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Michigan State University, Department of Food Science and Human Nutrition, E. Lansing, MI 48824-1224, USA
Received 21 June 2004; accepted 11 January 2005
Abstract
Five human intestinal Bidobacterium spp. (B. longum, B. adolescentis, B. breve, B. bidum, and B. infantis) and intestinal microorganism (Bacteriodes thetaiotaomicron, Clostridium perfringens, Eubacterium aerofaciens, and Enterococcus faecalis) were cultured
in De Man Rogosa Sharpe (MRS) medium or thioglycollate medium supplemented (5% w/v) with dierent unioral honeys (sourwood, alfalfa, or sage). Inoculated samples were incubated anaerobically at 37 C for 48 h. Samples were collected at 12 h intervals
and examined for specic growth rate. Levels of fermentation end products (lactic and acetic acids) produced by Bidobacterium
spp. were measured by high-pressure liquid chromatography. Growth of intestinal microorganisms co-cultured with Bidobacterium
spp. in the presence of dierent unioral honeys were also examined. All three honeys enhanced (p < 0.05) growth and activity of the
ve intestinal Bidobacterium spp. Their eect on other organisms of the intestinal microora was selective. Growth of C. perfringens and E. aerofaciens was inhibited (p < 0.05) in the presence of honey and further inhibited when co-cultured with Bidobacterium
spp. Bidobacterium spp. was not aected.
2005 Elsevier Ltd. All rights reserved.
Keywords: Bidobacteria; Intestinal microora; Honey; Growth
1. Introduction
The gastrointestinal (GI) microora is increasingly
being recognized as playing a key role in health and disease (Tannock, 2002). The GI microora is in a dynamic
equilibrium that may be altered by diet, medication,
stress, aging and various other environmental factors.
Over the past 10 years a variety of non-digestible carbohydrates have been identied and reported to modify the
balance of the GI microora by stimulating the growth
and/or activity of benecial microorganisms such as bif-
idobacteria and lactobacilli and suppressing the potentially deleterious bacteria (Kolida, Tuohy, & Gibson,
2002; Sanders, 1993). These non-digestible carbohydrates, termed prebiotics include lactulose, lactitol, a
variety of oligosaccharides and inulin (Crittenden,
1999). Commercially produced oligosaccharides are typically derived by enzyme catalyzed processes from a
variety of carbohydrates sources. These oligosaccharides
are not pure preparations, but a mixture of mono-,
di- and oligo-saccharides (Crittenden, 1999; Playne &
Crittenden, 1996). Non-digestible oligosaccharides also
occur naturally in foods such as fruits, vegetables, milk
and honey (Tannock, 1999).
Honey is a natural syrup containing primarily fructose (38.5%) and glucose (31.3%). Other sugars in honey
include maltose (7.2%), sucrose (1.5%) and a variety
722
of oligosaccharides (4.2%) (NHB, 1996). The oligosaccharides in honey vary in composition as well as degree of polymerization (DP). These oligosaccharides
result from the action of honeybee a-D-glucosidase,
which catalyzes the transfer of a-D-glucopyranosyl
groups from sucrose to an acceptor carbohydrate,
which results in fructooligosaccharides and a variety
of other oligosaccharides in varying amounts. Carbohydrate composition and content of honey is variable
and is dependent on the oral source of the honey
(Swallow & Low, 1990; Weston & Brocklebank,
1999). Use of sugar proles such as fructose, glucose,
sucrose, maltose contents, and glucose/water ratios
has been suggested for characterization of unioral
honeys (Mateo & Bosch-Reig, 1998). Dacosta-Leite
et al. (2000) suggested that the complex mixture of oligosaccharides in honey may be useful in determining
its oral origin.
Chick, Shin, and Ustunol (2001), Kajiwara, Gandhi,
and Ustunol (2002) and Ustunol and Gandhi (2001) reported on the ability of honey to enhance growth and
activity of commercially used bidobacteria in milk
and intestinal bidobacteria in vitro and suggested this
may be due to the unique carbohydrate composition
and complex mixture of oligosaccharides present in
honey. The average pH of honey is 3.9. Honey contains a variety of organic acids such as acetic, butyric,
citric, formic, gluconic, lactic, malic, pyroglutamic and
succinic acid (0.171.17%), which contribute to the
honeys low pH (NHB, 1996). Inhibitory properties
of honey against various pathogens have been demonstrated (Molan, 1992a, 1992b; Somal, Coley, Molan, &
Hancock, 1994). Honey may suppress the potentially
deleterious bacteria that are present among the gastrointestinal (GI) microora. Thus, it is possible that honey
may provide for the benecial management of the GI
microora.
