Bee bread - Perspective source of bioactive compounds for future

Abstract

Bee bread is product with long history used mainly in folk medicine. Nowadays, bee bread is growing in commercial interest due to its high nutritional properties. The objective of this study was to determine biological activity of ethanolic extract of bee bread obtained from selected region of Ukraine-Poltava oblast, Kirovohrad oblast, Vinnica oblast, Kyiv oblast, Dnepropetrovsk oblast. The antioxidant activity was measured with the radical scavenging assays using 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical as well as phosphomolybdenum assay. Total polyphenol content was determined with Folin-Ciocalteau reagent and total flavonoid content by aluminium-chloride method. Secondary was also evaluated antimicrobial activity in bee bread samples with disc diffusion method and minimum inhibitory concentrations. Antioxidant activity expressed as mg TEAC per g of dry weight (Trolox equivalent antioxidant capacity) was the highest in bee bread from Poltava oblast in DPPH and also phosphomolybdenum method. Samples of bee bread contained high levels of total polyphenols (12.36 - 18.24 mg GAE - gallic acid equivalent per g of dry weight) and flavonoids (13.56 - 18.24 μg QE - quercetin equivalent per g of dry weight) with the best values of bee bread from Poltava oblast. An elevated level of antioxidant potential in the bee bread determines its biological properties, which conditioned of the biological active substances. The best antibacterial activity of bee bred with disc diffusion method was found against Bacillus thuringiensis CCM 19. The antibacterial activity inhibited by the bee bread extract in the present study indicate that best minimal inhibition concentration was against bacteria Escherichia coli CCM 3988 and Salmonella enterica subs. enterica CCM 3807.

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Potravinarstvo® Scientific Journal for Food Industry
Volume 9 592 No. 1/2015
INTRODUCTION
Bee bread is a product of the hive obtained from pollen
collected by bees, to which they added honey and digestive
enzymes and subsequently stored in the combs, starting a
lactic fermentation which gives it greater power
conservation (Zuluaga et al., 2015). This type of lactic
acid fermentation is similar to that in yoghurts (and other
fermented milk products) and renders the end product
more digestible and enriched with new nutrients (Krell,
1996). The process of bee bread formation starts with
gathering of pollen, then a bee mixes it with flower nectar
or honey and saliva, and carries to the beehive, where non
flying bees fill the mixture into honeycomb cells for ¾ of
the cell volume. Residual cell volume is filled with honey,
thus protecting the pollen mass from oxygen. An anaerobic
lactic fermentation process takes place and bee bread is
forming. Bee bread differs from pollen by lower pH
(3.8 – 4.3), it contains less proteins and fats, but more
carbohydrates and lactic acid. Bee bread has a better
bioavailability because the walls of pollen, which cannot
be destructed by gastrointestinal liquids, have been partly
destructed by fermentation and the functionally and
energetically rich content of pollen can be assimilated and
used easier (Mizrahi and Lensky, 1997; Fatrcová-
Šramková et al., 2010). A proper hive management
promotes bee-bread collection, aimed at marketing it for
human consumption since it can be considered as food
supplement due to its content of a wide range of nutrients.
One of the contributions to their high nutritional value is
the presence of significant amounts of proteins, vitamins
and phenolic compounds as natural antioxidants. The
potential application of "bee bread" as a food and as a
nutraceutical supplement depends in large part on its
chemical composition which varies directly with the flora
of the region and the time of collection by the bees
(Čeksterytė et al., 2008). Bee bread differs from pollen by
lower pH (3.8 – 4.3), it contains less proteins and fats, but
more carbohydrates and lactic acid. Bee bread has a better
bioavailability because the walls of pollen, which cannot
be destructed by gastrointestinal liquids, have been partly
destructed by fermentation and the functionally and
energetically rich content of pollen can be assimilated and
used easier (Mizrahi and Lensky, 1997). Bee bread has
antimicrobial, antioxidant hepatoprotective, immuno-
modulating and antiradiation activity, adaptogenic
properties. It stimulates protective forces of a human body,
normalizes metabolism, has a positive influence on the
liver, nervous and endocrine system functions, and
enhances regeneration of tissues, physical and mental
persistence of a human body (Bogdanov, 2015).
