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BIOSCIENCES BIOTECHNOLOGY RESEARCH ASIA, March 2017. Vol. 14(1), 177-184
* To whom all correspondence should be addressed.
Tel.:00966504686549;
E-mail: abalghafari@kau.edu.sa
The Effect of Adding Different Concentrations of
Cows’ Milk on the Antioxidant Properties of Coffee
Ayat B. Al-Ghafari*, Rahaf H. Alharbi, Manal M. Al-Jehani,
Shoroq A. Bujeir, Huda A. Al Doghaither, and Ulfat M. Omar
Department of Biochemistry, Faculty of Science,
King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia.
http://dx.doi.org/10.13005/bbra/2433
(Received: 22 February 2017; accepted: 02 March 2017)
Polyphenols have been widely studied and considered as a health promoting
and disease preventive agents in humans. Several studies investigated the antioxidant
properties of polyphenols and their abilities to eliminate free radicals. In this study, the
antioxidant activity of coffee in the presence of different types of cows’ milk at different
concentrations, 10% or 20%, was investigated. Our results showed that milk could either
enhance the scavenging of DPPH or decrease the metal chelating and metal reducing
activity of polyphenol. However, more investigations are required to evaluate the
mechanisms by which fats in milk can alter the antioxidant activity of coffee.
Keywords: Whole milk; Semi-skimmed milk; Skimmed milk;
Scavenging activity; reducing power; metal chelating.
Oxygen is an essential element for the
survival of aerobic organisms. In many chemical
and biochemical reactions, oxygen-derived free
radicals are produced from oxidation-reduction
processes1. These free radicals are responsible for
oxidative stress that leads to variety of diseases
and disorders such as cancer2, cardiovascular
diseases3, neural disorders4, and Alzheimer’s
disease5. They are produced under normal
physiological conditions and are generated by
exogenous chemicals6. Reactive oxygen species
(ROS) can present in different forms of activated
oxygen such as hydrogen peroxide (H2O2) or as
singlet oxygen7. The redox homeostasis of cell is
maintained by the antioxidant protection
mechanism of the human body. If this mechanism
has been unbalanced, the reducing of oxidative
damage may be occurred by using antioxidant
supplement6.
Coffee is an exogenous antioxidant that
is widely consumed due to its desirable sensory
properties8. The proper consumption of coffee is
important to health as many negative and positive
effects can occur according to the dose. Negative
effects of coffee consumption include
hypertension, heart diseases, psychiatric disorders
and bone diseases. These side effects are mainly
attributed to increased caffeine consumption of
coffee9,10. On the other hand, a regular intake of
coffee has shown many positive effects on
metabolic, psychoactive and neurological
diseases11. The positive effects depend on the
phenolic and flavonoid compounds such as
chlorogenic acids (quinyl esters of
hydroxycinnamic acid)12, caffeic, ferulic, p-coumaric
acids13 and proanthocyanidins13,14. These
178 AL-GHAFARI et al., Biosci., Biotech. Res. Asia, Vol. 14(1), 177-184 (2017)
biological compounds are secondary metabolites
from plants6 and have potent strong antioxidant
and radical scavenging activities15,16, which help
the body to reduce the oxidative stress17.
Most of people prefer to drink coffee with
additives. Milk is one of the preferable choices of
additives because it contains various compounds
such as water, fats, sugars, minerals, vitamins, and
proteins18. When milk is added to coffee, the milk
proteins (caseins and whey proteins) can interact
with the coffee polyphenols and form a complex,
which might affect many properties of coffee
particularly the structural, functional, and
nutritional ones 19-21. Many studies have
investigated the antioxidant capacity of tea after
adding different types of milk to it. However, the
obtained results are contradictory. Some of them
have reported an inhibitory effect, while others
have shown no significant effect of adding milk22-
24. Milk fat exists in emulsified form coated by thin
layer known as a globule membrane25, and its effect
on antioxidant activity and bioavailability of coffee
polyphenols is unclear until now. Therefore, the
objective of this study was to assess the in vitro
effect of adding different concentrations of whole,
semi-skimmed, and skimmed cow’s milk on the
antioxidant activity of coffee.
MATERIALS AND METHODS
Preparation of samples
Red mug coffee (Nescafé) and different
types of long life (Almarai) cow's milk (whole, semi-
skimmed, and skimmed), the most popular
consumed milk brand according to the survey, were
purchased from a local supermarket. The types of
milk and the percentages used were selected
according to a survey performed on 900
participants in Jeddah, Western region of Kingdom
of Saudi Arabia.