However, these studies with honey cited above are
pure culture in vitro studies. Pure culture in vitro studies
are useful in comparative evaluations of metabolism and
are valuable in doing mechanistic studies, they do not
take into account the microbial competition that is present in the GI tract. The GI microora is made up of
400 known species (Tannock, 1999). Mixed culture
in vitro studies are important and necessary in determining the selectivity of fermentation of a particular
substrate.
Inuence of naturally occurring oligosaccharides
(found naturally in foods) on intestinal microora has
not been extensively studied and reported. The purpose
of this study was to investigate the carbohydrate composition of honey from dierent oral sources (sourwood,
alfalfa and sage). Furthermore, to determine in vitro the
eect of these honeys on growth and activity of bidobacteria and other predominant bacteria that make up
the intestinal microora to gain a better insight as to
and 0.1 g sodium sulte. Cultures were centrifuged at 4350g for 15 min at 4 C. Harvested cells were
washed twice with saline solution (0.13 M NaCl, pH 6.8)
and suspended in phosphate buer (0.01 M, pH 7) to
obtain 108 CFU/ml.
2.3. Growth determination
Propagation of Bidobacterium spp. was in MRS
medium (Difco) and thioglycollate medium (Difco)
was used to grow the other intestinal bacteria used in
this study. Sourwood, alfalfa, or sage honey was added
at 5% (w/v) level to MRS or thioglycollate media. The
mixtures were lter-sterilized with 0.45 lm lter (Millipore Corporation, Bedford, MA, USA). Control treatments had no honey added. Each solution was divided
into ve portions, MRS medium was inoculated with
Bidobacterium spp. and thioglycollate medium was
inoculated with the other intestinal organisms at a 2%
(v/v) level with the propagated cultures as described
above. Inoculated tubes were incubated anaerobically
at 37 C for 48 h. One gram of sample was withdrawn
every 12 h, mixed for 15 s using a vortex (Janke and
Kunkel, Stauten, Germany) and turbidity was measured
at 610 nm. Uninoculated sterilized medium was used as
the blank. Specic growth rate (l) for each organism
grown in dierent honeys was calculated using the following equation:
l ln X 2 ln X 1 =t1 t2 ;
where X2 and X1 are the cell densities at time t2 and t1,
respectively. Doubling time (Td) was calculated as
Td = ln 2/l (Desjardins, Roy, & Goulet, 1991).
2.4. Lactic and acetic acid determination
High performance liquid chromatography (HPLC)
was used to determine lactic and acetic acid production
by human intestinal Bidobacterium spp. grown in the
presence of dierent unioral honeys. Each strain
B. longum, B. adolescentis, B. breve, B. bidum, and
B. infantis were grown for 48 h in MRS medium containing sourwood, alfalfa, and sage honeys or no honey
added controls, as described previously. Samples were
prepared for HPLC analysis using the method described by Dubey and Mistry (1996). One hundred
microliters of 15.8 N HNO3 and 14.9 ml of 0.009 N
H2SO4 were added to 1.5 ml of each sample. The samples were centrifuged at 5000g for 10 min. The supernatant was ltered using 0.22 lm lter (Millipore) and
eluted through a reverse phase Supelclean tube (Supelco Inc., Bellefonte, PA, USA), and stored in HPLC
vials at 20 C until analysis. The HPLC system
(Waters Associates, Inc., Milford, MA, USA) consisted
of 1525 binary HPLC pump, 2487 dual k absorbance
detector, 717 plus autosampler. An Aminex ion exclu-
723
724
Sourwood
Alfalfa
Sage
Moisture
Fructose
Glucose
Maltose
Sucrose
Oligosaccharides
Ash
6.9 0.1
35.7 4.1
33.3 3.3
9.8 1.5
3.1 0.4
10.9 1.1
0.3 0.1
7.7 0.1
38.4 4.3
35.1 6.1
10.2 1.8
2.7 0.3
5.5 1.0
0.3 0.1
6.3 0.2
38.9 3.6
37.5 5.3
11.7 1.4
1.6 0.2
3.8 0.6
0.3 0
the honeys studied varied their total carbohydrate contents were similar.
