foods

Review
Modern Methods for Assessing the Quality of Bee
Honey and Botanical Origin Identification
Anna Puścion-Jakubik * , Maria Halina Borawska and Katarzyna Socha
Department of Bromatology, Medical University of Bialystok, Mickiewicza 2D St., 15-222 Białystok, Poland;
bromatos@umb.edu.pl (M.H.B.); katarzyna.socha@umb.edu.pl (K.S.)
* Correspondence: anna.puscion-jakubik@umb.edu.pl; Tel.: +48-857-485-469




Received: 5 July 2020; Accepted: 28 July 2020; Published: 31 July 2020


Abstract: This paper is a summary of the latest literature on methods for assessing quality of
natural bee honey. The publication briefly characterizes methods recommended by the International
Honey Commission, published in 2009, as well as newer methods published in the last 10 years.
Modern methods of assessing honey quality focus mainly on analyzing markers of individual
varieties and classifying them into varieties, using, among others, near infrared spectroscopy
techniques (NIR), potentiometric tongue, electronic nose, nuclear magnetic resonance (NMR),
zymography, polymerase chain reaction (PCR), DNA metabarcoding, and chemometric techniques
including partial least squares (PLS), principal component analysis (PCA) and artificial neural
networks (ANN). At the same time, effective techniques for analyzing adulteration, sugar, and water
content, hydroxymethylfurfural (HMF), polyphenol content, and diastase activity are being sought.
Modern techniques enable the results of honey quality testing to be obtained in a shorter time,
using the principles of green chemistry, allowing, at the same time, for high precision and accuracy
of determinations. These methods are constantly modified, so that the honey that is on sale is a
product of high quality. Prospects for devising methods of honey quality assessment include the
development of a fast and accurate alternative to the melissopalynological method as well as quick
tests to detect adulteration.

Keywords: adulteration; bee honey; quality assessment; variety

1. Introduction
Natural bee honey is a sweet product made by honeybees Apis mellifera L., both from the nectar of
plants and from the excreta of insects sucking the juice from living parts of plants or from secretions of
live parts of plants. Those are then combined with specific secretions of bees, stored in honeycombs,
evaporated, and left in honeycombs to mature. The Council of the European Union distinguishes
nectar and honeydew honey as well as filtered honey, pressed honey, extracted honey, drained honey,
comb honey, and chunk honey or cut comb honey [1].
Bee honey intended for human consumption should be of sufficiently high quality. Requirements
in individual countries include parameters such as variety determination, water content (no more than
20%; no more than 23% in heather and baker0 s honey; no more than 25% in baker0 s honey from heather),
HMF (general, no more than 40 mg/kg), proline (no less than 25 mg/100 g), diastase activity (general,
no less than 8 on the Schade scale), electrical conductivity (e.g., in honeydew honey, no less than
0.8 mS/cm), pH, insoluble impurities (no more than 0.1 g/100 g) and free acidity (in general, no more
than 50 milli-equivalents acid/1000 g). Other parameters include, for example, color and total phenolic
content (TPC). To assess honey quality, standard methods, including spectrophotometric, refractometric,
titration and melissopalynological methods, are used [2].

Foods 2020, 9, 1028; doi:10.3390/foods9081028 www.mdpi.com/journal/foods

Foods 2020, 9, 1028 2 of 21
Foods 2020, 9, x FOR PEER REVIEW 2 of 21

Growing
Growing scientific interest in
scientific interest in natural
natural bee
bee honey
honey is
is demonstrated by an
demonstrated by an increasing
increasing number
number of
of
publications available in the PubMed article database (Figure
publications available in the PubMed article database (Figure 1).1).

Figure 1. Number
Number of publications in the PubMed database with the word “honey” in the title.

Bee honey
honeyhashasbeen
beenused
usedfor forcenturies
centuries asas
a nutritional,
a nutritional,preventive,
preventive, andandmedicinal
medicinal product. Over
product. Overthe
past 10 year,
the past its use
10 year, itshas
usebeen
has studied in the following
been studied diseases:
in the following arthritisarthritis
diseases: [3], breast
[3],cancer
breast(cell line)(cell
cancer [4],
cervical cancer (cell
line) [4], cervical cancerline)
(cell[4],
line)colon cancercancer
[4], colon (cell (cell
line)line)
[5], [5],
coughcough[6],[6],
type
type2 2diabetes
diabetes[7],
[7], hepatic
steatosis [8], influenza [9], glioblastoma multiforme (cell line) [10], Helicobacter pylori [11], [11], mucositis
mucositis [12],
[12],
Pseudomonas [15],
osteoporosis [13], pressure ulcers [14], Pseudomonas [15], renal
renal cancer
cancer cell
cell line
line [16],
[16], and
and rosacea
rosacea [17].
[17].
Due to such diverse activity, honey must be of the highest quality
activity, honey must be of the highest quality [1,2,18,19].[1,2,18,19].
Market requirements
requirementsare arevery stringent:
very stringent:results should
results be obtained
should as soon
be obtained asassoon
possible; the method
as possible; the
should
methodbeshould
characterized by the highest
be characterized by theaccuracy
highestand precision;
accuracy andand, in addition,
precision; and, init should
addition,be inexpensive.
it should be
Therefore,
inexpensive. many authorsmany
Therefore, publish papers
authors on new
publish quality
papers on analysis
new qualitymethods.
analysis The aim of the
methods. Thepresent
aim of
study was to study
the present summarize
was the most important
to summarize the methods of assessing
most important honey of
methods quality published
assessing honeyin the last
quality
10 years. The
published novelty
in the of years.
last 10 these publication
The noveltylies in thepublication
of these collection ofliesdata
inregarding
the collectionbothof the evaluation
data regarding of
honey composition as well as adulteration.
both the evaluation of honey composition as well as adulteration.

