Evaluation of Honey Quality with Stored Time and Temperatures

Full text

Journal of Food and Nutrition Research, 2020, Vol. 8, No. 10, 591-599
Available online at http://pubs.sciepub.com/jfnr/8/10/8
Published by Science and Education Publishing
DOI:10.12691/jfnr-8-10-8
Evaluation of Honey Quality
with Stored Time and Temperatures
Wing-Ming Chou*, Hao-Chun Liao, Yuan-Chang Yang, Chi-Chung Peng*
Department of Biotechnology, National Formosa University, Yunlin, Taiwan
*Corresponding author:
Received September 21, 2020; Revised October 23, 2020; Accepted October 30, 2020
Abstract Honey from two sources, Bidens pilosa and Dimocarpus longan were stored at 35, 25, or 4°C under
dark or light for 3-24 months. They were evaluated for the nutrients, antioxidant activities and quality parameters
required in Codex Standard. Diastase activity of examined honey was reduced to less than 8 schade unit soon after
storage for 3-6 months, and its acidity was increased with long storage time at high temperature. Storage of honey at
4°C significantly diminished the loss of diastase activity and maintained the proper acidity. Hydroxymethylfurfural
(HMF) content retained low level as honey was stored up to two years at either 4 or 25°C, while its content was
quickly increased at 35°C. More phenolic content but less flavonoid content was found in honey after long storage
time. Along with longer deposit time and higher temperature, less antioxidant activities were detected in honey,
including scavenging activity of DPPH and superoxide radicals. In contrast, the reducing power was increased.
Storage of honey at 35°C caused the worst impact on its quality parameters of Codex Standard, while 4oC was the
best storage condition to maintain most quality parameters, nutrients and antioxidant functions. No apparent
difference was found between the storage conditions under dark and light.
Keywords: honey, storage, hydroxymethylfurfural, antioxidant
Cite This Article: Wing-Ming Chou, Hao-Chun Liao, Yuan-Chang Yang, and Chi-Chung Peng, “Evaluation
of Honey Quality with Stored Time and Temperatures.” Journal of Food and Nutrition Research, vol. 8, no. 10
(2020): 591-599. doi: 10.12691/jfnr-8-10-8.
1. Introduction
Since the ancient times, honey has been extensively
applied in food and medical product. The floral nectar, the
exudates of trees and honeydew were collected and
regurgitated to produce honey by bees [1]. Honey is well
known for its nutrient value, and comprises carbohydrates
and water mainly. It also contains minerals, free amino
acids, vitamins, proteins, and various substances including
pigments, flavonoids and antibacterial factors [2,3]. At
least 200 natural substances were found in honey [4]. The
composition of floral honey is associated with the floral
sources, along with external factors, environment, climate,
season and processing [5].
Honey has showed the therapeutic potential for heart
disease, cancer, cataracts, and several inflammatory
diseases [6]. It is attributed to its antioxidant capacity,
antimicrobial properties, and wound healing and anti-
inflammatory activities [7]. Many studies showed that its
antioxidant activity is related to the floral source [8]. Both
enzymatic and non-enzymatic antioxidants have been
found in honey, including glucose oxidase, catalase,
ascorbic acid, carotenoid derivatives, organic acids,
Maillard reaction products (MRP), phenolic compounds,
and free amino acids [9,10]. Among them, the phenolic
compounds are primarily responsible for the antioxidant
capacity [11,12]. And honey flavonoids are originally from
nectar, pollen or propolis. The identified flavonoids include
apigenin (belongs to flavone), such as kaempferol (a flavonol),
hesperetin (a flavavone) and diverse phenolic acids.
Honey is often preserved until use. To keep the
freshness and antioxidant function of stored honey is
critical for the retail and consumers. According to Codex
Standard [13], all retail honey should have the following
compositional and quality parameters: moisture content,
≤20 g/100 g; free acidity, ≤50 mequiv/ kg; diastase
activity, ≥8 schade unit; hydroxymethylfurfural (HMF)
content, ≤40 mg/kg (or ≤80 mg/Kg for honey from
tropical ambient temperatures).
Honey has an acidic pH value, ranging from 3.5 to 4.5,
owing to its organic acids. The acidity of honey is correlated
with its flavor and stability against microorganisms [14].
