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doi:10.2141/ jpsa.0130137
Copyright Ⓒ 2014, Japan Poultry Science Association.
Influence of Propolis Residue on the Bacterial Flora
in the Cecum of Nanbu Kashiwa
Kazumi Kita1, Ito R. Ken1, Chinami Akamine1, Wataru Kawada2,
Yoichiro Shimura2 and Tamio Inamoto2
1
2
Faculty of Agriculture, Iwate University, Morioka, Iwate 020-8550, Japan
Faculty of Bioresource Sciences, Akita Prefectural University, Shimoshinjo, Akita 010-0195, Japan
Since the growth promoter effect of antibiotics was found, many kinds of antibiotics have been used as feed
additives for the improvement of growth performance. Since January 1, 2006, however, European Parliament
prohibited the use of antibiotics as feed additives for promoting animal growth because of biosecurity threats arising
from the increasing resistance of pathogens to antibiotics. Thereafter, various natural substrates having the growth
promoter effect have been attempted to use as feed additives instead of antibiotics. Honeybees collect propolis from
the cracks in the bark of trees, and it has the versatile pharmacological activities including antibacterial effect.
Therefore, the present study aimed to examine the influence of propolis residue, which was the residue after ethanol
extraction, on the cecal bacterial flora in the meat-type chicken, Nanbu Kashiwa. Twenty-eight-d-old female Nanbu
Kashiwa were given an ordinary diet for 90 days. During last 10 days, an experimental diet including 2% of propolis
residue was provided. At the end of experimental period, ceca were removed and DNA was isolated from cecal
content. The V3 region of the bacterial 16S rRNA gene was amplified by PCR using the 357F-GC and 518R primers.
To indentify the bacterial diversity, denaturing gradient gel electrophoresis (DGGE) was applied, and the optical
intensity of DGGE bands was determined. The major bands were excised, and DNA was reamplified by PCR. The
sequencing data were analyzed by the Basic Local Alignment Search Tool (BLAST) of DNA Data Bank of Japan.
The bacteria identified by PCR-DGGE and BLAST were Lactobacillus aviaries, Lactobacillus, Olsenella,
Coriobacterium, Paraprevotella, Prevotellaceae, Clostridiaceae and Bacteroidaceae. The DGGE band of which the
optical intensity of the propolis residue group was lower than the control group was the family Prevotellaceae. The
successful use of propolis residue to reduce the family Prevotellaceae showed the possibility to use propolis residue as
alternative feed additives.
Key words: 16S rRNA, bacterial flora, cecum, denaturing gradient gel electrophoresis, Nanbu Kashiwa, propolis
residue
J. Poult. Sci., 51: 275-280, 2014
Introduction
In 1940’s, the growth promoter effect of antibiotics was
observed in animals fed dried mycelia of Streptomyces
aureofaciens containing chlortetracycline, and the mechanism of action of antibiotics as growth promoters is related to
interactions with intestinal microbial population (Dibner and
Richards, 2005; Niewold, 2007). Thereafter, many kinds of
antibiotics have been used as feed additives for the
improvement of growth performance. However, contemporary biosecurity threats arising from the increasing resistance
of pathogens to antibiotics elicited a call for the ban of
Received: July 16, 2013, Accepted: October 18, 2013
Released Online Advance Publication: November 25, 2013
Correspondence: Dr. K. Kita, Faculty of Agriculture, Iwate University,
Morioka, Iwate 020-8550, Japan. (E-mail: [email protected])
antibiotic growth promoters (Barton, 2000; Snel et al.,
2002). In 2003, Regulation 1831/2003 on additives for use
in animal nutrition was stated by the European Parliament.
This regulation stated that antibiotics used as feed additives
for promoting animal growth will have been prohibited until
December 31, 2005 (Castanon, 2007). Thereafter, various
natural substrates having the growth promoter effect have
been attempted to use as feed additives instead of antibiotics.
