Antibacterial Activity of Honey and Beebread of Different Origin Against S. aureus and S. epidermidis

Abstract

The study is aimed at the evaluation of antimicrobial properties of honey and beebread
products of different origin. The inhibitory action of 34 honey and 4 beebread samples
was tested against Staphylococcus aureus and Staphylococcus epidermidis by the agar well
diffusion method. Total antibacterial activity was evaluated by measuring the clear zone
around the well, and expressed in phenol concentration possessing equivalent activity. Honey
samples were tested after dilution to 50, 25 and 10 % (by mass per volume). The solutions
containing 10 % (by mass per volume) of honey did not have any effect on the growth of bacteria; some honey samples had no inhibitory activity on any of the concentrations used. The contribution of catalase and neutralization to the antimicrobial activity of honey was also assessed. It was found that the antibacterial activity of the tested honey samples was dependent on hydrogen peroxide formation, while such dependence was not observed for the beebread samples. Floral source of honey and bacterial culture were other two factors related to the antibacterial activity. However, the possible contribution of phytochemicals, which may be transferred to honey, should be assessed by using other methods.

Full text

ISSN 1330-9862 preliminary communication
(FTB-1533)
Antibacterial Activity of Honey and Beebread of Different
Origin Against S. aureus and S. epidermidis
Vilma Baltru{aityte
.1, Petras Rimantas Venskutonis1* and Violeta ^eksteryte
.2
1Kaunas University of Technology, Department of Food Technology, Radvilënøpl. 19,
Kaunas, LT-50254, Lithuania
2Lithuanian Institute of Agriculture, Dotnuva-Akademija, Këdainiødistrict, LT-58344, Lithuania
Received: July 18, 2005
Accepted: July 3, 2006
Summary
The study is aimed at the evaluation of antimicrobial properties of honey and bee-
bread products of different origin. The inhibitory action of 34 honey and 4 beebread sam-
ples was tested against Staphylococcus aureus and Staphylococcus epidermidis by the agar well
diffusion method. Total antibacterial activity was evaluated by measuring the clear zone
around the well, and expressed in phenol concentration possessing equivalent activity. Ho-
ney samples were tested after dilution to 50, 25 and 10 % (by mass per volume). The solu-
tions containing 10 %(by mass per volume) of honey did not have any effect on the
growth of bacteria; some honey samples had no inhibitory activity on any of the concen-
trations used. The contribution of catalase and neutralization to the antimicrobial activity
of honey was also assessed. It was found that the antibacterial activity of the tested honey
samples was dependent on hydrogen peroxide formation, while such dependence was not
observed for the beebread samples. Floral source of honey and bacterial culture were other
two factors related to the antibacterial activity. However, the possible contribution of phy-
tochemicals, which may be transferred to honey, should be assessed by using other meth-
ods.
Key words: honey, beebread, antibacterial activity, Staphylococcus aureus,Staphylococcus epi-
dermidis
Introduction
Honey is an important and unique food product
containing bioactive compounds derived from bees and
plants. Numerous studies demonstrate that honey pos-
sesses antimicrobial activity (1–7); it destroys and/or inhi-
bits the growth of some pathogenic vegetative microor-
ganisms (8). The unique composition of honey contributes
to its antimicrobial properties; however, its antibacterial
effect is not completely understood. Some researchers
believe that the main antimicrobial activity comes from
bee-origin, the others attribute antimicrobial honey pro-
perties to the components derived from flora (9). For in-
stance, Bogdanov (9) determined that antibacterial sub-
stances of honeydew honey samples are of bee-origin.
Allen et al. (1) and Molan (2,3) found that antimicrobial
activity of nectar honey and the mechanism of its action
are mostly dependent on the floral source from which
nectar honey has been collected. Mundo et al. (10)re
-
ported that honey inhibited bacteria due to a high sugar
concentration (lowering water activity), hydrogen perox-
ide, and proteinaceous compounds present in honey.
The enzymatic production of hydrogen peroxide is
considered to be a major factor of antimicrobial honey
effects (1,2,3,6,11). The amount of this bactericidal com-
pound in honey depends on the amount of catalase,
with different types of plants contributing to different
concentrations of catalase (12). Allen et al. (1) reported
201
V. BALTRU[AITYTËet al.: Antibacterial Activity of Honey and Beebread, Food Technol. Biotechnol. 45 (2) 201–208 (2007)
*Corresponding author; Phone: ++370 37 300 188; Fax: ++370 37 456 647; E-mail: rimas.venskutonis@ktu.lt

that the sensitivity of glucose oxidase to denaturation by
light could be influenced by unidentified substances pre-
sent in some floral sources (1). Therefore, the properties
of honey of multifloral origin, collected from different
types of flowers, differ from those of monofloral honey
samples.
