Evaluation of Biofunctional Compounds Content from Brewed Cofee

Evaluation of Biofunctional Compounds Content
from Brewed Cofee
Anca C. FĂRCAŞ, Sonia A. SOCACI*, Iulia BOCĂNICIU, Anamaria POP, Maria TOFANĂ, Sevastiţa MUSTE,
Dana FEIER
Faculty of Food Science and Technology, University of Agricultural Sciences and Veterinary Medicine
Cluj-Napoca, Romania
* Corresponding author e-mail: sonia.socaci@usamvcluj.ro
Bulletin UASVM Food Science and Technology 71(2) / 2014
ISSN-L 2344-2344; Print ISSN 2344-2344; Electronic ISSN 2344-5300
DOI: 10.15835/buasvmcn-fst:10474
Abstract
Coffee, one of the most popular beverages worldwide, is an infusion of ground, roasted coffee beans. Nowadays,
coffee is considered a functional food, especially due to its high content of compounds that exert antioxidant
and other beneficial biological properties. The annual consumption exceeds 5 billion kilograms of coffee, which
corresponds to 500 billion cups.
The aim of the present study was to evaluate the content in total phenolic compounds, flavonoids, caffeine as
well as the antioxidant activity of three brewed coffee samples in order to assess the amount of these bioactive
compounds in a cup of coffee.
The quantification of total phenolic compounds was achieved by Folin-Ciocalteu method, while the flavonoids
content was determined using a chromogenic system of NaNO2–Al(NO3)3–NaOH based spectrophotometric
method. The caffeine was extracted from brewed coffee samples with dichlormethane and then was quantified by
measuring the absorbance of the extract at 260 nm. The antioxidant capacity of each coffee sample was assessed
by evaluating their radical scavenging activity on DPPH radical.
Even though Arabica coffee variety is appreciated for its fine aroma profile, Robusta variety has proved to
be richer in phenolic compounds, flavonoids and caffeine. The larger amount of compounds with antioxidant
properties was also confirmed by the obtained antioxidant capacity of Robusta brewed coffee.
Keywords: Robusta, Arabica, antioxidant, caffeine, flavonoids, phenolic compounds.
INTRODUCTION
Coffee, one of the most popular beverages in
the world, has been consumed for thousands of
years for its alluring flavours and health benefits.
In 2010, coffee production reached 8.1 million tons
worldwide, the United States, Brazil, Germany,
Japan, and Italy being the major consumer
countries (www.ico.org).
Although more than 80 coffee species
have been identified (Clarke, 2003) only Arabica
(Coffea arabica) and Robusta (Coffea canephora)
are economically important. This two species of
coffee are different in many ways, including their
ideal growing climates, physical aspects, chemical
composition and characteristics of the brew made
with the ground roasted seeds. The popularity
of coffee consumption is due to its characteristic
taste and aroma, but also to its content in biological
active compounds: alkaloids (caffeine), phenolics,
vitamins,
lipids,
carbohydrates,
minerals,
nitrogenous compounds (Higdon and Frei, 2006).
Arabica trees produce the best quality coffee
and are the most widely cultivated (3/4 of the
world’s coffee), while Robusta beans are hardier,
contain 40-50% more caffeine, and are used
in many instant coffees. Compared to Arabica,
Robusta tends to be bitterer, with a telltale “burnt
rubber” aroma and flavour (Knox and Huffaker
1997).
115
Evaluation of Biofunctional Compounds Content from Brewed Cofee
In order to produce the specific and desirable
aroma, the consumer is offered not one pure coffee
variety but a blend of different varieties. One of
the most popular varieties, Arabica and Robusta,
mixed in different proportions, offer a wide
range of tastes and aromas of coffee beverages
(Kreicbergs et al., 2011).
