Food Control 123 (2021) 107857

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Food Control
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Caffeine, invertase enzyme and triangle test sensory panel used to

differentiate Coffea arabica and Vernonia amygdalina honey
Hawine Debela a, Abera Belay b, *
a
Department of Food Science and Applied Nutrition, Addis Ababa Science and Technology University, and Ethiopian Conformity Assessment Enterprise Addis Ababa, P O
Box 102049, Ethiopia
b
Department of Food Science and Applied Nutrition, Addis Ababa Science and Technology University, Addis Ababa, P O Box 16417, Ethiopia

A R T I C L E I N F O A B S T R A C T

Keywords: Honey is a highly consumed natural product, not only for the taste, but also for biofunctional benefit. Due to the
Botanical origin strong demand and special merit, the honey industry is under frequent deception worldwide, and the scarce
Caffeine branded honey replaced by the cheapest type of honey. The replacement of branded honey with inferior quality
Coffea arabica honey
honey is a global concern. Accordingly, identification and setting of a chemical marker for a specific type of
Invertase enzyme
Physicochemical property
honey is essential. In this study, Coffea arabica and Vernonia amygdalina honey investigated. Melissopalynology
Triangle test sensory Panel found that Coffea arabica (54.4–94.2%) and Vernonia amygdalina (47–68.8%) were dominant honey samples.
Coffea arabica honey had 96.11 ± 20.02 mg/kg caffeine, 7.64 ± 0.84◦Schade diastase, 14.12 ± 2.25 invertase
number (IN); and Vernonia amygdalina honey had 17.02 ± 0.29 mg/kg caffeine, 12.5 ± 0.55◦Schade diastase,
10.09 ± 0.08IN. There was a significant difference (p < 0.05) between Coffea arabica and Vernonia amygdalina
honey in caffeine. The association between Coffea arabica pollen count and the amount of caffeine had a positive
regression model (Caffeine = 1.3318(Pollen count of Coffea arabica(%))–3.9392), at r2 = 0.8746. In addition, the
invertase number was also regressed with a pollen count of Coffea arabica (%), (Invertase number = 0.081(Pollen
2
count of Coffea arabica(%))–8.6636) at r = 0.7399. Coffea arabica honey had 16.99 ± 1.01g/100g moisture, 5.6 ±
2.52 mg/kg hydroxymethylfurfural, and − 9.91 ± 3.09 [α]D20 specific rotation. Vernonia amygdalina honey had
19.02 ± 0.05g/100g moisture, 0.43 ± 0.01 mg/kg hydroxymethylfurfural, and − 5.98 ± 0.07 [α]D20 specific
rotation. Fructose, glucose, sucrose, turanose, and maltose for Coffea arabica honey were 36.26 ± 1.35, 31.24 ±
2.09, 0.93 ± 0.67, 0.08 ± 0.004, and 0.87 ± 0.79g/100g, respectively; and Vernonia amygdalina honey had
37.54 ± 0.82, 30.12 ± 0.87, 0.1 ± 0.01, 0.07 ± 0.002, 0.78 ± 0.09g/100g, respectively. Based on the triangle
test, 94.44% of the panelist identified the bitter taste immediately after swallowing for Vernonia amygdalina
honey. Thus, the level of caffeine and triangle test panel used to differentiate Coffea arabica honey and Vernonia
amygdalina honey. The amount of caffeine present in honey depends on the dominant level of coffee nectar,
which can be used as an indicator of Coffea arabica honey.

1. Introduction Worldwide, there are conventional honey quality parameters that

Honey, a natural biological plant product, is a nutritious food man­
ufactured by the honeybees from the nectar of plants or from secretions
of living parts of plants or excretions of plant-sucking insects (Belay,
Solomon, Bultossa, Adgaba, & Melaku, 2013; Codex Alimentarius,
2001). Honey has different properties and compositions, which depend
on its geographical floral origin, season, and environmental factors
(Kaškonienė, Venskutonis, & Čeksterytė, 2010). The nectar source pro­
vides different honey chemistry and sense of test (Belay, Solomon,
Bultossa, Adgaba, & Melaku, 2015).
used to authenticate honey. These major parameters are moisture con­
tent, reducing sugar, sucrose, free acidity, electrical conductivity, and
hydroxymethylfurfural (Codex Alimentarius, 2001). In addition to
these, honey also consists of minor components, such as different en­
zymes and caffeine. The most important honey enzymes are invertase
(α-glucosidase) and diastase (α-amylase) (Flanjak, Strelec, Kenjerić, &
Primorac, 2016).
There is a strong food deception in the world these days. Natural
food, like honey, is adulterated to increase the quantity and make more
profit. Due to the turnover motive and the scarcity of specific types of

* Corresponding author.
E-mail addresses: dhawidesire@yahoo.com (H. Debela), ab.berabelay@gmail.com (A. Belay).