Nevertheless, microbial alterations in honey may cause
total acidity over the legal limitation [15,16,17,18].
Diastase in honey catalyzes starch into short-chain sugars,
and its protein conformation may be destroyed after
heating. Maillard reaction (MR), a non-enzymic browning
reaction between sugars and free amino acids, occurs
during prolonged storage and heating [19]. HMF is one of
the major intermediate products of MR. Less diastase
activity and more HMF was detected in honey after
prolonged storage and heating [20,21,22,23,24].

592 Journal of Food and Nutrition Research
Herein, we studied the effect of storage conditions on
honeys from two floral sources, Bidens pilosa and
Dimocarpus longan in Taiwan. D. longan is the main
floral source of honey bee, and its fruits are edible and
popular in Taiwan. B. pilosa is used as ingredient of
herbal tea, as well as a folk medicine [25]. The climate in
the plains of Taiwan is often between 25 and 35oC. Thus,
the storage conditions of honey at 35, 25, or 4oC under
dark or light for 3-24 months were evaluated. The quality
parameters of honeys were examined and discussed, as
well as antioxidant activities, total flavonoids and phenolic
compound contents.
2. Materials and Methods
2.1. Honey Samples
B. pilosa and D. longan honey, produced by A.
mellifera bees were prepared and provided by Honey Bee
Town Company (Hualien, Taiwan). Each honey was
divided into six equal parts, and subjected to different
storage conditions, dark and light at temperature (35, 25,
4°C), followed by analyses of the physicochemical
parameters and antioxidant activities.
2.2. Diastase Activity Assay
The diastase activity was measured using the Phadebas
amylase test tablets purchased from Magle (Lund, Sweden),
according to the International Honey Commission [26].
Diastase Activity was referred to as diastase number in the
Schade scale, corresponding to the Gothe scale number, or
hydrolyzed starch (g) /100 g of honey/hour at 40°C.
2.3. Total Acidity
5.0 g of examined honey was dissolved in 100 ml
distilled water, and then subjected to determination of the
total acidity, according to method of AOAC method
962.19 [27]. A pH meter (Mettler Toledo SevenEasy
digital) was applied to measure pH value. The total acidity
was then calculated and expressed as meq/kg.
2.4. Determination of HMF Content in Honey
5 g of honey sample each was diluted to 50 mL, and
then filtered through a membrane (0.45 μm). Afterward,
HMF content was determined by high-performance
liquid chromatography (HPLC) under OD285 detection,
according to AOAC method 980.23 [28]. HPLC analysis
was performed using a Waters 1525 pumping system,
a Water 2489 detector, an RP-18 GP250 column
(4.6 mm), and a Waters 717plus autosampler. And the
isocratic mobile phase comprised 90% water, and 10%
methanol at a flow rate of 0.7 mL/min. HMF content of
the sample was calculated, according to the corresponding
peak of HMF standard solutions. A linear relationship
(R2=0.9981) between the concentration and the area of
HMF peak was achieved. Each sample was performed
three times, and the mean of HMF content was expressed
as mg/kg.
2.5. Determination of Total Polyphenol and
Total Flavonoid Contents in Honey
The total polyphenolic content of honey was measured
by Folin-Ciocalteu colorimetric method using gallic acid
as a calibration standard [8]. An aliquot of the honey
sample solution (0.1 mL) was diluted to 5 mL with water,
followed by adding Folin-Ciocalteu reagent (0.5 mL).
After mixing for 3 min, 1 mL of an aqueous Na2CO3
solution (35 g/L) was added and mixed throughly by
vortex. After placing the mixture at room temperature for
1 h, the absorbance of the mixture was measured at 725
nm against a blank.
The total flavonoid content was determined using an
aluminum chloride colorimetric method [8]. 0.5 ml of
honey sample was mixed with 1.5 ml of 95% alcohol, 0.1
ml of 1 M potassium acetate, 0.1 ml of 10% aluminum
chloride hexahydrate, and 2.8 ml of deionized water. After
incubation at room temperature for 30 min, the absorbance
of reaction mixture was measured at 415 nm. Quercetin
was applied to obtain a standard curve (0–100 μg/ml). The
total flavonoid content in each honey sample was
determined in triplicate.