2. Honey Composition
2. Honey Composition
Natural
Natural bee
bee honey
honey isis aa complex
complex product
product containing
containing several
several hundred
hundred compounds
compounds belonging
belonging toto
various
various chemical groups. In
chemical groups. In addition
addition to
to such
such basic
basic groups
groups asas those
those listed
listed in
in Table
Table 1,
1, i.e.,
i.e., glucose,
glucose,
fructose, sucrose, water,
fructose, sucrose, water, organic
organic acids,
acids, minerals,
minerals, or
or amino
amino acids, bee honey
acids, bee honey contains,
contains, among
among others,
others,
polyphenolic compounds, dyes, vitamins, essential oils, and other active
polyphenolic compounds, dyes, vitamins, essential oils, and other active substances. substances.
Twenty five different sugars are found in honey. In nectar honeys, the sugars include erlose,
maltose, sucrose, and turanose, while in honeydew honey-melezitose and raffinose. Dextrins are
found in Italian metcalfa honey. Among the acids in honey, there is mainly gluconic acid, and also in
smaller amounts: acetic, citric, formic, lactic, maleic, malic, oxalic, pyroglutamic, and succinic acids.
These acids influence the pH of honey, which is commonly between 3.3 and 4.6. Among the
main minerals in honey, potassium should be mentioned (it constitutes approximately 33% of all
elements present in honey). Others include barium (from 0.01 to 0.08 mg/100 g), boron (from 0.05
to 0.3 mg/100 g), chlorine (from 0.4 to 56 mg/100 g), cobalt (from 0.1 to 0.35 mg/100 g), fluoride
(from 0.4 to 1.34 mg/100 g), iodine (from 10 to 100 mg/100 g), lithium (from 0.225 to 1.56 mg/100 g),
molybdenum (up to 0.004 mg/100 g), nickel (up to 0.051 mg/100 g), rubidium (from 0.04 to 3.5 mg/100
g), silicium (from 0.05 to 24 mg/100 g), strontium (from 0.04 to 0.35 mg/100 g), sulfur (from 0.7 to 26
Foods 2020, 9, 1028 3 of 21

mg/100 g), vanadium (up to 0.013 mg/100 g), and zirconium (from 0.05 to 0.08 mg/100 g). The elemental
composition is influenced by the origin-honeydew honeys are characterized by a higher content of
elements. The most important and abundant amino acid in honey is proline. Moreover, enzymes such
as amylase (diastase), invertase, catalase, and glucose oxidase are present in honey. Enzymatic content
proves the freshness of honey, and proper heating and storage conditions. Volatile compounds, both
from the nectar and bee secretions, determine the smell of honey. Over 600 of them have been identified
to date. Dark honeys contain more flavonoids. When honey is stored at too high a temperature and in
the process of prolonged heating (particularly honeys with a lower pH), the content of the unfavorable
component, HMF, increases [20].

Table 1. Composition of honeydew and blossom honey [20].

Honeydew Honey Blossom Honey

Component (Min−Max) (Min−Max)
[g/100 g] [g/100 g]
Fructose 31.8 38.2
Glucose 26.1 31.3
Water 16.3 17.2
Sucrose 0.5 0.7
Other disaccharides 4.0 5.0
Melezitose 4.0 <0.1
Erlose 1.0 0.8
Other oligosaccharides 13.1 3.6
Acids 1.1 0.5
Minerals 0.9 0.2
Amino acids, proteins 0.6 0.3

3. Methods for Assessing Bee Honey Quality

Natural bee honey has been the subject of research for several decades. However, it still surprises
scientists with its diversity, nutritional, prophylactic, and biological activity. Therefore, techniques
that enable the assessment of its quality are continually modified and improved with the aim of
reducing analysis time, eliminating expensive and harmful reagents, decreasing workload, and
increasing accuracy.

3.1. Variety of Honey

The melissopalynological method is the principal technique used to determine the variety of
bee honey. This technique enables determination of the share of predominant pollen grains in a
particular honey, on the basis of which the honey variety is named, with the name derived from
the botanical name of the plant or plants. The method also allows for identification of adulterated
honeys [21].
The melissopalynological method was developed in 1895 by Pfister. It has been improved since
then and is currently the most widely used technique for correct determination of honey variety.
Beekeepers, however, determine the variety of honey on the basis of its organoleptic characteristics and
observations from which plants bees collect nectar [22]. Table 2 shows modern methods for identifying
the type and variety of honey. They are briefly characterized in a further part of the paper.
Determination of specific optical rotation is a method that distinguishes nectar honeys (usually
D
with negative values) from honeydew honeys (with positive values). Specific optical rotation [α] 20
◦
is the angle of rotation of polarized light at the wavelength of the D line of sodium, at 20 C, of an
aqueous solution containing 1 of the substance. The measurement is performed using the polarimetric
method and differences between honey types are due to their carbohydrate composition [2].
A number of researchers are seeking techniques that will not be as difficult and time-consuming
to perform as the melissopalynological method. One of the newest approaches to identifying
Foods 2020, 9, 1028 4 of 21

honey varieties uses an electronic tongue. An electronic tongue is a modern device which can be
used to distinguish between honey samples in conjunction with methods of optical spectroscopy,
Ultraviolet-Visable-Near Infrared (UV-VIS-NIR), and statistical analysis: cluster analysis (CA) and
principal component analysis (PCA). By combining these techniques and using with multidirectional
principal component analysis, correct classification of samples was obtained in 100% of cases [23].

Table 2. Selected methods for determining type and variety of honey.

Method Country Varieties of Honey or Origin Literature

Arbutus unedo L.—strawberry-tree (n = 4), Citrus
Electronic with spp.—orange blossom (n = 3), Helianthus annuus
Portugal [23]
UV—VIS—NIR L.—sunflower (n = 3), Lavandula stoechas L. — French
lavender (n = 3)
Castanea sativa Mill.—chestnut (n = 16),
Electronic tongue Croatia [24]
Robinia pseudoacacia L.—black locust (n = 49)
Representative samples from regions: Algarve regions,
Electronic potentiometric Alentejo, Beira Interior, Beira Litoral, Entre Douro e
Portugal [25]
tongue Minho, Estremadura e Ribatejo, Trás-os-Montes e Alto
Douro, and Pico and São Miguel islands (n = 65)
acacia flower (n = 3), buckwheat (n = 3), honeydew
Electronic nose Poland [26]
(n = 3), linden flower (n = 3), and rape (n = 3)
citrus (n = 1), fir honeydew (n = 1), honeydew (n = 1),
NMR Bulgaria oak honeydew (n = 15), polyfloral (n = 4), rapeseed (n = [27]
1), and spruce honeydew (n = 1)
Castanea sativa—chestnut (n = 4), Citrus—orange (n = 4),
Twodimensional zymography Italy Eucalyptus sp.—eucalyptus (n = 4), and Hedysarium [28]
coronarium—sulla (n = 4)
Apis carena: China (n = 3), Vietnam (n = 3),
Australia, Brazil, China,
PCR Apis mellifera: Australia (n = 3), Brazil (n = 3), China [29]
South Africa Vietnam
(n = 31), and South Africa (n = 2)
Arbatus unedo L.—strawberry tree (n = 42), Asphodelus
microcarpus Salzm. Et Viv—asphodel (n = 36), Brassica
napus L.—rapeseed (n = 14), Castanea sativa Mill.—sweet
chestnut (n = 14), Citrus spp.—citrus (n = 9), Erica
spp.—heather (n = 11), Eucalyptus spp.—eucalyptus
Croatia, France, (n = 22), Fagopyrum esculentum L.—buckwheat (n = 12),
CIE L * C * abh ab scale Germany, Hungary, Italy, honeydew (n = 22), Galactites tomentosa Moench—thistle [30]
Poland, Spain, Ukraine (n = 26), Hedysarum coronarium L.—sulla flower (n = 16),
Mentha spp.—mint (n = 12), Paliurus spina-christi
Mill.—garland thorn (Christ’s thorn) (n = 14), Robinia
pseudoacacia L.—black locust (n = 14), Tilia
spp.—lime (n = 12), Salvia officinalis L.—sage (n = 14),
Satureja spp.—savory (n = 15),
acacia (n = 37), fake honey (n = 14), linden (n = 10),
Fluorescence spectroscopy Serbia [31]
meadow mix (n = 23), and sunflower (n = 11).
DNA identification and
Italy Multifloral (n = 4) [32]
plastids
DNA metabarcoding USA Pollen from hives (n = 4) [33]
Acacia from six geographical regions of China:
Statistical analysis: PLS-DA China Gansu (n = 10), Henan (n = 12), Liaoning (n = 10), [34]
Shaanxi (n = 14), Shandong (n = 15), and Shanxi (n = 10)
Continuous flow mass
Hive frames with 64 seats [35]
spectrometry
CIE L * C * abh ab scale—Commission Internationale de l’Eclairage L * C * abh ab scale,
UV-VIS-NIR—Ultraviolet-Visable-Near Infrared.

