Our research work describes the properties of honey, which are the indicators of quality and possibly pollution of the environment, where the bees forage and therefore where the honey comes from. Absorption spectra of the analysed honey samples were decreasing from 200 to 700 nm, with a maximum between 250 and 280 nm. The results of quantitative hydroxylmethylfurfural (HMF) determination in natural honey samples were below the legal limit (40 mg/kg), higher values are commonly associated with elevated temperatures and light exposure. Treating the sample with microwaves causes a significant increase in the HMF concentration, which is consistent with the principles of microwave activation. Qualitative tests for specific components are a useful tool for determining the main components in honey and differentiating between natural and synthetic honey samples, and could be used as a basis for developing more accurate quantitative methods. #SciChallenge2017

1. “PRIMOŽ TRUBAR”
ELEMENTARY SCHOOL,
LAŠKO
Ivana ČAČIČ and Alja ŠPEC
FIELD: FOOD CHEMISTRY
Mentors: Marko JERAN, res. assoc. National Institute of Chemistry
dr. Eva MENART, univ. dipl. chem., National Institute of Chemistry
National museum of Slovenia
Co-mentor: Milena ŽOHAR, prof., Elem. school “Primož Trubarja Laško”
Laško and
Ljubljana,
2017
DETECTION OF PARAMETERS AND
ACTIVE COMPONENTS IN HONEY

2. BEES AND HONEYBees live in highly developed social
communities with 20.000 up to 80.000 members.
Among them are the queen bee,
a couple hundred drones,
and the rest are non reproductive working bees.
There are different types of honey, based on the geographical
and botanical source of nectar, collection method and season.
Throughout history honey has been used as a sweetener, in cosmetics, in varying
ceremonies, for embalming bodies...
Beekeeping is one of the oldest commercial activities. Originally, beekeeping took place in
troughs, made from wood. Later hives as we know them today were built for this purpose.
Beekeeping flourished in 18th and 19th century.

3. TYPES OF
HONEY
ACACIA HONEY
FLOWER HONEY
TILIA HONEY
CHESTNUT HONEY
SPRUCE HONEY
FOREST HONEY
SILVER FIR HONEY
CICADA HONEY

4. USE OF HONEY
ALTERNATIVE SWEETNER
CAN BE ADDED TO DRINKS
tea, lemonade, yoghurt, smoothies
IMPROVES DIGESTION
LOWERS CHOLESTEROL,
IMPROVES BREATH
EASES TOOTHACHE AND JOINT PAIN
HAS CALMING EFFECT
COSMETICS
Balm, facial masks, baths, natural remedy for
warts
HEALING OF WOUNDS, BURNS, INFECTIONS,
ACNE
BALANCES WHITE AND RED BLOOD CELLS
Nursing mothers and children of up to one year are
discouraged from consuming honey, since it can contain a
harmful bacteria Clostridium botulinum, which might be lethal
for more sensitive groups of people.

5. CHEMICAL COMPOSITION
• CARBOHYDRATES
• WATER
• AMINOACIDS
• ORGANIC COMPOUNDS
• MINERALS
• VITAMIS
• HYDROXYMETHYLFURFURAL (HMF)
PHYSICAL PROPERTIES
• DENSITY
• VISCOSITY
• HYGROSCOPY
• ELECTRICAL CONDUCTIVITY
• COLOUR
• CRYSTALLIZATION
• OPTICAL PROPERTIES
HMF:
• Potentially harmful
• Forms during
storage
• Forms at elevated
temperatures

6. EXISTING METHODS AND TEHNIQUES FOR
DETERMINATION OF COMPONENTS IN HONEY
REFRACTOMERY – water content in a sample
POLARIMETRY – specific angle of rotation of linearly polarized light, passing through a sample
HPAEC-PAD – presence of sugar (coupled chromatographic technique)
pH-METRY – pH value, overall acids and lactones
CONDUCTMETRY – electrical conductivity
TXRF – elemental analysis, X-ray fluorescence with total reflection
DRY COMBUSTION– ash content
CHROMATOGRAPHIC TECHNIQUES (HPLC) – advanced technique for detailed analysis
SPECTROPHOTOMETRY

7. Many scientific studies have reported the beneficial effects of honey
and the confirmed the presence of components which have a positive
effect on the human organism.
More and more attention today is dedicated to the
study of parameters, describing the quality of
honey, but we often forget about components that
can be formed over a longer period of time.
From the consumer point of view it is important to maintain the
honey‘s properties during storage.
We suppose that over a longer period of time honey can form higher
concentration of components, which can be unfavorable for humas
in higher doses.
One such example is hidroxymethylfurfural(HMF).
In this research work we studied examples of real and model
(synthetic) honey, and we believe our methods and results
can also be used for raising awareness of the general public.