2.6. Radical Scavenging Effect on 1,1-
Diphenyl-2-picrylhydrazyl (DPPH)
Radicals
DPPH assay was performed based on the method of
Liu et al. [8]. 0.3 mL of honey sample was mixed with 2.4
mL of ethanol (99%) and 0.3 mL of 1.0 mM DPPH radical
solution. The mixture was shaken vigorously and left to
stand for 30 min in the dark, before the absorbance was
measured at 517 nm. Ascorbic acid (0.1 mM and 1.0 mM)
were used as positive controls. The capability of the test
material to scavenge DPPH radicals was calculated as (%)
= 1 - (OD517 of the sample) / (OD517 of the control) × 100.
2.7. Radical Scavenging Effect on Superoxide
Radicals
The scavenging of superoxide anion radicals was
estimated according to the method of Robak and
Gryglewski [29]. The honey sample solution (500 μL) was
mixed with an equal volume of 80 μM PMS, 624 μM
NADH and 200μM NBT each. The mixture was shaken
vigorously and left to stand for 5 min. The absorbance of
reaction mixture was measured at 560 nm. Ascorbic acid
(0.1 mM, 1.0 mM and 10.0 mM) were used as positive
controls. The inhibition ratio was calculated as (%) =
(OD560 of the control - OD560 of the sample) / (OD560 of
the control) × 100.
2.8. Reducing Power
The reducing power of honey was determined
according to the method of Oyaizu [30]. The honey
sample solution (2.5 mL) was mixed with an equal
volume of 0.2 M sodium phosphate buffer (pH 6.6) and
1% potassium ferricyanide. The mixture was incubated at
50°C for 20 min. Afterward, an equal volume of 1%

Journal of Food and Nutrition Research 593
thrichloroacetic acid was added to the mixture, which was
then centrifuged at 1400g for 10 min. The upper layer was
mixed with distilled water and 0.1% ferric chloride at a
ratio of 1:1:2, and the absorbance of the incident radiation
by the solution was measured at 700 nm. The absorbance
was proportional to the reducing power, and dibutyl
hydroxytoluene (BHT) was used as a reference for
comparison.
2.9. Statistical Analysis
All results were analyzed using the general linear model
procedure available from Statistical Analysis System
software (Statistical Analysis System Institute, Cary, NC).
Duncan’s multiple range test [31] was used to detect
differences between means of the treatments. Each
experiment was conducted in triplicate. Differences
between means at the 95% (p < 0.05) confidence level
were considered as statistical significance.
3. Results and Discussion
3.1. Total Acidity Contents
The acidity of honeys under different storage conditions
were examined, including 35, 25, or 4°C for 3-24 months
under dark or light. As shown in Figure 1, the acidity of
honeys from B. pilosa and D. longan increased with the
duration of storage. Storage at high temperature would
increase total acidity in honeys significantly. The initial
acidity of B. pilosa honey was 43.06 meq./kg. After stored
for 9 months at 35oC, its acidity was higher than legal
limit, 50 mequiv/kg. After stored for 18 months at 25°C,
its acidity was out of legal limit as well. B. pilosa honey
kept at 4oC maintained its acidity in legal value up to
24 months. The initial acidity of D. longan honey was
18 meq./kg, less than B. pilosa honey. The increasing rate
of acidity of honey from D. longan was faster than
B. pilosa. After stored for 12 months at 35 or 25°C, its
acidity was higher than legal limit. And the acidity of
D. longan honey stored for 18 months at 4°C was out
of legal limit. Moreover, there was no significantly
difference between the conditions under dark and light.
According to the results, the storage of honey at 4°C is
recommended in order to hold its proper acidity. The
stored temperature and time had considerable effect on
acidity, which is coincided with the studied results by
Castro-Vázquez [32]. Among different honey types, the
variation in acidity may attribute to the variation of
organic and inorganic acids contents [33]. Nevertheless,
the compounds affecting acidity of honeys from B. pilosa
and D. longan remain further study.
3.2. Diastase Activity in Honey
The initial diastase activity was similar in both
examined honeys, as shown in Figure 2. Prolonged
storage under high temperature accelerated the loss of
diastase activity in honeys. After storage for 3 months
under our test conditions, diastase activity in the honeys
was less than legal limit, 8 schade unit, except for
D. longan honey stored at 4°C. Stored at 4°C may
significantly diminish the loss of diastase activity,
particularly for B. pilosa honey. There was no apparently
difference between dark storage and light storage.