Differences between the profiles of volatile compounds for 49 linden honeys

(Robinia pseudoacacia L.) and 16 chestnut honeys (Castanea sativa Mill.) provided the basis for the
development of an electronic tongue. It is a matrix with 22 sensors and pattern recognition software
which aims to imitate the human sense of taste to classify products. In a study by Čačić et al. [24],
honey samples were incubated at room temperature for 20 min, then the following phases were
programmed: standby (20 ◦ C, 10 min) and incubation (40 ◦ C, 5 min). Measurement time was 5 min.
PCA was used to show differences between honey samples from different geographical regions.
The five main components explained 66.26% of the variability. The analysis revealed similarities in
samples from geographic regions that were in close proximity to each other; therefore, it can be a tool
for determining the geographical origin of honey [24].
Foods 2020, 9, 1028 5 of 21

Scientists from Portugal used an electronic potentiometric tongue to identify honeys from the
following plants: Castanea sp., Echium sp., Erica sp., Lavandula sp., Prunus sp., Rubus sp., yielding 100%
classification compliance [25].
Dymerski et al. [26] published a paper on the classification of bee honeys from Poland using
an electronic nose. The authors based their research on the use of six semiconductor sensors and a
pneumatic system which caused honey samples to bubble, which together formed the electronic nose.
This technique makes it possible to distinguish, based on volatile compounds, between the following
varieties of honey: acacia, buckwheat, honeydew, linden, and rapeseed.
The nuclear magnetic resonance (NMR) technique has been used to distinguish honeydew and
nectar honeys obtained from oak (Quercus L.) [27]. The determinant of the botanical origin of this
variety is quercitol. A considerable advantage of this method is speed of analysis.
The subject of research by Rossano et al. [28] was the creation of a specific fingerprint by
two-dimensional zymography. Proteins present in honey can come from either nectar, pollen grains,
or gland secretions of the honeybee. The most important proteins found in honey are nine types
of Major royal jelly protein-1 (MRJP-1). The authors studied the proteolytic properties of varietal
honeys (orange, chestnut, eucalyptus, and sulla, which is obtained from Hedysarium coronarium L.)
and their protein profile. Eucalyptus honeys were characterized by the highest protein content
(0.91–1.24 mg protein/g honey), while chestnut honeys—by the highest total proteolytic activity
(25.29–36.43 mU/mg protein). By way of illustration, for orange honeys, 3 groups were obtained
in the image (first—19 kDa, isoelectric point 8.5–9.3; second—20 kDa, isoelectric point 4.2–4.4;
and third—24 kDa, isoelectric point 8.2–8.8). Two-dimensional zymograms can therefore provide the
basis for fast classification of natural varieties of bee honey and may also enable determination of the
origin of honey.
The MRJP2 gene has also been used to discriminate between two types of honey-honey produced
by Apis mellifera and that produced by Apis cerana. The latter is far more expensive, and therefore there
have been cases of mislabeling honey in terms of origin. In order to identify them correctly, the authors
designed two pairs of species-specific primers. The amplification products of A. mellifera and A. carena
honeys were 560 and 212 base pair (bp), respectively. The obtained primers were characterized by
high species specificity. The MRJP2 gene was detected using the PCR method and selected primers.
Differences in the gene formed the basis for establishing the origin of honey. The PCR method enabled
detection of the addition of A. mellifera honey which was as low as 1% [29].
Attempts have been made to distinguish between bee honey varieties using the CIE scale
(Commission Internationale de l’Eclairage) L * C * abh ab [30]. The authors Tuberoso et al. [30]
assessed the color of 17 monofloral honeys (n = 305). The advantage of the proposed method is
small sample size (approximately 2 g) and absence of any destructive effect (samples can be reused in
subsequent analyses). PCA and hierarchical clustering analysis (HCA) have been used to distinguish
between different honey varieties and classifications. In the case of reflection methods, the L * value for
light honeys is below 50 while for dark honeys, it is above 50. The authors emphasize the fact that this
technique may be particularly useful in the case of honeys with the protected designation of origin
(PDO) mark. This concept covers honeys produced, processed, and prepared in one area which have
distinct characteristics from this area. Their names are legally protected and listed on the EU protected
food name register [1]. The development of rapid tests which would assist in honey identification is
particularly important since bee honey is one of the 25 products at highest risk of adulteration [36,37].
Another method of honey classification is fluorescence spectroscopy and statistical analyses based
on parallel factor analysis (PARAFAC) and partial least squares method combined with discriminant
analysis (DAPLS) [31]. It was used in a study by Lenhardt et al. [31], which investigated acacia, sunflower,
lime, meadows, and artificial honey (n = 95). Fluorescence in the range of excitation (260–290 nm) and
emission (330–360 nm) comes from aromatic amino acids and is particularly important in differentiating
the botanical origin of honey samples. The authors found that phenolic compounds and Maillard
reaction products had the greatest impact on the distinction of individual varieties—emissions of these
Foods 2020, 9, 1028 6 of 21