Honeybees (Apis mellifera L.) collect the resin from the
cracks in the bark of trees and leaf buds. The resin is called
propolis, sometimes also referred to bee glue. Propolis is
defined by U.S. National Library of Medicine (2013) as a
resinous substance obtained from beehives that is used
traditionally as an antimicrobial and that is a heterogeneous
mixture of many substances. Because of its popularity in
folk medicine, propolis has become the subject of intense
Journal of Poultry Science, 51 (3)
276
pharmacological and chemical studies so far. A large number of studies have proven its versatile pharmacological
activities: antibacterial, antifungal, antiviral, antioxidant,
anticancer and anti-inflammatory activities (Grange and
Davey, 1990; Kujumgiev et al., 1999; Lotfy, 2006; Sforcin,
2007; Viuda-Martos et al., 2008; Mavri et al., 2012;
Sawicka et al., 2012). However, although it was reported
that the inclusion of propolis in poultry feeds affected some
hematological parameters, i.e. erythrocyte count, IgG concentration and IgM concentration (Cetin et al., 2010), the
influence of dietary propolis on bacterial flora in the cecum
has not been examined so far. Moreover, in most studies,
ethanol extract was used as propolis, and the antibacterial
effect of propolis residue after ethanol extraction has not
been investigated.
The PCR-DGGE analysis was employed to classify the
bacteria in the cecum of chickens. The DGGE is an
electrophoretic method to identify single base change in a
segment of DNA. In a denaturing gradient acrylamide gel,
double-strand DNA is subjected to an increasing denaturant
environment and will melt in discrete segment called melting
domeins. The melting temperature of these domeins is sequence specific. When the melting temperature of the lowest
melting domein is reached, the DNA will become partially
melted, creating branched molecules. Partial melting of the
DNA reduces its mobility in a polyacrylamide gel. Since the
melting temperature of a particular melting domein is sequence-specific, DNA containing different sequences will
encounter mobility shifts at different position in the gel.
In the present study, therefore, the influence of propolis
residue after ethanol extraction in poultry feeds on cecal
bacterial flora in meat-type chickens was examined by PCRDGGE.
Materials and Methods
Animals and Experimental Diets
Twenty-eight-d-old female meat-type chickens ‘Nanbu
Kashiwa’ ((Rhode Island Red×White Plymouth Rock) ×
(Game Fowl× (Colored Cornish×Iwate Native Fowl))) was
purchased from a prefectural research center (Animal Industry Research Institute, Iwate Agricultural Research
Center, Takizawa, Iwate, Japan), and they were given a diet
for meat-type chickens (grain 65%, oil seed meals 21%, fish
meal 6%, others 8%; CP 18%, ME 3,300 kcal/kg) for 90
days. Chickens were allowed free access to water. During
last 10 days of experimental period, an experimental diet
including propolis residue was fed to 9 chickens. Propolis
residue (Kakudate Apiary, Inc., Hachimantai, Iwate, Japan)
was added to the diet at the level of 2%. Propolis residue
used in the present study was immersed in edible alcohol
(ethanol) for 2 years and the residue after alcohol extraction
was used. As a control, the same feed for meat-type
chickens was fed to 9 birds. All chickens were raised in a
rural farm (Ludens Farm Inc., Nishine, Iwate, Japan). At the
end of experimental period, ceca were removed and stored at
−30℃ until analysis. Animal care was in compliance with
applicable guidelines from the Iwate University Animal Care
and Use Committee.
Linearity of PCR-amplified DNA to Bacteria Quantity
In the present study, PCR-amplified DNA of cecal bacteria
was supposed to be quantified by using a commercial image
analyzer (Gel-Pro Analyzer, version 3.1, Nippon Roper Co.
Ltd., Tokyo, Japan). To confirm the linearity of PCRamplified DNA to bacteria quantity, DNA purified from
Campylobacter jejuni was amplified by PCR and quantified
by GelPro Analyzer.