The diastase activity in honeys are more or less diverse,
depending on the sugar content of honeys, the floral and
geographical origins of honeys, the period of nectar
collected, the age of bees, and the bee colony [34]. Its
activity has been as an indicator to monitor whether the
honeys had been kept for too long time, or its product was
overheated, > 60°C [35,36].
3.3. HMF Contents
Both honeys from B. pilosa and D. longan contained a
low initial HMF content (1.21 mg/kg and 2.13 mg/kg,
respectively) (Figure 3). HMF was quickly generated in
these honeys after storage at 35°C. HMF contents in B.
pilosa honey stored at 35°C for 6 months was reached to
the legal limit, 80 mg/Kg for honey from tropical ambient
temperatures. After storage at 35°C for 9 months, HMF
contents in B. pilosa honey was exceeding the legal limit;
while HMF contents in D. longan honey was reached to
the legal limit. HMF contents in both honeys, stored at 4
and 25°C for 24 months, remain quite low levels that were
less than 10 mg/ml.
Figure 1. Acidity of honeys under different storage conditions, at 35, 25 or 4°C for 3-24 months under dark and light. (a) Bidens pilosa honey; (b) D.
longan honey; Each point is expressed as mean ± S.D. (n=3)

594 Journal of Food and Nutrition Research
Figure 2. Diastase activity of honeys under different storage conditions, at 35, 25 or 4°C for 3-24 months under dark and light. (a) Bidens pilosa honey;
(b) D. longan honey
Figure 3. HMF content of honeys under different storage conditions, at 35, 25 or 4°C for 3-24 months under dark and light. (a) Bidens pilosa honey; (b)
D. longan honey; Each point is expressed as mean ± S.D. (n=3)
HMF was reported to have genotoxic effects and
mutagenic potential [37]. Some studies indicated that heat
treatment or storage at room temperature caused a
considerable increase in HMF contents of honeys
[38,39,40]. Based on our results with HMF contents, it is
suggested to store honeys at 4-25°C, or consume honeys
within 6 months.
3.4. Total Phenolic and Total Flavonoid
Contents in Honey
The influence of storage condition on total phenolic and
total flavonoid contents of B. pilosa honey and D. longan
honey was examined and shown in Figure 4 and Figure 5.
Total phenolic content of B. pilosa honey was increased to
2-3 folds after storage for 12 months. Higher temperature
would more or less increase its phenolic content.
Compared with B. pilosa honey, D. longan honey had less
total phenolic compounds; however, its total phenolic
content was increased to 10-18 folds after storage for
12 months. The effect of heating on phenolic content of
D. longan honey was clearly observed. More phenolic
content of D. longan honey was detected as stored at
higher temperature.
B. pilosa honey was rich in total flavonoid compounds
(initially 180µg/ml). After storage for 3 months, around
half of its total flavonoid contents were reduced. Honey
from D. longan had less total flavonoid compounds
(initially 55 µg/ml) than B. pilosa in our previous and
current studies [8]. After storage for 9 months, around half
of total flavonoid contents of D. longan honey were
reduced. The decay rate of total flavonoid contents from
B. pilosa was much faster than D. longan in the first three
months. In general, storage at 4°C under dark would be
better for honeys to retain total flavonoid contents.
Honeys stored under dark had slightly less change in total
phenolic and total flavonoid contents, comparing to under
light.

Journal of Food and Nutrition Research 595
Figure 4. The total phenolic content of honeys under different storage conditions, at 35, 25 or 4°C for 3-24 months under dark and light. (a) Bidens
pilosa honey; (b) D. longan honey; Each point is expressed as mean ± S.D. (n=3)
Figure 5. The total flavonoid content of honey under different storage conditions, at 35, 25 or 4°C for 3-24 months under dark and light. (a) Bidens
pilosa honey; (b) D. longan honey; Each point is expressed as mean ± S.D. (n=3)
3.5. Scavenging Effect of Honey upon DPPH
Radicals
DPPH with stable radicals is often applied to
demonstrate the radical scavenging ability of natural
compounds [8]. The antioxidant potential of honey was
directly proportional to the amount of phenolic acids and
flavonoids present [7]. The higher DPPH radical-scavenging
activity may be attributed to its higher phenolic and
flavonoid content [8]. Compared to D. longan honey,
B. pilosa honey initially contained more phenolic
compound and flavonoid, and exhibited better DPPH
radical-scavenging activity.