Potravinarstvo, vol. 9, 2015, no. 1, p. 592-598
doi:10.5219/558
Received: 9 October 2015. Accepted: 30 November 2015.
Available online: 17 December 2015 at www.potravinarstvo.com
© 2015 Potravinarstvo. All rights reserved.
ISSN 1337-0960 (online)
License: CC BY 3.0
BEE BREAD – PERSPECTIVE SOURCE OF BIOACTIVE COMPOUNDS FOR
FUTURE
Eva Ivanišová, Miroslava Kačániová, Helena Frančáková, Jana Petrová, Jana Hutková,
Valeryii Brovarskyi, Serhii Velychko, Leonora Adamchuk, Zuzana Schubertová, Janette Musilová
ABSTRACT
Bee bread is product with long history used mainly in folk medicine. Nowadays, bee bread is growing in commercial
interest due to its high nutritional properties. The objective of this study was to determine biological activity of ethanolic
extract of bee bread obtained from selected region of Ukraine – Poltava oblast, Kirovohrad oblast, Vinnica oblast, Kyiv
oblast, Dnepropetrovsk oblast. The antioxidant activity was measured with the radical scavenging assays using
1,1-diphenyl-2-picrylhydrazyl (DPPH) radical as well as phosphomolybdenum assay. Total polyphenol content was
determined with Folin-Ciocalteau reagent and total flavonoid content by aluminium-chloride method. Secondary was also
evaluated antimicrobial activity in bee bread samples with disc diffusion method and minimum inhibitory concentrations.
Antioxidant activity expressed as mg TEAC per g of dry weight (Trolox equivalent antioxidant capacity) was the highest in
bee bread from Poltava oblast in DPPH and also phosphomolybdenum method. Samples of bee bread contained high levels
of total polyphenols (12.36 – 18.24 mg GAE – gallic acid equivalent per g of dry weight) and flavonoids (13.56 – 18.24 μg
QE – quercetin equivalent per g of dry weight) with the best values of bee bread from Poltava oblast. An elevated level of
antioxidant potential in the bee bread determines its biological properties, which conditioned of the biological active
substances. The best antibacterial activity of bee bred with disc diffusion method was found against Bacillus thuringiensis
CCM 19. The antibacterial activity inhibited by the bee bread extract in the present study indicate that best minimal
inhibition concentration was against bacteria Escherichia coli CCM 3988 and Salmonella enterica subs. enterica CCM
3807.
Keywords: antioxidant activity; pollen; flavonoids; polyphenols; antimicrobial activity

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Volume 9 593 No. 1/2015
The aim of study was to determine biological activity of
selected bee bread samples – antioxidant activity, total
polyphenols and flavonoids content. Secondary was also to
determine antimicrobial characteristic of these samples.
MATERIAL AND METHODOLOGY
Biological material
Bee bread was obtained from selected region of Ukaine
(Poltava oblast, Kirovohrad oblast, Vinnica oblast, Kyiv
oblast, Dnepropetrovsk oblast), by patent technology
developed by research teams Department of beekeeping,
National University of Life and Environmental Sciences of
Ukraine, Kyiv. Before the measurement samples were
crushed to the powder using mortar and store at 4°C in
refrigerator.
Chemicals
All chemicals were analytical grade and were purchased
from Reachem (Slovakia) and Sigma Aldrich (USA).
Sample preparation
0.1 g of bee bread was extracted with 20 mL of 80%
ethanol for 2 hours. After centrifugation at 4000 g (Rotofix
32 A, Hettich, Germany) for 10 min, the supernatant was
used for measurement (antioxidant activity, polyphenols,
flavonoids).
Antioxidant activity
Radical scavenging activity
Radical scavenging activity of samples was measured
using 2,2-diphenyl-1-picrylhydrazyl (DPPH) (Sánchéz-
Moreno et al., 1998). The extracts (0.5 mL) were mixed
with 3.6 mL of DPPH solution (0.025 g DPPH in 100 mL
ethanol). Absorbance of the sample extract was determined
using the spectrophotometer Jenway (6405 UV/Vis,
England) at 515 nm. Trolox (6-hydroxy-2,5,7,8-
tetramethylchroman-2-carboxylic acid) (10-100 mg.L-1;
R2 = 0.988) was used as the standard and the results were
expressed in mg.g-1 Trolox equivalents.
