Laboratory of Apidology and Apitherapy

Institute of Molecular Biology SAS

Dubravská cesta 21
845 51 Bratislava

Analysis of antibacterial efficacy of honey samples

Name of customer: Hannes Oberreiter
Address of customer: Reisstrasse 17, Weisskirhen
Year of harvesting honey: 2020
Honey samples:
Sample no. Type of honey
1401 Dandelion + spring mix
1401-1 Linden + raspberry
1701 Honeydew + spring mix
Forest honey Melezitose honey - extracted

Standardized honey (the base for medical grade honey): Manuka honey ORA UMF 15+
(Whakaari International, Whakatane, New Zealand)
Control honey solution: artificial honey (sugar solution where sugars are at same
concentrations like in natural honey to maintain identical osmotic properties)
Laboratory analysis performed at: Laboratory of Apidology and Apitherapy, Institute of
Molecular Biology, Slovak Academy of Sciences, Bratislava, Slovakia
Measured parameters and activities: a total antibacterial activity, overall protein profile of
honey

RESULTS OF ANALYSIS AND DISCUSSION

Antibacterial activity of honey samples:

Antibacterial activity of honey is depended on several different factors: high osmotic

pressure (high sugar content), low pH (due to gluconic acid formation), the presence of
antibacterial peptide defensin-1, production of hydrogen peroxide (generated by enzymatic
reaction in the presence of glucose oxidase enzyme), the presence of various phytochemicals
and polyphenols. Antibacterial activity of some specific honeys such as manuka and honeydew
honey has different mechanisms of action due to the presence of plant-derived components.
Such as manuka honey where methylglyoxal (MGO) plays a crucial role in its antibacterial
activity.
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 Laboratory of Apidology and Apitherapy
Institute of Molecular Biology SAS
Dubravská cesta 21
845 51 Bratislava

Honey antibacterial activity is expressed as a minimal inhibitory concentration (MIC), i.e. the
percentage of honey solution that is able to inhibit the bacterial growth. The lower the solution
percentage, the higher is the antibacterial activity of honey.

Results:

Figure 1. Antibacterial activity of honey samples determined by a minimal inhibitory concentration (MIC) assay
against two bacterial strain Staphylococcus aureus and Pseudomonas aeruginosa.

MIC analysis revealed that tested honey samples were more/equally effective against
Staphylococcus aureus in compared to Pseudomonas aeruginosa except sample Forest honey.
Sample Forest honey was melezitose honey which is a part of honeydew and due to hard
extraction honey was heated up and decreased antibacterial activity against both
Staphylococcus aureus and Pseudomonas aeruginosa was observed. Moreover, Pseudomonas
aeruginosa is more sensitive to high sugar concentration, so in the case of Forest honey
sample sugar concentration was the major antibacterial component. The antibacterial activity
of honey can vary adversely depending on various factors such as e.g. heating with high
temperature (more than 45°C). Components responsible for antibacterial activity are sensitive
to higher temperatures, therefore the antibacterial activity of heated honey is on level of
artificial honey (sugar solution).
Samples 1401, 1401-1 and 1701 were effective against both bacterial isolates with
high antibacterial activity but were less potent in compare to manuka honey where bacterial

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 Laboratory of Apidology and Apitherapy
Institute of Molecular Biology SAS
Dubravská cesta 21
845 51 Bratislava

growth inhibition was observed at concentration of 8% against Staphylococcus aureus and

14% against Pseudomonas aeruginosa. Honey samples 1401 and 1401-1 were from the same
hive but from different honeycombs. MIC values against Staphylococcus aureus were 12%,
16% and 14%, 16% against Pseudomonas aeruginosa. The differences may be caused by the
presence of different honey types in the same hive. Samples 1701 has even antibacterial
activity against both pathogens 12%. All measurements were performed in two biological
replicates.
In a case of artificial honey, only high sugar concentration is able to inhibit bacterial
growth. When the MIC values for honey sample are close to MIC values for artificial honey,
antibacterial activity of honey samples will depend mainly on sugar content. It happens when
honey was heated or prolonged storage.

Protein profile of tested honeys

M A1 A2 A3 1401 1401-1 1701 Forest honey

MRJP 1

Figure 2. Protein profile of various honey samples. A1-A3 – represent Austrian honey samples from other
beekeepers collected in 2020, samples 1401, 1401-1, 1701 are tested honey samples. M – Protein marker (a
mixture of pre-stained proteins with exact molecular weight). Red arrow represents the position of dominant
protein (MRJP1) in honey.

The overall protein profile of honeys was determined. According to the intensity of
particular bands it is possible to see whether the sample was diluted or not.
Honey samples 1401, 1401-1 and 1701 showed intense protein bands. Major royal jelly

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 Laboratory of Apidology and Apitherapy
Institute of Molecular Biology SAS
Dubravská cesta 21
845 51 Bratislava

protein (MRJP1) is the most dominant honey protein which is secreted by the bees from
hypopharyngeal glands during honey processing. Protein profile of Forest honey sample
combined with antibacterial activity shows that honey went through unappropriate processing
where its antibacterial activity was partially lost and the amount of bee proteins was decreased.

Conclusions

Obtained results from analysis of honey samples (1401, 1401-1, 1701 and Forest
honey) suggest that honey samples exhibited a high antibacterial activity against two wound
pathogens, Staphylococcus aureus and Pseudomonas aeruginosa except Forest honey.
Antibacterial activity of samples 1401, 1401-1, 1701 was less potent than activity of manuka
honey which represents the most expensive honey in the market, but still exhibited high
antibacterial potential.

In Bratislava, 27.08.2020 Ing. Juraj Majtan, DSc.

Head of Laboratory of Apidology and Apitherapy
Institute of Molecular Biology SAS

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