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Theabilityofblackcurrantextractstopositively
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ArticleinWorldJournalofMicrobiologyandBiotechnology·October2010
DOI:10.1007/s11274-010-0352-4
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MasseyUniversity
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World J Microbiol Biotechnol (2010) 26:1735–1743
DOI 10.1007/s11274-010-0352-4
ORIGINAL PAPER
The ability of blackcurrant extracts to positively modulate key
markers of gastrointestinal function in rats
Abdul-Lateef Molan • Zhuojian Liu
Marlena Kruger
•
Received: 11 November 2009 / Accepted: 11 February 2010 / Published online: 27 February 2010
Ó Springer Science+Business Media B.V. 2010
Abstract An animal model was used to assess the effects
of orally administered aqueous extract from two commercial health supplement ingredients; First Leaf (FL; composed of blackcurrant extract powder, lactoferrin and lutein
and is developed by the Four Leaf Japan Co. Ltd, Japan)
and Cassis Anthomix 30 (CAM30; blackcurrant extract
powder which is developed by Just the Berries Ltd, New
Zealand) on the proliferation of lactobacilli and bifidobacterial species and some undesirable bacteria in the caeca
of rats. Gavaging rats with CAM30, FL or inulin three
times weekly for 4 weeks resulted in a significant increase
in the numbers of bifidobacteria and/or lactobacilli and a
significant decrease in the numbers of bacteroides and
clostridia. Moreover, rats gavaged with FL, CAM30 and
inulin showed 31.5, 18 and 15% reduction in the activity of
b-glucuronidase and 26, 30.4 and 18% increment in the
activity of b-glucosidase when compared to the control
group gavaged with water, respectively. These benefits
may make these products good candidates as prebiotic
agents. In conclusion, this study has shown that FL and
CAM30 can positively modulate key markers of gastrointestinal function in rats.
Keywords Prebiotic activity Blackcurrant products Modulation of gut microbiota Rats
Abbreviations
FL
First Leaf product (composed of blackcurrant
extract powder, lactoferrin and lutein)
A.-L. Molan (&) Z. Liu M. Kruger
Human Nutrition and Physiology Division, Institute of Food,
Nutrition and Human Health, Massey University, Private Bag
11 222, Palmerston North, New Zealand
e-mail: [email protected]
BC
CAM30
FOS
NOVA
TAA
Blackcurrant concentrates
Cassis Athomix 30 (blackcurrant extract
powder)
Fructooligosaccharides
One way analysis of variance
Total antioxidant activity
Introduction
The human colon harbours a large and complex microbial
flora and most of its members are obligate anaerobes and
many of them (60–80%) have not been cultivated (Hayashi
et al. 2003). However, it is now generally accepted that the
composition of human intestinal microbiota has an
important role in health and disease (Gionchetti et al.
2000). Many definitions have been made for the probiotics
and the definition continues to be improved, and was
recently made official by the Food and Agriculture Organization (FAO) and the World Health Organization (WHO;
Araya et al. 2002) as ‘‘live microorganisms which when
administered in adequate amounts confer a health benefits
on the host’’. Although some probiotic bacteria can survive
the gastrointestinal tract transit and reach the colon safely,
some studies have shown that they did not colonize and
grow during short or even after prolonged feeding periods
(Tannock et al. 2000). Thus, continuous consumption
appears to be necessary to maintain a high population of
the probiotic strain in the colon. Accordingly, there is a
need for a strategy that enhances viability and promotes
growth of probiotic bacteria in the colon.
Some clinical studies have identified the complex
interactions between gut microflora and health such as the
relationship between the colonic bacteria and their
123
1736
metabolic characteristics to the risk of colon cancer
(Chadwick et al. 1992). It has been shown that some species are capable of generating carcinogens, mutagens, and
tumour promoting agents in the large bowel (Goldin 1990).
b-glucuronidase, nitroreductase, azoreductase and nitratereductase are synthesised by some pathogenic colonic
bacteria, and they are known to produce mutagens, carcinogens, and various tumour promoters (Gorbach and
Goldin 1990). The importance of b-glucuronidase activity
is based on its function of deconjugating with many
endogenous and exogenous compounds. b-glucuronidase
activity antagonises hepatic metabolism by hydrolysing the
biliary conjugates, and delays its excretion.