compounds differed most between honey varieties. Classification efficiency was approximately 90%.
Additionally, artificial honey was detected 100% correctly.
A method of identifying botanical species of plants in multifloral honey on the basis of DNA was
described by Bruni et al. [32]. The authors used rbcL and trn—psbA plastids as markers. The aim
of the study was to create a database of “bar codes” of selected plants and, on that basis, to identify
plants from which nectar was obtained. The application of this method allowed for, among others,
the identification of nectar from the genera Fagus, Quercus, Castanea and from toxic plants such as
Atropa belladonna (Wolfcream). Therefore, this method can be used indirectly to identify varieties of
bee honey.
In the same year, researchers from the USA [33] published a method of analyzing plant pollen,
based on the technique of DNA metabarcoding (testing of the so-called DNA bar codes). This technique
is based on the analysis of DNA barcodes, i.e., short sequences. Primers specific to the second internal
transcribed spacer were used in the experiment. Then, DNA amplicon, which was isolated from pollen
samples collected by bees, was sequenced. The ribosomal sequences served as a specific code for the
genetic identification of plant pollen. The proposed technique is particularly useful in the recognition
of pollen, present in bee honeys, which is difficult to identify and rare.
The study investigated 71 acacia honeys from China, from 6 different sources. The authors
analyzed 31 different factors using the partial least squares-discriminant analysis (PLS-DA) technique:
18 polyphenols, 9 oligosaccharides, and 4 carbon isotope ratio values. Based on those parameters,
correct classification regarding the geographical origin of honey was obtained in 94.12% cases [34].
Researchers from New Zealand modified the technique of detecting sugar adulteration in two
of New Zealand’s most famous honeys—manuka honey obtained from Leptospermum scoparium and
kanuka honey from Kunzea ericoides [35]. Adding sugar, sugar cane, or High-fructose corn syrup
(HFCS) to honey is a common technique used by manufacturers in order to generate higher profits.
Based on the recommendations of the International Organization of Chemical Food Analysts (AOAC,
Association of Official Agricultural Chemists), the authors improved the method, using the technique
of continuous flow mass spectrometry by testing the ratio of isotopes to determine d13C. However, this
method requires further refinement as some of the tested samples were incorrectly classified. It was
mainly samples which contained less than 75% of the predominant pollen that were affected.
Many authors are looking for botanical and geographical markers—compounds characteristic of
individual varieties of honey. These compounds allow for a specific variety to be clearly defined. By way
of illustration, markers for 17 varietal honeys have been identified using liquid chromatography with
diode array detection (LC-DAD) and/or gas chromatography mass spectrometry (GC-MS) methods.
The honeys came from the following countries: Croatia, France, Germany, Hungary, Italy, Poland,
Romania, Ukraine, and Spain. Selected ones are shown in Table 3. Confirmation of the presence of
specific markers is a starting point for further analysis and statistical inference.

Table 3. Markers for selected varieties of honey.

Honey Percent of
Botanical Origin Markers Method Literature
Varieties Specific Pollen
Fagopyrum LC-DAD,
Buckwheat 32–53 3-hydroxybenzoic acid; ferulic acid [30]
esculentum L. GC-MS
LC-DAD,
Citrus Citrus spp. 14–39 caffeine; methyl anthranilate [30]
GC-MS
4-methoxybenzaldehyde; 4-methoxybenzoic acid;
Heather Erica spp. 40–62 GC-MS [30]
methyl 4-hydroxy-3-methoxybenzoate
Lime Tilia spp. 11–47 1-(4-methylphenyl)ethanone; 4-terpinenol GC-MS [30]
LC-DAD,
Mint Mentha spp. 18–39 methylnsyringate; vomifoliol [30]
GC-MS
HPLC-MS,
Rape Brassica napus L. - kaempferol; morin with tandem [38]
ion detect
2-amino-4-hydroxypteridine-6-carboxylic acid,
Toran, Saha - - GC-MS [39]
methyl 3-hydroxyhexanoate
GC-MS—gas chromatography mass spectrometry, HPLC-MS—high performance liquid chromatography mass
spectrometry, LC-DAD—liquid chromatography with diode array detection.
Foods 2020, 9, 1028 7 of 21

3.2. Sugars
The carbohydrate composition of natural bee honey may be one of the key factors in establishing
its botanical origin and, indirectly, enabling its proper classification [40]. Sugars in honey are produced
by enzymatic sucrose hydrolysis and transglycosylation [41].
The content of fructose, glucose, sucrose, as well as sugars such as maltose, turanose, erlose,
raffinose, melezitose, and isomaltose is determined by high performance liquid chromatography
(HPLC) with infrared (IR) detection. Peaks are identified based on the standards used, their retention
times and peak heights. The column and detector should be thermostated at 30 ◦ C. Results are presented
in g/100 g [2]. Total glucose and fructose content should be no less than 60 g/100 g for nectar honeys,
while for honeydew and honeydew nectar it should be no less than 45 g/100 g. Sucrose content should
be no more than 5 g/100g, except for acacia (Robinia pseudoacacia L.) and alfalfa (Medicago sativa L.)
honey (the norm: no more than 10 g/100 g), and honey obtained from lavender (Lavandula spp. L.)
and borage (Borago officinalis L.) (the norm: no more than 15 g/100 g) [1]. The fructose:glucose ratio in
honeydew honey is normally higher than that in nectar honeys. In addition, melecytosis is a sugar
which is found exclusively in honeydew honey [42].
A less frequently used method is the titration technique in which methylene blue is used as an
internal indicator. The difference in invert sugar concentrations before and after inversion, multiplied
by a factor of 0.95, yields sucrose content [2].
Other methods recommended for determining sugar content in bee honey include gas
chromatography (GC) and HPLC with pulsed amperometric detection. In the GC method, sugars
are silylated and then the derivative fraction is quantified. Mannitol is used as an internal standard.
The latter method (HPLC) is based on the principle that at high pH levels sugars behave like very weak
acids—they are fully or partially ionized, and therefore they can be separated using the ion exchange
mechanism [2].
A novel method for determining the content of fructose, glucose, and sucrose in bee honey
samples has been proposed by scientists from Brazil [43]. Sugar content in their study was determined
by capillary electrophoresis. The advantages of this method include high resolution, short sample
preparation time, a small sample size, and short duration of the analysis itself. Within two minutes, the
three compounds tested were separated completely, ensuring repeatability and linearity. The detection
limits were 0.022–0.029 g/L.
High performance thin layer chromatography with image analysis was used by Puscas et al. [44] to
detect “adulterants” in bee honey. This technique allowed the authors to analyze basic sugars present
in honey: fructose, glucose, and sucrose. Moreover, the authors calculated the fructose:glucose ratio.
The study demonstrated that acacia honey was the most faked honey. The reason was probably the
producers’ desire to improve the organoleptic characteristics of this variety. The advantage of this
method is its simplicity and low cost of analysis. Therefore, it can be an alternative to more expensive
and more time-consuming techniques used to detect adulteration [44].
A different method of analyzing the content of glucose, fructose, sucrose, and additionally, maltose,
has been proposed by researchers from Turkey [45]. It is based on Raman spectroscopy, followed
by advanced statistical techniques of PLS, PCA, and ANN. The authors obtained a high correlation
coefficient between the values determined and those predicted by the models.
Another technique that enables the determination of monosaccharide and disaccharide content is
surface-assisted laser desorption with mass ionization spectrometry using HgTe nanostructures as a
matrix (SALDI-MS). Sucralose is used as the standard. The authors emphasize that this method does
not require time-consuming sample preparation and analysis time is only 30 min. In addition, it is
characterized by high repeatability [46].
Ma et al. [47] developed a method for assessing honey maturity on the example of 85 acacia
honeys for which 18 physicochemical parameters were analyzed in combination with chemometric
analysis. During the process of maturation, honey changes its chemical composition. Mature honey
has a moisture content below 18% and sugar concentration above the saturation point—it is enclosed in
Foods 2020, 9, 1028 8 of 21

honeycomb cells. The main differentiating variables revealed in the analysis of variance were total sugar
content (fructose, glucose, and sucrose), total protein content and total phenolic content. PCA, CA,
and orthogonal partial least squares-discriminant analysis (OPLS-DA) were used in the classification.