Table 2 shows the Td value of each intestinal bidobacteria strain grown in the presence of dierent unioral honeys and the unsupplemented control. A 5%
honey level was selected because in our previous studies
this was determined to be the optimum level in stimulating growth and activity of commercially used bidobacteria as well as human intestinal bidobacteria (Chick
et al., 2001; Kajiwara et al., 2002; Ustunol & Gandhi,
2001). All three honey types enhanced (p < 0.05) the
growth of the ve Bidobacterium spp. compared to
the unsupplemented control as evidenced by the shorter
Tds observed. However, there were no dierences among
the honeys from dierent oral sources. Shortest Td was
observed after 24 h for all ve Bidobacterium spp.
including when grown in the unsupplemented control.
However, at 36 and 48 h of incubation, Td was longer
(p < 0.05) for all Bidobacterium spp. grown in the
unsupplemented control compared to those grown in
the honey supplemented medium suggesting that the cultures reached a stationary phase in the control media.
The dierent unioral honeys were particularly eective
in enhancing (p < 0.05) the growth of Bidobacterium
spp. after 24 h of incubation. These results are consistent
with our previous results in which the Tds of intestinal
Bidobacterium spp. After 48 h of incubation were distinctively shorter when grown in reinforced clostridial
medium supplemented with 5% clover honey compared
Table 2
Doubling time (Td) of intestinal Bidobacterium spp. grown in the presence of dierent unioral honeys
Culture
Tda,b (h)
Control
Sourwood
Alfalfa
Sage
12
24
36
48
24.6 1.5
7.5 0.9a
25.5 0.8a
221.5 69.5a
6.4 0.7
5.2 0.7a
9.6 0.4b
13.7 0.7b
7.2 0.8
6.1 0.8a
11.2 0.6b
16.0 1.1b
9.0 0.4b
6.1 0.7a
11.4 0.5b
16.5 0.5b
12
24
36
48
23.2 0.5a
4.9 0.6a
68.3 10.4a
152.1 11.0a
5.8 0.5b
6.6 0.4a
12.0 0.5b
16.2 1.0b
6.2 0.5b
6.7 0.4a
15.2 0.5b
16.5 0.9b
6.2 0.6b
7.0 0.7a
16.4 1.5b
17.0 1.4b
12
24
36
48
12.4 2.0a
4.4 0.5a
52.5 8.1a
174.8 13.7a
4.2 0.5b
6.0 0.4a
9.1 0.6b
18.5 1.1b
5.3 0.4b
6.2 0.4a
9.7 0.8b
20.3 0.9b
6.5 0.7b
6.6 0.5a
11.2 1.0b
20.8 0.9b
12
24
36
48
27.5 3.9a
3.9 0.4b
46.7 8.0a
124.4 10.6a
7.5 0.8b
7.2 0.6a
8.8 1.2b
17.2 1.4b
10.3 0.8b
7.6 0.4a
9.0 0.8b
17.2 1.2b
10.3 0.5b
8.0 0.6a
9.0 0.6b
19.5 1.6b
12
24
36
48
14.8 1.4a
7.2 0.9a
93.2 7.8a
231.5 20.7a
6.1 0.6b
8.4 0.7a
8.9 0.7b
18.4 1.8b
8.9 0.8b
10.4 0.5a
12.3 1.1b
23.4 2.0b
11.4 0.7a
10.7 0.8a
13.4 1.0b
23.0 1.8b
a
b
Doubling times (Td) = ln 2/l (specic growth rate); l = (ln X2 ln X1)/(t1 t2). Means SDs; n = 3 for all treatments.
Means with the same letter in the same row are not signicantly dierent (p < 0.05). Comparisons are made only within the same row.
725
Table 3
Lactic acid production by intestinal Bidobacterium spp. grown in the presence of dierent unioral honeys
Strain
Sourwoodc
Alfalfac
Sagec
5.2 2.6a
6.6 1.7a
7.0 1.8a
5.0 1.3a
5.9 1.8a
40.3 4.7b
42.5 4.7b
31.4 3.3b
28.3 2.7b
36.2 3.8b
35.8 3.8b
34.1 3.1b
28.6 3.5b
26.3 2.5b
35.7 4.4b
35.0 4.0b
33.8 3.3b
28.0 3.0b
24.7 2.2b
33.9 4.6b
B.
B.
B.
B.
B.
a,b
Means with the same letter in the same row are not signicantly dierent (p < 0.05); n = 3 for all treatments.
Cultures were grown in MRS (37 C, 48 h ) supplemented with 5% honey.
Table 4
Acetic acid production by intestinal Bidobacterium spp. grown in the presence of dierent unioral honeys
Strain
Control
a
Sagec
b
B.
B.
B.
B.