https://doi.org/10.1016/j.foodcont.2020.107857
Received 21 August 2020; Received in revised form 13 November 2020; Accepted 24 December 2020
Available online 29 December 2020
0956-7135/© 2020 Elsevier Ltd. All rights reserved.
H. Debela and A. Belay
honey in the market, the cheapest type of honey is used to replace the
well-qualified and branded honey. Deception and substitution of
branded honey with lower price honey is a global concern in the honey
industry (Bertelli et al., 2010). Accordingly, the setting of a chemical
and sensorial marker for the identification of specific types of honey is
essential.
Honey from the particular floral source has specific sensorial char­
acteristics, in relation to color, texture, aroma, and flavor, taste, and
overall acceptance (Belay et al., 2015; Kumar et al., 2018). Sensory
evaluation enables to distinguish the botanical origin of honey, identify
fraud sources, and quantify certain defects (Foomaneechot & Sir­
inupong, 2018, pp. 1–6).
Melissopalynology analysis of honey showed that Coffea arabica and
Vernonia amygdalina are dominant honey plants in Ethiopia, and found
elsewhere in coffee-growing areas worldwide. Both honey plants, bloom
nearly at the same period, from January to March. The intensity of
flowering mostly depends on the strength of a short shower of seasonal
rain. Coffee is one of the most popular drinks all over the world. It
consists of more than 700 compounds, which are responsible for its ar­
omatic and unique flavor. Ethiopia is widely regarded as the birthplace
of coffee (Kebede & Bellachew, 2008). Honeybees are the frequent
visitor of coffee flowers to produce coffee honey (Ngo, Mojica, & Packer,
2011). In addition, bitter leaf (Vernonia) is also found in Ethiopia (Boyo,
Shitta, Oluwa, & Adeola, 2012). According to Fichtl and Admasu (1994,
p. 510), Vernonia Spp (Gerawa) is a valuable honey source in Ethiopia. In
warmer areas, the nectar secretion is abundant and bees produce a
significant surplus of dark aromatic honey (Degaga, 2017).
Coffea honey is not a common product. It is rare and almost unex­
ploited and contains elements of nutritional and biological interest
(Schievano et al., 2015). There is a consumer and processor interest to
collect and utilize coffee honey. However, the trader substitutes coffee
honey with other dark brown polyfloral and monofloral honey including
Vernonia honey that needs investigation. The detection of monofloral
honey using honey chemistry, allowed to authenticate its origin (Ohe,
Oddo, Piana, Morlot, & Martin, 2004).
Schievano et al. (2015) investigated the chemical characteristics of
Fig. 1. The study area.
2
Food Control 123 (2021) 107857
coffee honey from Colombia & Honduras. In addition, Belay et al.
(2017a) described the physicochemical, enzymes and amino acid profile
of Schefflera abyssinica, Croton macrostachyus, Eucalyptus globulus, Becium
grandiflorum, Acacia, Leucas abyssinica, Hypoestes, and Syzygium gui­
neense of Ethiopian origin. However, Coffea arabica and Vernonia
amygdalina honey of Ethiopian origin is not researched so far.
Thus, understanding the above-mentioned facts, the objective of this
study was to investigate Coffea arabica honey and Vernonia amygdalina
honey with the emphasis of differentiating these honeys, based on
botanical origin, caffeine content, enzymes, and physicochemical
properties.
2. Materials and methods
2.1. Study area
Honey samples were harvested during and after the Coffea arabica
and bitter tree (Vernonia amygdalina) flowering season, in the beginning
of January and to the end of March in the forest region of Ethiopia. The
samples were collected from four places, namely Mana, Gomma, Yayu
and Becho, Ethiopia (Fig. 1), where coffee and bitter tree plants are
dominantly found.
2.2. Sample collection
Twenty honey samples were collected from farm-gate, using an air
tighten, food-grade metallic container. The samples were filtered using
straining and settling, and transferred into a 500 g food grade honey jar.
The honey samples stored at − 20 ◦ C until analysis (Belay et al., 2015).
2.3. Botanical origin
Botanical origin was determined based on Belay, Solomon, Bultossa,
Adgaba& Melaku (2015), using pollen analysis. Accordingly, 10 g honey
weighed in a centrifuge tube (capacity ca. 50 mL) and dissolved in 20 mL
of distilled water, and centrifuged for 10 min at 2509 g (3500 rpm). The
H. Debela and A. Belay
supernatant was decanted and the residue spread evenly on a micro­
scope slide and allowed to dry. The slides examined through the mi­
croscope (ZEISS, Germany). The pollen source plant was identified using
reference slides and pollen atlas. The slides and the pollen atlas were
used as a reference to cross-check with the pollen morphology found in
the honey samples (Adgaba, 2007). The frequency of occurrences was
determined by counting 500 pollens from a particular slide. Then, the
pollen count was transformed into percent to measure the relative
dominance (Belay et al., 2015; Ohe et al., 2004).
2.4. Caffeine content
Caffeine content was determined according to Kadri, Zaluski, Lima,
Mazzafera & de Oliveira (2016). Honey sample (2 g) was diluted in 25
ml of hot distilled water, and heated (94 ◦ C) on a water bath until
completely dissolved. After cooling (24 ± 2 ◦ C), chloroform (10 mL) was
added and the mixture was mixed with a vortex mixer. The mixture was
centrifuged at 3400 g (4500 rpm) for 5min. The chloroform fraction was
recovered, and the aqueous phase was subjected to another round of
chloroform extraction. The two chloroform fractions were pooled and
dried in a rotatory evaporator at 35 ◦ C in a 25-mL flask, and solubilized
in 2 mL deionized water. The extract filtered and subjected to HPLC
(HPLC- 1260 Infinity Series Agilent Technologies, Germany).
Caffeine separation was performed using a C18 column (Supelco,
dimensions 4 mm × 250 mm, 0.5 μm particles) (ZORBAX SB-C18) using
50% methanol in water (containing 0.5% acetic acid), the flow rate of
0.8 ml/min. Caffeine was detected in a DAD detector operating at 272
nm, and the result was expressed as mg/kg.
2.5. Enzymes
2.5.1. Diastase activity
Diastase analysis was performed by Phadebas, based on the
Harmonized Method of the IHC using spectrophotometric method, in
which an insoluble blue-dyed starch hydrolyzed by the enzyme, yielding
blue water-soluble fragments (Bogdanov, 2009). One gram of honey
dissolved in the acetate buffer solution, and filled to the mark (100 ml).
5 ml of the solution transferred to a test tube and placed in the water
bath at 40 ◦ C. A blank was prepared by placing a 5 ml aliquot of the
acetate buffer (13.6 g of sodium acetate trihydrate in 1 L of distilled
water, pH adjusted to 5.2 by glacial acetic acid (1–2 mL)), and was
treated exactly like the sample solution. To both solutions, Phadebas
tablet added, and the solutions in the reagent mixer were stirred until
the tablets disintegrated (ca. 10 s) and returned them to the water bath.
The reaction terminated after exactly 15 min by adding 1 ml sodium
hydroxide solution. The mixture again stirred in the reagent mixer for
approximately 5 s. The solution filtered through filter papers (What­
man® Grade 541) and the absorbance measured in 1 cm cuvettes at 620
nm UV spectrophotometer (CECIL, CE 7500, 7000 series) using deion­
ized water as a reference.
Diastase activity was computed by regression equation for 0 to 6
DN (low) (DN = 35.2 ​ x ​ ΔA620 –0.46; and 8 to 40 DN (high) (DN =
28.2 ​ x ​ ΔA620 + 2.64). ΔA620 calculated by subtracting the absorbance
of the blank from the sample solution.
2.5.2. Invertase
Invertase activity of honey was determined based on Harmonized
Method of the IHC (Bogdanov, 2009). Five grams of honey was dissolved
in 0.1M buffer solution and filled up to the mark (25 ml). 5 ml of sub­
strate solution (p-nitrophenyl-α-D-glucopyranoside (pNPG) solution,
0.02 M) was placed in a test tube in the water bath (40 ◦ C) for 5 min 0.50
ml of honey solution was added (starting time) and the contents mixed
briefly in a mixer and incubated (40 ◦ C). After exactly 20 min, 0.5 ml of
the reaction-terminating solution (3 M, pH = 9.5 tris- (hydroxymethyl)
Aminomethane) was added and mixed again in a mixer (sample solu­
tion). For the blank, 5 ml of substrate solution incubated (40 ◦ C) at the
3
Food Control 123 (2021) 107857
same time. After 5 min, 0.5 ml of reaction-terminating solution added,
stopper the tube, mix well and then 0.5 ml of honey solution was added.
The solution cooled to room temperature and the absorbance was
measured at 400 nm using UV Spectrophotometer (CECIL, CE 7500,
7000 series). The honey invertase activity can be calculated in units/kg
(U/kg).
Invertase ​ U/kg = 158.94*ΔA400
where U = 1 international unit with a defined utilization of 1 μM
per minute; ΔA400 = difference in absorbance between the honey
sample and the blank.
2.6. Sensorial property
Sensory analysis was performed based on ISO 4120:2016. Sensory
panelists were selected according to ISO 8586:2012. The evaluation was
carried out using a triangle test (Lawless & Heymann, 2010). Thirty (30)
g of honey sample was served (room temperature). Assessors received a
set of three blindly labeled samples, which belongs to different floral
origins (Coffea arabica honey and Vernonia amygdalina honey). The as­
