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Effects of Royal Jelly and Bee Pollen on the Growth of Selected Probiotic Bacteria (Bf. animalis Spp. Lactis, L. acidophilus and L. casei)

  • Bursa Uludag University

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In this research article, the effects of bee pollen and royal jelly on the selected probiotic bacteria, as growth factors, were investigated. The probiotic cultures were activated in MRS broth at 37°C. Then, bee pollen and royal jelly (10 mg/100 μL, 25 mg/250 μL, 50 mg/500 μL, 75 mg/750 μL, and 100 mg/1000 μL) were added on the probiotic cultures in MRS broth and sampled at 0, 24, and 48 hours of incubation. The medias used for enumeration of the probiotic cultures were RCA (Reinforced Clostridial Agar) for
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J. APIC. SCI. Vol. 60 No. 2 2016
Metin Guldas
In this research article, the effects of bee pollen and royal jelly on the selected probiotic
bacteria, as growth factors, were investigated. The probiotic cultures were activated in
MRS broth at 37°C. Then, bee pollen and royal jelly (10 mg/100 μL, 25 mg/250 μL, 50
mg/500 μL, 75 mg/750 μL, and 100 mg/1000 μL) were added on the probiotic cultures
in MRS broth and sampled at 0, 24, and 48 hours of incubation. The medias used for enu-
meration of the probiotic cultures were RCA (Reinforced Clostridial Agar) for
Bf. animalis
, MRS (deMann, Rogosa and Sharpe) Agar with D-sorbitol for
Lb. acidophilus
MRS-Vancomycine Agar for
Lb. casei
. The lactic acid production by
Lb. acidophilus
Lb. ca-
, and
Bf. animalis
, and acetic acid production by
Bf. animalis
spp. l
, were
determined to compare the bacterial proliferation. The probiotic cultures were mainly
affected by the bee pollen and royal jelly during the rst 24 hours. The changes ob-
served in the number of probiotic counts between 24 and 48 hours were not signicant,
statistically (P<0.05). Generally, the probiotic bacterial counts increased parallel to the
concentration of bee pollen or royal jelly up to 75mg, and remained unchanged above this
concentration. In terms of lactic acid production and bacterial growth, the most signi-
cant growth was observed on
Lb. acidophilus
when bee pollen or royal jelly was added.
Keywords: bee pollen,
Bidobacterium animalis
Lactobacillus acidophilus
Lactobacillus casei
, royal jelly
Uludag University, Karacabey Vocational School, Department of Food
Processing Karacabey 16700 Uludag University, Bee Keeping Development,
Application and Research Center, Gorukle Campus Nilufer 16059 Bursa, Turkey
Royal jelly is a bee product secreted by the hy-
popharyngeal glands of young worker (nurse)
bees, to feed young larva and the adult queen
bee. This jelly is a thick, water-soluble and viscous
product. Royal jelly contains many proteins
and free amino acids, 18-52% total sugar, free
fatty acids, as well as water soluble vitamins,
minerals, and enzymes. It has been mentioned
that royal jelly has antibiotic activity due to high
concentration of lO-hydroxydecanoic acid in the
jelly (FAO, 2013a). Antibiotic effects of royal jelly
have been determined against the pathogenic
bacteria such as
Salmonella, Escherichia coli,
Micrococcus pyrogens, Proteus, Staphylococ-
cus aureus
, and
Bacillus subtilis
(Yatsunami &
Echigo, 1985; Bărnuţiu et al., 2011; FAO, 2013a).
In addition to antimicrobial properties, honey
may contain several pathogenic microorgan-
isms. For this reason, honey can be called a
reservoir for microorganisms (Olaitan et. al.,
Clostridium botulinum
is a signicant risk
factor for infants. The health problem caused
by this microorganism is called infant botulism.
Infant botulism is a toxigenic infection which is
harmful for infants under the age of one year
(Rudnicka et al., 2015).
Pollen is a natural product which consists of the
grain particles of the male gametophyte. These
particles are gathered and produced by bees
from owering plants. Pollen as a food source
for bees is rich in nutrients such as proteins and
amino acids, lipids, sugars (fructose, glucose,
and sucrose), vitamins (B, C, and E), minerals (Ca,
Mg, and P), trace elements (Fe, Cu, Mn, and Zn),
hormone-like growth factors (auxins and gibber-
ellin) as well as phytochemicals such as phytos-
corresponding author:
Received: 11 April 2016; accepted: 6 October 2016
DOI 10.1515/JAS-2016-0023
Original Article
J. APIC. SCI. VOL. 60 NO. 2 2016
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Effects of royal jelly and bee pollen on growth of probiotics
terols, carotenoids, and avonoids (Kacainova et
al., 2012; Komosinska-Vassev et al., 2015; FAO,
In recent years, consumption of probiotic
foods has increased due to the many health
benets such as reducing serum cholesterol
level, improving lactose utilisation to prevent
lactose intolerance, preventing gastrointestinal
cancers, keeping intestinal microora active, and
supporting immune system. Probiotics prevent
harmful pathogenic bacteria in the human body
and help to control intestinal infections due to
producing antimicrobial compounds such as
hydrogen peroxide, antibiotics, deconjugated bile
acids, and organic acids (Goderska & Czarneski,
2007; Baroutkoub et al., 2010; Kabeerdoss et
al., 2011; Wang et al., 2012; Shori & Baba, 2014).