Many studies have been focused on the
health effects of coffee and whether the overall
effects of coffee consumption have a positive or
negative impact on human health. Recent studies
have associated a moderate coffee consumption
with potential health benefits by lowering the
risk of several chronic diseases (Maranhao et al.,
2003) including cancer, type 2 diabetes mellitus,
Parkinson and liver disease (Higdon and Frei,
2006). Additionally, due to its high content in
chlorogenic acids, studies performed in Denmark,
United States, Mediterranean countries, Japan
and Brazil have reported that coffee is the most
important source of antioxidants this populations
diets (Saura-Calixto and Goni, 2006; Fukushima et
al., 2009; Torres and Farah, 2010).
Caffeine is an alkaloid responsible for no
more than 10% of the perceived bitterness of the
coffee beverage (Farah, 2012), being also found in
a number of other dietary sources, i.e. tea, coffee,
cocoa beverages, candy bars and soft drinks. The
content in caffeine of different food items ranges
from 70-220 mg/150 ml for coffee to 30-50
mg/150 ml for tea, 32-70 mg/330 ml for cola and
4 mg/150 ml for cocoa (Debry, 1994).
Moderate caffeine consumption leads very
rarely to health risks. Higher doses of caffeine
rather induce negative effects such as anxiety,
restlessness, insomnia and tachycardia, these
effects being seen primarily in a small subset of
caffeine sensitive individuals (Benowitz, 1990). A
review of the effects of caffeine on human health
commissioned by Health Canada also concluded
that moderate caffeine intakes up to 400 mg/day
are not associated with adverse health effects in
healthy adults. For women who are pregnant,
lactating or planning to become pregnant, the
caffeine intake limit should be no more than 300
mg/day (Nawrot et al., 2003). Several studies have
found that a high consumption of coffee (5-10 cups
daily) may be associated with an increased risk for
myocardial infarction (Sesso et al., 1999).
Today, coffee is considered a functional food,
primarily due to its high content of compounds that
exert antioxidant and other beneficial biological
properties. Research is showing that coffee is
much more than a combination of caffeine and
water. Polyphenols, particularly flavonoids and
phenolic acids are of great abundance in coffee
and contribute to its flavour and health properties
(Yu and Ho, 2009).
Several studies indicated that a high content
of polyphenols in coffee plays an important role
in its strong antioxidant activity (Charurin et
al., 2002, Sanchez-Gonzalez et al., 2005). The
concentration of highly active polyphenols in
green coffee beans is influenced by the species
and its origin, while in coffee beverages it depends
on the brewing procedure (Sanchez-Gonzalez et
al., 2005). During the roasting stage, exposure
to high temperatures can considerably modify
coffee’s chemical composition, some beneficial
compounds being degraded and new ones created.
Thus, the roasting process affects the composition
of the coffee polyphenols through the Maillard
reaction and confers to coffee its pleasant taste
and aroma (Richelle et al, 2001). However, the
high antioxidant activity of coffee is maintained
due to the formation during the roasting process
of non-enzymatic browning compounds (e.g.
melanoidins) which may behave as antioxidants
(Dvořáková et al., 2008). Factors that affect the
brew’s composition include the degree of roasting
and grinding, proportion of coffee to water,
hardness and temperature of water, length of
time coffee is in contact with water and the filter
material (Farah, 2012).
The aim of the present study was to evaluate
the content in total phenolic compounds,
flavonoids, caffeine as well as the antioxidant
activity of three brewed coffee samples in order to
assess the amount of these bioactive compounds
in a cup of coffee. The results were compared
with those obtained for a chicory sample, which
is a well known coffee substitute and sometimes
adulterant.
MATERIALS AND METHODS
The analysed coffee samples, Arabica, Robusta
and blended coffee (Robusta and Arabica) as well
as the chicory sample, were purchased from the
same manufacturer, from local supermarkets
(Cluj-Napoca, Romania). All the reagents used
were purchased from Sigma-Aldrich or Merck
(Darmstadt, Germany).
Bulletin UASVM Food Science and Technology 71(2) / 2014
116
FĂRCAŞ et al.
