Analysis of Caffeine and Antioxidant Content of Ethiopian Coffee Varieties from Different Growing Areas

Abstract

Quality coffee consumption is increasing nowadays; however, quality can only be achieved through appropriate cultivation and processing techniques. The study is aimed at using high-performance liquid chromatography (HPLC) and spectrophotometric techniques for evaluating different coffee varieties grown in Ethiopia based on caffeine, antioxidant, and polyphenol content. Despite coming from various coffee-growing regions, all of the tested coffee varieties (heirloom, forest coffee, heirloom 2, bourbon, mixed heirloom, and catuai) underwent wet processing. The data revealed that the caffeine content ranged from 0.14 to 0.37 mg/g, total antioxidant content from 63.75 to 78.05 mg AAE/g, and total polyphenol content from 27.08 to 31.04 mg GAE/g in the selected varieties. The findings show that the amount of the identified compounds is significantly (p ≤ 0 05) influenced by the variety type, growing area, and processing (intensity of roasting). The different coffee varieties were also tested for sensory quality, and the findings suggest that the typical coffee drinker prefers fruity flavors in their coffee, along with medium acidity and roasting intensity levels. The cultivation and processing technologies used in coffee production have a significant impact on the quality and sensory enjoyment of coffee.

Full text

Research Article
Analysis of Caffeine and Antioxidant Content of Ethiopian Coffee
Varieties from Different Growing Areas
Beatrix Sik , Erika Lakatos , Henrietta Bura, and Rita Székelyhidi
Department of Food Science, Széchenyi István University, Lucsony Street 15-17, 9200 Mosonmagyaróvár, Hungary
Correspondence should be addressed to Rita Székelyhidi; szekelyhidi.rita@sze.hu
Received 6 July 2023; Revised 22 August 2023; Accepted 2 September 2023; Published 11 September 2023
Academic Editor: Miguel Rebollo-Hernanz
Copyright © 2023 Beatrix Sik et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Quality coffee consumption is increasing nowadays; however, quality can only be achieved through appropriate cultivation and
processing techniques. The study is aimed at using high-performance liquid chromatography (HPLC) and spectrophotometric
techniques for evaluating different coffee varieties grown in Ethiopia based on caffeine, antioxidant, and polyphenol content.
Despite coming from various coffee-growing regions, all of the tested coffee varieties (heirloom, forest coffee, heirloom 2,
bourbon, mixed heirloom, and catuai) underwent wet processing. The data revealed that the caffeine content ranged from 0.14
to 0.37 mg/g, total antioxidant content from 63.75 to 78.05 mg AAE/g, and total polyphenol content from 27.08 to 31.04 mg
GAE/g in the selected varieties. The findings show that the amount of the identified compounds is significantly (p≤0 05)
influenced by the variety type, growing area, and processing (intensity of roasting). The different coffee varieties were also
tested for sensory quality, and the findings suggest that the typical coffee drinker prefers fruity flavors in their coffee, along
with medium acidity and roasting intensity levels. The cultivation and processing technologies used in coffee production have a
significant impact on the quality and sensory enjoyment of coffee.
1. Introduction
The world’sfifth-largest producer of coffee is Ethiopia. Cof-
fee is the primary source of income for countries that grow
coffee, which includes more than 80 nations, including Ethi-
opia [1]. Coffee is the second most traded good after oil [2].
Harar, Southern, Southwestern, and Northwestern are
the four main regions of Ethiopia where coffee is grown,
and in each of these regions, different environmental factors
and coffee cultivation techniques are used. In Ethiopia, cof-
fee is grown in four different ways: in the forest, semiforest,
gardens, and plantations [3]. Ethiopia has expanded its
coffee-growing areas in recent years, but no interventions
have been made to increase yield or quality [4]. However,
the climate significantly impacts the characteristics of the
coffee plant and product [5, 6].
Coffea arabica L. is a perennial plant species from the
Coffea genus that belongs to Rubiaceae family. It typically
grows in tropical and subtropical regions [7, 8] and is the
most widely grown coffee variety in Ethiopia. This variety
has high levels of heterogeneity [9], which allows the nation
to produce and market coffees with distinctive qualities. The
genetic component and cultivation and processing practices
significantly impact the physical characteristics and cup
quality of coffee [10]. Recent research has shown that the
yield, quality, and chemical makeup of Ethiopian coffees
are also influenced by the cultivation region [11], environ-
mental factors [12], harvest time [13], and postharvest pro-
cessing techniques [14, 15].
The study is aimed at comparing the sensory qualities
and caffeine, antioxidant, and polyphenol content of coffee
on the basis of varieties and growing regions in Ethiopia.
2. Materials and Methods
2.1. Chemicals. For the HPLC analysis, a caffeine (>99%,
Merck, Germany) standard was used for the calibration
curve, and a mixture of acetonitrile (Merck, Germany),
phosphoric acid (LiChrosolv, Merck, Germany), and high-
purity water was used as eluent. For the determination of
Hindawi
Journal of Food Processing and Preservation
Volume 2023, Article ID 8831024, 5 pages
https://doi.org/10.1155/2023/8831024