The microorganisms named as probiotics are
the subgroup bacteria belonging to genus of
, and
. Due
to organic acids (lactic, and acetic acids e.g.)
produced by lactic acid bacteria, the probiotics
lower pH and prevent the proliferation of
pathogenic and deteriorative bacteria (Simsek
et al., 2002; Strus et al., 2004; Valdez et al.,
2005; FAO/WHO 2006).
In the literature, it has been found that the
effects of honey on the probiotic bacteria
(Ustunol & Gandhi, 2001; Shin & Ustunol, 2005)
and yoghurt bacteria (Varga, 2006) were suf-
ciently investigated. But, the effects of pollen
and royal jelly on the growth of probiotic bacteria
have not been studied in detail (Nabas et al.,
2014; Yerlikaya, 2014). Different results have
been found due to the materials and methods
used, and the growth environment tested. The
analytical and microbiological results can differ
according to the chemical and microbiologi-
cal characteristics of yoghurt. The high acidity
of the yoghurt and other inhibitory factors
available in milk such as antibacterial peptides
liberated from the certain milk proteins (Fadaei,
2012; Vaj i h e h , 2012) , can interfere to obtain
actual test results.
There are a number of factors that can affect
the growth of
spp. in milk
products such as strains of probiotic bacteria,
pH, the presence of organic acids, interactions
with other microorganisms, storage tempera-
ture, and production conditions (Shah, 2000;
Boylston et al., 2004). Shin & Ustunol (2005)
stated that the growth of
was enhanced by the addition of honey while
E. aerofaciens
were inhibited if
they co-cultured with
. Bee pollen
also contains a number of microorganisms which
come from nature (Brindza et al., 2010).
The most signicant issue for the probiotic food
products is to contain enough viable probiotic
bacteria at the moment of consumption (108-109
log cfu ml-1). Probiotic foods that contain a
low number of viable probiotic organisms
do not provide expected health benets for
the consumers. Therefore, such prebiotic
compounds as inulin and fructooligosaccha-
rides (FOS) have been added to probiotic foods
to reach desired viable counts. Bee pollen and
royal jelly have known to contain rich nutrients
for the human diet. But, on the other hand, even
bee pollen or royal jelly also contains some anti-
microbial compounds.
In this research, the effects of bee pollen and
royal jelly containing a number of nutrients, on
the growth of three probiotic bacteria cultivated
in the selective media, were investigated.
Probiotic cultures
The probiotic cultures used in the research
Bidobacterium animalis spp. lactis
(BB12, DSM15954),
Lactobacillus acidophilus
(LA-5, DSM13241) and
Lactobacillus casei
ATCC55544) obtained from Chr. Hansen Co.,
Horsholm, Denmark. Ten grams of the probiotic
cultures were weighed, transferred into 20
mL sterilised water and shaken properly to
obtain a homogenised culture. Each culture
suspension (20 mL) was added and activated
in 50 mL of MRS (deMann, Rogosa and Sharpe)
broth containing 5 % (w/v) lactose at 3C for
24 h to obtain an approximately 108 cfu/mL
bacterial count (Popa & Ustunol, 2011). The re-
activation procedure and the estimation were
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J. APIC. SCI. Vol. 60 No. 2 2016
repeated on the same culture media (MRS
broth) in the above-mentioned conditions for
specied probiotic bacteria, until the targeted
initial bacterial count (approx. 108 cfu/mL) was
obtained as described by Guldas & Irkin (2010).
Then, 1 mL of each probiotic bacteria was trans-
ferred into 9 mL of MRS broth without pollen
and royal jelly to act as the control. Only
dobacterium animalis spp. lactis
was grown and
incubated in MRS broth at 37°C for 24 hours,
under anaerobic conditions using GasPacks (BBL
Microbiology Systems, Cockeysville, MD, USA).
Sample preparation
The pure royal jelly and bee pollen samples
were supplied from the Uludag University Bee
Keeping Development, Application and Research
Centre (AGAM, Bursa, Turkey). The samples
were kept in a refrigerator at 4°C. The bee
pollen samples were collected by pollen traps.
The pollen samples were provided by AGAM in
the rst half of May, 2015. The botanical origins
(plant taxa) of pollens identied in the same rural
area at the same seasonal time were given as
follows (Bilisik et al. 2008):
(29.35 %),
(21.05 %),
(10.37 %),
(1.91 %),
(1.75 %) ,
Trifolium pratense
(1.59 %),
(0.64 %), and
(0.64 %).
The bee pollen and royal jelly samples were
prepared for analyses in a laminar ow cabinet
(ESCO, Class II, Germany) to avoid contamina-
tion. The bee pollen and the royal jelly samples
were dissolved in sterilised water at room tem-
perature and 35°C and obtained 10 % of the bee
pollen and royal jelly solutions, respectively. From
these solutions, 100 µL, 250 µL, 500 µL, 750 µL,
and 1000 µL of the samples were pipetted into
the MRS broth with lactose (5 % w/v). Therefore,
concentrations of the bee pollen and royal jelly
added into the MRSL broth tubes were 10 mg /
100 µL, 25 mg / 250 µL, 50 mg / 500 µL, 75 mg
/ 750 µL, and 100 mg/1000 µL.