sessors reported which sample they believed is different. The number of
correct responses was counted and the significance was determined by
using a minimum number of correct responses (ISO 4120:2016).
2.7. Sugars
Sugars were determined using high-performance liquid chromatog­
raphy (HPLC- 1260 Infinity Series Agilent Technologies, Germany)
equipped with a differential refractive index (DRI) detector (AOAC,
1990). Honey sample (5 g) was dissolved in 25% methanol in water
solution, and the solution was transferred and makeup to a 100 ml
volumetric flask. The solution filtered through 0.45 μm syringe and
injected to HPLC. The separation was performed using NH2 column (4.6
× 250 mm) (ZORBAX NH2) with a particle size diameter of 5 μm, and the
column kept at 30 ◦ C. The mobile phase composition was 70% aceto­
nitrile in water. The injection volume of the sample was 10 μl, and the
flow rate was 1.3 ml/min. The retention time was identified using
standards of the peak. Sugar content (fructose, glucose, sucrose, tur­
anose, and maltose) was determined using a standard curve and the
value was calculated as g/100g of the honey sample.
2.8. Moisture
Moisture was determined with a digital refractometer (Abbe refrac­
tometer, Leica Mark II Plus) thermostated at 20 ◦ C. The honey sample
was homogenized and the surface of the prism covered evenly with the
sample. After 2 min, the refractive index was read with a refractometer.
The refractive index reading converted to moisture content using the
AOAC, 969.38 conversion table (AOAC, 1990).
2.9. Free acidity and pH
pH meter (HANNA, HI 2550, PH/ORP) was used to measure the free
acidity and pH of a 10% (w/v) solution of honey prepared in deionized
water. pH was determined by using a glass electrode after calibration
with a standard buffer solution (pH 4, 7, and 10). 10 g honey sample was
dissolved in 75 ml of carbon dioxide-free water. It was stirred with the
magnetic stirrer, then pH electrode immersed in the solution, and pH
was recorded. Free acidity was determined using 0.1M NaOH to pH
8.30, and the volume consumed recorded (AOAC, 1990 method 962.19).
2.10. Electrical conductivity
Electrical conductivity was measured using a conductivity meter
(HANNA, HI 2550, EC/TDS/NaCl meter). Twenty (20) g anhydrous
H. Debela and A. Belay
honey was dissolved in distilled water and makeup to 100 ml. Potassium
chloride solution (0.1M) was used for the cell constant and the electrical
conductance of this solution was read in mS after the temperature has
been equilibrated to 20 ◦ C (Bogdanov, 2009). The electrical conductivity
of the honey solution was calculated in mS.cm− 1 using the following
formula:
SH = K*G
where SH = electrical conductivity of the honey solution in mS/cm, K =
cell constant in cm− 1, and G = conductance in mS.
2.11. Ash
Ash content was determined according to AOAC (1990) method
920.181. Accordingly, 5 g of honey was placed in combustion pots,
which required preheating to darkness. Then, the samples incinerated at
high temperature (550 ◦ C) in a burning muffle (THERMO CONCEPT,
KLS 45/13, Germany) for 1 h. After cooling at room temperature, the
obtained ash weighed and the proportion of ash (Ac) in g/100g honey
was calculated using the following formula:
(m3 − m1)
Ash ​ (g/100g) ​ = X 100
m2
where m1 ¼ weight of ashing dish, m2 ¼ weight of honey taken, and
m3 ¼ weight of ashing dish þ ash.
2.12. Hydroxymethylfurfural
Hydroxymethylfurfural (HMF) was determined using AOAC (1990)
method 920.181 on high-performance liquid chromatography (HPLC-
1260 Infinity Series Agilent Technologies, Germany) at absorbance scale
of 285 nm, using DAD. Ten (10) g of honey was dissolved with 50 ml
deionized water. The solution was filtered through a 0.45 μm syringe
filter and read on HPLC using a C18 column (3.0 × 250 mm) (ZORBAX
SB-C18) with a particle size diameter of 5 μm. The column was kept at
30 ◦ C throughout the analysis. The mobile phase composition was 90%
deionized water (1% acetic acid) with methanol. The injection volumes
of the samples were 10 μl, with a flow rate of 0.7 ml/min (AOAC, 1990)
method 920.181. The HMF content of the samples was calculated by
comparing the corresponding peak areas of the samples.
2.13. Specific rotation
The specific rotation was determined by measuring the angular
rotation of a clear (clarified by Carrez I and Carrez II solutions), filtered
aqueous honey solution (10 g dissolved into 100 mL solution) by 2-dm
polarimeter (KRUSS, Germany) at 20 ◦ C as described in Harmonized
IHC method (Bogdanov, 2009). Carrez I (10 g K4Fe(CN)6.3H2O in 100
ml) and Carrez II (24 g Zn(CH3COO)2⋅2H2O and 3 g acetic acid in 100
ml) solutions were used for clarification. The specific rotation was
calculated using the following formula:
α ∗ 100
Specific ​ rotation ​ [α]20 D =
L∗g
where α = angular rotation found, L = length in decimeters of polar­
imeter tube, g = grams of dry matter sample mass taken, and D = sodium
D line.
2.14. Statistical analysis
Botanical origin and physicochemical data were generated from
multiple runs of samples measurements. All data were analyzed by SAS,
2002; using a one-way analysis of variance (ANOVA), and a significant
difference among the sample was identified at p < 0.05. The linear
regression model was developed to indicate the association between
4
Food Control 123 (2021) 107857
different quality merits.
3. Results and discussion
3.1. Botanical origin
Definition of honey, based on plant origin used to validate the spe­
cialty of honey. The pollen analysis result of honey samples collected
from four places (Mana, Gomma, Yayu, and Becho) of Ethiopia was
presented in Table 1. Honey samples from Mana, Gomma, and Yayu
belongs to Coffea arabica, and pollen dominance ranged from 54.4 to
94.2% (Table 1). The honey samples collected from Becho was found to
be Vernonia amygdalina, which had 47–68.8% dominance. This defini­
tion is based on the relative frequency of the pollen of taxon. Honey
considered monofloral, if the dominance is more than 45% (Belay et al.,
2015; Ohe et al., 2004). The other honey plant pollen, which is not
dominant and found in the honey at different frequencies were Termi­
nalia browni, Bersama ayssinica, Eucalyptus spp, and Schefflera abyssinica.
Description of botanical origin is useful to qualify and register honey for
protected designation of origin (PDO) that help to promote the forest
honey (Aliaño-González, Ferreiro-González, Espada-Bellido, Barbero, &
Palma, 2020).
3.2. Caffeine
The mean ± sd of caffeine in Coffea arabica honey and Vernonia
amygdalina honey was presented in Table 2. Coffea arabica honey and
Vernonia amygdalina honey had 96.11 ± 20.02 and 17.02 ± 0.29 mgkg− 1
caffeine, respectively. There was a significant difference (p < 0.05) in
caffeine content among the honey samples.
Coffea arabica and Vernonia amygdalina flowers can be foraged by the
honeybees from the same location. The difference between these plants
is accessibility of nectar for honeybees, in which the coffee plant blooms
and found earlier than Vernonia flowers, which is dependent on a short
shower (Degaga, 2017). Likewise, there was a rare chance for honeybees
to collect and process the nectar of Vernonia amygdalina together with
Coffea arabica (Addi & Bareke, 2019). For this reason, caffeine might
appear as a contaminant from a coffee plant in Vernonia amygdalina
honey. According to Swaileh and Abdulkhaliq (2013) and Jerković,
Tuberoso, Kuś, Marijanović, and Kranjac (2014), caffeine was also pre­
sent in other monofloral honey, but in much smaller amounts. In line
with this, the values of caffeine generated from HPLC-diode array de­
tector for Vernonia amygdalina honey (17.02 ± 0.29 mgkg− 1) was lower
than Coffea arabica honey (96.11 ± 20.02 mgkg− 1), which can possibly
be used as a mark to identify these monofloral honeys.
The relationship between pollen count of Coffea arabica honey (%)
with caffeine and invertase number was stated in Fig. 2. The association
between Coffea arabica honey pollen count and the amount of caffeine
had a positive regression model (Caffeine = 1.3318(Pollen count of Coffea
2
arabica (%)) – 3.9392), at r = 0.8746 (Fig. 2). The amount of caffeine
present in honey depends on the dominant level of coffee nectar, which
can be used as an indicator of Coffea arabica honey. The minor compo­
nents of the honey are good indicators and have consistent merit for
honey classification (Cimpoiu, Hosu, Miclaus, & Puscas, 2013; Sanz,
Gonzalez, De Lorenzo, Sanz, & Martınez-Castro, 2005). Contrary to this,
the association between pollen count of Vernonia amygdalina honey and
caffeine content inversely regressed (Caffeine = − 1.4436(Pollen count
2
ofvernonia amygdalin (%)) + 97.503) at r = 0.7869 (Fig. 3). Both caffeine and
invertase were not associated with the Vernonia amygdalina honey
(Fig. 3). In addition, the major components of honey (sugar) was not
associated with a pollen count of Coffea arabica honey and Vernonia
amygdalina honey (%) (Figs. 2 and 3). This could be due to the incon­
sistent behavior of honey sugars, which pass through different process
inside the hive and disintegrate into various components, by either the
honeybees or the effect of in-hive temperature.
Currently, there is a deception of one type of product over the other,
H. Debela and A. Belay Food Control 123 (2021) 107857