Reducing power
Reducing power of samples was determined by the
phosphomolybdenum method of Prieto et al., (1999) with
slight modifications. The mixture of sample extract
(1 mL), monopotassium phosphate (2.8 mL, 0.1 M),
sulfuric acid (6 mL, 1 M), ammonium heptamolybdate (0.4
mL, 0.1 M) and distilled water (0.8 mL) was incubated at
90°C for 120 min, then rapidly cooled and detected by
monitoring absorbance at 700 nm using the
spectrophotometer Jenway (6405 UV/Vis, England).
Trolox (10-1000 mg.L-1; R2=0.998) was used as the
standard and the results were expressed in mg.g-1Trolox
equivalents.
Total polyphenol content
Total polyphenol content of potato extracts was measured
by the method of Singleton and Rossi, (1965) using Folin-
Ciocalteu reagent. 0.1 mL of each sample extract was
mixed with 0.1 mL of the Folin-Ciocalteu reagent, 1 mL of
20% (w/v) sodium carbonate and 8.8 mL of distilled water
After 30 min. in darkness the absorbance at 700 nm was
measured using the spectrophotometer Jenway (6405
UV/Vis, England). Gallic acid (25-250 mg.L-1; R2=0.996)
was used as the standard and the results were expressed in
mg.g-1 gallic acid equivalents.
Total flavonoid content
Total flavonoids were determined using the modified
method of (Willett, 2002). 0.5 mL of sample extract was
mixed with 0.1 mL of 10% (w/v) ethanolic solution of
aluminium chloride, 0.1 ml of 1 M sodium acetate and
4.3 mL of distilled water. After 30 min. in darkness the
absorbance at 415 nm was measured using the
spectrophotometer Jenway (6405 UV/Vis, England).
Quercetin (0.01 – 0.5 mg.L-1; R2 = 0.997) was used as the
standard and the results were expressed in μg.g-1 quercetin
equivalents.
Antimicrobial activity
Microbial strains
Four strains of microorganisms were tested in this study,
including two Gram-negative bacteria (Escherichia coli
CCM 3988, Salmonella enterica subs. enterica CCM
3807, two Gram-positive bacteria (Bacillus thuringiensis
CCM 19, Staphylococcus aureus subs. aureus CCM 4223).
All tested strains were collected from the Czech Collection
of microorganisms. The bacterial suspensions were
cultured in the nutrient broth (Imuna, Slovakia) at 37 °C.
Disc diffusion method
Antimicrobial activity of each bee bred extract was
determined by a disc diffusion method. Briefly, 100 μL of
the test bacteria were grown in 10 mL of fresh media until
they reached a count of approximately 105 cells.mL-1.
Then 100 μL of the microbial suspension was spread onto
Mueller Hinton agar plates. The extracts were tested using
6 mm sterilized filter paper discs. The diameters of the
inhibition zones were measured in millimeters. All
measurements were to the closest whole millimeter. Each
antimicrobial assay was performed in at least triplicate.
Filter discs impregnated with 10 μL of distilled water were
used as a negative control.
Minimum inhibitory concentrations (MICs)
MICs were determined by the microbroth dilution
method according to the Clinical and Laboratory Standards
Institute recommendation (CLSI, 2014) in Mueller Hinton
broth (Biolife, Italy). Briefly, the DMSO plant extracts
solutions were prepared as serial two-fold dilutions
obtaining a final concentration ranging between
0.5-2048 μg.mL-1. After that each well was inoculated with
microbial suspension at the final density of 0.5 McFarland.
After 24 h of incubation at 37 °C, the inhibition of
microbial growth was evaluated by measuring the well
absorbance at 450 nm in an absorbance microplate reader
Biotek EL808 with shaker (Biotek Instruments, USA). The
96 microwell plates were measured before and after
experiment. Differences between both measurements were
evaluated as growth. Measurement error was established
for 0.05 values of absorbance. Wells without plant extracts
were used as negative controls of growth. Pure DMSO was
used as negative control. This experiment was done in
eight-replicates for a higher accuracy of the MICs of used
medical plant extracts.

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Statistical analysis
The basic statistical analyzes were realized in SAS
programming packages (THE SAS SYSTEM V 9.2.).
Correlation coefficients were calculated by CORR analysis
(SAS, 2009).