Media and growth conditions
For the bacterial enumeration, 1 mL from each
test tube containing MRSL broth was taken and
used to prepare serial dilutions before trans-
ferring into the petri plates. These petri plates
contained the selective media as described
Lactobacillus acidophilus
was enumerated se-
lectively in MRS (deMann, Rogosa and Sharpe)
D-sorbitol (10 g/100 mL) media (Tharmaraj and
Shah, 2003) at 37°C for 72 h. The selective enu-
meration of
Bf. animalis spp. lactis
was imple-
mented in RCA (reinforced clostridial agar) with
0.03 g/100 g aniline blue and dicloxacillin (2 mg/
mL, Sigma). The plates were incubated under
anaerobic conditions at 37°C for 48 h using
GasPacks (BBL Microbiology Systems, Cockey-
sville, MD, USA) according to Kailasapathy et al.
(2008). For enumeration of Lb. casei, MRS-Van-
comycine agar was used. The preparation of the
MRS Vancomycine agar was done by adding 2 ml
of 0.05 g vancomycine (Sigma)/100 ml solution
into 1 L of MRS broth to obtain 1 mg/L of the
nal concentration.
The average count of the duplicate plates or
tubes was used for statistical evaluations.
Determination of lactic and acetic acids
Lactic (D-/L-Lactic acids) and acetic acid pro-
ductions by the tested probiotic bacteria were
determined during 0, 24, and 48 hours of
incubation in MRS broth. For lactic and acetic
acid determinations, the enzymatic method
proposed by Popa & Ustunol (2011) was used.
Lactic acid productions by
L. acidophilus
DSM13241) and
L. casei
(431, ATCC5554 4);
acetic acid and lactic acid productions by
bacterium animalis spp. lactis
(BB12, DSM15954)
were determined at 340 nm by UV-VIS spectro-
photometer (Shimadzu UV-1800, double beam,
Japan) using test kits in terms of D- / L- lactic and
acetic acid (R-Biopharm Inc., Marshall, MI, USA).
The dened acids were determined as g/L.
Statistical analysis
The statistical analyses were done by using
SPSS 15.0 software for windows (SPSS Inc.,
Chicago, Illinois, USA). A one-way analysis of
variance (ANOVA) test was used to determine
the mean differences. Tukey HSD test was
achieved to determine the level of signicance
between the means.
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Effects of royal jelly and bee pollen on growth of probiotics
The analysis of the results presented on Figures
1 and 2, in which the effects of royal jelly and
bee pollen on the bacterial growth are reected,
showed that bee pollen or royal jelly exhibited
the strongest effect on probiotic bacterial pro-
liferation at the end of 24 h. The number of
animalis spp. lactis, Lb. acidophilus ve Lb. casei
during the mentioned period was between
8.4-8.8, 8.6-9.0 and 8.4-8.9 log cfu/ml with the
addition of the royal jelly; 8.6-9.0, 9.4-9.8, and
8.6-9.0 log cfu/ml with the addition of the bee
pollen, respectively.
The highest counts of
Bf. animalis spp. lactis
were obtained in the samples containing 75
mg/750 µL of bee pollen. During the 24 and
48 hours of cultivation,
Bf. animalis spp. lactis
counts increased 8.09 and 8.45%, while the
counts regarding the same probiotic bacteria
increased 4.61 and 5.10% in the control group,
respectively (Fig. 1a).
While the number of
Lb. acidophilus
in the
control group increased 8.70% at the end of 24
hours, it increased 13.3-15.9% in the bee-pollen-
added samples parallel to the elevated concen-
tration. The increase in the same bacteria was
13.8-15.6% after the bee pollen addition at the
end of 48 hours. As seen from Figure 1b, there
was no difference statistically (P<0.05) in
counts between 24 and 48 hours of
cultivation, after the addition of bee pollen.
The highest increase in the
Lb. casei
was approximately 10% and obtained from the
samples which had an addition of 75 mg/750 µL
of bee pollen (Fig. 1c). The number of
Lb. casei
did not change at the end of 48 hours nor did
the concentration elevate to 100 mg/1000µL
of bee pollen (P<0.05). The number of
Lb. casei
neither changed
at the end of 48 hours nor did
the bee pollen concentration was elevated up to
100 mg/1000µL (P<0.05).
The increase in
Bf. animalis spp. lactis
depended on the royal jelly concentration (Fig.
Bidobacterium animalis spp. lactis
approximately 7.5% in the samples which had 75
mg/750 µL of royal jelly added at the end of 24
hours. However, neither 75 mg/750 µL nor 100
mg/1000 µL of royal jelly caused a signicant
difference in the
Bf. animalis spp. lactis
The addition of royal jelly led to the highest
Lb. acidophilus
levels from among all the inves-
tigated probiotic bacteria (Fig. 2b). The number
Fig. 1a. Effect of bee pollen on the growth of *
Bf. animalis
spp. lactis*
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J. APIC. SCI. Vol. 60 No. 2 2016
Lb. acidophilus
reached the highest level
(9.81 log cfu/ml) after 24 hours, in the samples
in which 75 mg/750 µL of royal jelly had been
Lactobacillus casei
counts in the control
group in which no royal jelly had been added,
increased 1.46 and 2.19%.
Lactobacillus casei
counts increased 8.42 and 9.52% in the samples
in which 75 mg/750 µL of royal jelly had been
added after 24 and 48 hours, respectively (Fig.