It is thought that light, temperature, oxygen, proces-
sing, and storage may also affect the antibacterial activ-
ity of honey. Allen et al. (1) surveyed 345 samples of
New Zealand honey and found that the age does not af-
fect their antibacterial activity. Honey with high hydro-
gen peroxide activity is sensitive to heat and light (2,3,9)
because the main enzyme, which generates hydrogen per-
oxide, is inactivated.
Other factors, such as high osmotic pressure/low
water activity (aw), low pH/acidic environment, low
protein content, high carbon to nitrogen ratio, low redox
potential due to the high content of reducing sugars, vis-
cosity/anaerobic environment and other chemical agents/
phytochemicals are also likely to play some role in de-
fining antibacterial activity of honey (11).
The composition of active components in plants de-
pends on various factors, particularly plant cultivar and
chemotype and climatic conditions. Consequently, it can
be reasonably expected that honey properties from dif-
ferent locations should be different. Honey production
in Lithuania has a very long tradition tracing to ancient
times; however, its bioactive properties have not been
studied more comprehensively. The major purpose of this
work is to evaluate the antimicrobial activity of various
honey samples from Lithuania and some other honey
products. Such data would assist in more focused appli-
cation of honey as a possible natural remedy and/or
functional ingredient for the control of the most trivial
human pathogens in the food processing.
Materials and Methods
Melissopalynological analysis
The floral source of honey samples was determined
by the melissopalynological method (13,14). Determina-
tion of botanical origin of honey is based on the relative
frequency of the pollen from nectar secretion plants. Dif-
ferent opinions exist regarding the use of pollen present
in honey for the indication of its botanical origin (15),
however until the present date, this method has been
frequently used for this purpose. Pollen was identified
by using previously published data (16,17) and pollen
collection of well-known plants, which was prepared for
microscoping at the Apicultural Department of the Lith-
uanian Institute of Agriculture.
The prepared slides were examined using a micro-
scope with magnification of 400 for the identification of
pollen in honey and counting honeydew elements. At
least 500 pollen grains (PG) and honeydew elements
(HDE) were counted in 100 fields. All plant elements
were observed separately.
After the identification of PG and HDE in honey
samples, the pollen of nectarless plants and the HDE
were deducted from the total sum. The percentage of
pollen from nectar plants in botanical composition of
honey was calculated. HDE were calculated as percent-
age from total sum of PG and HDE. Unifloral spring ho-
ney from willow (K02, K03, K04, K05), polyfloral spring
honey (K01, K06, K24), summer honey from spring rape
(K07–K23) and polyfloral summer honey K25 were se-
lected for analysis. Unifloral honey met the botanical and
chemical composition requirements established by the In-
ternational Commission for Bee Botany, presently called
International Commission for Plant-Bee Relationships (13)
and recommended methods and standards (18–20). It
should be noted that the main honey plant in Lithuania
is spring rape (Brassica napus L. ssp. oleifera annua Metzg.)
and the pollen in honey from this plant is over-repre-
sented.
Botanical origin of samples is listed in Table 1.
Honey samples and their preparation
Honey samples were obtained from apiarists through-
out Lithuania. All samples were collected during the 2003
flowering season. Some honey samples were made when
bees were fed with pine (Pinus silvestris), birch (Betula
pendula) and stinging nettle (Urtica dioica) extract addi-
tives (the detailed botanical composition was not test-
ed). The samples of beebread were also tested. Beebread
is the mixture of pollen with honey to stick it together.
The commercial samples of beebread were used during
the analysis; detailed composition of beebread samples
was unknown. The only commercially available sample
of thermally processed beebread was also tested. The
sources and detailed characterization of honey samples
are listed in Table 1.
All samples were prepared aseptically and were
handled protected from direct sunlight. Honey solutions
were prepared in three fractions: 50, 25 and 10 % (by
mass per volume). The samples of each honey (10 g)
and sterile water were stored at 37 °C for 30 min before
mixing, to facilitate homogenization. The 50 % (by mass
per volume) solutions thus prepared were diluted to 25
and 10 %. The samples were assayed immediately after
dilution.
Beebread is not homogenous mixture of honey and
pollen, because pollen is not soluble in honey and water.
Therefore, the suspensions of 50, 25 and 10 % (by mass
per volume), which were produced by diluting the bee-
bread with distilled water, were tested for the antibacte-
rial activity.