Table 1
Relative pollen distribution of the honey samples (Percentage from 500 pollen counts).
Honey sample Coffea arabica Vernonia amigdalina Terminalia browni Bersama abyssinica Eucalyptus spp Schefflera abyssinica

M1 86.4 0.0 1.6 2.0 2.0 1.2

M2 75.2 0.0 7.4 0.0 4.6 0.0
M3 79.6 3.8 5.8 1.6 0.0 0.0
M4 79.0 1.0 11.0 0.0 3.0 2.4
M5 94.2 0.0 0.0 0.0 4.2 0.0
G1 70.8 0.0 0.0 0.0 5.4 2.4
G2 67.0 3.4 0.0 11.2 0.0 7.4
G3 54.4 0.0 0.0 27.0 3.0 7.0
G4 84.2 5.6 1.0 3.2 2.0 2.2
G5 71.0 4.0 0.0 2.4 5.8 5.2
Y1 71.0 0.0 5.4 0.6 2.4 4.2
Y2 76.2 0.0 4.4 6.2 8.6 1.0
Y3 73.8 0.0 3.8 8.4 6.8 2.2
Y4 67.8 2.0 7.2 9.6 2.4 1.0
Y5 75.6 0.0 13.0 0.0 4.4 0.0
B1 15.4 48.2 4.2 2.0 8.2 4.0
B2 19.4 51.4 2.0 1.0 0.0 0.0
B3 18.0 47.0 2.6 1.8 20.0 3.6
B4 18.2 58.0 0.0 2.4 11.0 0.0
B5 8.4 68.8 3.2 1.4 0.0 4.4

M = Mana, G = Gomma, Y= Yayu, B= Becho.

3.3. Enzymes
Table 2
Mean ± sd value for physicochemical properties, sugar compositions, Diastase,
3.3.1. Invertase activity
Invertase and Caffeine content of Coffea arabica and Vernonia amygdalina honey.
The enzyme content of honey may differ on nectar gathering time,
Honey samples the physiological period of the colony, quantity of nectar flow, and
Parameters Coffea arabica Vernonia amygdalina pollen consumption (Belay et al., 2017a). The mean ± sd value of
Moisture (g/100g) 16.99 ± 1.01b 19.02 ± 0.05a invertase activity was presented in Table 2. Coffea arabica honey and
Electrical conductivity (mS/cm) 0.52 ± 0.10a 0.52 ± 0.01a Vernonia amygdalina honey had an invertase value of 14.12 ± 2.25 and
Ash (g/100g) 0.28 ± 0.05a 0.28 ± 0.01a 10.09 ± 0.08 IN, respectively. There was a significant difference (p <
pH 3.93 ± 0.15b 4.25 ± 0.02a 0.05) in invertase activity among the monofloral honey. This could be
Free acidity (meq/kg) 33.74 ± 5.30a 20.75 ± 0.32b
Hydroxymethylfurfural (mg/kg) 5.60 ± 2.52a 0.43 ± 0.01b
due to the enzyme activity in honey that depends on the intensity of the
Specific rotation ([α]D20) − 9.91 ± 3.09b − 5.98 ± 0.07a nectar flow, and the amount of nectar processed by the honeybees.
Fructose (g/100g) 36.26 ± 1.35b 37.54 ± 0.82a Enzyme activity is intrinsic and closely associated with the pollen
Glucose (g/100g) 31.24 ± 2.09a 30.12 ± 0.87b dominance for some type of honey, which include Coffea arabica honey
Sucrose (g/100g) 0.93 ± 0.67a 0.10 ± 0.01b
(Escuredo, Seijo & Fernández-González, 2011). In addition to caffeine,
Turanose (g/100g) 0.08 ± 0.004a 0.07 ± 0.002b
Maltose (g/100g) 0.87 ± 0.79a 0.78 ± 0.09b invertase number was also regressed with a pollen count of Coffea
Diastase (◦Schade) 7.64 ± 0.84b 12.50 ± 0.55a arabica honey (%), (Invertase number = 0.081(Pollen count of Coffea arabica
2
Invertase (IN) 14.12 ± 2.25a 10.09 ± 0.08b (%)) – 8.6636) at r = 0.7399 (Fig. 2).
Caffeine (mg/kg) 96.11 ± 20.02a 17.02 ± 0.29b In the current study, the highest invertase number was observed in
“a,b” indicate significant differences (p < 0.05) among studied groups across the Coffea arabica honey. Based on the invertase classification of honey
row and are marked by different letters. (Oddo, Piazza, & Pulcini, 1999), invertase activity of the honey samples
fall in the medium to high (10–20 IN). The invertase activity in this
and honey adulteration is one of the main concerns of the honey in­ study was in agreement with the report of Escuredo, Seijo & Fernán­
dustry. It is emerging as a threat to the beekeeping industry worldwide dez-González (2011) (18.0 ± 5.9 IN) and Belay et al. (2017a) (11.6 IN).
(García, 2018). Various actions have taken to solve this problem, both
locally and internationally; however, no effective outcome was 3.3.2. Diastase activity
perceived in controlling the fraud of honey. Thus, setting caffeine con­ The diastase activity of Coffea arabica honey and Vernonia amygda­
tent as a marker can be used to differentiate Coffea arabica honey over lina honey was presented in Table 2. The mean ± sd of diastase activity
the other type of honey. The report of bib_Schievano_et_al_2015Schie­ for Coffea arabica honey and Vernonia amygdalina honey was 7.64 ± 0.84
vano et al. (2015) (97.8 ± 1.4 mgkg− 1) and Jerković et al. (2014) (83.59 and 12.5 ± 0.55◦Schade, respectively. The monofloral honey were
mgkg− 1) was in agreement with the findings of caffeine for this study. significantly different (p < 0.05) in diastase activity. This was in
The level of caffeine in Coffea arabica honey observed in this study was agreement with Draiaia et al. (2015), in which the diastase activity of
higher than the report of Kadri, Zaluski, Lima, Mazzafera & de Oliveira honey varied on the floral source.
(2016) (12.02 ± 0.81 mgkg− 1). Codex Alimentarius set a limit of ≥8◦Schade; however, honey with
Caffeine originated from a coffee plant can possibly use to differen­ low natural enzyme content can be also to the standards (diastase
tiate monofloral honey. According to Kadri, Zaluski, Lima, Mazzafera & ≥3◦Schade) (Codex Alimentarius, 2001). In the current study, the dia­
de Oliveira (2016) the level of caffeine found in Coffea arabica honey stase activities of Coffea arabica honey was below 8◦Schade, which was
was higher than the nectar (1.64 mg/kg) in coffee flower. The higher the below the limit of Codex Alimentarius (2001) and fulfill the limit set by
concentration of caffeine in coffee honey was due to the evaporation of Codex (not less than 3◦Schade) as naturally low enzyme honey. Honey
water present in the coffee nectar, which the honeybees concentrate the from tropical regions and some monofloral honey naturally have a low
caffeine during honey processing. diastase activity (Wang & Li, 2011). Fresh honey, which did not expose
to heat, had lower diastase activity (Belay et al., 2017a). In the current
study, the diastase activity of Vernonia amygdalina honey was higher