Coffee brews have been prepared as
people in the survey commonly drink it. Each
sample was prepared by adding 2g of instant coffee
to 200 ml boiling water and then either 20 ml or 40
ml of different types of cow milk was added to
have a final concentration of 10% or 20%,
respectively.
Determination of total phenolic and flavonoid
contentsIn a clean test tube, 0.5ml of each coffee
sample (with or without milk) was incubated at
room temperature for 5 minutes with 5ml deionized
water and 5ml Folin-Ciocalteu’s reagent (BDH,
Poole, England). Then, in a dark place, all tubes
were incubated with 1ml of anhydrous sodium
carbonate (CDH, New Delhi, India) for 1 hour before
the absorbance was measured at 750 nm. A
standard curve was prepared from a serial
concentration (0 to 1000µg/ml) of Gallic acid (Sigma-
Aldrich, Poole, UK) which was prepared in
methanol: water (50: 50 v/v) 26,27.
On the other hand, the total flavonoid
content was determined by incubating 250µl of
each coffee sample (with or without milk) with 1.25
ml deionized water and 75µl sodium nitrite (Sigma-
Aldrich, Poole, UK) for 5 minutes. Then, 150µl of
(10%) aluminum chloride (loba chemie PVT.LTD.,
Mumbai, India), 0.5 ml of (1M) sodium hydroxide
(AppliChem Panreac, Missouri, USA) and 275µl of
deionized water were added to the previous mixture.
After that, the absorbance was read at 510 nm and
a standard curve was prepared from a serial dilution
(0 to 500µg/ml) of β-Catechin (Sigma-Aldrich,
Poole, UK) 28.
Determination of radical scavenging activity with
H2O2 and DPPH assays
In test tubes, 1 ml of sample was
incubated for 10 minutes at room temperature with
0.6 ml of (40 mM) hydrogen peroxide (Sigma-
Aldrich, Poole, UK). Then, the absorbance was
recorded at 230 nm and the radical scavenging
activity for H2O2 was calculated by the following
equation: inhibition % = [(Absorbance of blank –
Absorbance of sample) / Absorbance of blank] ×
100 29,30.In the DPPH-radical scavenging assay,
ethanol (Sigma-Aldrich, Poole, UK) and DPPH
reagent (Sigma-Aldrich, Poole, UK) were added to
water extract solutions of samples and were
incubated in dark place for 1 hour at room
temperature. The absorbance was measured at 517
nm and the DPPH radical scavenging activity was
calculated using the same equation used to
calculate the inhibition percentage of H2O2 31.
Determination of metal chelating and reducing
power activity
To estimate the ferrous ion chelating
activity of samples32, 5µl of each sample (with or
without milk) was incubated for 30 seconds with
50µl of (2mM) ferrous chloride (Sigma-Aldrich,
179AL-GHAFARI et al., Biosci., Biotech. Res. Asia, Vol. 14(1), 177-184 (2017)
Poole, UK) and 1.5ml distilled water. Then, 100µl of
(5 mM) ferrozine (Sigma-Aldrich, Poole, UK) was
added to samples and was incubated at room
temperature for 10 minutes. The absorbance was
measured at 562 nm and the following equation
(Chelating Activity % = (Absorbance of control –
Absorbance of sample)/Absorbance of control)
×100) was used to estimate the percentage of
ferrozine-Fe2+ complex.
Regarding the reducing power assay33,
1ml of sample was mixed with phosphate buffer
(0.2M, pH 6.6) (Oxoid, Hampshire, England) and
potassium ferricyanide (1%) (Koch-light,
Colnbrook Bucks, UK) before they were incubated
in 50°C for 30 minutes. Then, trichloroacetic acid
(TCA) (10%) (Sigma-Aldrich, Poole, UK) was
added to stop reaction and then tubes were
centrifuged at 6000 rpm for 10 minutes. The
supernatant was mixed with equal amount of
distilled water and ferric chloride (1%) (BDH, Poole,
England) and the absorbance was immediately
measured at 700 nm.
Statistical analysis
GraphPad Prism 7 software was used to
analyze the results. Three independent experiments
were performed for each assay and the means were
assessed using one-way ANOVA test with
Bonferroni’s test correction. The data were
expressed as mean ± SD and results were
considered statistically significant when P<0.05.