Seventy five percent of all honey samples were neu-
tralized with 0.1 M NaOH to pH=(7.0±0.1) (measured with
potentiometer) and were diluted to 50 % (if it was nec-
essary).
Chemicals
The concentration of 2 mg/mL of catalase (2000
units/mg, Sigma-Aldrich, Steinheim, Switzerland) from
bovine liver in pure distilled water was freshly prepared
every day. Honey samples were diluted to 50 and 25 %
with this catalase solution and assayed in the same way
as other honey samples. The final concentration of cata-
lase corresponded to 0.1 and 0.15 %, respectively.
Reference solutions of phenol (99 %, Novomoskov-
skij zavod, Moscow, Russia) of 8, 10, 12, 14, 16, 18 and
20 % (by mass per volume) were prepared in purified
water. The antibacterial activity of each solution was then
tested five times by adding 100 mL of phenol solution to
the well.
202 V. BALTRU[AITYTËet al.: Antibacterial Activity of Honey and Beebread, Food Technol. Biotechnol. 45 (2) 201–208 (2007)

203
V. BALTRU[AITYTËet al.: Antibacterial Activity of Honey and Beebread, Food Technol. Biotechnol. 45 (2) 201–208 (2007)
Table 1. Characterization of the tested honey samples and beebread
Sample
code
Collection
dates Botanical composition/% Municipality Location
K01 03. 06. 2003
fruit tree 35.6, willow (Salix alba L., Salix caprea L.) 35.5, spring rape (Bras-
sica napus L. ssp. oleifera annua Metzg.) 18.8, dandelion (Taraxacum officinale
L.) 6.7, chestnut (Aesculus hippocastanum L.) 3.4
Kedainiai Akademija
K02 09. 06. 2003 willow (Salix alba L., Salix caprea L.) 76.1, fruit tree 10.3, mustard (Sinapis
arvensis L.) 7.1, dandelion (Taraxacum officinale L.) 3.7, alder (Frangula L.) 2.8 Kedainiai Krakes,
Janiunai
K03 09. 06. 2003 willow (Salix alba L., Salix caprea L.) 68.9, fruit tree 24.6, dandelion
(Taraxacum officinale L.) 6.5 Kedainiai Uzupe,
Lazai
K04 08. 06. 2003 willow (Salix alba L., Salix caprea L.) 67.4, fruit tree 21.7, dandelion
(Taraxacum officinale L.) 10.9 Kedainiai Voluciai
K05 14. 06. 2003 willow (Salix alba L., Salix caprea L.) 55.3, fruit tree 28.3, wild mustard
(Sinapis alba) 8.0, dandelion (Taraxacum officinale) 8.4 Kedainiai Medininkai
and Spitole
K06 13. 06. 2003
fruit tree 33.3, willow (Salix alba L., Salix caprea L.) 32.5, dandelion
(Taraxacum officinale L.) 24.4, spring rape (Brassica napus L. ssp. oleifera
annua Metzg.) 4.9, hawthorn (Crataegus L.) 4.9
Kedainiai Degesiai
K07 30. 06. 2003 spring rape (Brassica napus L.ssp. oleifera annua Metzg.) 96.8, other plants*
3.2, honeydew 3.1 Kedainiai Voluciai
K08 07. 07. 2003 spring rape (Brassica napus L.ssp. oleifera annua Metzg.) 96.6, other plants*
3.4, honeydew 3.3 Kedainiai Gudziunai
K09 09. 07. 2003 spring rape (Brassica napus L.ssp. oleifera annua Metzg.) 92.0, fruit tree 3.5,
willow (Salix alba L., Salix caprea L.) 2.5, dandelion (Taraxacum officinale) 2.0 Kedainiai Degesiai
K10 22. 07. 2003
spring rape (Brassica napus L.ssp. oleifera annua Metzg.) 48.5, willow (Salix
alba L., Salix caprea L.) 30.0, cornflower (Centaurea cyanus L.) 7.2, red clover
(Trifolium pratense L.) 7.0, wild mustard (Sinapis alba L.) 5.3, bird’s foot tre-
foil (Lotus corniculatus L.) 2.0
Kedainiai Paberze
K11 24. 07. 2003
spring rape (Brassica napus L.ssp. oleifera annua Metzg.) 45.9, red clover
(Trifolium pratense L.) 24.7, fruit tree 8.8, white clover (Trifolium repens L.)