The number of probiotic bacteria in the samples
in which bee pollen was added, increased or
Fig. 1b. Effect of bee pollen on the growth of *
Lb. acidophilus
Fig. 1c. Effect of bee pollen on the growth of *
Lb. casei
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Effects of royal jelly and bee pollen on growth of probiotics
decreased slightly between 24 and 48 hours
of incubation. But, these changes observed in
the probiotic counts were not signicant, sta-
tistically (P<0.05). The concentration in which
the maximum observed probiotic growth
was 75 mg/750 µL and counts of probiotic
bacteria (
Bf. animalis spp. lactis, Lb. acidophi-
Lb. casei
) increased only 0.36, 0.50,
and 0.60% between 24 and 48 hours in this
concentration, respectively. In other words, the
elevation of the concentration above 75
mg/750 µL even for bee pollen or royal jelly, did
Fig. 2a. Effect of royal jelly on the growth of *
Bf. animalis
Fig. 2b. Effect of royal jelly on the growth of *
Lb. acidophilus
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J. APIC. SCI. Vol. 60 No. 2 2016
not cause a signicant increase in the number
of the probiotic bacteria (P<0.05)
The highest growth among the investigated
probiotic bacteria was observed in
Lb. acidophi-
when there was an addition of bee pollen
or royal jelly. When 75 mg/750 µL concentration
was considered, the increase in
Lb. acidophilus
counts was approximately 15 and 10% when
using bee pollen and royal jelly, respectively.
As seen from the tables, the organic acid pro-
ductions of the investigated probiotics were
parallel to the bacterial growth (Tab. 1, 2, 3, and
4). Even the lactic acid or acetic acid productions
increased when there were elevated concen-
trations of bee pollen and royal jelly. Secondly,
organic acid productions and growth stimula-
tion when the bee pollen was used, were higher
than the royal jelly addition. During the fermen-
tation of food products, organic acid production
is one of the main criterions for monitoring the
microbial growth. Lactic acid in fermented milk
products is not only the signicant indicator of
bacterial growth (Bouzas et al., 1991) but also
one of most signicant taste parameters (Popa
& Ustunol, 2011). The lactic acid content of the
control tubes ranged between 2.88 and 3.81g/L
in my research. As a comparison, Popa & Ustunol
(2011) found the lactic acid produced by the bi-
dobacteria and
Lb. acidophilus
to be 3.08 and
3.59 g/L in the control tubes that contained
MRSL broth, respectively.
The lactic acids produced by
Bf. bidum
in the control tubes were measured
as 23.69 and 23.56 g/L at 24 hours of incubation,
respectively, in my experiment. As a comparison,
Popa & Ustunol (2011) found the production of
the lactic acids to be 28.32 and 28.28 g/L in
the same growth media (MRSL broth) and at 24
hours of incubation, respectively.
As seen from Table 1, 2, and 3, a distinct
increase in the production of the organic acids
was observed when comparing to the control
groups. The changes observed in the lactic acid
(D-/L- Lactic acids) contents between these
groups were statistically signicant (P≤0.05).
Most of the changes in the organic acid contents
between 24 and 48 hours of incubation were
not statistically signicant, (P≤0.05). The differ-
ences between the pronounced periods was ap-
proximately equal to 1 g/L or lower in terms of
the production of the organic acids (Tab. 1, 2, 3,
and 4).
In addition, among the three probiotic bacteria,
lactic acid produced by
Lb. acidophilus
slightly higher (Tab. 2) than the other two
probiotics (Tab. 1 and 3). This nding can also
Fig. 2c. Effect of royal jelly on the growth of *
Lb. casei
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Effects of royal jelly and bee pollen on growth of probiotics
be conrmed by the microbial growth of the
same bacteria, as can be seen in Figure 1b
and 2b. The lowest amount of lactic acid was
produced by
Lb. casei.
The production of lactic
acids in the tubes containing bee pollen and
royal jelly ranged between 23-28 g/L produced
Bf. bidum
Lb. acidophilus
(Tab. 1 and 2)
at 24 or 48 hours of incubation. While the same
organic acid content produced by
Lb. casei
between 21.53 and 23.89 g/L
(Tab. 3).
Acetic acid production by
Bf. bidum
in the
control tubes was 0.94 and 1.03 g/L at 0 and
Table 1
Effect of bee pollen and royal jelly on production of D-/L-Lactic acids by
Bf. animalis
0 h 24 h 48 h 0 h 24 h 48 h
The Control 3.11± 0 .17a 23.69±0.08a 23.45±0.29a 3.11±0 .17a 23.69±0.08a 23.45±0.29a
Bee Pollen Royal Jelly
10 m g /
100 µL 3.72±0.39b 24.91±0.34b 25.20±0.36b 3.28±0.28b 23.55±0.45a 23.1 0.35 a
25 mg/
250 µL 3.34±0.34c 26.67±0.23c 26 . 3 3±0.17c 3 . 25±0. 31b 26.19±0.41b 25.89±0.51b
50 mg/
500 µL 3.81± 0.21b 25.46±0.59b 25.92±0.41b 3 . 3 0 .11c 24.88±0.21a 25.07±0.22b
75 mg/
750 µL 3.25±0.26c 28.28±0.14d 28.66±0.04d 3.64±0.36d 28.77±0.62c 28.44±0.07c
1000 µL 3.40.07c 2 7.7 1± 0 .1 8 d 2 7. 5 4± 0 . 2 5 d 3.42±0.03c 28.64±0.18c 28.15±0.23c
*Means with the same superscripts in the same column are not signicantly different (P≤0.05), n=3
Table 2
Effect of bee pollen and royal jelly on production of D-/L-Lactic acids by
Lb. acidophilus
0 h 24 h 48 h 0 h 24 h 48 h
The Control 3.27±0.33a 23.56±0.08a 23.86±0.04a 3.27±0.33a 23.56±0.08a 23.86±0.04a
Bee Pollen Royal Jelly
10 m g /
100 µL 2. 8 8 ± 0.51b 24.28±0.41b 24.75±0.39b 3.16 ± 0 . 37a 23.33±0.23a 23.48±0.52a
25 mg/
250 µL 3.03±0.02a 26.41±0.47c 26.73±0.55c 3.09±0.06b 26.09±0.19b 26.72±0.29b
50 mg/
500 µL 2.94±0 .24b 27.17±0.12 d 27.65±0.26c 3.02±0.32b 26.19±0. 21b 26.38±0.06b
75 mg/
750 µL 3.20±0.35a 28.45±0.05e 28.76±0.08d 3.42±0.48c 27.87±0.07c 27.13 ±0 . 32c
1000 µL 3. 0 7±0.18a 28.22±0.42e 28.54±0.44d 3.75±0.04d 27.95±0.33c 27.10±0.05c
*Means with the same superscripts in the same column are not signicantly different (P≤0.05), n=3
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J. APIC. SCI. Vol. 60 No. 2 2016
24 hours of incubation, respectively (Tab. 4). In
the ndings of Popa & Ustunol (2011), the acetic
acid content was slightly higher: 1.16 and 1.34
g/L, in the control tubes in the same measure-
ment periods.