RESULTS
Estimation of total phenolic and flavonoid contents
In this study, a survey was performed on
900 participants that live in Jeddah, Kingdom of
Saudi Arabia to assess the most consumed coffee
type, the average daily consumption of coffee-
cups, and the type of additives used commonly
when drink coffee. Results from survey revealed
that (7%) of the participants prefer to add milk (with
either 10% as minimum concentration or 20% as
maximum concentration) to coffee. Therefore, this
study was interested in studying the effect of
adding different milk types with different
concentrations on the phenolic compounds
bioavailability and the antioxidant activities of
coffee. To test all these objectives, many samples
were prepared and used in this study [Nescafé red
mug coffee, Nescafé red mug coffee with whole
milk (10% and 20%), Nescafé red mug coffee with
semi-skimmed milk (10% and 20%), and Nescafe
red mug coffee with skimmed milk (10% and 20%)].
The total phenolic content was
determined with Folin-Ciocalteu assay and was
expressed as Gallic acid equivalent whereas; the
total flavonoid content was determined with
aluminum chloride assay and was expressed as
Catechin equivalent. Table (1) represents the total
phenolic and flavonoid contents in the samples.
Generally, results showed that adding milk to coffee
improves the contents of total phenolic and
flavonoid compounds especially with the whole
milk, either at 10% or 20% concentration.
Estimation of radical scavenging activity
To determine the radical scavenging
activity of coffee samples, two assays were
performed. The first assay, H2O2 radical scavenging
assay, measured the ability of samples to scavenge
hydroxyl radical at 320 nm. Hydrogen peroxide is
one of the most reactive oxygen species (ROS)
that can cross cell membranes rapidly34. Once it is
inside the cell, H2O2 can form hydroxyl radical
through reaction with (Fe2+) and (Cu2+) 35.
Polyphenols of coffee can scavenge free radical
by donating electron as illustrated in the equation:
2 H2O2→2 H2O + O2
In the second assay (DPPH radical
scavenging assay), the scavenging reaction
between (DPPH) and an antioxidant (H-A) can be
expressed by the equation:
(DPPH) + (H-A)→DPPH-H + (A)
(Purple) (Yellow)
Antioxidants in coffee can react with
DPPH and reduce it to the DPPH-H form (which
can be determined by a reduction in the
absorbance)36. Table 2 represents the results from
H2O2 and DPPH radical scavenging assays. The
results in Fig 1 showed that adding any type of
milk (whole, semi-skimmed, and skimmed) at any
concentration, 10% or 20%, had a paradoxical effect
on the radical scavenging activity. For H2O2
scavenging activity, adding any type of milk at low
concentration (10%) did not show any significant
effect on the scavenging activity, whereas; at high
concentration (20%), the activity of coffee to
scavenge H2O2 reduced significantly when milk is
added. In contrast, the addition of milk improves
180 AL-GHAFARI et al., Biosci., Biotech. Res. Asia, Vol. 14(1), 177-184 (2017)
Table 1. Total phenolic and flavonoid contents
Samples 10% Milk 20% Milk
Total phenolic Total flavonoid Total phenolic Total flavonoid
content content (µg of content (µg of content (µg of
(µg Gallic Chatecin/2g Gallic acid/2g Chatecin/2g
acid/2g coffee)* coffee)* coffee)* coffee)*
Coffee (no milk added) 695 ± 2.5 506 ± 1.5 647 ± 3.4 506 ± 4.2
Coffee with whole milk 1181 ± 3.0 977 ± 6.0 1678 ± 7.0 1353 ± 5.0
Coffee with semi-skimmed milk 836 ± 5.0 822 ± 7.0 1061 ± 3.0 907 ± 2.5
Coffee with skimmed milk 732 ± 7.0 694 ± 5.0 897 ± 6.0 639 ± 5.0
* Data were represented as Mean±SD of three independent experiments
Table 2. Estimation of the radical scavenging activity
Samples 10% Milk
H2O2 radical scavenging activity DPPH radical scavenging activity
Mean ± SD P-value Mean ± SD P-value
Coffee (no milk added) 77% ± 3.82 REFERENCE 45% ± 1.54 REFERENCE
Coffee with whole milk 80% ± 4.96 P>0.05 (NS) 94% ± 3.0 ***≤0.001
Coffee with semi-skimmed milk 88% ± 5.0 P>0.05 (NS) 84% ± 1.0 **≤0.01
Coffee with skimmed milk 85% ± 6.5 P>0.05 (NS) 65% ± 7.0 *≤0.05
20% Milk
Samples H2O2 radical scavenging activity DPPH radical scavenging activity
Mean ± SD P-value Mean ± SD P-value
Coffee (no milk added) 76% ± 3.0 REFERENCE 45% ± 1.5 REFERENCE
Coffee with whole milk 72% ± 2.0 P>0.05 (NS) 55% ± 7.0 P>0.05 (NS)
Coffee with semi-skimmed milk 62% ± 1.6 **P≤0.01 80% ± 7.0 **P≤0.01
Coffee with skimmed milk 38% ± 5.0 ***P≤0.001 83% ± 5.0 **P≤0.01
-All data were represented as Mean ±SD calculated by one-way ANOVA test followed by Bonferroni’s test correction.