6.5, willow (Salix alba L., Salix caprea L.) 5.3, buckwheat (Fagopyrum esculentum
Moench) 4.7, cornflower (Centaurea cyanus L.) 4.1, honeydew 27.0
Kedainiai Daumantai
K12 28. 07. 2003
spring rape (Brassica napus L.ssp. oleifera annua Metzg.) 62.5, wild mustard
(Sinapis alba L.) 17.5, cornflower (Centaurea cyanus L.) 7.6, willow (Salix alba
L., Salix caprea L.) 6.8, dandelion (Taraxacum officinale L.) 3.6, fruit tree 2.0
Kedainiai Medininkai
K13 29. 07. 2003
spring rape (Brassica napus L. ssp. oleifera annua Metzg.) 77.5, thistle
(Cirsium L.) 8.5, willow (Salix alba L., Salix caprea L.) 5.4, dandelion
(Taraxacum officinale L.) 2.7, linden (Tilia cordata L.) 3.2, fruit tree 2.7
Kedainiai Spitole
K14 30. 07. 2003
spring rape (Brassica napus L.ssp. oleifera annua Metzg.) 63.7, willow (Salix
alba L., Salix caprea L.) 8.8, cornflower (Centaurea cyanus L.) 7.6, wild mus-
tard (Sinapis alba L.) 7.2, caraway (Carum carvi. L.) 5.0, fruit tree 4.8, linden
(Tilia L.) 2.9
Kedainiai Uzupe
K15 07. 2003
spring rape (Brassica napus L.ssp. oleifera annua Metzg.) 84.7, wild mustard
(Sinapis alba L.) 10.5, cornflower (Centaurea cyanus L.) 3.5, other plants* 1.3,
honeydew 2.7
Marijampole unknown
K16 25. 07. 2003 spring rape (Brassica napus L.ssp. oleifera annua Metzg.) 89.2, linden (Tilia
cordata L.) 7.7, beans (Vicia faba L.) 1.7, other plants* 1.4 Kedainiai Slapaberze
K17 05. 08. 2003 spring rape (Brassica napus L.ssp. oleifera annua Metzg.) 88.0, wild mustard
(Sinapis alba L.) 6.9, cornflower (Centaurea cyanus L.) 2.2, other plants* 2.9 Kedainiai Degesiai
K18 06. 08. 2003
rape (Brassica napus L.ssp. oleifera annua Metzg.) 54.8, wild mustard
(Sinapis alba L.) 24.2, cornflower (Centaurea cyanus L.) 6.7, willow (Salix alba
L., Salix caprea L.) 4.0, raspberry (Rubus idaeus L.) 4.0, fruit tree 3.2, other
plants* 3.1
Kedainiai Krakes,
Janiunai
K19 07. 08. 2003
spring rape (Brassica napus L.ssp. oleifera annua Metzg.) 71.5, wild mustard
(Sinapis alba L.) 14.7, willow (Salix alba L., Salix caprea L.) and fruit tree 4.1,
cornflower 2.6, (Centaurea cyanus L.) 2.6, other plants* 0.4
Kedainiai Lazai
K20 22. 07. 2003
spring rape (Brassica napus L.ssp. oleifera annua Metzg.) 78.5, willow (Salix
alba L., Salix caprea L.) 6.2, fruit tree 6.2, red clover (Trifolium pratense L.)
3.8, cornflower (Centaurea cyanus L.) 2.4, wild mustard (Sinapis alba L.) 2.9
Kedainiai Terespolis
K21 08. 08. 2003
spring rape (Brassica napus L.ssp. oleifera annua Metzg.) 74.8, fruit tree 6.9,
wild mustard (Sinapis alba L.) 5.7, raspberry (Rubus idaeus L.) 4.0, caraway
(Carum carvi L.) 3.3, willow (Salix alba L., Salix caprea L.) 3.0, other plants* 2.3
Kedainiai Siaudyne

Assessment of antibacterial activity
Antibacterial activity of honey samples and reference
solutions was tested by an agar diffusion test (1). Two
undesirable species in food products, namely S. aureus
and S. epidermidis, were used as test cultures. Bacteria
were grown in nutrient broth (Merck KGaA, Darmstadt,
Germany) at 37 °C for 24 h. After cultivation 100 mLof
the culture were poured into 150 mL of sterilized nutri-
ent agar (Oxoid, CM3, Merck KGaA, Darmstadt, Germa-
ny) cooled to 50 °C. The agar was mixed and poured in
four 90-mm diameter Petri dishes immediately after mix-
ing and stored at 4–6 °C overnight before being used the
next day.
Five wells of 6 mm in diameter were punched in the
solid agar with the mouthpiece of a sterile Pasteur pi-
pette (five in each plate). The bottom of each well was
covered with a drop of melted nutrient agar in order to
prevent infiltration of the sample between the culture me-
dium and the bottom of the plate, which would interfere
with the test. The wells were numbered at random.