As a consequence, modification of the human intestinal
microbiota has currently become an important objective in
nutritional science. This goal can be achieved in three
ways: either by inclusion in the diet of a significant proportion of beneficial bacteria, mainly Bifidobacterium and
Lactobacillus species, with the expectation that they will
be able to colonize the intestinal tract (probiotics); to give
non-digestible carbohydrates (prebiotics), like fructo-oligosaccharides, which have shown an ability to promote the
growth of desirable bacteria; or to give a mixture of both
(synbiotics; Gopal et al. 2003).
Prebiotics are generally carbohydrates that bypass the
small intestine. Prebiotics are often especially intended to
stimulate the growth of bifidobacteria because of the presumed beneficial role of bifidobacteria on gastrointestinal
health (Grizard and Barthomeuf 1999). Presently available
prebiotics (such as Fructooligosaccharides and inulin) can
aid in the survival and proliferation of probiotic bacteria,
but are limited by some drawbacks (Apajalahti et al. 2002).
Therefore, there remains a need for alternative prebiotics
with fewer or no side effects that could either be incorporated in a probiotic food matrix, or used as a stand-alone
prebiotic to enhance proliferation of lactic acid bacteria
naturally present in the intestine.
Blackcurrant berries are rich sources of various phenolic
substances with antioxidative, antibacterial, antiviral and
anti-inflammatory properties including flavonoids and
anthocyanidins as well as vitamin C. Consumption of fresh
blackcurrant (Ribes nigrum) is limited, but is used commercially to manufacture marmalade, jelly, jam, juice, etc.,
including a very popular liquor in France called ‘Cassis’
(Ibarz et al. 1992). Miller and Rice-Evans (Miller and RiceEvans 1997) studied the total antioxidant activity (TAA) of
orange juice, apple juice and blackcurrant drink and found
that blackcurrant drink (Ribena) has the highest TAA and
apple juice the lowest. The blackcurrant concentrate (BC),
which had been prepared from blackcurrant juice, is
reportedly known to increase resting forearm blood flow
(Matsumoto et al. 2005) and to improve the peripheral
circulation disorder caused by the cold water loading in
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World J Microbiol Biotechnol (2010) 26:1735–1743
human volunteers (Takenami et al. 2004). Furthermore, BC
intake effectively restored the decreased oxy-hemoglobin
level during and after the typewriting workload leading to
an improvement of muscle fatigue (Takenami et al. 2003).
It has been reported that oral intake of BC significantly
improves the peripheral circulation in human volunteers
(Matsumoto et al. 2005; Takenami et al. 2004; Takenami
et al. 2003). Recently, Werlein et al. (Werlein et al. 2005)
studied the effect of concentrates from blackcurrant and a
purified anthocyanin mix on the growth of some Grampositive and Gram-negative bacteria and yeast and found
that blackcurrant concentrates inhibited the growth of
Staphylococcus aureus DSM 799 and Enterococcus faecium DSM 2918, whilst Saccharomyces cerevisiae ATCC
9763 was slightly stimulated by the fruit concentrates.
In the present study, the influence of FL, CAM30 and
inulin on the numbers of bifidobacteria, lactobacilli, bacteroides, and certain clostridial numbers in the caecal
contents of rats gavaged orally with these products was
evaluated using fluorescence in situ hybridization (FISH)
molecular method. In addition, the effect of these products
on food intake and body weight gain and on the activity of
selected metabolic indexes (b-glucuronidase and b-glucosidase enzymes) in rats was also investigated.
