This document discusses honey adulteration, including direct adulteration by adding sugars like corn or cane sugar, and indirect adulteration by feeding bees adulterating substances. It outlines various detection tests for different types of adulteration, such as testing density for sugar solutions or using Fiehe's test and HMF content to detect invert sugar. The health effects of adulteration and pesticide residues in honey are also mentioned. Finally, the document lists several journal articles and references on topics related to honey adulteration detection and analysis.

1. Honey
Apis mellifera L.

2. Adulteration of Honey
Presented by
S.Keerthana
17FT1D7815

3. CONTENTS
• Introduction
• Adulteration of honey
• Detection test
• Effects
• Journals
• Related articles
• Video clippings
• Bibliography

4. Introduction
• Adulteration:
Adulteration is a legal term meaning that a food product fails to meet
federal or state standards.
• Honey:
Honey means the natural sweet substance produced by honey bees
from the nectar of blossoms or from secretions of plants which honey
bees collect, transform store in honey combs for ripening.

5. Composition of Honey
Sr.
No.
Component Amount
(in %)
1. Water (moisture) 20
2. Fructose (Laevulose) 37
3. Glucose (Dextrose) 34
4. Sucrose (Non Reducing Sugar) 5.0
5. Total Ash (Minerals) 0.25
6. Acids Organic 0.20
7. Proteins, Amino Acids, etc. 1.50
8. Other ingredients including
pollen grains
1.05

6. Standards For Honey As Per BIS, India
Amendment No.1 April 2011 to Is 4941: 1994 Extracted Honey Specification
Sr.
No.
Characteristics Special
Grade
A Grade Standard
Grade
1. Specific Gravity at 27 C, (min) 1.37 1.37 1.37
2. Moisture, % by mass, (Max) 20 22 25
3. Total Reducing Sugar, % by mass, (min.) 70 65 65
4. Sucrose, % by mass, (max.) 5.0 5.0 5.0
5. Fructose-Glucose ratio, (min.) 1.0 1.0 1.0
6. Ash, % by mass, (max.) 0.5 0.5 0.5
7. Acidity (expressed as Formic Acid), % by mass (max.) 0.2 0.2 0.2
8. Fiehe’s Test1 Negative
9. Hydroxymethyl Furfural (HMF), mg/kg, (max.) 80 80 80
10. Total counts of pollens and plant elements/g of honey, (max.) 50,000 50,000 50,000
11. Optical Density at 660 nm, %, (max.) 0.3 0.3 0.3
1 If Fiehe’s test is positive, carry out the determination of hydroxymethyl furfural (HMF) content. If it is more
than 80 mg/kg, then fructose glucose ratio should be more than 1.00.

7. Adulteration of Honey methods
• Honey adulteration can be direct or indirect.
Direct adulteration means that a substance is added directly to
honey.
• Adulteration with corn sugar
• Adulteration with cane sugar
• Adulteration with rice syrup
• Adulteration with individual sugars
Indirect adulteration happens when honeybees are fed adulterating
substance.

8. Detection Tests

9. Adulteration of Honey with sweeteners and sugars

10. Contd..

11. Detection of sugar solution

13. Density of
fructose=1.69 g/cm³
Density of sucrose
=1.59g/cm³
Detection of sugar solution
SUGAR : At room
temperature, it is highly
water-soluble (1800
grams will dissolve in 1
liter of water).

16. Contd..

17. Detection of invert sugar
• Fiehe’s Test: Add 5 ml. Of solvent ether to 5 ml. Of honey. Shake well and
decant the ether layer in a petri dish. Evaporate completely by blowing the
ether layer. Add 2 to 3 ml. Of resorcinol (1 gm. Of resorcinol resublimed in
5 ml. Of conc. HCl.) Appearance of cherry red colour indicates presence of
sugar/jaggery.
• Hydroxy Methyl Furfural (HMF) occurs in honey due to acid-catalyzed
dehydration of hexose sugars. Its value in natural fresh honey varies from
10 to 14 mg/ kg, but it increases upon storage, depending on the pH of
honey and on the storage temperature.
• HMF content of honey also increases upon its adulteration by invert sugars.
Presence of invert sugar in honey is assessed by Fiehe's test where HMF
reacts with resorcinol and gives a red coloured complex. According to PFA
Act, Fiehe's test should be negative for honey, whereas Codex Standards
states that HMF content of honey should not be more than 60 mg/kg.