3.3. Water
The harvesting of unripe honey will result in it having too high a water content. This will
cause faster fermentation as a result of microbial development, including Zygosaccharomyces.
Water, by collecting in higher layers of honey causes thinning, followed by foaming, an acidic smell,
and a characteristic taste. On the other hand, yeast from the genus Torulopsis cause fermentation which
is manifested, for example, by honey leaking from its packaging [42]. The susceptibility of honey to
the development of microorganisms increases in samples with a water content above 17% [40].
Moisture content in honey is measured by refractometry. This parameter is determined in honey
melted at 50 ◦ C and then cooled to room temperature. The principle of this method is founded on
measurement of the refractive index, based on which water content in honey is determined. The higher
the solids content, the higher the refractive index. The result is expressed in g/100 g [2]. Requirements
in European Union countries are based on the regulation that the water content in honey should be no
more than 20 g/100 g with the exception of baker’s and heather (Calluna (L.) Hull.) honey (the norm:
no more than 23 g/100 g) and baker’s honey from heather (the norm: no more than 25 g/100 g) [1].
New methods of assessing moisture content in honey are based on evaluating the way of
crystallization by studying crystal size and shape. Tappi et al. [48] utilized Differential scanning
calorimetry (DSC) and time domain magnetic resonance (TD-NMR) methods. The TD-NMR method
allowed the authors to distinguish two pools of protons, whose relative intensity was approximately
55% and 45%. It was also observed that static crystallization is divided into two stages, with the
second partially reversing the effects of the first. The study confirmed that in a dynamic process with
continuous stirring, crystallization time of honey is reduced 5–6 times [48].
The fructose: glucose ratio affects the speed and manner of honey crystallization. In exemplary
honeys from India studied by Naik et al. [49], the ratio was 0.931, 1.17, 1.18, 1.23, and 1.54. The most stable
crystals were formed with the fructose: glucose ratio of 1.18, which was confirmed by simulations
using ANN. Other ingredients that influence the manner of crystallization are maltose, sucrose,
and water. This study proves that the recommended ratio (1.18) allows for a delay or even avoidance
of the crystallization process. It also enables us to understand the interactions of sugars present in
honey using molecular dynamics [49].

3.4. Enzymes: Diastase and Invertase

Enzymes present in honey include invertase, amylases (α- and β-amylase), maltase, phosphatases,
catalase, glucose oxidase [40], β-fructofuranosidase, and ascorbinoxidases [42]. What is particularly
important in regard to enzymes, their content in bee honey is influenced by, among others, storage
conditions (including high temperatures), and decrystallization process [40].
A unit of diastase activity is the amount of enzyme contained in bee honey that converts 0.01 g
of starch (insoluble starch conjugated with blue dye is used as a substrate) within 1 h at 40 ◦ C.
Determination is performed by spectrophotometric reading at 620 nm. The result is expressed in units
of Schade/g honey [2]. Good quality honey should have no less than 8 Schade units/gram (except
baker’s honey). Honeys with a low enzyme content (citrus honeys with an HMF content of no more
than 15 mg/kg) can have a diastase number of no less than 3 [1].
One unit of invertase activity is the number of micromoles of a substrate that are destroyed
per minute. The substrate used is p-nitrophenyl-α-D-glucopyranoside, which breaks down into glucose
and p-nitrophenol under the influence of α-glucosidase (also called invertase). At pH 9.5, the reaction
is trapped and the nitrophenol is converted to the nitrophenol anion—the amount of a substrate is
determined based on its amount. Spectrophotometric reading is taken at 400 nm [2].
Foods 2020, 9, 1028 9 of 21

An alternative method for determining diastatic activity of bee honey has been developed by
Sakač & Sak-Bosnar [50]. The proposed method uses a platinum redox sensor and is based on the
potentiometric measurement of free triiodide, which is released from the triiodide–starch complex
during degradation. The authors compared the new, fast measurement technique with the Schade
method and the method based on incubation with Phadebas tablets. For most samples, there were
no significant differences in activity determination between the three methods studied. Moreover,
the analysis time of one sample in the proposed technique is only 5 min, and therefore much shorter
than, e.g., that of the aforementioned method of incubation (where incubation time is around 60 min).
In 2019, a paper on the determination of diastase number by Visable-Near Infrared (VIS/NIR)
spectroscopy was published. The Gaussian Filter Smoothing-standard Normal Variate (GF-SNV) and
least squares-support vector machine (LS-SVM) were used. The root-mean-square error (RMSE) was
0.2 [51].
In addition, the enzymes β-fructofuranosidase and β- and γ-amylase are used in the production
of syrups from starch and therefore their high activity indicates the presence of sugars originating
from starch syrups in honey [52].

3.5. pH and Free Acidity

Organic acids, present in bee honey, are the key factor responsible for its taste. Among the most
common organic acids are citric and gluconic acid, as well as succinic, malic, butyric, lactic, formic,
acetic, and pyroglutamic acids. These acids affect the overall acidity of honey (called titration) [40].
The negative logarithm of hydrogen ion concentration is pH, while free acidity is the sum of
all free acids present in honey. The principle of the method is based on the dissolution of bee honey
in water, pH measurement, followed by titration with sodium hydroxide solution (0.1 M) to obtain a
pH of 8.3 [2]. Free acidity should be no be more than 50 milliequivalents/kg of honey with the exception
of baker’s honey (the norm: no more than 80 milliequivalents/kg) [1].
Acidity level and water content in honey are parameters that affect the development of yeast and
mold in this bee product. For example, an analysis of honeys from Brazil, published in 2014, showed
that 45.7% of honeys were of inferior quality [53].
With regard to pH and free acidity determination, no recent data on faster methods for analyzing
these parameters is available. As far as free acidity is concerned, newer techniques would make
analysis easier as titration has to be performed quickly—within 2 min—which, in laboratory practice,
is not always successful and requires the analysis to be repeated several times, which leads to the
destruction of valuable samples. An innovative approach in the case of these parameters consists in
data presentation. In 2020, Ratiu et al. published a study in which correlations between the analyzed
parameters, including pH, were shown using heat maps [54].