Materials and methods
Preparation of extracts
The aqueous extracts from First Leaf (FL; blackcurrant
powder, lactoferrin and lutein) obtained from Four Leaf
Japan Co., Ltd and Cassis Anthomix 30 (CAM30; blackcurrant extract powder) obtained from Just the Berries Ltd,
were prepared by dissolving the powdered extracts in
sterile distilled water. Both products are commercial and
are already on the market (Japan and United States). Inulin
powder (Sigma, Australia) was dissolved in sterile distilled
water. All the extracts were prepared on the same day of
gavaging.
Experimental design/animals and housing
Forty Sprague–Dawley male rats aged 8 weeks that had
been weaned onto a balanced semi-synthetic diet were
housed individually in plastic cages in a room with a
temperature of 22 ± 1°C and a 12 h light: dark cycle (light
on at midnight) with free access to water and food
throughout the study. The rats were obtained from the
Animal Unit, Massey University, Palmerston North, New
Zealand. The experimental protocol was approved by the
Massey University Animal Ethics Committee (Palmerston
North, New Zealand; Protocol No: 07/141; 14/12/2007).
World J Microbiol Biotechnol (2010) 26:1735–1743
Administration of extracts
This study involved gavaging rats with *2 ml of sterile
distilled water, inulin, FL or CAM30 extracts delivered to
the back of each rat’s mouth via a soft silicon rubber tube
attached to a 3 ml syringe. So that the rats were comfortable with this procedure, during the 7 day acclimation
period the rats were handled daily and each rat was gavaged orally with 2 ml of sterile water (at room temperature) on alternative days to match the experimental
procedure.
For the remainder of the study (from day 8 to the end)
the rats were allocated randomly into four equal groups
(n = 10). The rat groups were gavaged with 2 ml of sterile
water (negative control), 2 ml of inulin extract (positive
control), 2 ml of FL extract (containing 30 mg FL/kg body
weight) or 2 ml of CAM30 extract (containing 13.4 mg
CAM30/kg body weight), respectively, three times weekly
(Monday, Wednesday and Friday) for four consecutive
weeks. The same experimental diet was offered to all
treatment groups during the feeding period. It was formulated to meet the nutrient requirements of growing rats.
Food intake was determined three times weekly as the
difference in weights of food before and after each feeding
period. Similarly, the water intake was determined by
weight difference. The rats were weighed at the beginning
of the experiment and then thrice weekly and at the end of
the trial. At the end of the experiment, rats were euthanized
by CO2 inhalation according to recommendations for
euthanasia of experimental animals. After dissection, the
secum with contents was removed. The cecal contents were
transferred to tubes and stored at -80°C until being
processed.
The amount of gavaging dose was based on a dose rate
of 30 mg FL and 13.4 mg of CAM30/kg rat body weight,
respectively as recommended by the client. For inulin, the
dose rate was equivalent to 1% of food intake.
Fluorescence in situ hybridization (FISH) analysis
of microbiota in the cecal contents
The probes used in the study were Bif164 Bac303, Chis150
and Lab158, specific for bifidobacteria, bacteroides, clostridia (perfingens/histolyticum subgroup) and lactobacilli/
enteroccocus, respectively. These were commercially synthesised and labelled with the fluorescent dye Cy3 (GeneWorks, Australia). The samples were prepared for FISH
analysis as described by Molan et al. (2009). In short,
caecal content samples were prepared by mixing 1 g of the
digesta collected from the middle portion of the cecae with
9 ml of sterile-filtered phosphate buffer (PBS; pH 7.2). The
mixture was homogenized by vortexing with a dozen glass
beads for 3 min, the caecal debris was removed by
1737
centrifugation at low speed (700g), and the bacteria containing supernatant was fixed in 4% (w/v) paraformaldheyde in PBS (pH 7.2) overnight at 4°C. Fixed
samples were washed in PBS and stored in a known volume of 50% (v/v) ethanol-PBS at -20°C until time of
hybridization. Aliquots (5 ll) of fixed bacterial cells were
applied to Teflon-coated microscopic slides (BIOLAB,
New Zealand; 10 wells) and air dried. The bacterial cells
were then dehydrated with a series of solutions containing
50, 80, and 99.5% ethanol (3 min for each concentration).