18. Adulteration using sulphite-ammonia caramel
• Dark honey is usually referred to as “forest honey” and quite often
the dye sulphite ammonia caramel (E150d) is added.
• This is mainly due to the higher sale price of dark honey.
• Determination of the content of ammonia sulphite caramel is possible
with liquid chromatography tandem mass spectrometry (LC-MS/MS).
• A level of caramel E150d above the limit of quantification methods (>
5 mg/kg) is considered an infringement of regulations (Bartošová
2013).

19. Methods of detecting indirect adulteration

20. Analysis methods
Gas Chromatography (GC) and Liquid Chromatography (LC) analysis:
• This method may be considered as a replacement of isotopic analysis, which has some
limitations.
Near Infrared Transflectance Spectroscopy (NIR):
• It is a rapid, non-destructive and relatively inexpensive method which may be suitable for
use as a screening technique in the quality control of honey.
Fourier Transform Infrared (FTIR) spectroscopy with Attenuated Total Reflectance (ATR):
• In contrast to the time-consuming carbon isotope ratio analysis techniques, these FTIR
spectroscopic procedures can be performed in very short time.
Protein characterization:
• The major proteins in honey have different molecular weights depending upon the
honeybee species. Therefore, the measurement of major proteins in honey is a useful
method to discriminate the honey that produced from different honeybee species.
High-Performance Anion-Exchange Chromatography with Pulsed Amperometric
Detection (HPAEC-PAD):
• It is an efficient tool for the characterization of the honey floral species. This method is
less time consuming and less expensive than other methods.

21. Contd..
Liquid Chromatography Coupled to Isotope Ratio Mass Spectrometry (HPLC-IRMS):
• The new procedure has advantages over existing methods in terms of analysis time, sensitivity, lack of
sample preparation, reduced consumption of reagents, and simplicity of the operative procedure. In
addition, it is the first isotopic method developed that allows beet sugar addition detection.
Calorimetric methods (Application of DSC):
• Application of DSC showed the possibility of using the glass transition temperature to distinguish
between honeys and syrups and is a powerful technique for characterizing the thermal behavior of
honeys and for detecting the effect of adulteration on physicochemical and structural properties of
samples.
Stable Carbon Isotope Ratio Analysis (SCIRA): It is determined by the isotope ratio
Fourier Transform (FT) Raman spectroscopy:
• FT-Raman spectroscopy is successfully applicable to detect beet and cane invert syrups. This method
can also be used to discriminate between the types of adulterants irrespective of its floral origin.
Microscopic detection:
• Microscopic analysis of adulterated honeys with cane sugar exhibited parenchyma cells, single ring
vessels and epidermal cells. Overall the microscopic procedure is a good screening method for the
detection of adulteration of honey with cane sugar products.

22. • Cordella et al. (2005) describe the development of high performance
anion exchange chromatography with Pulsed Amperometric
Detection (HPAEC-PAD) for the analysis of honey to detect
adulteration combined with chemometric techniques for processing
chromatograms for better discrimination of pure and adulterated
honey.
• This method was investigated using honey samples containing
between 10% and 40% of different industrial sugar syrups used for
the feeding of honey bees.
Journal Papers

23. Contd..
• Ruiz-Matute et al. (2010a) studied the sugar composition of high-fructose
corn syrup (HFCS) using gas chromatography coupled with mass
spectrometry (GC-MS). Sucrose syrups were analysed in parallel as a
control. HFCS was shown to contain fructosyl-fructose and other unknown
sugars that could be fructosyl-glucose.
• Guler et al. (2014) investigated the sensitivity of methods for the analysis
of carbonisotope ratios. They analysed a total of 100 samples of
unadulterated honey, honey made by bees fed with different amounts (5,
20 and 100 litres/colony) of sugar syrups.
• These syrups included corn syrups with high fructose-85 (HFC-85%), with
moderate fructose-55 (HFC-55%), bee-feeding syrups (BFS), glucose syrups
(GMS), and sucrose syrups (SS).