-P-values were calculated by a comparison made versus the (coffee only) value, which was referring to it as
reference.
NS: Not Significant
significantly the activity of coffee to scavenge any
radicals (as shown by DPPH assay) at both
concentration of milk (10% or 20%).
Estimation of metal chelating and reducing power
activity Two assays were performed to estimate
the ability of antioxidants, present in the samples,
to act as metal chelating agents and as reducing
agents. In ferrous chelating activity assay, the dark
color which is formed by the interaction of ferrozine
with Fe2+ is changed into a lighter color in the
presence of a chelating agent in the sample37. On
the other hand, ferric reducing power assay used
the Fe3+ reduction as an indicator of electron-
donating activity which is an important mechanism
of any phenolic antioxidant38.
Table 3 represents the results from metal
chelating and reducing power assays.
Interestingly, the results in Fig 2 showed that
adding any type of milk (whole, semi-skimmed, and
skimmed) at any concentration, either 10% or 20%,
reduces significantly the metal chelating and the
metal reducing power activities of the coffee
compared to the coffee alone (with no milk added).
181AL-GHAFARI et al., Biosci., Biotech. Res. Asia, Vol. 14(1), 177-184 (2017)
Table 3. Estimation of metal chelation and reducing power activity
Samples 10% Milk
Ferrous chelating activity Ferric reducing power activity
Mean ± SD p-value Mean ± SD P-value
Coffee (no milk added) 37% ± 5.50 REFERENCE 0.62 ± 0.05 REFERENCE
Coffee with whole milk 23% ± 1.89 **P≤0.01 0.38 ± 0.03 **P≤0.01
Coffee with semi-skimmed Milk 18% ± 1.42 **P≤0.01 0.43 ± 0.09 *P<0.05
Coffee with skimmed milk 17% ± 2.84 ***P≤0.001 0.42 ± 0.06 *P<0.05
20% Milk
Samples Ferrous chelating activity Ferric reducing power activity
Mean ± SD P-value Mean ± SD P-value
Coffee (no milk added) 38% ± 3.40 REFERENCE 0.61 ± 0.04 REFERENCE
Coffee with whole milk 22% ± 2.80 **P≤0.01 0.35 ± 0.02 ***P≤0.001
Coffee with semi-skimmed milk 23% ± 5.40 **P≤0.01 0.37 ± 0.02 ***P≤0.001
Coffee with skimmed milk 27% ± 0.86 *P<0.05 0.33 ± 0.04 ***P≤0.001
-All data were represented as Mean ±SD calculated by one-way ANOVA test followed by Bonferroni’s test correction.
-P-values were calculated by a comparison made versus the (coffee only) value, which was referring to it as reference.
DISCUSSION
Coffee is one of the most popular
beverages in the world. Many of the studies
indicated that adding some additives to coffee can
alter the bioactive compounds present in it, and
therefore, may affect the biological activity of
coffee as an important antioxidant. In many cases,
coffee is consumed with milk. Milk protein may
form interaction with coffee polyphenols, therefore,
the present study aimed to investigate the total
phenol and flavonoid contents and the antioxidant
activity of bioactive components when different
types of milk (whole, semi-skimmed, and skimmed)
at both concentration (10% and 20%) were added
to coffee. Our results indicated that adding milk to
coffee increased the total phenol and flavonoid
contents. This might be explained by the presence
of other compounds in milk that can act as an
antioxidant such as lactoferrin, ascorbic acid,
tocopherols and tocotrienols39.
Hydrophobic interaction and hydrogen
bond are the major reactions that derive
polyphenol-protein complex formation. Several
factors can influence the extent of these binding
forces such as structure of polyphenols and
proteins as well as some physical conditions
particularly (temperature, pH, and ionic strength)40.