total antioxidant content with the FRAP assay, sodium ace-
tate (Merck, Germany), 2,4,6-tripyridyl-s-triazine (TPTZ)
(Sigma-Aldrich, USA), anhydrous iron chloride (Merck,
Germany), ascorbic acid (Sigma-Aldrich, USA), acetic acid
(Reanal, Hungary), and 37% hydrochloric acid (Reanal,
Hungary) were used. Anhydrous sodium carbonate (Rie-
del-de Haën, Germany), gallic acid (Sigma-Aldrich, USA),
and Folin-Ciocalteu reagent (Fischer Chemical, USA) were
used for the determination of total polyphenol content with
the Folin-Ciocalteu method.
2.2. Coffee Samples. The coffee samples were obtained by
ordering them from an online store which provides the con-
sumer with all the cultivation and processing characteristics
of the sold coffees. In the study examined only Arabica cof-
fee varieties that are grown in Ethiopia. All investigations
were performed in three replicates. The green coffee beans
were roasted for 5 to 15 minutes at 200
°
C. The characteristic
features and growing regions of the coffee under investiga-
tion are listed in Table 1.
2.2.1. Examination of the Sensorial Properties of Coffee
Varieties. Sensorial testers were average consumers with
average sensory sensitivity. Testers used a questionnaire to
assess the samples. The study involved 21, with 16 women
and 5 men participating. The respondents were 37 years
old on average. 20 g of roasted and ground coffee samples
was combined with 500 mL of water to create the coffee
drinks using an espresso coffee maker [16]. The Food Indus-
try Manual’s Part 7 served as the basis for creating the
assessment sheet [17].
2.2.2. Sample Preparation for HPLC Analysis. Based on the
methodology of Nhan and Phu [18], caffeine extraction
and HPLC determination were carried out, with some
adjustments made by Shrestha et al. [19]. The coffee beans
were finely ground using a coffee grinder (Bosch), and 0.3 g
of the sample was weighed into 250 mL Erlenmeyer flasks
with an analytical balance (TE 214S, Sartorius). The samples
were added to 200 mL of ultrapure water (Zener Power I
Scholar-UV, Human Corporation) and then submerged for
30 minutes in a 100
°
C water bath. After the sample solutions
had cooled, they were centrifuged (Z206A, Hermle) at
6000 RPM for 20 minutes. 1 mL of the supernatant was then
pipetted into volumetric flasks marked at 10 mL, and the
remaining used eluent was added to the mark. About 1 mL
of the diluted solutions was filtered through a 0.22 μm
hydrophilic syringe filter and placed into 1.8 mL HPLC vials.
2.2.3. Sample Preparation for Spectrophotometric Analysis.
The active compounds were extracted from the matrix using
solvent extraction to calculate the levels of polyphenols and
antioxidants in coffee samples [20]. 3.5 g of the ground cof-
fee beans was weighed and put into 100 mL Erlenmeyer
flasks on an analytical balance. The samples were centri-
fuged (Z206A, Hermle) at 6000 RPM for 20 minutes after
being centrifuged for 5 minutes in 60 mL of high-purity
water. In order to calculate the antioxidant content, 1 mL
of the supernatant was pipetted into 10 mL volumetric flasks
and completely filled with high-purity water. The samples
did not require dilution after centrifugation to determine
the total polyphenol content.
2.3. Determination of Caffeine Content with HPLC-UV
Equipment. Reverse-phase liquid chromatography was used
to determine the amount of caffeine in the coffees. The sta-
tionary phase was a LiChrospher 100 C-18 column (5 μm),
which had the following specifications: a length of 250 mm,
an inner diameter of 4 mm, and a temperature of 40
°
C.
Acetonitrile-0.05% phosphoric acid solution (10 : 90 V/V%)
was the mobile phase, flowing at a rate of 1 mL/min. The
amount of the injected sample was 5 μL, and the detection
was done using a UV detector at a wavelength of 275 nm.
As a standard, caffeine solutions (0.2–30 g/mL) were used.
2.4. Determination of Total Antioxidant and
Polyphenol Content
2.4.1. FRAP Assay. The process used for the FRAP assay is
based on the approach outlined by Benzie and Strain [21].
100 mL of water, 3 mL of FRAP solution, and 50 μLofthe
extracted coffee sample were pipetted into a test tube. After
being left in the dark for 5 minutes, the finished solutions
were compared to a blank solution made up only of water
and FRAP solution using a Spectroquant Pharo 100 spectro-
photometer (Merck, Germany). The results were expressed
as ascorbic acid equivalent (AAE)/g dry matter using ascor-
bic acid as standard (40-500 mg/L).
2.4.2. Folin-Ciocalteu Assay. Total polyphenol content was
determined using a modified version of the Folin-Ciocalteu
method as described by Singleton et al. [22, 23]. 1.5 mL of
ultrapure water was pipetted into test tubes with 50 μLof
coffee extract before adding the reagents. 2 mL of a 7.5%
Na
2
CO
3
solution should be added after 2.5 mL of 10%
Folin-Ciocalteu reagent. The mixture-containing tubes were
left in a dark location for 90 minutes, after which the absor-
bance at 725 nm was compared to the blank, which con-
tained the reagents and water. The used standard solutions
were made with gallic acid (25–1000 mg/L).
2.5. Data Analysis. By using the equation of the second-
order least squares analytical curve fitted to the measure-
ment solutions by the nonlinear least squares method, the
absorbance values measured for samples were converted into
the caffeine, total antioxidant, and polyphenol contents. This
information was then entered into Microsoft Office Excel to
be calculated. In order to compare the significant difference
in the data (p<005), analyses of variance (ANOVA) were
used. All results are expressed as means n=3 ± standard
deviation.
3. Results and Discussion
3.1. Sensorial Test Results. The results of the sensorial test are
shown in Figure 1. Due to its dark color, heirloom coffee had
the lowest popularity and catuai coffee had the highest. Cus-
tomers favor chocolate brown, medium-roasted coffees over
black, heavily roasted samples.
2 Journal of Food Processing and Preservation