Coffee brews preparation
The coffee and chicory brews were prepared by
solid-liquid extraction by infusing 1g of ground coffee
or chicory in 100 ml of hot deionised water for 2
minutes. After filtration, the extracts obtained were
used for total polyphenols, flavonoids, antioxidant
activity and caffeine determination. All analyses were
performed with freshly prepared coffee brews.
The total phenolic assay
The content of total phenolics was determined
following a modified Folin-Ciocalteu method
(Singleton et al., 1999). An aliquot of 0.1ml of extract
was mixed with 6 mL of water and 0.5mL of Folin–
Ciocalteu reagent. After 4 min, 1.5 ml Na2CO3 solution
(7.5%) was added and the samples were dilluted to
a final volume of 10 ml with distilled water. After
incubation for 120 min at room temperature, the
absorbance was read at 750 nm, using a Shimadzu
UV-1700 PharmaSpec spectrophotometer, against
the blank, in which the sample was replaced with
methanol. Standard curve was performed using
different concentration solution of gallic acid and
the results were expressed as mg GAE/g (means ±
standard deviation of triplicate analysis).
The total flavonoid assay
The total flavonoid content of the extracts was
determined using a modified aluminum chloride
colorimetric method (Zhu H. et al., 2010). An
aliquot of 10 ml coffee extract were homogenized
with 10 ml methanol and were centrifuged
at 4000 rpm for 15 min. Then, 1 ml of the
supernatant was mixed with 4 ml distilled water,
0.3 ml NaNO2 (5%) and 0.3 ml AlCl3 (10%). After
5 min, 2 ml NaOH 1 N and 6.4 ml distilled water
were added and the absorbance was measured
against the sample solution without coloration
at 510 nm. Standard curve was performed using
different concentrations of quercetin solution and
the results were expressed as mg QE/g (means ±
standard deviation of triplicate analysis).
Determination of 2,2-diphenylpicrylhydrazil
radical scavenging capacity (DPPH)
The DPPH scavenging activity assay was
performed according to a method reported by
Odriozola-Serrano et al., 2008. A volume of 3.9 ml
of methanolic DPPH solution was allowed to react
in darkness, for 30 minutes with 10μL of sample
and 90 μL of H2O. The absorbance was measured
at 515 nm against methanol. The antioxidant
activity was calculated as follows: % Radical
scavenging activity (RSA) = (A0-A1/A0) 100, where
A0 was the absorbance of DPPH solution and A1 the
absorbance of the sample.
The caffeine determination
The caffeine was extracted from brewed
coffee samples with dichloromethane and then
was quantified by measuring the absorbance of
the extract at 260 nm (Jenway, Application Note).
An aliquot of 25 ml brewed coffee was placed into
a separating funnel and 25ml of dichloromethane
was added. The caffeine was extracted by inverting
very slowly the funnel three times, venting the
funnel after each inversion. The dichloromethane
layer was removed into a clean flask and the
extraction procedure was repeated twice more
and the solvent layers combined. A 1000ppm
standard stock solution was prepared in water.
Working standard solutions of 10, 25, 50, 100, 150
and 200 ppm were prepared from the standard
stock solution. The obtained calibration curve of
standards solutions was then used to quantify the
caffeine concentration of each coffee sample.
RESULTS AND DISCUSSION
The content in polyphenols, flavonoids,
caffeine and antioxidant activity of the analysed
coffee and chicory brews samples are shown in
Table 1.