The increase observed in the probiotic bacterial
counts during the rst 24 hours, and following
stationary growth in the later 24 hours, is
probably related to the consumption rate of
Table 3
Effect of bee pollen and royal jelly on production of D-/L-Lactic acids by
Lb. casei
0 h 24 h 48 h 0 h 24 h 48 h
Control 3.38±0.15a 21.18±0. 5 6 a 21.58±0.31a 3.38±0.15a 21.18±0.56a 21.58±0.31a
Bee Pollen Royal Jelly
10 m g /
100 µL 3.23±0.04a 23.09±0.05b 23.16±0.33b 3.56±0.35a 22.46±0.07b 22.77± 0.20b
25 mg/
250 µL 3 . 40.28 a 2 2 . 9 0 .11c 22. 8 2 ± 0 .15 b 3 . 0 5 ± 0 .14 b 21.53±0.29a 21. 8 0 . 2 6 a
50 mg/
500 µL 3.15±0.34 a 22.74±0. 4 6 c 22.67±0.28b 3.45±0.48a 21.5 0.44a 21.13± 0 .13 a
75 mg/
750 µL 3.60±0.03b 23.78±0.27d 23.86±0.42c 3.17± 0 . 0 7 b 22.43±0.32b 22.27±0. 37b
1000 µL 3.19±0.47a 23.89±0.33d 23.57±0.06c 3.29±0.16b 22.95±0.05b 22.6 0 . 51b
*Means with the same superscripts in the same column are not signicantly different (P≤0.05); n=3
Table 4
Effect of bee pollen and royal jelly on acetic acid production by
Bf. animalis
0 h 24 h 48 h 0 h 24 h 48 h
Control 0.94±0.03a 1.0 0.35 a 1.11± 0 .13a 0.94±0.03a 1.03±0.35a 1.09±0.13a
Bee Pollen Royal Jelly
10 m g /
100 µL 0.69±0.26b 0.85±0.09b 1. 20.16 b 0.77±0.07b 0.92±0.20b 1.13±0 . 0 9a
25 mg/
250 µL 0.87±0.11a 1.17±0.0 6 a 1. 0 8±0.47a 1. 02±0.10 a 0.87±0.25b 0.94±0.22b
50 mg/
500 µL 1.09±0. 4 8 a 1. 2 0.17a 1.32±0.29c 0.96±0.08a 1.19±0.06 c 1. 0 5±0.41a
75 mg/
750 µL 1.13± 0 . 21b 1.46±0.43c 1.35±0.26 c 1.11±0 . 0 5 a 1.22±0.33c 1.17±0.19 a
1000 µL 0.92±0.35a 1.38±0.33c 1.27±0.23c 1.04±0.28a 1.23±0.08c 1.2 0.09c
*Means with the same superscripts in the same column are not signicantly different (P≤0.05), n=3
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Effects of royal jelly and bee pollen on growth of probiotics
the added nutrients by the probiotic bacteria.
The majority of bee pollen and royal jelly which
are rich in terms of nutritious compounds were
probably consumed in the rst 24 hours by
the probiotic bacteria. Even royal jelly or bee
pollen has rich nutrient contents. Most of them
are of simple and small molecules to stimulate
microbial growth. Probably, the chemical compo-
sition of the bee pollen and royal jelly caused
a synergistic effect on the probiotic bacterial
growth. The chemical composition available in
the dairy products is very important in terms of
metabolic activity of probiotic bacteria. Types
and quantities of such nutrients as carbohy-
drates, peptides, and amino acids are signicant
factors in terms of the synergistic effect (Dave
& Shah, 1998; Heller, 2001; Vajiheh, 2012).
On the other hand, bee products also contain
several inhibitor components affecting microbial
growth (Kacainova et al., 2012). The positive
results concerning the symbiotic effects of royal
jelly with
L. acidophilus
Bf. animalis
to produce antioxidant compounds, were
observed by Nabas et al. (2014). Even royal jelly
or bee pollen contains glucose and fructose,
Vitamin B and its derivates, trace elements such
as Fe, Cu, Zn, and Mn. Most them are signicant
food sources for the probiotic bacterial growth.