The bacterial cells fixed on the glass slides were hybridized
by addition of 8 ll of hybridization buffer (0.9 M NaCl,
0.01% sodium dodecyl sulfate, 20 mM Tris–HCl, 20%
deionized formamide; pH 7.2) with 0.5 ll of Cy3-labeled
oligonucleotide specific probes (50 ng/ll). The slides were
hybridized at 46°C for 2 h in a plastic box containing wet
sponges (soaked in hybridization buffer). After hybridization, the slides were rinsed with warm hybridization buffer
at 48°C and washed in pre-warmed washing buffer
(225 mM NaCl, 0.01% sodium dodecyl sulfate, 20 mM
Tris–HCl; pH 7.2) for 20 min at 48°C. After washing step,
the slides were rinsed with ice-cold distilled water and
thoroughly dried before being observed using a fluorescence scanning microscope.
The dried slides were examined with an Olympus BX51
microscope, under 4009 magnification. The images were
captured using an Optronics MagnaFIRE SS99802 digital
camera with MagnaFIRE frame-grabbing software on a
Pentium IV computer. Fluorescent cells were counted
automatically in five randomly selected fields/slide using
ImageJ.
Bacterial enzyme activities
The samples were prepared as described by Gudiel-Urbano and Goni (2002) with some modifications. In short,
cecal content samples were prepared by mixing 1 g of the
digesta collected from the middle portion of the cecae
with 9 ml of 0.1 M phosphate buffer (pH 7). The mixture
was homogenized by vortexing and the caecal suspension
(1 ml) was mixed with equal volume of 0.1 M phosphate
buffer (pH 7) containing 0.2% Triton-X-100 to disrupt the
bacterial cells. The mixture was centrifuged for 10 min at
5,000g at room temperature. The supernatant was assayed
for the enzymatic activity of both b-glucosidase and
b-glucuronidase.
The enzyme activities were measured as described by
Djouzzi et al. (1997) with some modifications, by the rate
of release of p-nitrophenol from p-nitrophenyl-b-D-glucoside (N-7006, Sigma–Aldrich, New Zealand) as substrate
for b-glucosidase and p-nitrophenyl-b-D-glucuronide
(N-7009, Sigma–Aldrich, New Zealand) as substrate for
b-glucuronidase. Briefly, the reaction mixture containing
123
1738
The pH of each caecal sample was measured in order to test
if there is any link between the levels of b-glucuronidase,
b-glucosidase, the numbers of lactobacilli and bifidobacteria and the caecal pH. The pH was measured by a digital
pH-meter at room temperature (20–22°C).
Data analysis
Caecal concentrations of bacteria were expressed as log
number of bacterial cells/g caecal content wet weight.
Logarithmically-transformed data were analysed by one
way analysis (ANOVA) of variance using SAS system with
the level of significance set at P \ 0.05. For average
comparisons, data comparisons were made using the student’s t test.
Results
Prebiotic and antimicrobial activities
The probe technology can provide information on bacterial
populations at the genus, group, and even species levels in
terms of identification and enumeration. Fluorescence in
situ hybridization is a powerful method for the enumeration
of bacteria in complex habitats such as the gut. Most
notably, it does not require cultivation of the target
organisms. This method was used in this study to enumerate the good and bad bacteria in caecal contents of rats
gavaged with the tested products.
Gavaging rats with FL and CAM30 led to a significant
increase (P \ 0.0001) in bifidobacterial numbers when
compared with the rats gavaged with water (negative control; Fig. 1). In addition, bifidobacterial numbers significantly increased in rats gavaged with inulin (P = 0.0016).