As shown in Figure 6, B. pilosa honey and D. longan
honey lost DPPH radical-scavenging activity with the
duration and temperature of storage. In the beginning,
B. pilosa honey had better DPPH radical-scavenging
activity than D. longan honey. B. pilosa honey still had
better activity than D. longan honey after storage under
the same condition for two years. At 25°C, B. pilosa
honey lost 50% of activity within ~18 months; while
D. longan honey lost ~50% of activity after storage for 9
months. When honeys were stored at 4°C, their decline
rates of DPPH radical scavenging activity were retarded.
No apparent difference was found between the storage
conditions under dark and light.
3.6. Radical Scavenging Effect upon
Superoxide Radicals
Superoxide radical damaging the cell and tissues could
be produced via autoxidation and enzymatic reactions in
our bodies [8]. NBT method was applied to examine the
scavenging rate of superoxide radicals by honeys. Honeys
from B. pilosa and D. longan could inhibit superoxide
radical formation with an inhibition rate of 60-75% initially,
but lost such ability during storage (Figure 7). B. pilosa
honey lost ≧50% of the superoxide radical-scavenging
ability soon after 3 months storage, while D. longan honey
lost 15-40% of the ability. Unlike B. pilosa honey, such
ability in D. longan honey gradually decreased with the
prolong storage and high temperature. According to
superoxide radical scavenging rate, it is suggested to
store D. longan honey at 4°C. Regarding to prolong
storage for more than 12 months, B. pilosa honey stored
at 35°C under light had unusually better superoxide
radical scavenging ability than those stored at other
conditions.

596 Journal of Food and Nutrition Research
Figure 6. The DPPH scavenging rate of honey under different storage conditions, at 35, 25 or 4°C for 3-24 months under dark and light. (a) Bidens
pilosa honey; (b) D. longan honey; Each point is expressed as mean ± S.D. (n=3)
Figure 7. The superoxide radical scavenging rate of honey under different storage conditions, at 35, 25 or 4°C for 3-24 months under dark and light.
(a) Bidens pilosa honey; (b) is D. longan honey; Each point is expressed as mean ± S.D. (n=3)
Figure 8. The reducing power of honeys under different storage conditions, at 35, 25 or 4°C for 3-24 months under dark and light. (a) is Bidens pilosa
honey; (b) is D. longan honey; Each point is expressed as mean ± S.D. (n=3)
3.7. Reducing Power of Honey
The reducing power is mainly associated with the
phenolic contents in honey. The reducing power of
honey increased after prolong storage (Figure 8). It may
due to the amount of total phenolic contents increased in
both D. longan and B. pilosa honey (Figure 4). Similar
results were reported that there was good correlation
between reducing power and polyphenolic contents in
honey [8].

Journal of Food and Nutrition Research 597
Table 1. The correlation coefficient between physicochemical properties, antioxidant capacity and storage conditions in Bidens pilosa honey
Storage condition
25°C
25°C/ dark
35°C
35°C/ dark
4°C
4°C/dark
Acidity
0.473
0.591
0.987
0.758
0.105
0.245
HMF content
0.483
0.329
0.866
0.793
0.116
0.102
DPPH scavenging activity
-0.927
-0.902
-0.926
-0.996
-0.881
-0.840
Superoxide radical scavenging activity
-0.501
-0.385
-0.698
-0.501
-0.379
-0.446
Total phenolic content
0.919
0.974
0.997
0.958
0.825
0.910
Total flavonoid content
-0.860
-0.730
-0.980
-0.970
-0.705
-0.743
Reducing power
0.960
0.940
0.972
0.988
0.817
0.764
Table 2. The correlation coefficient between physicochemical properties, antioxidant capacity and storage conditions in D. longan honey
Storage condition
25°C
25°C/dark
35°C
35°C/dark
4°C
4
℃
/dark
Acidity
0.839
0.940
0.953
0.966
0.581
0.362
HMF content
0.173
0.104
0.961
0.910
0.010
-0.021
DPPH scavenging activity -0.882 -0.959 -0.953 -0.923 -0.804 -0.839
Superoxide radical scavenging activity
-0.890
-0.800
-0.927
-0.921
-0.824
-0.721
Total phenolic content
0.873
0.857
0.937
0.840
0.713
0.788
Total flavonoid content -0.904 -0.896 -0.940 -0.998 -0.846 -0.784
Reducing power
0.873
0.852
0.892
0.975
0.800
0.862
3.8. Correlation Analysis
The relationships between storage condition and
the parameter analyzed in this study (total phenolic
content, total flavonoid content, antioxidant activity, and
physicochemical properties) are presented in Table 1 and
Table 2.