Campylobacter jejuni were incubated at 40℃ for 48 h in
micro anaerobic nutrient agar called by brain heart infusion
agar (PEALCORE®, Eiken Chemical Co., Ltd., Tochigi,
Japan) including defibrinated horse blood (Nippon Bio-Test
Laboratories Co., Ltd., Tokyo, Japan) and Campylobacter
selective supplement (Butzler) (SR0085E, Oxoid Co., Ltd.,
Basingstoke, Hants, England). Micro anaerobic nutrient agar
plates were maintained in a BBLTM GasPak 100TM
Anaerobic System (Becton Dickinson Co., Ltd., Franklin
Lakes, NJ. USA) with AnaeroPack®-MicroAero (Mistubishi
gas chemical Co., Ltd., Tokyo, Japan). To collect campylobacter, 4 mL of Dulbecco’s phosphate-buffered saline
(DPBS, Gibco®, Life Technologies Japan Ltd., Tokyo,
Japan) was put onto agar plates, and 3 mL of suspension
containing campylobacter was recovered. The absorbance of
campylobacter suspension was measured at 600 nm, and the
suspension was diluted with DPBS at the level of 0.05, 0.1,
0.2, 0.4, and 0.8 of absorbance (600 nm). DNA was extracted from suspension by using ISOPLANT II DNA
extraction kit (Nippon Gene Co. Ltd., Tokyo, Japan) in
accordance with the manufacturer’s protocol. Total DNA
was dissolved in 200 μL of TE (10 mM Tris-HCl (pH 7.5), 1
mM EDTA) buffer. Using the extracted bacterial DNA, 16S
rRNA gene amplification was performed by PCR using the
following primers to amplify the V3 region of the bacterial
16S rRNA gene: 357F-GC, 5′
-CGC CCG CCG CGC GCG
GCG GGC GGG GCG GGG GCA CGG GGG GCC TAC
GGG AGG CAG CAG-3′
; 357F primer was used to add a
40-nucleotide GC rich sequence at the 5′end) and 518R (5′
GTA TTA CCG CGG CTG CTG G-3′
; Muyzer et al., 1993).
The concentration of each primer was 10 pmol/μl. The 20 μl
using GoTaq Green Master Mix (Promega, Madison, WI,
USA) was mixed with 2 μl of primer solution and 5 μl of
bacterial DNA solution (50 μg/ml). The PCR reactions were
performed under the following conditions: 94℃ for 4 min;
20 cycles of 94℃ for 0.5 min, 65-56℃ for 0.5 min, and 72℃
for 0.5 min, with a stepwise decrease of 1℃ after every
second cycle; 10 cycles at 94℃ for 0.5 min, 55℃ for 0.5
min, and 72℃ for 1 min; 1 cycle at 72℃ for 4 min, and 4℃
for storage. The presence and yield of PCR products were
assessed using agarose gel electrophoresis. After PCR,
optical density of amplified DNA bands was measured by
Gel-Pro Analyzer. The regression equation of optical intensity of PCR-amplified DNA bands to the absorbance at
600 nm was calculated using the General Linear Model
Procedures of SAS (SAS/STAT version 6) (SAS Institute,
1999).
Kita et al.: Propolis Residue and Cecal Bacterial Flora
PCR-DGGE of Cecal Bacteria Flora of Chickens Given
Dietary Propolis Residue
Before isolation of total DNA from cecal content, less than
1 g of cecal content was washed by passing through a strong
nylon mesh (Cell strainers, 100 μm mesh, BD Falcon®, Japan
BD Co. Ltd., Fukushima, Japan) with DPBS. After passing
through the mesh, samples were centrifuged at 3, 300×g,
4℃ for 10 min. The supernatant was discarded, and the pellet was suspended in 35 ml of DPBS. The suspension was
centrifuged, and the supernatant was discarded. The pellet
was suspended in 35 ml of TE buffer and centrifuged. After
discarding the supernatant, the pellet was suspended in 10 ml
of TE buffer. The 1 ml aliquots of suspension were centrifuged at 9,300×g , 4℃ for 10 min, and the supernatant was
discarded. Washed cecal content was stored at −80℃.
Total DNA was extracted from washed cecal content by
using ISOPLANT II DNA extraction kit in accordance with
the manufacturer’s protocol. Total DNA was dissolved in
200 μL of TE buffer.