Haddadin et al. (2012) found that royal jelly
supported the growth of
L. acidophilus
animalis spp. lactis
and obtained about 9.0 log
cfu/ml probiotic counts when 2 and 5% of royal
jelly was added directly to the milk.
That there was an uncertainty concerning the
bee pollen addition on the probiotic bacteria (
L. reuteri, L. rhamnosus
B. lactis
) was also
mentioned by Rosendale et al. (2008). They
stated that bee pollen showed a biphasic
response, and that the effect of bee pollen on
probiotic bacteria was not clear. They also added
that the mechanism on bacterial growth by the
bee pollen was not yet sufciently explained.
But, Yerlikaya (2014) also found a dose-depend-
ent increase in the growth of probiotic bacteria
with the addition of pollen, as was in agreement
with my research.
Controversially, no sugar or sugar source as
honey was added into the growth media in my
research whereas different sugars and honey
were used in the experiment by Popa & Ustunol
(2011). But, the growth stimulation factors
(bee pollen and royal jelly) in my research and
the same broth (MRSL broth containing 5 %
lactose) as the growth media in both research
experiments, were used. As can be seen from
the Tables (1-4), the lactic and acetic acid values
were close, but slightly lower in the control
tubes compared to the ndings by Popa &
Ustunol (2011). This was probably related to the
same broth being used.
Generally, the dose-dependent increase was
observed in the probiotic counts due to the
bee pollen and royal jelly addition. The probiotic
bacterial counts increased less than 1% in most
of the samples between 24 and 48 hours after
the addition of bee pollen and royal jelly. The
bee pollen addition was more effective on the
growth of the investigated probiotic bacteria in
terms of lactic and acetic acid productions. The
incubation time did not make a difference on
the acid production of the investigated probiotic
bacteria between 24 and 48 hours of incubation.
The differences observed in the organic acid
productions, mainly D-/L- Lactic acids, were sig-
nicant between the control and the pollen/
royal jelly added groups (P≤0.05).
The author would like to thank Assoc. Prof. Dr.
Reyhan Irkin from Balikesir University, Engi-
neering Faculty, Food Engineering Department
for her technical assistance and Assist. Prof. Dr.
Serdar Duru from Uludag University, Faculty of
Agriculture, Department of Animal Science for
his support about statistics.
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... This is evidence of the positive effect of RJ on the growth of bacteria until a certain dose. Similar synergistic activity at a given RJ concentration was reported by Guldas (2016) against L. casei, L. acidophilus and B. animalis spp. lactis. ...
... Among tested probiotic bacteria, L. plantarum was the most resistant bacteria with the highest estimated MIC value of 56 mg/mL. Several previous studies evaluated the inhibitory activity of RJ or compounds against mostly pathogenic bacteria (Bílikova et al., 2015;Isidorov et al., 2015;Moselhy et al., 2013) and rarely lactic acid or probiotic bacteria (Guldas, 2016;Haddadin et al., 2012;Nabas et al., 2014). They mentioned that RJ or its derivative components showed higher inhibitory activity against Gram-positive bacteria than Gram-negative bacteria. ...
Royal jelly (RJ) is attractive functional food due to health promoting effect and rich nutrient content. The aim of this study was to produce a probiotic dairy dessert (Keskul) at an optimal RJ dose which is no adverse effect on physicochemical, rheological, sensory properties and functionality of the product. Initially, the growth parameters of Lactobacillus plantarum, Lactobacillus rhamnosus and Bifidobacterium animalis subspp. lactis in presence of RJ (0.001 - 10.0 mg/mL) were evaluated with Gompertz model. Then, the experimental (100 mg/mL) and estimated (56 mg/mL) Minimum Inhibitory Concentration (MIC) of tested probiotic bacteria were determined according to predictive microbiology approach. Among probiotics, L. plantarum was found distinctively proper for producing dairy dessert with RJ. In this study, a new dairy dessert was developed consisting of RJ and L. plantarum. The control, including probiotic (D1), probiotic and RJ (1 mg/mL) (D2), probiotic and RJ (5 mg/mL) (D3) were analyzed during shelf life of dessert. The combination of L. plantarum with RJ (1 mg/mL) provided probiotic properties (>6 log CFU/g) and limited the mold count. Thus, the dairy dessert developed in this study may be a good alternative to innovative functional food using RJ with acceptable sensorial attributes.
... At present, universally accepted biomarkers and preventive strategies of AD are quite limited [214]. Imaging studies state that an extremely long preclinical phase paves the way for the active symptomatic phase of AD. ...
... Therefore, detecting and treating cognitive decline at an early stage is necessary to prevent the development of dementia [204]. Attention has been focused on the use of simple, low-cost interventions to manage modifiable risk factors for AD, such as metabolic dysfunctions (e.g., diabetes mellitus, hypertension, hyperlipidemia, and obesity), sedentary life style, and unhealthy diet [214]. Depressive disorders, especially treatment resistant ones, may represent another AD prevention target. ...