The coffee samples from catuai and heirloom were rated
as having the best aromas and a mildly fruity flavor. Forest
coffee, which some respondents said smelled like tobacco,
was the least preferred coffee scent.
Catuai coffee was the most popular coffee in terms of
acidity, while heirloom 2 was less well liked due to its low
acid content and forest coffee was less well liked due to its
high acidity.
In terms of bitterness, reviewers rated mixed heirloom
and catuai coffees as the best. In this regard, heirloom 2
and forest coffee performed less well. There are similarities
in how coffee’s acidity and bitterness are rated.
The most well-liked flavors were fruity-tasting catuai
and heirloom coffee. The least preferred coffee flavor was
forest coffee, which many tasters claimed had a tobacco fla-
vor, possibly explaining its floral undertones.
The catuai coffee was the most well liked overall. The
sensorially evaluated coffees that received the least favor-
able ratings from the participants were forest coffee and
heirloom 2.
3.2. Caffeine, Antioxidant, and Polyphenol Content of
Ethiopian Coffee Samples. The findings from the analysis of
samples of Ethiopian coffee’scaffeine, antioxidant, and poly-
phenol content are displayed in Table 2.
According to the findings, the heirloom coffee variety
stands out because its caffeine content is significantly
(p≤0 05) lower than that of the other coffee varieties at
0.14 mg/g. This might be a result of the coffee being chosen
to come from a particularly low altitude (1500 m), where it
is likely that it did not need to be protected from insects in
the growing area, unlike the other coffee plants. Because cof-
fee beans contain more caffeine before roasting, the low caf-
feine content is also a result of the very dark roasting. The
amounts of caffeine in the coffee varieties forest coffee, bour-
bon, mixed heirloom, and catuai were all 0.36 mg/g, while
those in heirloom 2 were 0.37 mg/g. The investigated caf-
feine level values for the various species are consistent with
Miłek et al. [24] published data.
The FRAP technique was successful in detecting the total
antioxidant content. With the exception of bourbon-type
coffee, which had a value of 64.75 mg AAE/g, the antioxidant
content of the coffee samples decreased with altitude. The
bourbon (64.75 mg AAE/g) and catuai (63.75 mg AAE/g)
coffee varieties from the Sidamo and heirloom (78.05 mg
AAE/g) and forest coffee (71.84 mg AAE/g) from the Kaffa
region showed the difference in TAC value, which can be
attributed to their region of cultivation. According to the
article by Tasew et al. [25], the degree of roasting is another
factor that influences the total antioxidant content in
Table 1: Cultivation and physicochemical parameters of the examined coffee varieties (n=3).
Coffee varieties Region Altitude (m) Taste Processing Acidity Roasting
Heirloom Kaffa 1500 Fruity, winey Wet High Very dark
Forest coffee Kaffa 1500 Floral, grapey Wet High Medium
Heirloom 2 Oromia 1800 Floral, fruity Wet Low Dark
Bourbon Sidamo 1500-2200 Chocolate Wet Medium Medium
Mixed heirloom Yirgacheffe 2200 Floral Wet Medium Dark
Catuai Sidamo 2150-2500 Blueberry, pineapple, plum Wet Medium Medium
5.00
6.04
4.71
5.09
4.80
4.90
5.19
4.95
3.57
3.80
3.14
3.85
6.00
5.23
3.38
3.52
3.38
4.04
6.57
5.80
4.19
4.19
4.66
4.85
6.90
5.66
4.76
5.42
4.71
4.90
8.00
6.14
5.42
5.38
4.85
5.38
0
1
2
3
4
5
6
7
8
9
Color Fragrance Acidity Bitterness Taste Total
impression
Heirloom
Forest coee
Heirloom 2
Bourbon
Mixed heirloom
Catuai
Figure 1: Results of the sensorial examination of coffee samples.
3Journal of Food Processing and Preservation