Using the extracted bacterial DNA, 16S rRNA gene amplification was performed by PCR as described above. The
primers to amplify the V3 region of the bacterial 16S rRNA
gene were 357F-GC and 518R. The presence and yield of
PCR products were assessed using agarose gel electrophoresis.
In the present study, to indentify the bacterial diversity,
denaturing gradient gel electrophoresis (DGGE) was applied.
The DGGE analysis was conducted in accordance with the
manufacturer’s protocol of The DCodeTM Universal Mutation Detection System (Bio-Rad, Hercules, CA, USA). Polyacrylamide gel (8%) containing gradients of 20 to 60%
denaturant (100% denaturant corresponds to 7 M urea and
40% deionized formamide) was prepared using a gradient
maker (Model 475 Gradient Delivery System, Bio-Rad).
The gel to which DNA samples (5 μL of PCR reactant and 2
μL of 70% glycerol in each lane) was applied was run for 5 h
at 130 V at 60℃ in 1× TAE (40 mM Tris-acetate, 1 mM
EDTA; pH 8.0) buffer. DGGE marker II (Nippon Gene) was
used as a marker. After electrophoresis, the gel was stained
with SYBR Gold (Invitrogen, Carlsbad, CA, USA) nucleic
acid gel stain, and band images were detected using a luminoimage analyzer (LAS-4000, FUJIFILM Co. Ltd., Tokyo,
Japan). The optical intensity of DGGE bands was analyzed
by using a commercial image analyzer.
The major bands were excised with a sterile razor, and the
DNA was eluted by incubating a gel slice in TE buffer at 4℃
overnight. The eluted DNA was purified using Wizard SV
Gel and a PCR Clean-UP System (Promega), and then used
as a template for the following PCR reamplification with
primers 357F (5′
-CCT ACG GGA GGC AGC AG-3′
) and
518R under the conditions described above. The reamplified
16S rRNA gene was ligated into the pGEM-T Easy Vector
(Promega), and then the plasmids were introduced into
Escherichia coli DH5α. The 16S rRNA gene on the purified
plasmids was sequenced using primers M13F and M13R
(Yanisch-Perron et al., 1985) with the BigDye Terminator
Cycle Sequencing Kit (Applied Biosystems, Foster City, CA,
277
USA) according to the manufacturer’s instructions. Sequences were then analyzed using an Applied Biosystems
3100 Genetic Analyzer. The sequencing data were analyzed
by BLAST of DDBJ (http://blast.ddbj.nig.ac.jp/blastn?lang=
ja) on 16S rRNA gene for Bacteria and Archeae database.
Statistical Analyses
Results are expressed as means±SE. Statistical analysis
of data was performed by one-way ANOVA followed by
student’s t-test using the General Linear Model Procedures of
SAS (SAS/STAT version 6) (SAS Institute, 1989).
Results
Linearity of PCR-amplified DNA to Bacteria Quantity
The regression equation of optical intensity of PCRamplified DNA bands (Y) to the absorbance at 600 nm (X)
was Y＝78.1X＋96.1 (Fig. 1). The absorbance at 600 nm of
the suspension of Campylobacter jejuni was significantly and
linearly related to the optical intensity of PCR-amplified
DNA bands.
PCR-DGGE of Cecal Bacteria Flora of Chickens Given
Dietary Propolis Residue
PCR-DGGE profiles of 16S rRNA gene fragments derived
from bacterial flora in the cecum of meat-type chickens
(Nanbu Kashiwa) fed a diet containing propolis residue were
represented in Fig. 2. The DGGE bands with arrows and
numbers had the significant difference in the optical intensity
of bands between propolis residue and control groups. The
values for optical intensity of DGGE bands of 16S rRNA
bacterial gene with significant difference between propolis
residue and control groups are shown in Table 1. The optical
intensity of 2 bands (band No. 1 and 3) of chickens fed a diet
containing propolis residue was higher than that of birds fed
a control diet. On the other hand, the optical intensity of a
band (band No. 2) of chickens given the control diet was
higher than that of birds given propolis residue.