RESULTS AND DISCUSSION
Antioxidant activity
In the DPPH radical-scavenging method, a compound
with high antioxidant potential effectively traps the radical,
thereby preventing its propagation and the resultant chain
reaction (Brand-Williams et al., 1995). DPPH is a stable
free radical that is dissolved in ethanol and its purple color
shows a characteristic absorption at 515 nm. Antioxidant
molecules scavenge the radical by hydrogen donation and
the colour from the DPPH assay solution becomes light
yellow resulting in a decrease in absorbance (Silva et al.,
2012). As shown Fig. 1 all tested samples had effect to
trap DPPH radical, with the best value in bee bred from
Poltava oblast (15.78 mg TEAC.g-1) and Vinnica oblast
(14.62 mg TEAC.g-1). High antioxidant activity also
reported (Zuluaga et al., 2015), which evaluated
polyfloral Colombian bee bread with ABTS method;
values from their study range from 46.1 to 76.3 μmol
Trolox/g. In spite of the relevance of bee bread as an
antioxidant substance, there is not enough systematic
information about the antioxidant activity and profile of
bioactive compounds of bee bread.
Phosphomolybdenum method is used to measure the
reductive ability of antioxidant, and it is evaluated by the
transformation of Mo(VI) to Mo(V) where, the ability of
samples to reduce Mo may be attributed from hydrogen
donation from phenolic compounds which is also related to
presence of reducing agent (Huda-Faujan et al., 2009).
The reducing ability of the bee breads (Fig. 1) was in the
order: bee bread from Poltava oblast > bee bread from
Kyiv oblast > bee bread from Vinnica oblast > bee bread
from Kirovohrad oblast > Dnepropetrovs oblast. Similar
like DPPH method, the best values were determined in
sample from Poltava region. Barros et al., (2007)
demonstrated that the reducing properties are generally
associated with the presence of reductones, which had
been shown to exert antioxidant action by breaking the
free radical chain by donating the bread hydrogen atom.
Higher level of polyphenols in bee bread could act as
reductone where these compounds could react with free
radicals by converting them to more stable products and
terminating the radical chain reaction (Oh et al., 2013).
Siddiqui et al., (2012) claimed antioxidants chelate and
disengage transition metals, thereby preventing such
metals from participating in the initiation of lipid
peroxidation and oxidative stress through metal catalyzed
reaction.
On the basis of the above findings, bee bread seems to be
attractive as an important source of antioxidants for the
food and pharmaceutical industries. The differences
observed between the antioxidant activities of the tested
samples may be attributed to the presence of natural
antioxidants, mainly phenolic compounds that differed
depending on the region where they were collected (Sati et
al., 2013; Tlili et al., 2014).
Total polyphenol and flavonoid content
Phenolic compounds are considered among the largest
contributors to the antioxidant potential of natural food
products. Total polyphenol content (Table 1) in bee-bread
ranged from 12.36 to 25.4 mg GAE.g-1. The highest value
was observed in sample from Poltava region. Nagai et al.,
(2004) also determined high level of total polyphenols in
bee bread and also reported that bee bread can be applied
more as health food and medicine. Zuluaga et al., (2015)
determined in Colombian bee bread values from 2.1 to
13.7 mg GAE.g-1 of polyphenols. The information about
spectrum of polyphenol compounds in bee bread is
missing, but we can expect, that bee bread contains similar
polyphenols like bee pollen. It is also potential, that and
bee bread can contain new type of polyphenols. According
to Fanali et al., (2013) in bee pollen, polyphenolic
compounds are commonly glycosylated, esterified, present
in free forms or combined with other pollen components.
Bonvehi et al., (2001) reported that bee pollen is rich for
gallic acid, vanillic, protocatechuic, p-coumaric acid,
hesperidin, rutin, luteolin, apigenin, kaempferol, quercetin
and isorhamnetin.