In our study, our results revealed that at 10% milk
concentration, there was no significant difference
in hydrogen peroxide scavenging activity between
the coffee with and without milk. Interestingly,
adding milk at higher concentration (20%)
decreased the H2O2 scavenging activity for coffee
especially with skimmed milk. Some flavonoids
have a high affinity to bind to proteins40. This
affinity makes many phenol groups of flavonoids
occupied and thus H2O2 scavenging do not occur
properly. The formation of polyphenol-protein
complex can result in protein unfolding. In the
presence of lipids, which are classified as
Amphiphilic molecules, hydrophobic lipid phase
interacts with hydrophobic part of the protein,
which has been exposed to the outer phase due to
denaturation. This prevents the association
between hydrophobic sites of polyphenols with
the aromatic groups and aliphatic side chains of
hydrophobic amino acids41. However, those
polyphenols when bind with the polar head groups
of lipid can probably make phenol groups somehow
in a position that allows phenols to scavenge H2O2,
while polyphenol-protein complex without lipid
presence require the involvement of many
hydrogen and hydrophobic bonds in addition to
the high affinity of flavonoids toward protein
binding40. These findings may explain why whole
milk sample has H2O2 scavenging activity higher
than semi-skimmed and skimmed milks.
182 AL-GHAFARI et al., Biosci., Biotech. Res. Asia, Vol. 14(1), 177-184 (2017)
Fig. 1. Radical scavenging activity of red mug coffee
with and without various milk types and concentrations.
Three different types of milk were tested (whole, semi-
skimmed, and skimmed) at two concentrations (10%
and 20%). A & B) represents the H2O2 and DPPH
radical scavenging activity of samples at 10% milk
concentration, whereas; C & D) represents the H2O2
and DPPH radical scavenging activity of samples at
20% milk concentration. Results revealed that the
addition of milk at any concentration or type decreases
the H2O2 radical scavenging activity of the coffee,
whereas; increases the DPPH radical scavenging activity.
The values expressed as mean±SD (n=3). Comparisons
of means were made using one-way ANOVA followed
by a correction with Bonferroni’s test (*P<0.05,**
P≤0.01, and *** P≤0.001).
Fig. 2. Metal chelating and reducing power activity of
red mug coffee with and without various milk types
and concentrations. Three different types of milk were
tested (whole, semi-skimmed, and skimmed) at two
concentrations (10% and 20%). A & B) represents the
metal chelating and reducing power activity of samples
at 10% milk concentration, whereas; C & D) represents
the metal chelating and reducing power activity of
samples at 20% milk concentration. Results revealed
that the addition of milk at any concentration or type
decreases the metal chelating and reducing power
activity of the coffee. The values expressed as mean±SD
(n=3). Comparisons of means were made using one-
way ANOVA followed by a correction with
Bonferroni’s test (*P<0.05,** P≤0.01, and ***
P≤0.001).
On other hand, in the present study,
results from DPPH radical scavenging activity
experiments are in contrast with that of H2O2 for all
tested samples. In addition to the probabilities that
mentioned above about the difference of phenols,
some amino acids with aromatic and bulky side
groups are considered as effective radical
scavengers because they can act as H-donors due
to the presence of special groups in their side
chains. For example, the phenolic group in tyrosine,
indolic group in tryptophan, imidazole group in
histidine, and sulfur hydrogen42 in methionine and
cysteine. Moreover, several studies refer to the
correlation between the high concentration of
flavonoids and polyphenols with the high DPPH
scavenging activity43,44. In our study, the
polyphenol and flavonoid contents were higher in
all coffee with milk samples compared to the plain
coffee sample.
In general, many studies, reported that
the polyphenols interaction with proteins, not only
affects the scavenging activity but also affects
the chelating and metal reducing activity of
coffee18,45. The hydrophobic stacking of the
aromatic groups of the protein and polyphenols,
or the interaction of the –OH groups of the
polyphenols with the protein chain can resulted in
the formation of aggregates40. Our results showed
that the ferrous chelating activity increased when
coffee is not mixed with milk. This might be due to
the presence of free –OH groups of polyphenols
or might be related to the binding of polyphenols
to the milk caseins by covalent and non-covalent
interactions either in a multi-site or in a multi-
dentate interaction 24. Our results are in agreement
with other similar studies22,46 that used FRAP assay
to assess the ferrous chelating activity.
In conclusion, the results of the current
study revealed that milk might either enhance the
scavenging of DPPH or decrease the chelating and
metal reducing activity of polyphenol. However,
183AL-GHAFARI et al., Biosci., Biotech. Res. Asia, Vol. 14(1), 177-184 (2017)
more investigations are required to evaluate the
mechanisms by which fats in milk can alter the
antioxidant properties of coffee.
ACKNOWLEDGEMENTS
This research was supported by the
Science Research & Innovation Unit at the Faculty
of Science, King Abdulaziz University in Jeddah,
Saudi Arabia.
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