8. H Y P O T H E S I S
• Using simple chemical tests we can determine the main components in honey (e.g. sugars) and
to an extent differentiate between different types of honey.
• With UV-Vis spectrometry we can see the difference between real and synthetic honey
samples.
• Different honey samples have different HMF contents, based on the conditions of storage and
preparation (for synthetic honey samples, e.g. temperature and microwaves)

9. SYNTHESIS OF ARTIFICIAL HONEY
FEHLING‘S TEST
TOLLENS‘ TEST
BIURET TEST
XANTHOPROTEIC REACTION
UV-VIS SPECTROSCOPY
SPECTROPHOTOMETRIC
DETERMINATION OF HMF
A = log = ε × c × b
oI
I
RCHO + 2 Cu2+ + 5 OH- → Cu2O + RCOO- + 3 H2O
RCHO + 2 Ag(NH3)2
+ + 3 OH- → RCOO- + 2 Ag + 4 NH3 + 2 H2O
EXPERIMENTAL PART

10. RESULTS
AND
DISCUSSION

11. FEHLING‘S TEST RESULTS
Samples, which contained monosaccharides, were coloured
intensely yellow, some even intensely orange (e.g.
purchased forest honey).
The test was shown to be selective and results as expected, as
disaccharides did not form the yellow colouring, indicative
of a positive result.

12. TOLLENS‘ TEST RESULTS
In synthetic honey from glucose and lactic acid under classic
heating conditions the amount of aldehydes and ketones
appears to be higher compared to most samples; the test
result was a very obvious gray colour.
The most intense colouring, which suggests the most
aldehydes and ketones, was observed in homemade flower
honey.
Judging from the colour intensity, we can assume that the
smallest amount of aldehydes and ketones was present in
synthetic honey from sucrose and lactic acid under classic heating
conditions.
The test result in this case was a slight gray hue.

13. BIURET TEST RESULTS
Results of the biuret test for aminoacids confirmed our
expectations – most samples gave a negative result
(no colour change).
*
We noticed a slight colour change in the samples of
homemade and purchased honey. The result is
justified, since this was natural honey.
**
To reliably prove the presence of aminoacids in the
samples, a different detection technique would be
required – possibly quantitative and with a lower
detection limit.

14. The xanthoproteic reaction for proteins gave a very slight
positive result for homemade and purchased honey samples.
The most colour change was observed in homemade mixed
honey samples.
As expected, no colour change was observed in samples of
synthetic honey, which do not contain proteins.
XANTHOPROTEIC REACTION RESULTS
Real honey samples contain a mixture of mostly natural
substances, which can chemically react and form complex
products. Based on the results, the content of these
substances seems very low.

15. RESULTS OF RECORDING UV/VIS SPECTRE IN SAMPLES OF HONEY
UV/VIS spectra were recorded in the
range from 200 nm to 500 nm.
We observed differences
mainly between real and
synthetic honey samples.

16. RESULTS OF QUANTITATIVE HMF
DETERMINATION WITH SPECTROPHOTOMETRY
Highest allowed
concentration of HMF is
40mg/kg!
Sample Content of HMF
[mg/kg]
Purchased flower honey 27,08
Homemade flower honey 13,80
Purchased forest honey 24,19
Homemade mixed honey 27,95
Synthetic honey from surcose and lactic
acid under microwaves
21,28
Synthetic honey from sucrose and lactic
acid under conventional heating conditions
9,17
Synthetic honey from glucose and lactic
acid under microwaves
11,57
Synthetic honey from glucose and lactic
acid under conventional heating conditions
9,71
Synthetic honey from glucose and citric
acid under conventional heating conditions
43,41
Synthetic honey from glucose and citric
acid under microwaves
58,23

17. C O N C L U S I O N

18. HYPOTHESIS CONFIRMED!
We were able to differentiate between different types of honey using
simple chemical tests.
The differences were well visible with UV-Vis spectrometry, particularly
between real and synthetic honey samples.
Analysed natural honey (purchased and homemade) did not
exceed the critical value of HMF in honey
based on legislation.
Synthetic honey samples contained more HMF if they were prepared using
microwaves.
Relatively high and unsuitable amounts of HMF
are linked to storage of honey – long exposure to light sources or high
temperature.
We can minimize the concentration of HMF
in honey by storing it in dark, cool places.

19. A
C
K
N
O
W
L
E
D
G
E
M
E
N
T
MENTORS:
Res. assoc. Marko JERAN, National Institute of Chemistry
dr. Eva MENART, National Institute of Chemistry
National museum of Slovenia
CO-MENTOR:
Milena ŽOHAR, Elm. school Primož Trubar Laško
Dr. Samo HOČEVAR, National Institute of Chemistry
Asist. prof. dr. Katja ŽMITEK, Higher school for applied sciences
Asist. prof dr. Igor PRAVST, Insitute for nutrition
Mavrin GAŠPERŠIČ, BIC Ljubljana
Jaka KRAŠEVEC, BIC Ljubljana
Lidija TOPILIŠEK, Elm. school Primož Trubar Laško
Marko SAJKO, Elm. school Primož Trubar Laško
Pupils of “9. c”
Our families
Sincere gratitude to everyone who supported
us and led us in the right direction (to fame and glory).
Thank you once again!

20. THANK YOU
FOR
YOUR
ATTENTION!