3.6. Ash and Elements

Mineral substances, amino acids, and organic acids (e.g., citric acid), present in bee honeys, form
ionic forms in honey aqueous solutions, which consequently affects the conduction of electrical current
and the measurable parameter referred to as electrical conductivity [40]. Minerals, after burning honey,
remain as ash [42].
Determining ash content is helpful in assessing the type of honey. Ash is the residue after burning
a sample at 350–400 ◦ C, for at least 1 h, repeated until a constant weight is obtained. Its amount is
expressed in g/100 g [2].
Electrical conductivity is determined on a 20% honey solution, calculated on dry matter.
Determination of electrical conductivity is based on the measurement of electrical resistance, which is
the inverse of conductivity. The result is expressed in mS/cm [2]. This parameter should be no more
than 0.8 mS/cm with the exception of, among others, honeydew honey [1].
Newer methods allow for detailed examination of the composition of honey, including the content
of macroelements, microelements, and toxic elements.
Foods 2020, 9, 1028 10 of 21

The capillary electrophoresis method with UV detection used by Shi et al. [55] determined
11 metal cations present in bee honeys (Ba2+ , Ca2+ , Cd2+ , Cr3+ , Cu2+ , Fe2+ , K+ , Li+ , Mg2+ , Na+ ,
and Zn2+ ), which affected the quality of bee honey. Soil and bee food are mentioned as sources
of cations. The varied composition can be the basis for distinguishing honey varieties as well as for
safety assessment and detection of adulteration. The advantages of this technique include a short
analysis time (around 8 min) and a detection limit of 0.01–0.21 mg/L [55].
The determination of 14 elements (Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Na, Ni, P, Pb, and Zn) in
honeys obtained from Poland and various European regions (n = 66) was conducted by Grembecka and
Szefer [56]. Statistical analyses were conducted resulting in dendrograms discriminating dark (heather,
buckwheat, and honeydew) from light honey (rape, linden, acacia, and multifloral). This confirmed a
higher content of elements in dark honeys.
The content of lead, cadmium, and chromium was determined by scientists from Brazil in
2014 by Electrothermal Atomic Absorption Spectrometry (ET-AAS) technology [57]. Determining
the concentration of these elements in honey can be useful as a bioindicator of environmental
contamination. The advantages of this method, in relation to honey analysis, include the following: no
need to pre-treat samples, precision and accuracy of determination. In addition, honeys can act as a
bioindicator of environmental pollution in regard to radioactive elements [58,59].
The content of microelements and trace elements in bee honey was the subject of research by
Ribeiro et al. [60]. The authors used the Total Reflection X-ray Fluorescence Spectroscopy (TXRF)
technique. Honeys were obtained in 4 seasons: spring, summer, autumn, and winter. Seasonal changes
in potassium (e.g., winter: 98.7 mg/100 g ± 12, autumn: 194.5 mg/100 g ± 20) and calcium (e.g., winter:
375.2 mg/100 g ± 38, autumn: 46.4 mg/100 g ± 6) levels were demonstrated. However, no significant
changes were found in the content of Cr, Ti, Se, and Ni.

3.7. Proline
Proline is the major amino acid in bee honey, constituting 49% in nectar honey and 59% in honeydew
honey of the total amino acid content. Other important amino acids present in nectar and honeydew
honey include phenylalanine (32% and 6%) and glutamic acid (3% and 11%), respectively [40].
Determination of proline content in bee honey is performed, among others, to confirm the
authenticity of honey. The content of this amino acid is determined on the basis of its colored complex
formed with ninhydrin; absorbance is read at 510 nm [2]. The proline content of honey should be no
less than 25 mg/100 g [42].
The assessment of amino acid or protein content in bee honey and use of these ingredients by
bees is the subject of modern research.
Proteins in honey constitute from 0.1 to 0.5%. The main ones are invertase (α-glucosidate)
and MRJP. To date, methods such as dialysis against distilled water and lyophilisation of the resulting
dialysate have been used to isolate proteins as the so-called gold standard. Bocian et al. [61] developed
a faster and cheaper extraction method using saturated phenol. It was tested on 3 types of honey:
black locust, buckwheat, and rapeseed. The main advantages of the method are an almost threefold
reduction in protein isolation time from 32 to 12 h and no need for expensive equipment (including
a freeze dryer). Electrophoresis demonstrated that the proteomes of individual varieties differ from
each other, which allows for distinguishing varieties of bee honey.

3.8. Polyphenols and Other Antioxidant Composition

Phenolic substances, which are phenol derivatives, are synthesized by plants. They are divided
into simple phenols and polyphenols. Polyphenols are characterized by the presence of more than one
aromatic ring in the structure of the molecule [62].
The principle of determining total TPC is as follows: phenolic compounds that are present
in honey are oxidized under the influence of the Folin–Ciocalteu reagent. The components of this
solution, phosphomolybdic acid, and phosphotungstic acid, are reduced to molybdenum and tungsten
Foods 2020, 9, 1028 11 of 21

oxides, which yield a blue color. The color intensity is directly proportional to the content of phenolic
compounds present in the sample [63]. Differences in the content of phenolic compounds in individual
honey varieties were used by Zhang et al. [64]. The authors developed a method for the determination
of chlorogenic, gallic, ferulic, caffeic, p-hydroxybenzoic, p-coumaric, protocatechuic, syringic acids,
and rutin.
In 2014, a study investigating differences in the content of antioxidant compounds in honey
samples was also published. The content of phenolic acids (benzoic, chlorogenic, gallic, coffee,
and trans-cinnamic acids) as well as flavonoids (catechin, kemferol, myricetin, and naringenin) was
determined [65]. The authors indicated that differences in compound concentration between individual
varieties were caused, among others, by the botanical origin of honey. The highest levels of benzoic
and caffeic acid were found in bee honeys from Bangladesh.
Both older and more recent publications are based on the color assessment method in which the
absorbance of a 50% honey solution is measured at a wavelength of 635 nm [66]. Additionally, the color
intensity of honey is assessed by measuring the color intensity ABS450—the absorbance of a 50% honey
solution at 450 and 720 nm [67]. Many authors correlate the obtained data with other parameters,
looking for dependencies and explaining the biological activity of honey. Publications from recent years
that evaluate the antioxidant properties of honey are based on previously developed methodologies and
focus on, inter alia, determining the content of various compounds including flavonoids, ascorbic acid,
carotenoids, β-carotene, lycopene, reducing sugar, as well as assessing radical scavenging activity
with 2,2-diphenyl-1-picrylhydrazyl (DPPH) [68], ferric-reducing/antioxidant power (FRAP) assay [69],
and total antioxidant capacity (TAC) [70]. These methods are based on spectrophotometric absorbance
measurements (Table 4). They are fairly fast and the obtained results are repeatable and therefore
they constitute the basis of modern research on antioxidant properties. These properties are crucially
important from the point of view of the prophylactic abilities of honey and their use in supporting
the treatment of many diseases, which has been confirmed by numerous publications within the last
10 years [71–73].