As shown in Table 1, the correlation coefficient has a
great difference of acidity and HMF content at different
temperatures, it’s clearly indicated that HMF content in
honey samples significantly correlated with storage duration,
and total acidity. Storage duration showed a very strong
correlation with HMF level, indicating that time and
temperature are the most important factors that affect
HMF formation. The other physicochemical parameters such
as total acidity showed strong correlations with HMF
formation. Since both experimental data and statistical
analysis indicated that storage duration and total acidity
levels significantly correlated with HMF formation, conducting
additional tests such as measuring free acids and total
acidity may provide ways to quickly assess honey quality.
A positive correlation between the reducing power,
phenolic content, and storage condition (Table 1) was observed.
The high correlation coefficient indicates that phenolics
are one of the main components responsible for the
reducing behaviour of honey but not storage conditions. A
negative correlation between the DPPH scavenging activity,
superoxide radical scavenging activity, total flavonoid
content, and storage condition (Table 1) was observed.
The correlation coefficient indicates that flavonoids are
one of the main components responsible for the proton
radicals scavenging ability of honey but not storage
conditions. Gheldof et al. [11] addressed that the
antioxidant activity of honey contributed by phenolic
compounds significantly, but in spite of this, it seems that
antioxidant activity appears to be a result of the combined
activity of honey phenolics, peptides, organic acids,
enzymes and Maillard reaction products. The correlation
coefficient of total phenolic content, total flavonoid
content and antioxidant capacity showed insignificant
difference with different temperature after two years
storage, which may indicate the storage time was one
reason of the parameters’ change.
Table 2 showed similar result with Table 1, suggesting
that long term storage has similar influence on B. pilosa
and D. longan honey.
4. Conclusion
Long term storage under high temperature affected the
quality of honeys from both sources. Upon storage for
longer time and higher temperature, acidity was increased,
more total flavonoid contents and radical scavenging ability
was loss in honey. B. pilosa honey had around 3.5 folds
more flavonoid contents than D. longan honey initially.
B. pilosa honey lost lots of flavonoid, and exhibited
almost the same flavonoid contents as D. longan honey
after one year storage. HMF content was quickly increased at
35°C, while it retained low level up to two years at either 4
or 25°C. According to our study, 4°C was the best storage
condition to maintain most quality parameters, nutrients and
antioxidant functions. It is suggested to store honey at lower
temperature less than one year. Considering HMF content,
honey should not be stored at temperature higher than 35°C.
Abbreviations
HMF: Hydroxymethylfurfural; DPPH: 1, 1-diphenyl-2-
picryl-hydrazyl; MRP: Maillard reaction products;
HPLC: high-performance liquid chromatography;
NBT: nitroblue tetrazolium chloride; BHT: dibutyl
hydroxytoluene;
Data Availability
All data used in the manuscript are already included in
the manuscript.

598 Journal of Food and Nutrition Research
Conflicts of Interest
The authors report no potential conflicts of interest.
Acknowledgments
This work was supported by a grant from the Ministry
of Science and Technology, Taiwan, ROC (MOST
108-2320-B-150 -001-MY2). The authors are thankful for
Mr. Jen-Chieh Li of the Honey Bee Town Co., Ltd. for
preparing honey samples.
References
[1] Bassey, I.H. In Bee keeping, digest of selected literature on bee
keeping, honey and bee wax processing; Libriservice limited:
Lagos, Nigeria, 1986; pp. 22.
[2] Pérez, R.A. Analysis of volatiles from Spanish honeys by
solid-phase microextraction and gas chromatography-mass
spectrometry. J. Agri. Food Chem. 2002; 50: 2633-2637.
[3] Terrab, A.; Gonzále, M.M.L.; González, A.G. Characterisation of
Moroccan unifloral honeys using multivariate analysis. European
food research and technology. 2003; 218: 88-95
[4] da Silva, P.M.; Gauche, C.; Gonzaga, L.V.; Costa, A.C.O.; Fett, R.