204 V. BALTRU[AITYTËet al.: Antibacterial Activity of Honey and Beebread, Food Technol. Biotechnol. 45 (2) 201–208 (2007)
Sample
code
Collection
dates Botanical composition/% Municipality Location
K22 12. 08. 2003
spring rape (Brassica napus L.ssp. oleifera annua Metzg.) 73.4, cornflower
(Centaurea cyanus L.) 7.3, red clover (Trifolium pratense L.) 6.5, willow (Salix
alba L., Salix caprea L.) 5.6, wild mustard (Sinapis alba L.) 6.2, fruit tree 1.0 Kedainiai Gudziunai
K23 20. 08. 2003
spring rape (Brassica napus L.ssp. oleifera annua Metzg.) 71.4, wild mustard
(Sinapis alba L.) 13.9, willow (Salix alba L., Salix caprea L.) 9.5, dandelion
(Taraxacum officinale L.) 5.2 Kedainiai Medininkai
K24 26. 08. 2003
willow (Salix alba L., Salix caprea L.) 38.5, fruit tree 13.6, raspberry (Rubus
idaeus L.) 12.4, dandelion (Taraxacum officinale L.) 11.2, chestnut (Aesculus
hippocastanum L.) 10.7, spring rape (Brassica napus L.ssp. oleifera annua
Metzg.) 7.7, honeydew 6.1, thistle (Cirsium arvense Scop.) 5.9
Neringa Pervalka
K25 24. 07. 2003
spring rape (Brassica napus L.ssp. oleifera annua Metzg.) 42.5, white
clover (Trifolium repens L.) 18.1, bird’s foot trefoil (Lotus corniculatus L.)
16.6, honeydew 9.4, linden (Tilia cordata L.) 9.8, raspberry (Rubus idaeus L.)
8.0, willow (Salix alba L., Salix caprea L.) 3.0, beans (Vicia faba L.) 2.0
Taurage Pasesuvys
E26 07. 2003
honey with pine (Pinus silvestris) extract: thistle (Cirsium arvense Scop.)
37.8, spring rape (Brassica napus L.ssp. oleifera annua Metzg.) 40.6, honey-
dew 42.2, raspberry (Rubus idaeus L.) 21.6 unknown unknown
E27 07. 2003
honey with birch (Betula pendula) extract: thistle (Cirsium arvense Scop.)
29.4, spring rape (Brassica napus L.ssp. oleifera annua Metzg.) 41.2, honey-
dew 34.6, raspberry (Rubus idaeus L.) 20.6, wild mustard (Sinapis alba L.) 8.8 unknown unknown
E28 07. 2003
honey with stinging nettle (Urtica dioica) extract: thistle (Cirsium arvense
Scop.) 32.0, spring rape (Brassica napus L.ssp. oleifera annua Metzg.) 24.0,
honeydew 32.0, raspberry (Rubus idaeus L.) 20.0, white clover (Trifolium
repens L.) 12.0, wild mustard (Sinapis alba L.) 12.0
unknown unknown
C29 08. 2003 unknown (honey from local apiarist) Vilkaviskis Svitrunai
C30 07. 2003 unknown (grassland and forest honey, EKO agros, R. Maciene, Lithuania) unknown commercial
sample
C31 08. 2003 unknown (grassland and forest honey, EKO agros, R. Maciene, Lithuania) unknown commercial
sample
C32 07. 2003 unknown (grassland and forest honey, EKO agros, R. Maciene, Lithuania) unknown commercial
sample
C33 05. 2003 unknown (grassland and forest honey, EKO agros, R. Maciene, Lithuania) unknown commercial
sample
C34 07. 2003 unknown (grassland and forest honey, EKO agros, R. Maciene, Lithuania) unknown commercial
sample
C35 2003 unknown (beebread after thermal processing, EKO agros, R. Maciene,
Lithuania) Pakruojis commercial
sample
C36 17. 09. 2003 unknown (beebread with honey and honeycomb, EKO agros, R. Maciene,
Rockaiciai, Lithuania) Pakruojis commercial
sample
C37 09. 2003 unknown (beebread with honey and honeycomb, EKO agros, R. Maciene,
Rockaiciai, Lithuania) Pakruojis commercial
sample
C38 17. 09. 2003 unknown (beebread with honey and honeycomb, EKO agros, R. Maciene,
Isdagieciai, Lithuania) Pakruojis commercial
sample
*percentage of pollen in the honey was less than 1.0 %
Table 1. – continued

Each sample was tested in quadruplicate by adding
100 mL of the test solution to four wells. Total activity,
non-peroxide activity and the activity of neutralized ho-
ney were evaluated. The blank sample containing sterile
distilled water was tested in the same way as the honey
samples.