The sequencing data of 16S rRNA gene were analyzed by
a BLAST search of NCBI database (16S ribosomal RNA
Fig. 1. The regression equation of optical intensity of
PCR-amplified DNA bands (Y) to the absorbance at 600
nm (X). Circles are means±SE. n＝3 (one missing value
at the lever of 0.8 of absorbance).
Journal of Poultry Science, 51 (3)
278
PCR-DGGE profiles of 16S rRNA gene fragments derived
from bacterial flora in the cecum of chickens (Nanbu Kashiwa) fed a
diet containing propolis residue. DNA samples were extracted from 9
samples of cecum content. The 16S rRNA gene fragment was amplified
by universal PCR primer sets (forward, 357F-GC; reverse, 518R). Six
samples were randomly selected from 9 samples of PCR products. The
DGGE bands with arrows and numbers had the significant difference in
the optical intensity analyzed by an image analyzer (GelPro analyzer)
between propolis residue and control groups.
Fig. 2.
The optical intensity of DGGE bands of 16S
rRNA gene of bacteria in the cecum of meat-type
chickens (Nanbu Kashiwa) fed a diet containing propolis residue
Table 1.
DGGE bands
1
2
3
Dietary propolis residue level
0%
2%
32 . 0b,*±7 . 0
42 . 7a±6 . 9
4 . 4b±1 . 3
181 . 8a,*±25
22 . 4b,*±4 . 7
18 . 5a,*±3 . 7
The values were the optical intensity measured by a commercial
image analyzer (GelPro Analyzer) with the significant difference
between propolis residue and control groups. Means not sharing a
common superscript letters within the same row are significantly
different at P＜0.05. Values are means±SE from 6 chickens in
each group. * One missing value.
gene (Bacteria and Archea)), and when the sequence was
identical to the database at the level of more than 95% and
90-95%, the rank was regarded as family and genus,
respectively.
DNA sequencing results of DGGE bands were deposited
in DDBJ database under accession numbers AB817937
(uncultured Lactobacillus sp.), AB817938 (uncultured Bacteroidaceae bacterium), AB817939 (uncultured Lactobacillus sp.), AB817940 (uncultured Clostridiaceae bacterium),
AB817941 (uncultured Lactobacillus sp.), AB817942
(uncultured Prevotellaceae bacterium), AB817943 (uncul-
Sequence analysis of 16S rRNA gene of bacteria in the cecum of meat-type chickens (Nanbu
Kashiwa) given dietary propolis residue
Table 2.
DGGE bands
Closet relative
Rank
Identity (%)
1
2
3
Clostridiaceae
Prevotellaceae
Coriobacterium
family
family
genus
92
93
97
The sequencing data were analyzed by a Blast search of Data Bank
of Japan (DDBJ). When the bacterial sequence was identical to the
database at the level of more than 95% and 90-95%, the rank was
regarded as family and genus, respectively.
tured Clostridium sp.), AB817944 (uncultured Olsenella
sp.), AB817945 (uncultured Olsenella sp.), AB817946
(uncultured Coriobacterium sp.), AB817947 (uncultured
Bacteroidaceae bacterium), AB817948 (uncultured Paraprevotella sp.), AB817949 (uncultured Bacteroidaceae
bacterium), AB817950 (uncultured Prevotellaceae bacterium), AB817951 (uncultured Bacteroidaceae bacterium).
The bacteria identified in the present study were Lactobacillus aviaries (identity 100%), Lactobacillus (identity
100%), Olsenella (identity 99%), Coriobacterium (identity
97%), Paraprevotella (identity 96%), Prevotellaceae (identity 93%), Clostridiaceae (identity 92%) and Bacteroidaceae
(identity 92%). Sequence analysis of 16S rRNA gene with
significant difference in the optical intensity of DGGE bands
between propolis residue and control groups is shown in
Kita et al.: Propolis Residue and Cecal Bacterial Flora
Table 2. Two bands (band No. 1 and 3) of which the optical
intensity of the propolis residue group was higher than that of
chickens fed a control diet were identified to the genus
Coriobacteriumand the family Clostridiaceae, respectively.