Table 4. Selected methods of assessing the antioxidant properties of honey.

Method Wavelength Unit Literature

Ascorbic acid 515 nm mg ascorbic acid/kg [68]
Carotenoids content 453, 505, and 663 nm mg of carotenoid/kg [68]
Color intensity ABS450 450 and 720 nm mAU [67]
Color in Pfund scale 635 nm mm [66]
% radical scavenging activity,
DPPH (scavenging activity) 517 nm [67]
ICE50
Flavonoid contents 510 nm mg (+)-catechin equivalents /kg [68]
FRAP assay 593 nm µM Fe(II) [67]
Emission: 535 nm Excitation: 485
ORAC trolox equivalent/g [67]
nm
Phenol content 750 nm mg gallic acid/kg [67]
Reducing power 700 nm EC50 [68]
Ascorbic acid equivalents/g
TAC 695 nm [70]
or gallic acid equivalents/g
DPPH—2,2-diphenyl-1-picrylhydrazyl, FRAP—reducing/antioxidant power assay, ORAC—oxygen radical
absorbance capacity, TAC—total antioxidant capacity.

3.9. Hydroxymethylfurfural
5-hydroxymethylfurfural is a component resulting from the Millard reaction. It is formed under
the conditions of increased temperature during the reaction of dehydration of sugar. This reaction
takes place in an acidic environment. There are also reports in the available literature regarding HMF
formation during caramelization and degradation of hexoses [40].
The principle of the method is based on the measurement of 5-(hydroxymethyl-)
furan-2-carbaldehyde content in honey by HPLC with reversed phase and UV detection. HMF content
in the standard is determined by spectrophotometry at 285 nm. The result is expressed in mg/kg [2].
Foods 2020, 9, 1028 12 of 21

Honey available for sale may contain no more than 40 mg/kg, except for baker’s and tropical honeys
(the norm: no more than 80 mg/kg) [1]. Other methods (White and Winkler) are less frequently used [2].
An alternative method for determining HMF content was proposed in 2013 by Hošt’alková et al. [74].
The authors used high-performance thin-layer chromatography (HPTLC) and the Reflectoquant
spectrophotometric test. In both techniques, short analysis time (2.5 min) was obtained and
deviation between the methods was 15%. The HPTLC method was characterized by higher precision.
The advantage of both techniques is the absence of harmful reagents, which is in line with the principles
of green chemistry.
NIR spectroscopy is gaining importance in food sample analysis. Stőbener et al. [75] used Fourier
Transform Attenuated Total Reflection Infrared Spectroscopy (ATR FTIR) to determine the content of
5-hydroxymethylfurfural in bee honey. This compound is a cyclic aldehyde formed in the Millard
reaction by dehydrating hexoses, catalyzed by acid, at a high temperature. The authors added a HMF
solution to honey at a concentration of 9 to 100 ppm, every 5 ppm. HMF alone produces vibrating bands
in the spectrum from 3500 to 2700 and from 1850 to 600 cm−1 . After adding the HMF standard to honey,
no vibrational characteristic of pure analyte was observed. The authors, using modelling and several
calibration models, developed a method for determining HMF in honey. They used chemometric
techniques such as non-linear iterative partial least squares (NIPALS). The authors observed that for
the proper development of a method used to determine trace compounds, it is necessary to use a large
number of calibration samples in a wide range of concentrations. The best model was a model based
on spectrum averaging—it allowed to reduce noise and improve forecasting parameters. Using this
technique, it is possible to detect the HMF concentration of 13 ppm in bee honey.

3.10. Insoluble Matter and Contaminants

Impurities in bee honey can enter the final product during preparation, centrifugation, or packaging.
The source of insoluble substances may be an improperly performed process of straining honey during
extraction from honeycombs. During this process, the bee product flowing from the honey extractor
passes through a series of sieves [42].
Ingredients insoluble in water (at 80 ◦ C), present in honey, constitute the residue left after the
filtration of honey solution. The residue is dried at 135 ◦ C, to a constant weight. The result is expressed
in weight percent [2]. Honey should contain no more than 0.1 g/100 g of insoluble ingredients, except for
pressed honey (the norm: no more than 0.5 g/100 g) [1].
Due to the prevalence of diseases causing massive extinction of bees, techniques for
detecting undesirable residues of various compounds in natural bee honey are gaining popularity.
Among xenobiotics, sulfonamide residues should be mentioned [76,77]. They are used to control 3
major threats to honeybees: European foulbrood caused by Melissococcus plutonius, American foulbrood,
which is caused by Paenibacillus larvae ssp. Larvae and Varroa, which is based on Varroa destructor.
However, the presence of these substances in bee products may present a risk to consumers [78].
Sajid et al. [77] performed determination of sulfonamide residues by extraction using a short C-18
column, utilizing high-performance liquid chromatography with fluorescence detection. The advantage
of this method is its low cost (including reagent utilization), speed of execution and short extraction
time [77]. Szcz˛esna et al. [79] demonstrated the presence of sulfonamide residues in honey in the range
of 5–2891 µg/kg, revealing that 17% of tested honeys exceeded the permissible concentration limits.
High resolution mass spectrometry has also been used to detect xenobiotics, combined with data
mining analyses and statistical tools useful in metabolomic techniques. A study by Cotton et al. [80]
revealed that the concentrations of 35 compounds in tested honeys were above the permitted limits.
This technique can also be used to classify honeys (acacia, orange, lavender, and multifloral).
Azzouz & Ballesteros [81] developed a method for determining 22 pharmacologically active
substances present in honey (including non-steroidal anti-inflammatory drugs, antibacterial agents,
β-blockers, lipid regulators, antiseptics, and anti-epileptics) by GC-MS, using the solid phase extraction
(SPE) module. Some of these substances may be present in honey as a result of fighting honeybee
Foods 2020, 9, 1028 13 of 21