5
H. Debela and A. Belay Food Control 123 (2021) 107857

Sugar (g/100g)

Caffeine (mg/kg)

Invertase (IN)
Pollen count of Coffea arabica (%)

Fig. 2. Association between pollen frequency with caffeine, invertase number, and sugar content of Coffea arabica honey.

Sugar (g/100g)
Caffeine (mg/kg)

Invertase (IN)

Pollen count of Vernonia amygdalina (%)

Fig. 3. Association between pollen frequency with caffeine, invertase number, and sugar content of Vernonia amygdalina honey.

than 8◦Schade. This indicates that the source of honey could influence
the diastase activity of honey (Singh & Bath, 1997).
3.4. Sensory evaluation
Sensory analysis encompasses a set of techniques for a correct
judgement of human responses to foods and traces the conceivable
biasing effects of fraud, brand identity, and quality that influences
consumer perception. Like other analytical test procedures, sensory
evaluation is concerned with precision, sensitivity, and avoid false-
positive results (Lawless & Heymann, 2010). The sensory judgement
of honey can possibly complement the physicochemical and pollen an­
alyses, in order to confirm quality, verify the absence of defects, evaluate
the conformity to established sensory profiles of monofloral honeys
(Marcazzan, Mucignat-Caretta, Marina Marchese, & Piana, 2018). A
sensory evaluation carried out using a triangle test method in which a
proportion of correct choices above that expected is considered evidence
for a perceivable difference between products (Lawless & Heymann,
2010). This was based on accepting or rejecting the null hypothesis (Ho)
and the alternative hypothesis (Ha). The triangle test sensory panel
result, for thirty-six judges, was presented in Table 3. The triangle test
showed that 94.44% of assessors clearly identified and revealed to have
a perceptible difference between Coffea arabica honey and Vernonia
Table 3
Triangle test sensory panel result (%) (n = 36).
Description Yes
Different 94.44a
Same 5.56b