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The Conyza sp. is an annual or short-lived perennial weed of the Asteraceae family. Fructans are linear fructose polymers used in the food industry as a prebiotic nutrient, fat, and sugar substitute and in the pharmaceutical industry as a drug delivery system. The objective of this study was to chemically characterize preliminarily fructans from Conyza sp. roots: fructooligosaccharides (FOS) and inulin-type and evaluated the prebiotic potential activity of these molecules in vitro. Chemical analyses (¹H NMR, ¹³C NMR, GC-MS, FT-IR, and off-line ESI-HRMS) confirmed the presence of the fructans molecules in the aqueous crude extracts of Conyza sp. roots. The study of the prebiotic effect of Conyza sp. fructans in vitro showed an ability to stimulated lactobacilli strains with growth slightly similar (Conyza FOS) and smaller (Conyza inulin) than the commercial prebiotic inulin available (Orafti® GR). These data suggest that Conyza sp. a weed that causes problems agronomic can be used as a promising source to obtain fructans, compounds with potential in the food and technological industries.
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The main purpose of this research was to monitor the influence of the powdered Cyanobacteri-um Spirulina platensis addition to plain yoghurt and the yoghurt containing Lactobacillus acidophilus on survival of the microbiota during the refrigerated storage. The cell viability of yoghurt starter cultures (Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus) and Lactobacillus acidophilus under refrigeration conditions in yoghurts prepared with (0.5 or 1.0 (w/w) %) and without the addition of Spirulina powder was investigated. The yoghurts were prepared under hygienic laboratory conditions and their pH and acidity were controlled during the process. The samples of yoghurts were stored at 4 °C and investigated on days 1, 5, 10,15, 20,25 and 30. Viable counts of the lactic acid bacteria were above 6 cfu g-1 of all "spirulina powder" added samples whereas control yoghurt samples contained lower lactic acid bacteria count at the end of the storage period. Addition of 1 % Spirulina platensis powder into the yoghurts did not cause significant differences on the viable lactic acid bacteria (p≤0.05). The results showed the positive effect of S. platensis powder on the survival of the lactic acid bacteria during storage of yoghurt (P≤0.05). The sensory analysis was also performed for the yoghurt samples. Sensory scores of 0.5 % spirulina powder added yoghurt samples were better than 1 % spirulina powder added ones. It was determined that spirulina powder added yoghurt is a good medium of lactic acid bacteria during the 30 days of refrigerated storage.
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Honey is a natural, sweet substance produced by honey bees Apis millifera. In spite of its antimicrobial properties, honey may contain certain microbes, most of which are harmless to humans. However, the presence of Clostridium botulinum in honey is considered a risk factor for infant botulism development. Infant botulism is a toxicoinfection occurring in infants under the age of one year, after C. botulinum spores consumption. This disease is extremely rare, however, in recent years there has been an increase in the number of infant botulism cases. According to CDC (Centres for Disease Control and Prevention) infant botulism constitutes 76% of all botulism cases and more than half occurs as a consequence of honey consumption. C. botulinum is easily transmitted from soil to blossom, pollen, surface of honey bees and then to honey. The Polish legal system states that any integral components cannot be excluded from honey during processing. However, it is impossible to eliminate the spores from honey without deactivating its enzymes or removing pollen. The EU together with CDC recommend that infants under one year of age should not be fed with honey. They obligate the companies to provide the consumer with the information about the risk of honey consumption by infants. In Poland, infant botulism is not registered, is underestimated and may be misdiagnosed as a sudden infant death syndrome. The producers of honey are not obligated to label honey with the proper information about the microbiological risk of its consumption by infants.
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In this research, effect of bee pollen supplement on antimicrobial, chemical, rheological, sensorial properties and probiotic viability of fermented milk beverages was studied. Bee pollens were added in the rate of 2.5 mg·mL-1 (B), 5 mg·mL-1 (C), 7.5 mg·mL-1 (D), 10 mg·mL-1 (E), and 20 mg·mL-1 (F). Control sample (A) was not supplemented with bee pollen. Control and supplemented milk samples were fermented by a commercial ABT1 starter culture (Chr. Hansen, Hørsholm, Denmark) containing Lactobacillus acidophilus La 5, Bifidobacterium animalis subs. lactis Bb 12, and Streptococcus thermophilus. While no antimicrobial impact was observed against L. monocytogenes, S. aureus, P. fluorescens, P. aeruginosa and A. hydrophilia upto 7.5 mg·mL-1 pollen addition, addition between 10 mg·mL-1 to 20 mg·mL-1 resulted in activity, and positive effect only in inhibition rates against bacteria such as S. thyphimurium and E. coli. Bee pollen supplements has shown a positive effect on probiotic viability and occurred on increase apparent viscosity, but their effect on sensorial properties was negative. Furthermore an improvement with increasing concentration of pollen addition that yielded no negative effect on physicochemical properties was detected.
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Bee pollen is a valuable apitherapeutic product greatly appreciated by the natural medicine because of its potential medical and nutritional applications. It demonstrates a series of actions such as antifungal, antimicrobial, antiviral, anti-inflammatory, hepatoprotective, anticancer immunostimulating, and local analgesic. Its radical scavenging potential has also been reported. Beneficial properties of bee pollen and the validity for their therapeutic use in various pathological condition have been discussed in this study and with the currently known mechanisms, by which bee pollen modulates burn wound healing process.