123
(A) 9.5
Log number/g cecal contents
pH values of caecal samples
Significant increases in lactobacilli numbers were seen
in rats gavaged with CAM30 (P = 0.03075) while gavaging rats with FL led to a slight but insignificant increase
(P = 0.0964) in lactobacilli when compared with the
control rats gavaged with water (Fig. 1b). Similarly, rats
gavaged with inulin showed insignificant increase in lactobacilli in comparison with control rats. It is important to
mention that the number of bifidobacteria in the caeca of
rats gavaged with CAM30 was significantly higher than
that in the caeca of rats gavaged with FL or inulin (Fig. 1).
When compared with control rats gavaged with water,
bacteroides numbers decreased to significant levels in rats
gavaged with CAM30 (P = 0.0106), FL (P = 0.0115) and
Bifidobacteria
*
***
***
9
8.5
8
Control
(B)
Log number/g cecal contents
0.2 ml of a 5 mM substrate solution and 0.2 ml of caecal
suspension from control and rats gavaged with inulin, FL
or CAM30 was incubated at 37°C. The reaction was
stopped at 30 min after the addition of 1.6 ml of 0.25 M
Na2CO3 (Gudiel-Urbano and Goni 2002). The released
p-nitrophenol was measured at 400 nm. The amount of
p-nitrophenol released was determined by comparison with
a standard nitrophenol curve.
Enzyme activities were calculated as nanomoles of
transformed substrate per minute per gram of cecal content
wet weight (nmol/min/g).The % reduction in the activity of
b-glucuronidase activity and % increase in the activity of
b-glucosidase were calculated relative to the enzymatic
activity in control rats gavaged with water.
World J Microbiol Biotechnol (2010) 26:1735–1743
Inulin
FL
CAM30
##
Lactobacilli
$$
9.5
***
9
8.5
8
Control
Inulin
FL
CAM30
Fig. 1 Enumeration of Bifidobacterium species (a) and Lactobacillus
species (b) [Log10 cells/g of wet caecal contents] from the four groups
of rat caecal samples hybridized with Bifidobacterium genus-specific
oligoneucleotide probes in FISH analysis. The rat groups were
gavaged with water (negative control), inulin (positive control), FL or
CAM30 three times weekly for four consecutive weeks. The data are
expressed as means ± standard errors of the means (n = 10 rats/
group). * P B 0.05; *** P B 0.0001 by analysis of variance versus
the negative control group. ## P \ 0.01 by analysis of variance versus
the inulin group. $$ P \ 0.01 by analysis of variance versus the FL
group
World J Microbiol Biotechnol (2010) 26:1735–1743
1739
inulin (P = 0.0311; Fig. 2a). Similarly, clostridial (perfingens/histolyticum sub. grp.) numbers significantly
decreased in rats gavaged with CAM30 (P = 0.0481), FL
(P = 0.0346) and in inulin-treated rats (P = 0.0168) in
comparison with rats gavaged with water (Fig. 2b).
Concerning the activity of b-glucosidase, rats gavaged
with FL, CAM30 and inulin showed 26, 30.4 and 18%
increment in the activity of this enzyme when compared to
the control group gavaged with water, respectively (Fig. 4).
pH of caecal samples
In rats gavaged with FL, CAM30 and inulin, the activity of
b-glucuronidase decreased by 31.5, 18 and 15%, respectively relative to control rats gavaged with water (Fig. 3).
Moreover, the FL-treated rats were characterized by a
significantly lower (P \ 0.0001) activity of b-glucuronidase compared with positive control group gavaged with
inulin and those gavaged with CAM30 (Fig. 3). This may
explain the significant reduction in the numbers of bacteroides and clostridia in the same caecal samples.
Log number/g cecal contents
(A) 9.5
Bacteroides
*
*
*
9
Figure 5 shows the pH values of the caecal samples collected from rats gavaged with water, inulin, FL and
CAM30. The present study showed that the caecal pH in
rats gavaged with inulin, FL and CAM30 was significantly
% inhibition relative to control
incubation
Activity of microbial enzymes (b-glucuronidase
and b-glucosidase activity)
β-glucuronidase
40
***
$
30
20
10
0
Inulin
FL
CAM30
Fig. 3 % reduction in the activity of b-glucuronidase in caecal
contents of rats gavaged with inulin, FL or CAM30 relative to the
control group gavaged with water. The data are expressed as
means ± standard errors of the means (n = 10 rats/group).