Total flavonoid content (Table 1) in observed samples of
bee bread ranged from 13.56 to 18.24 μg QE.g-1. The
highest value, similarly like polyphenol content was
observed in sample from Poltava region. Flavonoids are
the secondary components of most importance in bee
bread and influence the visual appearance of the grain
(pigmentation) and flavour (astringency and bitterness)
(DeGrandi-Hoffman et al., 2013). In pollen grains, most
of flavonoids exist as glycosides, known as aglycones,
being quercetin the major compound. Although there is not
a recommended daily ingest for flavonoids, it is suggested
an intake of about 200 – 100 mg per day. Zuluaga et al.,
(2015) determined total flavonoid content in Colombian
bee bread from 1.9 to 4.5 mg QE.g-1. It is very difficult
determine average total flavonoid content in bee bread
generally. Zuluaga et al., (2014) reported that bee pollen
contains higher content of total flavonoids with compare to
bee bread due to possible differences in botanical origin of
pollen and also the fact that a degradation of the outer
layer of the grain makes more available bioactive
compounds to degrade by environmental conditions. These
authors also published that in Colombian region was
established average content of flavonoids in 5.16 mg.g-1
(QE) of bee pollen. The separation of the individual
polyphenols and flavonoids and detection of the other
antioxidants will be necessary for evaluate of biological
activity of bee bread in future.
Antimicrobial activity
Bee bread samples showed a potential activity against the
growth of both gram positive and gram negative bacteria
which was resistant to antibiotics. This would be a very
interesting approach to control more dangerous species of
micro-organism in medical sciences. Because of the
development of resistance by the microorganisms to
common antibiotics, it has become necessary to search for
an alternative approach dealing with this situation. It had
been suggested that natural products are preferable to
synthetic ones (Abouda et al., 2011).
Results of antibacterial testing with disc diffusion method
(Figure 2) showed that higher antibacterial activity was

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found against Bacillus thuringiensis in sample from
Vinnica oblast, Kyiv oblast and Dnepropetrovsk oblast.
The higher inhibition zone was found in sample from
Kirovohrad oblast against bacteria Escherichia coli. The
higher antimicrobial activity against Salmonella enterica
subs. enterica was found in sample from Kyiv oblast.
Samples of natural bee-bread from different aromatic and
medicinal plants were studied for their antimicrobial
activities on antibio-resistant bacterial strains isolated from
human pathology. Four samples of bee-bread were
collected from different regions in Morocco. Dilutions of
bee-bread from 1/2, 1/4, 1/8 and 1/16 were tested by the
agar well diffusion method on various strains of bacteria
including Escherichia coli, Staphylococcus aureus,
Bacillus cereus and Pseudomonas aeruginosa. Results
revealed that most of strains were inhibited by the dilution
1/2 and 1/4. The gram positive bacteria were more
sensitive to bee-bread and bee-pollen than gram negative
bacteria. All the samples showed strong antimicrobial
activities on the bacterial strains, which were first tested
for their resistance to antibiotics (Abouda et al., 2011).
The best antimicrobial activity (Tab. 2) MIC50 was found
in sample from Poltava region where minimal inhibition
concentration (6.40 μg.mL-1) against gram negative
bacteria; very good antibacterial activity were also found
in same sample against bacteria in MIC90 (6.40 μg.mL-1).
In generally all tested samples against all tested bacteria
had antibacterial influence.
Statistical analysis
Using Pearson correlation coefficients was verified
correlation (Table 3) between antioxidant activity
determined by DPPH and phosphomolybdenum method
and total polyphenol and flavonoid content. The strong
correlation dependence (0.95) was found between
antioxidant activity (DPPH) and polyphenol content and
also between flavonoid content and antioxidant activity
(phosphomolybdenum method) (0.89). Between two
different methods for determining the antioxidant activity,
was determined the mean linear relationship (0.54). Based
on these results, it can be concluded that polyphenols and
flavonoids have a strong impact on the antioxidant activity
of bee bread.
CONCLUSION
In conclusion, the results of this study demonstrate that
bee bread is very good source of bioactive compounds not
only with antioxidant but also antimicrobial effect. The
best results were observed in most of parameters in sample
from Poltava oblast. Bee bread can be use more in future
not only in medicine, pharmacy but also in food industry.
For confirmation of biologically effect is necessary more
and intensive study, in vivo test for evaluating bioactive
components and digestibility properties; very important is
also determining some negative compounds which can
Figure 1 Radical scavenging activity and reducing power of bee bread (TEAC – Trolox equivalent antioxidant capacity
PO – Poltava oblast, KiO – Kirovohrad oblast, VO – Vinnica oblast, KO – Kyiv oblast, DO – Dnepropetrovsk oblast.