6
H. Debela and A. Belay
amygdalina honey samples, and 5.56% of assessors were not differenti­
ated the honey samples. Accordingly, there was a significant difference
(p < 0.05) between Coffea arabica honey and Vernonia amygdalina
honey, based on the minimum number of correct responses (24 judges)
needed to determine the perceptible difference (Lawless & Heymann,
2010). All the honey seem to be uniform in taste, and sightlessly defined
as sweet in sensory perception; however, one monofloral honey differ­
entiates from the other based on sensorial perception. Honey from
different geographical and botanic origins vary based on their sensory
profile (Belay et al., 2015; Castro-Vázquez, Díaz-Maroto,
González-Viñas, & Pérez-Coello, 2009).
A wide range of aroma descriptors, which range from bitter, rancid to
sweet, and flowery, can describe honey. Sensory evaluation, based
mainly on the attributes of taste, is one of the most valuable tools in
honey characterization. Bitterness is one of the four basic tastes (sweet,
salty, sour, umami), which can be viewed as a positive attribute in a
number of foods (Castro-Vázquez, Díaz-Maroto, De Torres, &
Pérez-Coello, 2010). The bitter taste, which is observed at the back of
the tongue during swallowing, was used to identify the bitterness of
honey. According to the panelists’ remark, the bitter taste immediately
after swallowing, noted in Vernonia amygdalina honey, used to differ­
entiate the monofloral honey. Thus, floral origin had a contribution to
the sensorial perception of honey. This was in line with the report of
Kumar et al. (2018).
3.5. Sugar profile
The mean ± sd of the sugar compositions in Coffea arabica and
Vernonia amygdalina honey were presented in Table 2. The mean ± sd of
fructose for Coffea arabica honey and Vernonia amygdalina honey was
36.26 ± 1.35 and 37.54 ± 0.82 g/100g, respectively (Table 2). There
was a significant difference (p < 0.05) in fructose content among the
honey samples. Fructose is the most abundant sugar in honey. The
fructose content in the current study was in agreement with the report of
Ouchemoukh, Schweitzer, Bey, Djoudad-Kadji, and Louaileche (2010)
(35.99–42.57g/100g) and Kaškonienė et al. (2010) (32.92–40.0
g/100g); however, lower than Primorac, Flanjak, and Topolnjak (2011)
(42.0 ± 3.3 g/100g) report.
The mean ± sd of glucose content in Coffea arabica honey and Ver­
nonia amygdalina honey was 31.24 ± 2.09 and 30.12 ± 0.87 g/100g,
respectively (Table 2). The monofloral honey was significantly differed
(p < 0.05) in glucose content. Glucose was the second most abundant
sugar in this finding and was in agreement with Ouchemoukh et al.
(2010) (24.63–35.06 g/100g) and Belay, Haki, Birringer, Borck, Lee,
Cho et al. (2017b) (29–37.2 g/100g) report. The significant difference in
fructose and glucose among the honey samples may be due to the dif­
ference in the floral source as stated by Molan (1996). There are no
limits that have been fixed for fructose and glucose individual values,
but their sum (Fructose + glucose) have the values corresponding to the
limits required of the international standard for honey established by
Codex Alimentarius Commission (not less than 60 g/100 g) (Codex
Alimentarius, 2001). The mean total monosaccharide (Fructose +
glucose) of Coffea arabica and Vernonia amygdalina honey samples in this
study were 67.5 and 67.66 g/100g, respectively. This indicated that all
the honey were in compliance with Codex Alimentarius.
Coffea arabica honey and Vernonia amygdalina honey had mean
fructose to glucose (f/g) ratio of 1.16 and 1.25, respectively. Many re­
searchers reported a mean value of f/g ratio, around 1.2 (Kaškonienė
et al., 2010; Manzanares, García, Galdón, Rodríguez, & Romero, 2011).
This ratio depends largely on the source of the nectar from which the
honey was extracted (Tornuk et al., 2013), which was in agreement with
this study.
Sucrose content of Coffea arabica honey and Vernonia amygdalina
honey was presented in Table 2. The mean ± sd of sucrose for Coffea
arabica honey and Vernonia amygdalina honey was 0.93 ± 0.67 and 0.10
± 0.01g/100g, respectively. There was a significant difference (p <
7
Food Control 123 (2021) 107857
0.05) in sucrose content among the honey samples. Lower sucrose
content indicated the honey is ripened (Ouchemoukh et al., 2010). The
sucrose content in this study was in agreement with Kaškonienė et al.
(2010) (0.7–2.5 g/100g) and Ouchemoukh et al. (2010) (0.48–5.26
g/100g) report. Codex Alimentarius set a limit for sucrose content (not
more than 5 g/100g). All the honey samples in this study were within
the acceptable limit set by the Codex Alimentarius (2001).
The mean ± sd of turanose and maltose for Coffea arabica honey and
Vernonia amygdalina honey were presented in Table 2. The mean ± sd of
turanose for Coffea arabica honey and Vernonia amygdalina honey was
0.08 ± 0.004 and 0.07 ± 0.002 g/100g, respectively. A significant dif­
ference (p < 0.05) was observed in turanose content among the honey
samples. Turanose content in this study was lower than Ouchemoukh
et al. (2010) (0.29–2.02g/100g); however, in close agreement with
Kaškonienė et al. (2010) (0.1–0.61 g/100g) report.
The mean ± sd of maltose for Coffea arabica honey and Vernonia
amygdalina honey was 0.87 ± 0.79 and 0.78 ± 0.09 g/100g, respectively
(Table 2). It was observed that there was a significant difference (p <
0.05) in maltose content among the honey samples. Maltose content in
this study was in agreement with Primorac et al. (2011) (0–5.8 g/100g)
and Ouchemoukh et al. (2010) (0.47–3.42g/100g) report.
3.6. Moisture content
The moisture content of Coffea arabica honey and Vernonia amyg­
dalina honey was presented in Table 2. In this study, the mean ± sd of
moisture content for Coffea arabica honey and Vernonia amygdalina
honey was 16.99 ± 1.01 and 19.02 ± 0.05 g/100g, respectively. It was
observed that there was a significant difference (p < 0.05) in moisture
content among the honey samples. This was in agreement with the
report of Kumar et al. (2018). Moisture content is related to the botan­
ical origin of the honey sample, harvesting techniques and extraction
from the comb in relation to the ripening process by the bees, and can
vary from season to season and from year to year (Belay et al., 2013;
Kumar et al., 2018). Moisture content is one of the most important
quality parameters of honey. The Codex Alimentarius established a limit
of 20 g/100g of honey (Codex Alimentarius, 2001). All the honey were
within the acceptable limit set by Codex Alimentarius.
The moisture content of Coffea arabica honey of this finding was
lower than the moisture content of Coffea arabica honey (17.93 ± 0.45g/
100g) reported by Kadri, Zaluski, Lima, Mazzafera & de Oliveira (2016).
The moisture content of Vernonia amygdalina honey of this study was
higher than the moisture content (17.30 g/100g) reported by Belay,
Haki, Birringer, Borck, Lee, Cho et al. (2017b); however, in close
agreement with moisture content (18.64 g/100g) reported by Legesse
(2014).
3.7. Ash content
Ash content is one of the parameters that have been associated with
botanical and geographical origins of honey samples. The ash content in
honey is generally small and depends on the nectar composition of
predominant plants in their formation (Kahraman, Buyukunal, Vural, &
Altunatmaz, 2010). The ash content of the present study was presented
in Table 2. The mean ± sd of ash content for Coffea arabica honey and
Vernonia amygdalina honey samples was 0.28 ± 0.05 and 0.28 ± 0.01
g/100g, respectively. There was no significant difference (p > 0.05)
observed in ash content among the honey samples. This could be due to
the geographical position, in which the honey samples were collected.
According to Yücel and Sultanog (2013) the percentage of ash content
has been associated with the mineral content, botanical and geograph­
ical origins of honey samples.
Kadri, Zaluski, Lima, Mazzafera & de Oliveira (2016) reported a
mean value for Coffea arabica honey (0.17 ± 0.03 g/100g), which was