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The present study was done to evaluate the effect of three different royal jelly samples on the kinetic growth of two isolates of lactic bacteria; Lactobacillus acidophilus and Bifidobacterium bifidum. The results showed that the addition of royal jelly supported and improved the growth of L. acidophilus and B. bifidum. The highest count of L. acidophilus was 9.01 (log 10 cfu/mL) when 2% (w/v) of the royal jelly sample 3 was added to milk. The highest count of B. bifidum was 9.07 (log 10 cfu/mL) when 5% (w/v) of the royal jelly sample 1 was added to milk. Based on the obtained results, royal jelly showed the capability of prebiotic activity and increasing the activity of L. acidophilus and B. bifidum. Royal jelly promotes SCFAs productions which are believed to have an antitumor effect. The results showed the presence of significant synbiotic effect of fermented milk and royal jelly on the intestinal microflora. This effect is translated by the reduction in the faecal enzyme activities of β-glucuronidase, arylsulphatase, and β-gluconsidase which are involved in colon carcinogenesis. © Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences.
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This study was carried out to investigate the antioxidant properties of synbiotic product, Lactobacillus acidophilus supplemented with 2.5% royal jelly in skim milk and Bifidobacterium bifidum supplemented with 7.5% royal jelly in skim milk, using DPPH (1,1-Diphenyl-2-picrylhydrazyl) radical scavenging assay, reducing power, total antioxidant in linoleic acid system and formation of diene-conjugation assay. Results showed that the synbiotic effect of royal jelly and probiotic bacteria provided substantial antioxidant activities. Milk samples fermented by B. bifi dum supplemented with 7.5% royal jelly and L. acidophilus supplemented with 2.5% royal jelly exhibited high scavenging activity with 96.8 and 93.3%, respectively, at a concentration of 500 μg/mL. IC50 values were estimated at 226.7 μg/mL for B. bifidum supplemented with 7.5% royal jelly and at 210.2 μg/ml for L. acidophilus supplemented with 2.5% royal jelly. On the other hand, L. acidophilus supplemented with 2.5% royal jelly and B. bifidum supplemented with 7.5% royal jelly exhibited significantly high reducing power at a concentration of 1000 μg/mL. The percentages of peroxide inhibition of L. acidophilus supplemented with 2.5% royal jelly and B. bifidum with 7.5% royal jelly were 52% and 42%, respectively. Significant inhibitions were found in the formation of conjugated diene at 66.9% and 65.8% for L. acidophilus with 2.5% royal jelly and B. bifidum with 7.5% royal jelly, respectively. These results were compared with standards BHT, ascorbic acid and Trolox.
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Pollen samples collected in the spring of 2002 in 8 south-western Slovakia localities and 40 live individuals of bumblebee were analyzed for the presence of bacteria and microscopic fungi. Microorganisms occurring on pollen and bumblebees were identified using cultivation and microscopic methods supplemented with biochemical tests. In the pollen were found fermenting and non-fermenting Gram-negative rods, Gram-positive sporulating cocci and non-haemolytic Gram-positive cocci. Analyses of microscopic fungi on bumblebee bodies showed the presence of only four species -Acremonium murorum, Aspergillus penicilloides, Fusarium oxysporum, Harpografium fasciculatum representing Fungi imperfecti. The highest amount of microscopic fungi occurred on drone, lower numbers on queen and worker bees. In the pollen samples 21 fungal species forming 13 genera of microscopic fungi were detected. The highest number of mould species was classified in the genera of Mucor, Rhizopus, Aspergillus, Alternaria and Paecilomyces, the species of other genera occurred in lower frequency. As the majority of the identified micromycetes represent the mitosporic fungal group of saprophytic microorganisms inhabiting soil or the organic residues of plants lacking pathogenic effects, it could be concluded, the tested pollen samples may be declared for safe resource of food and/or feed.
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The effects of Allium sativum and Cinnamomum verum water extracts on the survival of Bifidobacterium bifidum during 21 days of refrigerated storage and after simulated gastrointestinal digestion (SGD) were investigated. Two types of yogurt (cow- and camel-milk yogurts) were prepared in the presence of A. sativum or C. verum. The viable cell counts (VCC) of B. bifidum in fresh A. sativum- or C. verum-cow milk yogurt (1 day) were higher (8.1 × 109 cfu/ml and 6.6 × 109 cfu/ml, respectively; p < 0.05) than plain-yogurt (1.9 × 109 cfu/ml). In contrast, B. bifidum VCC in fresh plain-camel milk yogurt was 1.99 × 109 cfu/ml whereas the presence of A. sativum or C. verum in yogurt increased (p < 0.05) VCC to 19.61 × 109 cfu/ml and 25.55 × 109 cfu/ml, respectively. The VCC of B. bifidum in both herbal-yogurts decreased (p < 0.05) during refrigerated storage for both types of yogurt. The VCC of B. bifidum was ∼1.3 × 109 cfu/ml in all fresh cow milk yogurts after 1 h gastric digestion. Intestinal digestion (1 h) increased VCC of B. bifidum in all fresh yogurts but not in 7 day old yogurts (plain- and A. sativum-yogurts). However, prolonged digestion to another 1 h in intestine reduced (p < 0.05) VCC of B. bifidum in all fresh and storage yogurts. In contrast, all fresh camel milk yogurts showed VCC of B. bifidum ⩽1 × 109 cfu/ml after SGD. Seven day old A. sativum – camel milk yogurt showed the lowest survival of B. bifidum after gastric digestion compared to plain- and C. verum-yogurt. The VCC reduced (p < 0.05) in all camel milk-yogurts after 2 h intestinal digestion. In conclusion, A. sativum or C. verum water extract enhanced the growth of B. bifidum in both types of yogurt during refrigerated storage. However, these herbs did not influence B. bifidum survival after SGD.