The plates were incubated overnight at 37 °C and
afterwards were placed over the black template to mea-
sure the clear zone of inhibition diameter (mm). The di-
ameter was measured along the horizontal and vertical
lines on the template. The mean radius of the clear zone
around each honey sample was calculated (diameter of
the well was 6 mm) and measured in mm.
The mean radius of the clear zone around each well
of phenol reference solution was measured simultaneou-
sly and a graph of phenol concentration against the mean
radius of the clear zone was plotted. After linear appro-
ximation of the obtained curve, the equation was dedu-
ced; this was used to determine the activity of each ho-
ney sample from the mean radius around the clear zone.
The activity was expressed as the equivalent phenol frac-
tion (in %). Calibration curves were prepared for each
culture.
It was found that the relationship between antimi-
crobial agent concentration and clear zone radius is lin-
ear over the full range tested. Most likely, the calibration
curves gave straight lines because small wells in the agar
were used; thus the antibacterial substances in them be-
came easily depleted as diffusion into the surrounding
agar occurred. Calibration curves and the equation ob-
tained with phenol solutions are provided in Fig. 1.
Statistical analysis
All values are expressed as the mean±standard devi-
ation. Standard deviations were calculated using spread-
sheet software (Excel®). Correlation coefficients to deter-
mine the relationship between antimicrobial activity and
the amount of one plant were calculated using MS Ex-
cel®software (CORREL statistical function).
Results
In the first series of experiments honey samples were
tested after their dilution to 50, 25 and 10 % (by mass
per volume). The results revealed remarkable variations
in antibacterial activity of the tested honey samples. How-
ever, some of the 38 tested honey samples at the frac-
tions applied did not have any antimicrobial activity
against the tested microorganisms (Table 2); for instance,
15 samples did not inhibit S. aureus and 11 samples had
no effect against S. epidermidis. Five honey samples lost
their inhibitory activity against both tested bacteria after
dilution to 25 %.
The results of the assay of the total antimicrobial ac-
tivity of untreated honey and beebread are summarized
in Table 3. No values are shown for 10 % (by mass per
205
V. BALTRU[AITYTËet al.: Antibacterial Activity of Honey and Beebread, Food Technol. Biotechnol. 45 (2) 201–208 (2007)
y = 0.3942x + 0.4384
R=0.98
2
0
2
4
6
8
10
12
14
8 10121416182022
Phenol concentration/%
Inhibition radius/mm
y = 0.6683x + 0.058
R=0.98
2
0
2
4
6
8
10
12
14
Inhibition radius/mm
B
A
8 10121416182022
Phenol concentration/
%
Fig. 1. Calibration curves for the phenol reference solutions used
in the agar well diffusion assay of antibacterial activity (the value
of each point is the mean of five determinations); A – inhibition
of S. aureus, B – inhibition of S. epidermidis
Table 2. Number of samples that inhibited the growth of the tested bacteria at 50 and 25 % (by mass per volume) solutions
Plant source
Total
no. of
samples
Total antibacterial activity/% Non-peroxide
activity
After
neutralization/%
S. aureus S. epidermidis Both tested
bacteria
S. aureus S. epidermidis
50 25 50 25 50 50
spring rape (more than 45 %) 17 14 11 16 13 0 16 17
willow (more than 45 %) 4 2 1 4 3 0 4 4
multifloral 10 3232 0 6 4
honey with birch extract 1 0000 0 0 0
honey with pine extract 1 1010 0 1 1
honey with stinging nettle extract 1 0000 0 1 1
beebread 4 4444 4 4 4

volume) solutions since both tested bacteria were resis-
tant to all honey and beebread samples at this fraction.
The inhibition effect considerably decreased when honey
samples were diluted from 50 to 25 % (by mass per vol-
ume). The inhibition activity of 50 % honey and bee-
bread solutions was higher than that of 25 % solutions.
The antimicrobial activity of 50 and 25 % honey solu-
tions was equivalent to the inhibitory effect of phenol
solutions of 1.73–6.97 and 0.59–4.44 % fractions, respec-
tively. The solutions of beebread were active in both
tested cultures at 50 and 25 % fractions. In general, the
samples of beebread showed stronger inhibition of the
growth of bacteria than honey solutions (Table 3).