lower than this report. The mean ash content of Vernonia amygdalina
honey in the current study was lower than ash content (0.38 g/100g)
H. Debela and A. Belay
reported by Belay, Haki, Birringer, Borck, Lee, Cho et al. (2017b). Both
of the monofloral honey satisfies the Codex Alimentarius limit (not more
than 0.6 g/100g) in ash content. This also indicated that the honey
samples were from floral nectar (Codex Alimentarius, 2001).
3.8. Electrical conductivity
The electrical conductivity (EC) of honey is closely related to the
origin of honey (Belay et al., 2013). The mean ± sd of electrical con­
ductivity values for Coffea arabica honey and Vernonia amygdalina honey
sample was 0.52 ± 0.10 and 0.52 ± 0.01 mS/cm, respectively (Table 2).
There was no significant difference (p > 0.05) observed in electrical
conductivity among the monofloral honey. The mean electrical con­
ductivity of Vernonia amygdalina honey in the current study was in close
agreement with electrical conductivity (0.58 ms/cm) reported by Belay,
Haki, Birringer, Borck, Lee, Cho et al. (2017b). The value of electrical
conductivity was related to the value of the ash. Electrical conductivity
depends on the ash content of the specific type of honey (Belay et al.,
2013).
Electrical conductivity is used to distinguish between floral and
honeydew honey. Codex Alimentarius (2001) established a limit for
electrical conductivity of floral honey not more than 0.8 mS/cm.
Accordingly, both of the honey samples defined as floral honey.
3.9. Free acidity and pH
The mean ± sd of free acidity for Coffea arabica honey and Vernonia
amygdalina honey was 33.74 ± 5.30 and 20.75 ± 0.32 meqkg− 1,
respectively (Table 2). It was observed that there was a significant dif­
ference (p < 0.05) in free acidity among the honey samples. The free
acidity of Coffea arabica honey was higher than the report of Kadri,
Zaluski, Lima, Mazzafera & de Oliveira (2016) (17.86 ± 1.22 meq/kg).
The free acidity of Vernonia amygdalina honey of the current study was in
agreement with the report of Legesse (2014) (22.72 ± 10.27 meqkg− 1).
The mean ± sd of pH for Coffea arabica honey and Vernonia amyg­
dalina honey were 3.93 ± 0.15 and 4.25 ± 0.02, respectively. There was
a significant difference (p < 0.05) in pH among the monofloral honey.
According to Kadri, Zaluski, Lima, Mazzafera & de Oliveira (2016), the
mean ± sd of pH for Coffea arabica honey was 3.94 ± 0.07, which was in
close agreement with the current study. The pH of Vernonia amygdalina
honey of the current study was in agreement with the pH value (4.08)
reported by Belay, Haki, Birringer, Borck, Lee, Cho et al. (2017b).
The acidic nature of honey is attributed to the presence of organic
acids (Oroian, 2012) and the variation in acidity among the honey could
be the variation in those constituents. The presence of different organic
acids, geographical origin and harvest season can affect the honey
acidity (da Silva, Gauche, Gonzaga, Costa, & Fett, 2016). Codex Ali­
mentarius (2001) established a maximum limit of free acidity (40
meq/kg) and all the honeys were within the limit of this standard.
3.10. Hydroxymethylfurfural (HMF)
The HMF content is widely recognized as parameter indicating the
freshness of honey (Kahraman et al., 2010). The HMF content of Coffea
arabica honey and Vernonia amygdalina honey was presented in Table 2.
The mean ± sd of HMF content for Coffea arabica honey and Vernonia
amygdalina honey samples were 5.60 ± 2.52 and 0.43 ± 0.01 mgkg− 1,
respectively. The study showed that there was a significant difference (p
< 0.05) in HMF content among the honey samples.
Schievano et al. (2015) reported HMF value of 26.2 mg/kg for Coffea
arabica honey, which was higher than the current study. The HMF
content of Vernonia amygdalina honey in the current study was lower
than Legesse (2014) (14.26 mg/kg) report. According to international
standards of Codex Alimentarius, 5-HMF content of honey should be
lower than 40 mgkg− 1. All honey samples analyzed in this study were
considered as fresh honey and meet the standards established by Codex
8
Food Control 123 (2021) 107857
Alimentarius (2001).
3.11. Specific rotation
The specific rotation of honey is the result of the rotation of linearly
polarized light by carbohydrates. Nectar honey containing small
amounts of sucrose has a negative specific rotation, resulted from the
predominance of fructose (Primorac et al., 2011). In this study the mean
± sd of specific rotation for Coffea arabica honey and Vernonia amyg­
dalina honey was − 9.91 ± 3.09 and − 5.98 ± 0.07[α]20 D, respectively
(Table 2). There was a significant difference (p < 0.05) in specific
rotation among the honey samples. All the honey samples of this study
have negative rotation, which can be defined as nectar honey. This was
in agreement with the report of Primorac et al. (2011) and Dinkov
(2003) reports.
4. Conclusion
In this study, caffeine content, enzyme activity, physicochemical and
sensory properties of Coffea arabica honey and Vernonia amygdalina
honey were investigated. Melissopalynology identified honey samples as
Coffea arabica honey and Vernonia amygdaline honey. All the honey
samples meet the requirements of the Codex Alimentarius standard.
Coffea arabica honey and Vernonia amygdalina honey significantly
differed (p < 0.05) in caffeine content and invertase number. The tri­
angle test sensory panel showed a significant difference (p < 0.05) be­
tween Coffea arabica honey and Vernonia amygdalina honey. Thus, the
level of caffeine, invertase number, and triangle test sensory panel used
to differentiate Coffea arabica and Vernonia amygdalina honey.
Authors’ contributions
All of the two authors conducted the research. Hawine Debela and
Abera Belay designed the research, develop the parameters, analyze the
data, and wrote the manuscript. All authors read and approved the final
version of the manuscript.
CRediT authorship contribution statement
Hawine Debela: Develop the parameters, Sample collection, Formal
analysis, Data Formal analysis and interpretation, Writing – original
draft. Abera Belay: Develop research concept, Develop the parameters,
Sample collection, Formal analysis, Data Formal analysis and interpre­
tation, Writing – original draft, Writing – review & editing, Project
administration, All authors read and approved the manuscript.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
We have a special thanks to Addis Ababa Science and Technology
University (AASTU)- Research Directorate and the Department of Food
Science and Applied Nutrition for supporting the project; and Ethiopian
Conformity Assessment Enterprise (ECAE) for laboratory analysis.
Beekeeping Experts, technicians and forest beekeepers from Jima zone
(Gera and Mana) and Illubabor zone (Yayu and Becho) are highly
appreciated. A kind provision of Phadebas tablet from Phadebas -AB
Sweden for enzyme analysis is highly acknowledged.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
H. Debela and A. Belay
org/10.1016/j.foodcont.2020.107857.
References
Addi, A., & Bareke, T. (2019). Floral resources diversity of honeybees in important types
of vegetation of Ethiopia. Asian Journal of Forestry, 3(2), 64–68.
Adgaba, N. (2007). Atlas of pollen grains of major honeybee flora of Ethiopia. Holetta,
Ethiopia: Holetta bee research centre.
Aliaño-González, M. J., Ferreiro-González, M., Espada-Bellido, E., Barbero, G. F., &
Palma, M. (2020). Novel method based on ion mobility spectroscopy for the
quantification of adulterants in honeys. Food Control, 114, Article 107236.
AOAC (Association of Official Analytical Chemists). (1990). Official methods of the
analysis of official analytical chemists (15th ed., Vol. II). USA: Virginia.
Belay, A., Haki, G. D., Birringer, M., Borck, H., Lee, Y. C., Kim, K. T., & Melaku, S.
(2017a). Enzyme activity, amino acid profiles and hydroxymethylfurfural content in
Ethiopian monofloral honey. Journal of Food Science and Technology, 54(9),
2769–2778.
Belay, A., Haki, G. D., Birringer, M., Borck, H., Lee, Y. C., Kim, K. T., & Melaku, S.