B.
a,b
Means with the same letter in the same row are not signicantly dierent (p < 0.05); n = 3 for all treatments.
Cultures were grown in MRS (37 C, 48 h) supplemented with 5% honey.
17.3 2.3
13.5 2.5a
15.7 2.6a
12.7 2.8a
20.3 3.4a
Alfalfac
83.3 8.1
91.5 11.0b
76.8 8.2b
52.6 7.3b
82.6 10.3b
73.1 6.6b
83.6 8.7b
70.9 8.6b
47.4 6.9b
75.4 8.5b
76.3 7.1
84.7 9.1b
72.7 7.7b
49.5 7.6b
77.4 9.0b
Table 5
Doubling time (Td) of various intestinal bacteria grown in the presence of dierent unioral honeys
Culture
Tda,b (h)
Control
Sourwood
Alfalfa
Sage
12
24
36
48
9.4 0.8
9.0 0.5a
13.3 0.8a
18.0 0.7a
9.0 0.6
8.7 0.5a
12.7 0.4a
17.7 0.5a
9.2 0.5
8.7 0.7a
13.0 0.6a
17.9 0.6a
9.2 0.5a
8.9 0.5a
13.4 0.5a
18.0 0.5a
12
24
36
48
3.5 0.4a
5.9 0.6a
9.3 0.9b
12.3 1.4b
4.1 0.5a
6.6 0.7a
13.9 1.4a
18.2 1.3a
4.0 0.5a
6.7 0.4a
13.5 0.6a
18.0 0.9a
4.2 0.6a
6.5 0.7a
14.1 1.0a
17.9 1.4a
12
24
36
48
3.8 0.5a
7.0 0.6a
12.6 1.3a
14.2 1.4b
4.2 0.4a
7.4 0.7a
14.3 0.9a
19.3 0.8a
4.0 0.4a
7.2 0.5a
14.7 0.8a
20.3 1.5a
4.2 0.3a
7.4 0.5a
12.9 1.0a
18.8 1.6a
12
24
36
48
3.1 0.4a
6.9 0.6a
9.2 0.8a
13.5 1.0a
2.9 0.5a
6.3 0.5a
9.2 0.8a
14.0 0.4a
3.0 0.4a
6.4 0.5a
9.4 0.6a
14.2 0.8a
3.2 0.6a
6.7 0.4a
9.4 0.5a
14.2 0.6a
a
b
Doubling times (Td) = ln 2/l (specic growth rate); l = (ln X2 ln X1)/(t1 t2). Means SDs; n = 3 for all treatments.
Means with the same letter in the same row are not signicantly dierent (p < 0.05). Comparisons are made only within the same row.
726
Table 6
Growth of Clostridium perfringens and Eubacterium aerofaciens alone
and co-cultured with Bidobacterium spp. in the presence of sourwood
honey after 48 h incubation
Table 8
Growth of Clostridium perfringens and Eubacterium aerofaciens alone
and co-cultured with Bidobacterium spp. in the presence of sage
honey after 48 h incubation
cfu/ml
cfu/ml
8a
Table 7
Growth of Clostridium perfringens and Eubacterium aerofaciens alone
and co-cultured with Bidobacterium spp. in the presence of alfalfa
honey after 48 h of incubation
cfu/ml
C.
C.
C.
C.
C.
C.
perfringens
perfringens
perfringens
perfringens
perfringens
perfringens
:
:
:
:
:
B.
B.
B.
B.
B.
longum
adolescentis
breve
bidum
infantis
7.1 108a
3.0 108b
3.5 108b
2.9 108b
2.4 108b
3.3 108b
E.
E.
E.
E.
E.
E.
aerofaciens
aerofaciens
aerofaciens
aerofaciens
aerofaciens
aerofaciens
:
:
:
:
:
B.
B.
B.
B.
B.
longum
adolescentis
breve
bidum
infantis
6.2 108a
3.6 108b
3.9 108b
3.1 108b
2.7 108b
3.7 108b
a,b
Means with the same letter in the same column are not signicantly
dierent (p < 0.05); n = 3 for all treatments.
C.
C.
C.
C.
C.
C.
perfringens
perfringens
perfringens
perfringens
perfringens
perfringens
:
:
:
:
:
B.
B.
B.
B.
B.
longum
adolescentis
breve
bidum
infantis
5.8 10
2.9 108b
1.4 108b,c
3.1 108b
3.6 108b
3.1 108b
C.
C.
C.
C.
C.
C.
perfringens
perfringens
perfringens
perfringens
perfringens
perfringens
:
:
:
:
:
B.