As it was previously mentioned, the concentration of biologically active compounds in green
Tab.1. The content of total phenols, flavonoids, caffeine and antioxidant activity (mean ± sd) of
Arabica, Robusta, blended coffee and chicory samples
Compounds
Total phenols (mg GAE/g)
Flavonoids (mg QE/g)
DPPH (%)
Caffeine (mg/g)
Robusta (Rb)
47.85 ± 0.77
18.81 ± 0.32
43.63 ± 1.12
51.62 ± 0.96
(Means ± standard deviation of triplicate analysis)
Bulletin UASVM Food Science and Technology 71(2) / 2014
Coffee samples
Arabica (Ar)
Rb+Ar
33.9 ± 0.56
41.95 ± 0.49
15.58 ± 0.08
17.86 ± 0.29
36.18 ± 1.34
40.22 ± 0.89
37.52 ± 0.47
50.38 ± 1.02
Chicory
13.2±0.42
0.72±0.14
9.55±0.55
1.18±0.04
117
Evaluation of Biofunctional Compounds Content from Brewed Cofee
coffee beans is influenced mainly by the species
and its origin while in coffee beverages it depends
on the brewing procedure. Considering the fact
that in our study the brewing conditions was the
same for all analyzed coffee samples, decisive
influence on the content of bioactive compounds
is closely dependent on the coffee variety used
(pure Robusta, pure Arabica and a mix of these
two varieties).
The highest concentration in phenolic compounds was determined for Robusta variety (47.85
mg GAE/g), followed by the blended sample (41.95
mg GAE/g) and Arabica variety (33.90 mg GAE/g).
The same order was also observed for flavonoids,
Robusta variety having the highest content (18.81
mg QE/g) followed by the blended sample (17.86
mg GAE/g) and Arabica (15.58 mg GAE/g). The
larger amount of compounds with antioxidant
properties found in Robusta brewed coffee was
also confirmed by the obtained antioxidant
capacity values. Thus, the same pattern as for
phenolic compounds was noticed for the radical
scavenging activity, the Robusta coffee sample
having the greatest antioxidant activity. The
coffee substitute, chicory sample, had the lowest
content in antioxidant compounds, its capacity
of inhibiting free radicals being up to 4.6 times
smaller than that of coffee samples.
Figure 1 represents the comparative content
of polyphenols, flavonoids and caffeine contained
in a cup of brewed coffee, considering a standard
coffee (5g of grounded coffee brewed with 100 mL
of boiled water).
Ch
Flavonoids
3.6
Rb+Ar
89.30
77.90
Ar
94.06
Rb
According to the literature, Robusta can
contain up to two times more caffeine than
Arabica (Knox and Huffaker 1997), our results
being in agreement with those data, the caffeine
concentration in the Robusta sample being 1.37
times higher than in Arabica. If we consider a
moderate caffeine intake as 400 mg/day (as
recommended by Health Canada), this can be
easily achieved by consuming 2 up to 3 cups of
coffee/day. However, one should take into account
that coffee is not the only source of caffeine in the
diet. Teas (black and green), energizing drinks,
sweets, etc are also contributing to the caffeine
intake. Regarding the analysed chicory sample,
this contained a very small amount of caffeine
compared with the coffee brews.
Even though Robusta variety has proved to
be richer in phenolic compounds, flavonoids and
caffeine, Arabica coffee variety is appreciated for
its fine aroma profile. Therefore, by mixing these
two varieties of coffee, the product obtained can
be more balanced in terms of the ratio between
the biologically active compounds and sensorial
properties. Nevertheless, based on the obtained
results for the determined bioactive compounds,
we can presume that the blended sample contains
a larger percent of Robusta variety.
CONCLUSION
The content in bio functional compounds as
well as the antioxidant capacity of different brewed
coffee samples was assessed. According to the
obtained results, beside the caffeine intake, coffee
is a valuable source of antioxidants with a great
potential health benefits. Instead, coffee substitute
such as chicory have not only a small amount of
caffeine but also a low content of compounds with
antioxidant properties.
Total phenols
66.00
209.75
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169.50
239.25
5.92
Caffeine
251.9
187.6
258.1
0
30
60
90
120
150
mg/cup
180
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240
270
Fig. 1. The total amount of polyphenols, flavonoids and
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coffee or chicory (5 g coffee or chicory per 100 ml
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