(2017b). Sugar profile and physicochemical properties of Ethiopian monofloral
honey. International Journal of Food Properties, 20(11), 2855–2866.
Belay, A., Solomon, W. K., Bultossa, G., Adgaba, N., & Melaku, S. (2013).
Physicochemical properties of the Harenna forest honey, Bale, Ethiopia. Food
Chemistry, 141(4), 3386–3392.
Belay, A., Solomon, W. K., Bultossa, G., Adgaba, N., & Melaku, S. (2015). Botanical
origin, colour, granulation, and sensory properties of the Harenna forest honey, Bale,
Ethiopia. Food Chemistry, 167, 213–219.
Bertelli, D., Lolli, M., Papotti, G., Bortolotti, L., Serra, G., & Plessi, M. (2010). Detection
of honey adulteration by sugar syrups using one-dimensional and two-dimensional
high-resolution nuclear magnetic resonance. Journal of Agricultural and Food
Chemistry, 58(15), 8495–8501.
Bogdanov, S. (2009). Harmonised methods of the international honey commision. Bern, Swiss
bee research center, international honey commission. World Network of Honey Science,
63 str.
Boyo, A. O., Shitta, M. B. O., Oluwa, T., & Adeola, S. (2012). Bitter leaf (Vernonia
amygdalin) for dye sensitized solar cell. Trends in Applied Sciences Research, 7(7),
558–564.
Castro-Vázquez, L., Díaz-Maroto, M. C., De Torres, C., & Pérez-Coello, M. S. (2010).
Effect of geographical origin on the chemical and sensory characteristics of chestnut
honeys. Food Research International, 43(10), 2335–2340.
Castro-Vázquez, L., Díaz-Maroto, M. C., González-Viñas, M. A., & Pérez-Coello, M. S.
(2009). Differentiation of monofloral citrus, rosemary, eucalyptus, lavender, thyme
and heather honeys based on volatile composition and sensory descriptive analysis.
Food Chemistry, 112(4), 1022–1030.
Cimpoiu, C., Hosu, A., Miclaus, V., & Puscas, A. (2013). Determination of the floral origin
of some Romanian honeys on the basis of physical and biochemical properties.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, 100, 149–154.
Codex Alimentarius. (2001). Revised Codex standard for honey. Codex stan 12-1982: Rev.
1, 1987. Rev 2, 2001.
Degaga, A. H. (2017). Identification of honey source bee floras during major and minor
honey harvesting seasons in Jimma Zone, Southwest Ethiopia. Journal of Environment
and Earth Science, 7(3), 25–32.
Dinkov, D. (2003). A scientific note on the specific optical rotation of three honey types
from Bulgaria. Apidologie, 34(3), 319–320.
Draiaia, R., Dainese, N., Borin, A., Manzinello, C., Gallina, A., & Mutinelli, F. (2015).
Physicochemical parameters and antibiotics residuals in Algerian honey. African
Journal of Biotechnology, 14(14), 1242–1251.
Escuredo, O., Seijo, M. C., & Fernández-González, M. (2011). Descriptive analysis of
Rubus honey from the north-west of Spain. International Journal of Food Science and
Technology, 46(11), 2329–2336.
Fichtl, R., & Admasu, A. (1994). Honeybee flora of Ethiopia. Weikersheim, Germany:
Margraf Verlag.
Flanjak, I., Strelec, I., Kenjerić, D., & Primorac, L. (2016). Croatian produced unifloral
honey characterized according to the protein and proline content and enzyme
activities. Journal of Apicultural Science, 60(1), 39–48.
Foomaneechot, O., & Sirinupong, N. (2018). Physicochemical, biochemical properties
and sensory evaluation of Thai blossom honey. The international conference on food
and applied bioscience. proceeding book.
Food Control 123 (2021) 107857
García, N. L. (2018). The current situation on the international honey market. Bee World,
95(3), 89–94.
Jerković, I., Tuberoso, C. I. G., Kuś, P. M., Marijanović, Z., & Kranjac, M. (2014).
Screening of Coffea spp. honey by different methodologies: Theobromine and
caffeine as chemical markers. RSC Advances, 4(105), 60557–60562.
Kadri, S. M., Zaluski, R., Lima, G. P. P., Mazzafera, P., & de Oliveira Orsi, R. (2016).
Characterization of Coffea arabica monofloral honey from espírito santo, Brazil. Food
Chemistry, 203, 252–257.
Kahraman, T., Buyukunal, S. K., Vural, A., & Altunatmaz, S. S. (2010). Physico-chemical
properties in honey from different regions of Turkey. Food Chemistry, 123(1), 41–44.
Kaškonienė, V., Venskutonis, P. R., & Čeksterytė, V. (2010). Carbohydrate composition
and electrical conductivity of different origin honeys from Lithuania. LWT-Food
Science and Technology, 43(5), 801–807.
Kebede, M., & Bellachew, B. (2008). Phenotypic diversity in the hararge coffee (Coffea
arabica L) germplasm for quantitative traits. East African Journal of Sciences, 2(1),
13–18.
Kumar, A., Gill, J. P. S., Bedi, J. S., Manav, M., Ansari, M. J., & Walia, G. S. (2018).
Sensorial and physicochemical analysis of Indian honeys for assessment of quality
and floral origins. Food Research International, 108, 571–583.
Lawless, H. T., & Heymann, H. (2010). Sensory evaluation of food: Principles and practices.
New York Dordrecht Heidelberg London: Springer Science & Business Media.
Legesse, G. (2014). Identification and characterization of major mono-floral honeys in
Ethiopia. On the Proceedings of the Annual National Review Workshop on Results of
Livestock Research, 13-15 June 2013 EIAR, 297–306. Addis Ababa.
Manzanares, A. B., García, Z. H., Galdón, B. R., Rodríguez, E. R., & Romero, C. D. (2011).
Differentiation of blossom and honeydew honeys using multivariate analysis on the
physicochemical parameters and sugar composition. Food Chemistry, 126(2),
664–672.
Marcazzan, G. L., Mucignat-Caretta, C., Marina Marchese, C., & Piana, M. L. (2018).
A review of methods for honey sensory analysis. Journal of Apicultural Research, 57
(1), 75–87.
Molan, P. C. (1996). Authenticity of honey. In Food authentication (pp. 259–303). Boston,
MA: Springer.
Ngo, H. T., Mojica, A. C., & Packer, L. (2011). Coffee plant–pollinator interactions: A
review. Canadian Journal of Zoology, 89(8), 647–660.
Oddo, L. P., Piazza, M. G., & Pulcini, P. (1999). Invertase activity in honey. Apidologie, 30
(1), 57–65.
Ohe, W. V. D., Oddo, L. P., Piana, M. L., Morlot, M., & Martin, P. (2004). Harmonized
methods of Melissopalynology. Apidologie, 35, 18–25.
Oroian, M. (2012). Physicochemical and rheological properties of Romanian honeys.
Food Biophysics, 7(4), 296–307.
Ouchemoukh, S., Schweitzer, P., Bey, M. B., Djoudad-Kadji, H., & Louaileche, H. (2010).
HPLC sugar profiles of Algerian honeys. Food Chemistry, 121(2), 561–568.
Primorac, L., Flanjak, I., & Topolnjak, Z. (2011). Specific rotation and carbohydrate
profile of Croatian unifloral honeys. Czech Journal of Food Sciences, 29(5), 515–519.
Sanz, M. L., Gonzalez, M., De Lorenzo, C., Sanz, J., & Martınez-Castro, I. (2005).
A contribution to the differentiation between nectar honey and honeydew honey.
Food Chemistry, 91(2), 313–317.
Schievano, E., Finotello, C., Mammi, S., Illy Belci, A., Colomban, S., & Navarini, L.
(2015). Preliminary characterization of monofloral Coffea spp. honey: Correlation
between potential biomarkers and pollen content. Journal of Agricultural and Food
Chemistry, 63(25), 5858–5863.
da Silva, P. M., Gauche, C., Gonzaga, L. V., Costa, A. C. O., & Fett, R. (2016). Honey:
Chemical composition, stability and authenticity. Food Chemistry, 196, 309–323.
Singh, N., & Bath, P. K. (1997). Quality evaluation of different types of Indian honey.
Food Chemistry, 58(1–2), 129–133.
Swaileh, K. M., & Abdulkhaliq, A. (2013). Analysis of aflatoxins, caffeine, nicotine and
heavy metals in Palestinian multifloral honey from different geographic regions.
Journal of the Science of Food and Agriculture, 93(9), 2116–2120.
Tornuk, F., Karaman, S., Ozturk, I., Toker, O. S., Tastemur, B., Sagdic, O., & Kayacier, A.
(2013). Quality characterization of artisanal and retail Turkish blossom honeys:
Determination of physicochemical, microbiological, bioactive properties and aroma
profile. Industrial Crops and Products, 46, 124–131.
Wang, J., & Li, Q. X. (2011). Chemical composition, characterization, and differentiation
of honey botanical and geographical origins. Advances in Food and Nutrition Research,
62, 89–137.
Yücel, Y., & Sultanog, P. (2013). Characterization of honeys from Hatay Region by their
physicochemical properties combined with chemometrics. Food Bioscience, 1, 16–25.