On the other hand, a band (band No. 2) of which the optical
intensity of the propolis residue groups was lower than that
of the control group was the family Prevotellaceae.
Discussion
As represented in Fig. 2, the regression equation of optical
intensity of PCR bands (Y) to the absorbance at 600 nm (X)
was calculated as Y＝78.1X＋96.1. This equation confirmed that PCR-DGGE followed by the measurement of
optical intensity of amplified DNA bands using GelPro
Analyzer can be a tool to quantify the amount of cecal bacteria of chickens.
The PCR-DGGE analysis followed by DNA sequence
analysis and BLAST search revealed the presence of various
types of bacteria, i. e. Lactobacillus, Olsenella, Coriobacterium, Paraprevotella, Prevotellaceae, Clostridiaceae and
Bacteroidaceae. The presence of Lactobacillus, Clostridiaceae and Bacteroidaceae in the chicken was also reported
by other researchers (Geier et al., 2009a, b; Lumpkins et al.,
2010), which was in good agreement to our observation.
Since the PCR-DGGE analysis was developed by Muyzer
et al. (1993) using a 16S rRNA gene of bacteria, this method
has been beneficially used in the field of poultry science. At
the beginning, the DGGE analysis was applied to examine
the characteristics of bacteria. Johnston and Fernando
(1995) analyzed the genetic variability between species and
strains of Eimeria spp. This method was also applied to
evaluate the subtype of Campylobacter jejuni isolated from
poultry (Nielsen et al., 2000) and to examine the influence of
growth on the spatial variation of the intestinal bacterial
community of broiler chickens (Van der Wielen et al., 2002).
Thereafter, the influence of dietary manipulation (Thompson
et al., 2008; Geier et al., 2009a, b; Janczyk et al., 2009;
Nava et al., 2009) and feed additives like antibiotics
(Knarreborg et al., 2002; Pedroso et al., 2006; Zhou et al.,
2007) on the microbial flora in the intestine of chickens was
studied. In the present study, propolis residue was used as
feed additives to anticipate the antibacterial effect against
intestinal microbial flora. As expected, dietary propolis residue successfully reduced the family Prenvotellaceae (Tables
1 and 2). The successful use of propolis residue to reduce the
family Prevotellaceae showed the possibility to use propolis
residue as alternative feed additives.
As represented in Tables 1 and 2, in contradiction to
Prevotellaceae, gram-possitive bacteria Clostridiaceae and
Coliobacterium were significantly increased by dietary
propolis residue additive treatment. As Kačániová et al.
(2012) reported that feeding of propolis to chickens increased
enterococcus and lactobacillus in excreta, propolis residue
seems to have the potency to increase the growth of some
kinds of bacteria.
Boyanova et al. (2006) reported that Bulgarian propolis
was highly active against the anaerobic bacteria including
279
Prevotellaceae. As shown in Tables 1 and 2, the antibacterial effect against the family Prevotellaceae was also
found in the propolis residue collected from Iwate prefecture
in Japan. Interestingly, in spite of the great differences in the
chemical composition of propolis from different geographic
locations, all samples exhibited similar antibacterial activity
(Kujumgiev et al., 1999), and this finding points out that
analyses of chemical composition of propolis to identify one
individual substance or a particular substance class could not
be responsible for the antibacterial activity (Cetin et al.,
2010). However, the propolis residue used in the present
study was collected in the region of ‘Hachimantai’ of Iwate
prefecture of Japan. In this area, the prior vegetation is
Japanese Beech, in which several antibiotics have been found
(Taguri et al., 2004, 2006). Therefore, some kinds of
antibacterial in Japanese Beech seem to be affective to the
bacteria growth, and this issue needed to be elucidated in the
future.
Acknowledgmetns
This study was financially supported by A-STEP (Adaptable & Seamless Technology Transfer Program through
Target-driven R&D) at the feasibility study (FS) stage of
Japan Science and Technology Agency. Technical support
was provided by Nikunoyokosawa Co. Ltd. (Hachimantai,
Iwate, Japan). DNA sequencing was supported by Biotechnology Center in Faculty of Bioresource Science at Akita
Prefectural University.
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