. (2009). Study of Maillard reaction products derived from
aqueous model systems with different peptide chain lengths. Food Chemistry,
116(4), 846853.
Lan, X., Liu, P., Xia, S., Jia, C., Mukunzi, D., Zhang, X., et al. (2010). Temperature effect
on the non-volatile compounds of Maillard reaction products derived from
xylose-soybean peptide system: Further insights into thermal degradation and
cross-linking. Food Chemistry, 120(4), 967972.
Laroque, D., Inisan, C., Berger, C., Vouland, ., Dufoss, L., & Gurard, F. (2008).
Kinetic study on the Maillard reaction. Consideration of sugar reactivity. Food
Chemistry, 111(4), 10321042.
Lerici, C. R., Barbanti, D., Manzano, M., & Cherubin, S. (1990). Early indicators of
chemical changes in foods due to enzymic or nonenzymic browning reactions.
1: Study on heat treated model systems. Lebensmittel-Wissenschaft &
Technologie, 23(4), 289294.
Li, Y., Lu, F., Luo, C., Chen, Z., Mao, J., Shoemaker, C., et al. (2009). Functional
properties of the Maillard reaction products of rice protein with sugar. Food
Chemistry, 117(1), 6974.

3845

Lopez-Exposito, I., Minervini, F., Amigo, L., & Recio, I. (2006). Identication of
antibacterial peptides from bovine kappa-casein. Journal of Food Protection,
69(12), 29922997.
Matthaus, B. (2002). Antioxidant activity of extracts obtained from residues of
different oilseeds. Journal of Agricultural and Food Chemistry, 50(12), 34443452.
Morales, F. J., & Jimnez-Prez, S. (2001). Free radical scavenging capacity of
Maillard reaction products as related to colour and uorescence. Food
Chemistry, 72(1), 119125.
Obrien, J., & Morrissey, P. A. (1989). Nutritional and toxicological aspects of the
Maillard browning reaction in foods. Critical Reviews in Food Science and
Nutrition, 28(3), 211248.
Pihlanto-Leppala, A., Antila, P., Mantsala, P., & Hellman, J. (1994). Opioid peptides
produced by in-vitro proteolysis of bovine caseins. International Dairy Journal,
4(4), 291301.
Ruan-Henares, J. A., & Morales, F. J. (2007). Angiotensin-I converting enzyme
inhibitory activity of coffee melanoidins. Journal of Agricultural and Food
Chemistry, 55(4), 14801485.
Suetsuna, K., Ukeda, H., & Ochi, H. (2000). Isolation and characterization of free
radical scavenging activities peptides derived from casein. Journal of Nutritional
Biochemistry, 11(3), 128131.
Sun, Q., & Luo, Y. (2011). Effect of Maillard reaction conditions on radical scavenging
activity of porcine haemoglobin hydrolysate-sugar model system. International
Journal of Food Science and Technology, 46(2), 358364.
Tessier, F. J., & Birlouez-Aragon, I. (2012). Health effects of dietary Maillard reaction
products: The results of ICARE and other studies. Amino Acids, 42(4),
11191131.
Vagnarelli, P., Desario, A., Cuzzoni, M. T., Mazza, P., & Decarli, L. (1991). Cytotoxicity
and clastogenic activity of riboselysine browing model system. Journal of
Agricultural and Food Chemistry, 39(12), 22372239.
Wang, H., Li, Y., Cheng, Y., Yin, L., & Li, L. (2013). Effect of the Maillard reaction on
angiotensin I-converting enzyme (ACE)-inhibitory activity of douchi during
fermentation. Food and Bioprocess Technology, 6(1), 297301.
Wang, H. Y., Qian, H., & Yao, W. R. (2011). Melanoidins produced by the Maillard
reaction: Structure and biological activity. Food Chemistry, 128(3), 573584.
Wijewickreme, A. N, & Kitts, D. D (1998). Metal chelating and antioxidant activity of
model Maillard reaction products. In F. Shahidi, C. T. Ho, & N. vanChuyen (Eds.).
Process-induced chemical changes in food (Vol. 434, pp. 245254). .
Yen, G. C., & Hsieh, P. P. (1995). Antioxidative activity and scavenging effects on
active oxygen of xyloselysine Maillard reaction products. Journal of the Science
of Food and Agriculture, 67(3), 415420.
Yoshimura, Y., Iijima, T., Watanabe, T., & Nakazawa, H. (1997). Antioxidative effect
of Maillard reaction products using glucoseglycine model system. Journal of
Agricultural and Food Chemistry, 45(10), 41064109.
Zeng, Y., Zhang, X., Guan, Y., & Sun, Y. (2011). Characteristics and antioxidant
activity of Maillard reaction products from psicoselysine and fructoselysine
model systems. Journal of Food Science, 76(3), C398C403.
Zeng, Y., Zhang, X., Guan, Y., & Sun, Y. (2012). Enzymatic hydrolysates from tuna
backbone and the subsequent Maillard reaction with different ketohexoses.
International Journal of Food Science and Technology, 47(6), 12931301.