addition to the region of origin. The presented results, with
the exception of the catuai variety, also support the literary
data of Daniel and Workneh [26] that the antioxidant con-
tent of Ethiopian coffees ranges from 64 to 97 mg AAE/g,
depending on the region.
Plants needed to produce a lot of polyphenols to protect
themselves from strong UV radiation in the desert, so next
to the lowest acidity the Heirloom 2 coffee variety had the
highest polyphenol content (31.04 mg GAE/g), this may be
explained with the origin- Oromo region. The lowest poly-
phenol content (27.08 mg GAE/g) was found in the heirloom
coffee variety, which may be attributed to the very dark
roasting that reduced the amount of phenolic compounds.
The results are further supported by the study of Dybkowska
et al. [27], which found that the amount of total polyphenols
differed between the light and dark roasts of the same Ethi-
opian coffee sample by up to 14 mg GAE/g.
4. Conclusion
The panelists preferred coffee drinks with a medium level of
acidity, softer fruit flavors, and lighter chocolate brown cof-
fee, which is influenced by the degree of roasting. The eval-
uation of the bitter taste’s results showed a strong
correlation with acidity. Coffee drinks with floral notes did
not fare well in the taste test; they were compared to the
smell of tobacco.
Five grams of caffeine is the lethal dose. The findings
show that the examined coffee varieties do not contain
enough caffeine to harm the body when consumed in the
amount of 3-4 cups per day. The caffeine levels in the tested
coffee varieties were typical of the region where they were
grown.
The samples contained a significant amount of antioxi-
dants and polyphenols, which can be used to refute the myth
that coffee is unhealthy. The degree of roasting determines
the concentration of these compounds. With altitude, anti-
oxidant content exhibited a trend toward decreasing. The
results also reveal significant differences, which might be
brought on by variations, growing regions, and altitude.
Data Availability
The data used to support the findings of this study are
included within the article.
Conflicts of Interest
The authors have declared no conflict of interest.
Acknowledgments
This study is funded by the Széchenyi István University.
Open access funding is enabled and organized by the Elec-
tronic Information Service National Programme.
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b
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