brood diseases European, such as European brown rot. Therefore, the monitoring process of these
substances seems necessary. The advantages of this method include accuracy, sensitivity, and low
matrix interference, as evidenced by a recovery of 86–102%.
Pesticides are one of the factors responsible for weakening bees and increasing mortality of
their populations [82]. The technique for determining 22 insecticides belonging to 3 chemical
groups (neonicotinoids, pyrethroids, and pyrazoles) was developed by Paradis, Bérail, Bonmatin and
Belzunces [83]. The study material were samples of rapeseed, acacia, chestnut, and multifloral honey.
Most compounds (acrinathrin, bifenthrin, cypermethrin, deltamethrin, esfenvalerate, fipronil,
fipronil desulfinyl, fipronil sulfide, fipronil sulfone, λ-cyhalothrin, permethrin, pyraclofos, resmetrin,
tebufenpyrad, τ-fluvalinate, and tolfenpyrad) were determined by GC-MS/MS, while the remaining
6 were determined by liquid chromatography coupled with mass spectrometer (LC-MS/MS, liquid
chromatography–mass spectrometry) (acetamiprid, clothianidin, ethiprole, imidacloprid, thiacloprid,
and thiamethoxam). The advantage of this technique was the detection of pesticides which were
present in honey in very low concentrations (below 1 ng/g).
The liquid chromatography technique with mass spectrometer and ion trap is also used to
detect pyrrolizidine alkaloids (PAs). These alkaloids, which are secondary plant metabolites,
have hepatotoxic effects. They are found in plants from the families Asteraceae, Fabaceae and
Boraginaceae. Among the 11 alkaloids analyzed, lycopsamine (182–4078 µg/kg) was the most
common [84].
A description of another technique, also used to detect pyrrolizidine alkaloids, was published
in 2014. It was based on an enzyme-linked immunosorbent assay (ELISA). The authors developed a
method whose detection capacity for Jacobin, heliotrine, lycopsamin, and sennesionin was 25 µg/kg.
The advantages of this method include low cost, short analysis time (up to 2.5 h), and the possibility of
testing 21 samples simultaneously [85].
Methods for detecting contaminants in honey are still evolving as confirmed by data collected in
Table 5 which contains selected impurities detected in the last 10 years.

Table 5. Selected contaminants detected in bee honey over the last 10 years.

Component Year of Publication Literature

aminoglycosides 2015 [86]
aflatoxin 2013 [87]
Bacillus subtilis and Bacillus cereus 2014 [88]
benzimidazole derivatives 2015 [89]
bromine and iodine 2015 [90]
cadmium 2019 [91]
Candida lundiana sp. nov. i Candidia
2012 [92]
suthepensis sp. nov.
cannabinoids 2019 [93]
chloramphenicol 2013 [94]
chlorophenol 2015 [95]
cobalt 2012 [96]
fluoroquinolones 2013 [97]
lead 2014 [98]
lincomycin 2014 [99]
mayanotoxin from Rhododendron ponticum 2014 [100]
mercury 2012 [101]
metronidazole 2011 [102]
nickel 2015 [103]
penicillin 2013 [51]
polycyclic aromatic hydrocarbons 2012 [104]
sulfonamides 2012 [105]
tetracycline 2015 [106]
137 Cs 2013 [59]
Foods 2020, 9, 1028 14 of 21

3.11. Adulterations
Due to the health-promoting properties of bee honey, the consumer demand for it has increased in
recent years, which has resulted in the necessity to import honey from outside the European Union (EU).
One way of falsifying bee honey is by adding cheaper HFCS. Bee honey is one of the most frequently
falsified foods [107,108]. Çinar et al. [109] searched for a method of detecting adulteration of bee honey
with HFCS by a method of calculating the 13 C/12 C isotope ratio. This proportion is influenced by
the type of photosynthesis performed by plants: C3 (plants convert CO2 to a 3-carbon compound),
C4 (conversion to a 4-carbon compound) and CAM (plants use both cycles) [110]. Representatives of
the C4 group are, among others, corn plants, and sugar cane [108]. It has been shown that the higher
the HFCS concentration, the higher the C4 sugars. Calculation of the amount of sugars in C4 plants,
based on the determination of the ratio of 13 C/12 C isotopes in the C atom derived from CO2 by mass
spectroscopy, is the basis for detecting adulteration of HFCS honey [109].
Research into honey adulteration based on the 13 C/12 C isotope ratio is continuing as evidenced by
a study published in 2019 by authors researching honeys from Taiwan [111].
An innovative method was developed to identify this type of adulteration based on head space-ion
mobility spectrometry (HS-IMS). For testing purposes, honey samples were adulterated with syrup in
the range of 10 to 50%. The following statistical methods were used: HCA and PCA. The advantage of
this method is low detection limit, ability to monitor the amount of the adulterant in real time as well
as absence of toxic solvents and low gas consumption, which is consistent with the principles of green
chemistry. The authors optimized 5 parameters: incubation time (15 min), incubation temperature
(50 ◦ C), sample size (0.15 g honey), volume of injection (0.91 mL), and heating time (22 min) [112].
The technique of three-dimensional fluorescence, associated with multidimensional calibrations,
was used to determine the concentration of rice syrup in bee honeys and thus to assess adulteration with
these syrups [113]. The analyses were conducted, among others, on rape, buckwheat, and sunflower
honey samples. Statistical methods such as PCA, PLS, and ANN were used. The aim was to develop
an accurate, fast, and non-invasive method of detecting adulteration.
The HPLC technique is used to detect adulteration with starch syrup [114]. A characteristic peak
with a retention time of 15.25 min, derived from starch syrup, is observed. The authors of the method
emphasize its low cost and simplicity of implementation.
The methods for detecting adulteration of honey are constantly being improved. In 2019, a paper
on the use of VIS-NIRS in combination with chemometric methods was published. Counterfeiting was
performed with the following substances: rice syrup, brown cane sugar, fructose syrup, and inverted
sugar in concentrations from 5 to 50%. Full distinction between adulterated and unadulterated
multifloral honey was obtained by the LDA (Linear Discriminant Analysis) method [115].

4. Conclusions
Methods enabling the examination of several parameters which would prove the quality of bee
honey within a short period of time are needed. Modern techniques which have been developed and
validated over the past few years provide high precision and accuracy but need further improvement
so that the natural bee honey available on the market can be tested as quickly and reliably as
possible. Future research into methods of honey quality assessment should focus on developing
techniques of rapid evaluation of the botanical origin of honey since the principal method currently
in use, the melissopalynological method, is very time consuming, requires considerable experience,
botanical knowledge as well as knowledge of the honey production process. It produces inconclusive
results which can be difficult to interpret. Moreover, it is necessary to devise quick, reliable,
and inexpensive methods for detecting honey adulteration. As an overview of current methods
of honey quality assessment, this paper can help shape the direction of future research in this field.
Foods 2020, 9, 1028 15 of 21

Author Contributions: Conceptualization, A.P.-J. and M.H.B.; methodology, A.P.-J. and K.S.; writing—original
draft preparation, A.P.-J.; writing—review and editing, K.S. and M.H.B.; supervision, K.S.; and project
administration, A.P.-J. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by Medical University of Bialystok, grant number N/ST/MN/15/002/2216.
Conflicts of Interest: The authors declare no conflict of interest.

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