B.
B.
B.
B.
longum
adolescentis
breve
bidum
infantis
7.0 108a
2.9 108b
3.2 108b
3.1 108b
2.6 108b
3.1 108b
E.
E.
E.
E.
E.
E.
aerofaciens
aerofaciens
aerofaciens
aerofaciens
aerofaciens
aerofaciens
:
:
:
:
:
B.
B.
B.
B.
B.
longum
adolescentis
breve
bidum
infantis
6.5 108a
4.6 108b
2.8 108b
5.2 108a
5.7 108a
4.9 108b
E.
E.
E.
E.
E.
E.
aerofaciens
aerofaciens
aerofaciens
aerofaciens
aerofaciens
aerofaciens
:
:
:
:
:
B.
B.
B.
B.
B.
longum
adolescentis
breve
bidum
infantis
5.8 108a
3.4 108b
3.6 108b
3.0 108b
2.3 108b
3.6 108b
a,b
Means with the same letter in the same column are not signicantly
dierent (p < 0.05); n = 3 for all treatments.
a,b
Means with the same letter in the same column are not signicantly
dierent (p < 0.05); n = 3 for all treatments.
727
Table 9
Inhibition of Clostridium perfringens and Eubacterium aerofaciens co-cultured with Bidobacterium spp. in the presence of dierent unioral honeys
after 48 h incubation
Strain
Inhibition (%)a
Sourwood
B.
B.
B.
B.
B.
Alfalfa
Sage
C. perfringens
E. aerofaciens
C. perfringens
E. aerofaciens
C. perfringens
E. aerofaciens
50 4.2
76 5.5
46 2.1
38 4.3
46 4.0
30 2.1
57 4.7
20 1.2
12 1.8
25 3.2
56 2.9
51 3.3
59 2.7
66 5.1
55 4.6
42 2.3
37 3.0
50 3.3
56 3.9
40 1.8
59 4.7
54 4.5
56 3.5
37 2.2
56 4.4
41 3.6
62 5.3
48 2.6
60 5.1
38 2.4
spp. remained unchanged in all of the co-culture experiments (Tables 68). In the presence of sourwood honey,
growth of C. perfringens was inhibited by 50%, 76%,
46%, 38%, and 46% when co-cultured with B. longum,
B. adolescentis, B. breve, B. bidum, and B. infantis,
respectively. Whereas, growth of E. aerofaciens was
inhibited by 30%, 57%, 20%, 12% and 25% when grown
in the presence of sourwood and co-cultured with B. longum, B. adolescentis, B. breve, B. bidum and B. infantis,
respectively. In the presence of alfalfa honey, growth of
C. perfringens was inhibited by 56%, 51%, 59%, 66%,
and 55% when co-cultured with B. longum, B. adolescentis, B. breve, B. bidum, and B. infantis, respectively.
Growth of E. aerofaciens in the presence of alfalfa honey
was inhibited by 42%, 37%, 50%, 56% and 40%, respectively, by the same Bidobacterium spp. In case of sage
honey, growth of C. perfringens was inhibited by 59%,
54%, 56%, 37%, and 56% when co-cultured with B. longum, B. adolescentis, B. breve, B. bidum, and B. infantis,
respectively. Growth of E. aerofaciens was inhibited by
41%, 62%, 48%, 60% and 38%, respectively, by the same
Bidobacterium spp. These results suggest that the
growth of C. perfringens and E. aerofaciens were not
only inhibited in the presence of honey, but further inhibition in growth was probably due to the presence of
Bidobacterium spp. which its growth, lactic and acetic
acid production were enhanced by the dierent unioral
honeys, as reported in the previous study. Although, the
degree of inhibition of C. perfringens and E. aerofaciens
was not inuenced by honey type, it was signicantly
(p < 0.05) inuenced by bidobacteria strain (Table 9).
These results, although preliminary in nature provide
important information with regards to potential modication of the gut microora with dietary components.
These results will need to be veried in vitro and in vivo
studies and human trials.
4. Conclusions
Although limited in scope our results indicate that,
overall, honey should provide many of the proposed
health benets provided by prebiotic agents by selec-
tively supporting the growth of indigenous bidobacteria in the GI tract and reducing the intestinal pH as a
result of the production of lactic and acetic acids and
inhibiting growth of C. perfringens and E. aerofaciens.
It is possible that honey may provide for the benecial
management of the GI microora, however, honey
could also be a source of Clostridium botulinum spores
and should be avoided by infants under one year of age.
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