Honey: Chemical composition, stability and authenticity. Food
Chemistry 2016; 196: 309-323.
[5] Persano, O.L.; Piro, R. Main European unifloral honeys:
descriptive sheets. Apidologie. 2004; 35: 38-81.
[6] Hossen, M.S.; Ali, M.Y.; Jahurul, M.H.A.; Abdel-Daim, M.; Gan,
S.H.; Khalil, M.I. Beneficialroles of honey polyphenols against
some human degenerative diseases. Pharmacological Reports.
2017; 69: 1194-1205.
[7] Basualdo, C.; Sgroy, V.; Finola, M.S.; Marioli, J.M. Comparison
of the antibacterial activity of honey from different provenance
against bacteria usually isolated from skin wounds. Vet. Microbiol.
2007; 124: 375-381.
[8] Liu, J.R.; Yeh, Y.L.; Lin, T.Y.; Wang, Y.W.; Peng, C.C. Effect
of floral sources on the antioxidant, antimicrobial, and
anti-inflammatory activities of honeys in Taiwan. Food Chem.
2013; 139: 938-943.
[9] Khalil, M.I.; Sulaiman, S.A.; Boukraa, L. Antioxidant properties
of honey and its role in preventing health disorder. The Open
Nutraceuticals Journal. 2010; 3: 6-16.
[10] Al-Mamary, M.; Al-Meery, A.; Al-Habori, M. Antioxidant
activities and total phenolic of different types of honey. Nutr Res.
2002, 22, 1041-1047
[11] Gheldof, N.; Engeseth, N.J. Antioxidant capacity of honeys from
various floral sources based on the determination of oxygen
radical absorbance capacity and inhibition of in vitro lipoprotein
oxidation in human serum samples. J. Agri. Food Chem. 2002; 50:
3050-3055.
[12] Gheldof, N.; Wang, X.H.; Engeseth, N.J.; Buckwheat honey
increases serum antioxidant capacity in humans. J. Agri. Food
Chem. 2003; 51: 1500-1505.
[13] CODEX STANDARD FOR HONEY CODEX STAN 12-1981,
Adopted in 1981, Revisions 1987 and 2001.
[14] Malika, N.; Mohammed, F.; Chakib, E. Microbiological and
Physico- chemical properties of Moroccan honey. Int. J. Agr. Bio.
2005; 7: 773-776.
[15] Pérez-Arquillué, C.; Conchello, P.; Ariño, A.; Juan, T.; Herrera, A.
Quality evaluation of Spanish rosemary (Rosmarinus officinalis)
honey. Food Chem. 1994; 51: 207-210.
[16] Pérez-Arquillué, C.; Conchello, P.; Ariño, A.; Juan, T.;
Herrera, A. Physico chemical attributes and pollen spectrum
of some unifloral Spanish honeys. Food Chem. 1995; 54:
167-172.
[17] López, B.; Latorre, M.J.; Fernández, M.J.; García, M.A.;
Herrero, C. Chemometric classification of honeys according to
their type based on quality control data. Food Chem. 1996; 55:
281-287.
[18] Costa, L.S.M.; Albuquerque, M.L.S.; Trugo, L.C.;
Quintero, L.M.C.; Barth, O.M.; Ribeiro, M.; De María,
C.A.B. Determination of non-volatile compounds of different
botanical origin Brazilian honeys. Food Chem. 1999; 65:
347-352.
[19] Lund, M.N.; Ray, C.A. Control of maillard reactions in foods:
strategies and chemical mechanisms. J. Agri. Food Chem. 2017;
65: 4537-4552.
[20] Ramíre Cervantes, M.A.; Gonzále Novelo, S.A.; Sauri Duch, E.
Efecto del tratamiento térmico temporal de la miel sobre la
variación de su calidad durante el almacenamiento. Apiacta. 2000;
35: 162-170.
[21] Shapla, U.M.; Solayman, M.; Alam, N.; Khalil, M.I.; Gan, S.H.
5-Hydroxymethylfurfural (HMF) levels in honey and other food
products: effects on bees and human health. Chem Cent J. 2018;
12: 35.