Non-peroxide antimicrobial activity was evaluated by
the addition of catalase to the honey and beebread solu-
tions. Phenol equivalent coefficients for beebread sam-
ples after the addition of catalase varied from (2.20±0.65)
to (5.78±0.70) % (by mass per volume, N=4) and were
only slightly lower compared with the same coefficients
of the untreated beebread samples (Table 3).
The antibacterial activity of neutralized honey was
equivalent up to (12.13±0.70) % (mass per volume, N=4)
phenol solution (C29, undefined honey), while the high-
est activity of untreated honey was equivalent to the phe-
nol solution of (6.97±0.80) % (by mass per volume, N=4)
(K21, rape honey).
The antimicrobial effect of honey samples against S.
aureus and S. epidermidis was different, indicating that the
sensitivity of these bacteria to the antimicrobial activity
of honey is different. The correlation coefficients between
the amount of rape and willows in honey and their anti-
bacterial activity against S. aureus were 0.08 and 0.14, re-
spectively. It indicates that there was no correlation be-
tween these two parameters. The correlation coefficients
between equivalent concentration of phenol solution of an-
tibacterial activity against S. epidermidis and the amount of
rape and willows in honey were –0.08 and –0.435.
Discussion
Total antimicrobial activity of untreated honey and
beebread
Literature sources indicate that antibacterial activity
of honey considerably depends on the floral source (1);
consequently the honeys could be distinguished by their
predominant plant composition. It is interesting to note
that botanical composition of some honeys (e.g. K02 and
K03, K07 and K08) was quite similar (Table 1), however
their antimicrobial activity was different. For instance,
the sample K02 did not inhibit the growth of S. aureus,
while the sample K08 was not active against both cul-
tures. It is worth noting that the colour of these honey
samples was different; K02 and K07 honey samples were
darker than K03 and K08. This suggests that botanical
source of honey might be less important for their anti-
bacterial activity; consequently, something else, most likely
bee-origin metabolism products, defines antimicrobial pro-
perties of honey. Possible deviations in the identification
of the floral source of these honey samples should also
be considered (15). It was well established that the hy-
drogen peroxide activity in honey correlates with floral
sources (3).
Only three of the ten tested multifloral honey sam-
ples (K24, K25 and C29) inhibited the growth of S. au-
reus and S. epidermidis. It is interesting that commercially
available honey samples did not exhibit antibacterial pro-
206 V. BALTRU[AITYTËet al.: Antibacterial Activity of Honey and Beebread, Food Technol. Biotechnol. 45 (2) 201–208 (2007)
Table 3. Antibacterial activity of 50 and 25 % natural and 50 % neutralized honey solutions (only the samples of each type with the
highest activity, as well as medium activity of each group are shown)
Plant source
Maximum total activity/% phenol Maximum activity after
neutralization/% phenol
S. aureus S. epidermidis S. aureus S. epidermidis
50 25 50 25 50 50
spring rape (more than 45 %) 6.97±0.80 4.28±0.26 4.12±0.58 3.14±0.71 11.18±0.95 7.16±0.56
willow (more than 45 %) 3.96±0.45 2.14±0.48 3.28±0.79 1.88±0.58 9.83±1.20 6.37±0.83
multifloral 6.97±0.66 3.80±0.80 5.62±0.62 3.70±0.58 12.13±0.70 6.93±0.32
honey with birch extract –––– ––
honey with pine extract 1.11±0.86 –2.67±0.28 –2.30±0.48 3.09±0.15
honey with nettle extract –––– 4.76±0.83 2.81±0.39
bee bread 6.02±0.61 4.44±0.61 4.31±0.62 3.42±0.41 8.64±0.48 4.50±0.76
Medium total activity/% phenol Medium activity after
neutralization/% phenol
spring rape (more than 45 %) 4.76 ±0.62 3.08±0.58 2.83±0.49 1.89±0.51 5.90±0.68 4.05±0.64
willow (more than 45 %) 3.68±0.42 2.14±0.48 2.89±0.60 1.74±0.52 6.44±1.35 3.96±0.77
multifloral 4.12±0.64 2.49±0.75 3.70±0.59 2.62±0.55 7.44±0.71 4.59±0.36
honey with birch extract –––– ––
honey with pine extract 1.11±0.86 –2.67±0.28 –2.30±0.48 3.09±0.15
honey with nettle extract –––– 4.76±0.83 2.81±0.39
bee bread 5.57±0.66 3.57±0.67 3.98±0.52 2.51±0.65 7.55±0.61 3.79±0.63
The values represent average±standard deviation of four replicates

perties. Most likely, processing and/or storage conditions
had negative influence on the antimicrobial properties
of these honey samples. It is known that honey for sale
can be heated from 45 to 80 °C. Therefore, the loss of an-
tibacterial activity in heat processed commercial honey
samples could be accounted for denaturation of glucose
oxidase.