Table 1 Total polyphenol and flavonoid content in bee bread.
Sample
Total polyphenol content
(mg GAE.g-1)
Total flavonoid content
(μg QE.g-1)
Poltava oblast
25.44 ±0,22
18.24 ±0.08
Kirovohrad oblast
19.96 ±0.59
15.25 ±0.04
Vinnica oblast
20.88 ±0.34
13.56 ±0.04
Kyiv oblast
12.36 ±0.34
15.35 ±0.09
Dnepropetrovsk oblast
13.47 ±0.56
14.04 ±0.03
Note: GAE – gallic acid equivalent; QE – quercetin equivalent; ± standard deviation.
0
5
10
15
20
PO KiO VO KO DO
mg TEAC.g-1
DPPH method
0
50
100
150
200
250
300
PO KiO VO KO DO
mg TEAC.g-1
Phosphomolybdenum method

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decrease the quality of bee bread (heavy metal,
radionuclide, and microbes). Results in this work can be an
important tool for recognizing bee bread as being a
beneficial source of natural nutrients.
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Figure 2 Antimicrobial activity of bee bread against bacteria.
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CCM, SA- Staphylococcus aureus subs. aureus CCM 4223); PO – Poltava oblast, KiO – Kirovohrad oblast, VO –
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PO KiO VO KO DO
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EC SE SA BT

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Acknowledgments:
This work was supported by grant VEGA 1/0456/12.
Contact address:
Eva Ivanišová, Slovak University of Agriculture in Nitra,
Faculty of Biotechnology and Food Sciences, Department

Potravinarstvo® Scientific Journal for Food Industry
Volume 9 598 No. 1/2015
of Plant Storage and Processing, Tr. A. Hlinku 2,
949 76 Nitra, Slovakia, E-mail: eva.ivanisova@uniag.sk.
Miroslava Kačániová, Slovak University of Agriculture
in Nitra, Faculty of Biotechnology and Food Sciences,
Department of Microbiology, Tr. A. Hlinku 2, 949 76
Nitra, Slovakia, E-mail: miroslava.kacaniova@uniag.sk.
Helena Frančáková, Slovak University of Agriculture in
Nitra, Faculty of Biotechnology and Food Sciences,
Department of Plant Storage and Processing,
Tr. A. Hlinku 2, 949 76 Nitra, Slovakia, E-mail:
helena.frančáková@uniag.sk.
Jana Petrová, Slovak University of Agriculture in Nitra,
Faculty of Biotechnology and Food Sciences, Department
of Microbiology, Tr. A. Hlinku 2, 949 76 Nitra, Slovakia,
E-mail: jana.petrova@uniag.sk.
Jana Hutková, Slovak University of Agriculture in Nitra,
Faculty of Biotechnology and Food Sciences, Department
of Microbiology, Tr. A. Hlinku 2, 949 76 Nitra, Slovakia,
E-mail: rapcanova.pk@gmail.com.
Valeryii Brovarskyi, National University of Life and
Environmental Sciences of Ukraine, Kyiv, Ukraine,
Department of beekeeping, Heroiv Oborony St, 15, Kiev,
Ukrajina, 03041. E-mail: valeryii.brovarskyi@gmail.com.
Serhii Velychko, National University of Life and
Environmental Sciences of Ukraine, Kyiv, Ukraine,
Department of beekeeping, Heroiv Oborony St, 15, Kiev,
Ukrajina, 03041. E-mail: serhii.velychko@gmail.com.
Leonora Adamchuk, National University of Life and
Environmental Sciences of Ukraine, Kyiv, Ukraine,
Department of beekeeping, Heroiv Oborony St, 15, Kiev,
Ukrajina, 03041. E-mail: leonora.adamchuk@gmail.com.
Zuzana Schubertová, Slovak University of Agriculture in
Nitra, Faculty of Agrobiology and Food Reources,
Institute of Biodiversity and Biological Safety,
Tr. A. Hlinku 2, 949 76 Nitra, Slovakia, E-mail:
zuzana.schubertovaniag.sk.
Janette Musilová, Slovak University of Agriculture in
Nitra, Faculty of Biotechnology and Food Sciences,
Department of Chemistry, Tr. A. Hlinku 2, 949 76 Nitra,
Slovakia, E-mail:janette.musilova@uniag.sk.