[22] Huidobro, J.F.; Santana, F.J.; Sanchez, M.P.; Sancho, M.T.;
Muniategui, S.; Simal-Lozano, J. Diastase, invertase and
β-glucosidase activities in fresh honey from north-west Spain.
J. Apic. Res. 1995; 34: 39–44.
[23] Persano Oddo, L.; Pulcini, P.; Piazza, M.G. Invertase content of
Italian unifloral honeys. Apic. Mod. 1996; 87: 25-29.
[24] Persano Oddo, L.; Piazza, M.G.; Pulcini, P. Invertase activity in
honey. Apidologie 1999, 30, 57-65.
[25] Silva, F.L.; Fischer, D.C.H.; Tavares, J.F.; Silva, M.S.; de
Athayde-Filho, P.F.; Barbosa-Filho, J.M. Compilation of
secondary metabolites from Bidens pilosa L. Molecules 2011; 16:
1070-1102.
[26] Bogdanov, S. Harmonised Methods of the International
Honey Commission. Swiss Bee Research Centre, FAM, Liebefeld.
2002.
[27] AOAC. Office methods of analysis (15th edition) Association of
Official Analytical Chemist Washington, DC, USA. 1990; No 969.
12.
[28] AOAC. Office methods of analysis (15th edition) Association of
Official Analytical Chemist Washington, DC, USA. 1990; No 980.
23.
[29] Rock, J.; Gryglewski, R.I. Flavonoids are scavengers of
superoxide anions. Biochem. Pharma. 1988; 37: 837-841
[30] Oyaizu, M. Studies on products of browning reaction antioxidative
activities of products of browning reaction prepared from
glucoseamine. Jpn. J. Nutr. 1986; 44: 307-315.
[31] Montgomery, D.C. Design and analysis of experiments (8th ed.).
New York, NY: John Wiley and Sons. 2012.
[32] Castro-Vázquez, L.; Díaz-Maroto, M.C.; González-Viňas, M.A.;
Fuente, D.L.E.; Pérez-coello, M.S. Influence of storage conditions
on chemical composition and sensory properties of Citrus honey. J.
Agri. Food Chem. 2008; 56: 1999-2006.
[33] EI Sohaimy, S.A.; Masry, S.H.D.; Shehata, M.G. Physicochemical
characteristics of honey from different origins. Annals of
Agricultural Sciences. 2015; 60(2): 279-287.
[34] Oddo LP, Piazza MG, Pulcini P. Invertase activity in honey.
Apidologie. 1999, 30, 57-65.
[35] Ahmed, M.; Djebli, N.; Aissat, S.; Khiati, B.; Meslem, A.; Bacha,
S. In vitro activity of natural honey alone and in combination with
curcuma starch against Rhodotorula mucilaginosa in correlation
with bioactive compounds and diastase activity. Asian Pac. J.
Trop. Med. 2013; 3: 816-821.
[36] Yücel, Y.; Sultanoglu, P. Characterization of honeys from Hatay
region by their physicochemical properties combined with
chemometrics. Food Biosci. 2013; 1: 16-35.
[37] Janzowski, C.; Glaab, V.; Samimi, E.; Schlatter, J.; Eisenbrand, G.
5- Hydroxymethylfurfural: assessment of mutagenicity, DNA-
damaging potential and reactivity towards cellular glutathione.
Food Chem. Toxicol. 2000; 38: 801-809.
[38] Mouhoubi-Tafinine, Z.; Ouchemoukh, S.; Bachir bey, M.;
Louaileche, H.; Tamendjari, A. Effect of storage on
hydroxymethylfurfural (HMF) and color of some Algerian honey.
International Food Research Journal 2018; 25(3): 1044-1050.
[39] Singh, N.; Bath, P.K. Relationship between heating and
hydroxymethylfurfural formation in different honey types. J. Food
Sci. Technol. India. 1998; 35(2): 154-156.

Journal of Food and Nutrition Research 599
[40] Turhan, K. Effects of Thermal Treatment and Storage on
Hydroxymethylfurfural (HMF) Content and Diastase Activity of
Honeys Collected from Middle Anatolia in Turkey. In Innovations
in Chemical Biology.; Sener, B., Eds.; Springer: New York City,
New York, 2009; pp. 233-239.
© The Author(s) 2020
. This article is an open access article distributed under the terms and conditions of the Creative Commons
Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).