24. Health effects
Although the adulteration of honey is not injurious to health, problems of honey fraud
negatively influence market growth by damaging consumer confidence.

25. Effects
• Pesticides residues include acaricides, organic acids, insecticides, fungicides,
herbicides, and bactericides. Many of these contaminants are banned
because of their well-documented health hazards such as carcinogenic effect
on humans.
• Lactam antibiotics cause cutaneous eruptions, dermatitis, gastrointestinal
symptoms and anaphylaxis at very low doses
• Long-term effects of exposure to antibiotic residues include microbiological
hazards, carcinogenicity, reproductive effects, and teratogenicity.
Microbiological effects are one of the major health problems in human
beings. Certain drugs like nitrofurans and nitroimidazoles can cause cancer in
human being. Similarly, some drugs can produce reproductive and
teratogenic effects at very low doses.

26. Articles
• Over 150 different pesticides have been found in colony samples
• The maximum limits of pesticide residues in honey are not included in
the Codex Alimentarius.
• The European Union legislation has regulated the MRLs for three
amitraz, coumaphos, and cyamizole, which are 0.2, 0.1, and 1 mg·,
respectively.
• The US Environmental Protection Agency has established MRLs for
amitraz (1 mg·), coumaphos (0.1 mg·), and fluvalinate (0.05 mg·)

27. • In India, a study was carried out to explore the extent of pesticide residue
in honey produced in the various parts of Himachal Pradesh. It was found
that HCH and its isomers were the most frequently detected followed by
dichlor-diphenyl-trichlorethylene (DDT) and its isomers.
• Malathion’s residue was found exceeding the MRLs (5 ppb) proposed by
the Ministry of Commerce, Government of India. The results showed that
honey from natural vegetation contained lesser residues.
• In Turkey, 24 organochlorine pesticide residues in 109 different honey
samples collected from stores and open markets in Konya, Turkey, were
analyzed by gas chromatography-electron capture detection. Aldrin, cis-
chlordane, trans-chlordane, oxy-chlordane, 2,4(′)-DDE, and 4,4(′)-DDE were
found in all honey samples.
• In Spain, residues of thymol were found in honey collected from the
beehives, ranged from 0.75 to 8.20 μg·

28. News articles

32. Bibliography
• Commission Regulation (EC) No. 2377/90 of 26 June 1990 laying down a Community Procedure
for the stablemen of maximum residue limits of veterinary medicinal products in foodstuff of
animal origin (as amended by regulations) ECC No.2034/96 (OJ L272 25.10.1996, p 2), No2686/98
(OJ L337 12.12.1998, p 20) No. 1931/99 (OJ L240 10.09.1999, p 3), and No. 239/99(OJ L 290
12.11.1999, p 5).
• Food and Drug Administration of the United States, Pesticide tolerances,
2003, http://www.cfsan.fda.gov/.
• D. Beyoǧlu and G. Z. Omurtag, “Occurrence of naphthalene in honey consumed in Turkey as
determined by high-pressure liquid chromatography,” Journal of Food Protection, vol. 70, no. 7,
pp. 1735–1738, 2007. View at Google Scholar · View at Scopus
• S. Adamczyk, R. Lázaro, C. Pérez-Arquillué, P. Conchello, and A. Herrera, “Evaluation of residues of
essential oil components in honey after different anti-varroa treatments,” Journal of Agricultural
and Food Chemistry, vol. 53, no. 26, pp. 10085–10090, 2005. View at Publisher · View at Google
Scholar · View at Scopus
• Mehryar, Laleh, and Mohsen Esmaiili. "Honey and honey adulteration detection: A
Review." Proceeding 11th International Congress on Engineering and Food. Athens, Greece,
Elsevier Procedia. Vol. 3. 2011.
• Al-Waili, Noori, et al. "Antibiotic, pesticide, and microbial contaminants of honey: human health
hazards." The Scientific World Journal 2012 (2012).