Non-peroxide antimicrobial activity
Non-peroxide activity can be detected by adding ca-
talase to honey solution. Catalase destroys hydrogen per-
oxide, which is slowly formed in honey as a result of
the glucose oxidase action. All tested samples, except
beebread, lost their antibacterial properties after the ad-
dition of catalase (compared with total antibacterial ac-
tivity), indicating that antibacterial activity of the tested
honey samples was due to the hydrogen peroxide for-
mation, while non-peroxide activity can be attributed to
the beebread samples. According to the previously pub-
lished data, in some honey samples from New Zealand,
other than peroxide-based mechanisms were prevalent
in honey antibacterial properties (1,6).
Effect of pH on antibacterial activity of honey
and beebread
The effect of pH on the antimicrobial activity of ho-
ney and beebread samples was assessed by neutralizing
the products with 0.1 M NaOH. The pH of the honey
solutions under study (50 % mass per volume fraction)
was from 3.9 to 4.8; some literature sources indicate that
the average pH value for honey is 3.9 (8,12). It should
be noted that the pH of the samples increased slightly
upon dilution. Dilution may have a complex effect on
honey, since it may increase glucose oxidase activity,
leading to the formation of H2O2and gluconic acid as
well as diluting the other organic acids present in honey.
Neutralization of honey and beebread samples was aimed
at establishing possible effect of low pH of Lithuanian
honey samples on their antibacterial properties. For in-
stance, it was reported that the pH of S. aureus growth
varies from 4.0 to 9.8 (21); consequently pH of the neu-
tralized honey should be favourable for bacterial growth.
It is probable that neutralization of honey will affect the
activity of glucose oxidase, since the optimal pH for this
enzyme is in the range of 5.0−8.0. Therefore, neutraliza-
tion of honey to pH=7.0 should promote the activity of
glucose oxidase, resulting in the increase of hydrogen
peroxide production. Most likely, it is the main factor
explaining the fact that antibacterial activity of the tested
neutralized honey samples was higher by 1.5−2.5 times
as compared to honey total activity before neutralization.
Furthermore, after their neutralization eight samples be-
came active against S. aureus and three against S. epider-
midis. Published data on the activity of the neutralized
honey is rather scarce. Snow and Manley-Harris (22) in-
vestigated the stability of antibacterial activity of honey
to alkaline solutions. They found that at pH=11 antibac-
terial activity of manuka (Leptospermum scoparium)ho
-
ney was irreversibly lost. In our case, neutralized honey
acquired higher antibacterial activity.
The results obtained suggest that stronger antibacte-
rial activity of neutralized Lithuanian honey samples is
due to the hydrogen peroxide formation by glucose oxi-
dase. The increase of enzyme activity in the neutral media
promotes the production of hydrogen peroxide, which in-
hibits the growth of bacteria. This assumption is sup-
ported by the fact that the results obtained are in agree-
ment with those obtained after the addition of catalase.
Thermally processed beebread (C35) retained uniform
activity after catalase addition and product neutraliza-
tion. This indicates that inhibition properties of bee-
bread samples did not depend on hydrogen peroxide;
most likely the activity of thermally processed beebread
was due to the presence of other, thermally stable com-
pounds. Neutralization had less significant effect on the
increase of antibacterial properties of other beebread so-
lutions (with honey and honeycomb) as compared to ho-
ney solutions. It was reported that formation of hydro-
gen peroxide in beebread was lower than in rape, clover
and heath origin honey samples by 3.20, 3.17 and 2.10
times, respectively (23).
Conclusions
Assessment of antimicrobial activity of different Li-
thuanian honey samples and beebread against S. epider-
midis and S. aureus showed that inhibitory effects are not
inherent to all the selected honey samples; to achieve the
inhibition of bacterial growth, the concentration of ho-
ney should be sufficiently high, usually higher than 25
% (by mass per volume). S. epidermidis was more resis-
tant to the antimicrobial effects of honey than S. aureus.
The results obtained after product neutralization and its
treatment with catalase revealed that antibacterial activ-
ity of the tested honey samples is mainly due to the en-
zymatic formation of hydrogen peroxide. However, the
samples of beebread possessed residual non-peroxide
activity as well. The study indicates that the use of ho-
ney and beebread in food formulations can help to con-
trol some food pathogens, however, further investigations
are needed to establish possible effects of honey origin
on the inhibitory properties against food microorganisms.
Acknowledgements
This research was supported by Lithuanian State
Foundation of Science and Studies.
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