*** P B 0.0001 by analysis of variance versus the inulin group.
$
P \ 0.05 by analysis of variance versus the CAM30 group
8.5
8
Inulin
Log number/g cecal contents
(B) 9.5
FL
CAM30
Closteridia
9
*
*
*
8.5
8
Control
Inulin
FL
CAM30
Fig. 2 Enumeration of bacteroides (a) and clostridia (b) [Log10 cells/
g of wet caecal contents] from the four groups of rat caecal samples
hybridized with Bacteroides genus-specific oligoneucleotide probes
in FISH analysis. The rat groups were gavaged with water (negative
control), inulin (positive control), FL or CAM30 three times weekly
for four consecutive weeks The data are expressed as means ± standard errors of the means (n = 10 rats/group). * P B 0.05 by analysis
of variance versus the negative control group
% increase in enzyme activity relative to
control incubation
Control
β-glucosidase
40
**
35
30
*
25
20
15
10
5
0
Inulin
FL
CAM30
Fig. 4 % increase in the activity of b-glucosidase in caecal contents
of rats gavaged with inulin, FL or CAM30 relative to the control
group gavaged with water. The data are expressed as means ± standard errors of the means (n = 10 rats/group). ** P B 0.0001 by
analysis of variance versus the inulin group; * P \ 0.05 by analysis
of variance versus the inulin group
123
1740
World J Microbiol Biotechnol (2010) 26:1735–1743
compared with the control group gavaged with water
(Table 1).
There were no significant differences detected in the
body weight of rats gavaged with inulin, FL and CAM30
when compared with rats in the control group (Table 2).
Overall, water intake in rats gavaged with inulin, FL or
CAM30 was not affected in comparison with the rats gavaged with water only (data not shown).
pH value
8
***
***
***
pH value
6
4
2
Discussion
0
Control
Inulin
FL
CAM30
Fig. 5 Caecal pH in rats gavaged with water (control), inulin, FL and
CAM30. *** P B 0.0001 by analysis of variance versus the control
group gavaged with water only (negative control)
lower (P \ 0.0001) than that in control rats gavaged with
water.
Effect on food intake, water intake and body
weight gain
There were no significant differences detected in the food
intake of rats gavaged with inulin, FL and CAM30 when
The results show for the first time that aqueous extracts
from FL and CAM30 were effective at promoting the
growth of lactobacilli and bifidobacteria in the caecum of
rats gavaged orally with these extracts. From a health
perspective, this finding is very important due to the
growing interest in probiotic bacteria and the perceived
benefit of increasing their numbers in the gastrointestinal
tract, namely the enhancement of gut health by competitively excluding pathogens from the occupation of adhesion sites or by influencing the gut environment via
secretion of simple or complex molecules. Increasing the
numbers of lactic acid bacteria (lactobacilli and bifidobacteria) in the colon was found to reduce the formation of
ammonia, skatole, harmful amines and other procarcino-
Table 1 Food intake in Sprague–Dawley male rats fed chow pellets and gavaged orally water, FL, CAM30 or inulin three times weekly for four
consecutive weeks
Measurement
Food intake (g/day/rat)
Water group
Inulin group
FL group
CAM30 group
Week 1
30.86 ± 0.77
31.28 ± 0.44
30.48 ± 0.47
32.09 ± 0.66
Week 2
31.4 ± 0.89
30.98 ± 0.72
31.4 ± 0.54
31.74 ± 0.65
Week 3
Week 4
32.5 ± 0.96
33.86 ± 1.1
32.3 ± 0.62
33.76 ± 0.85
31.99 ± 0.58
33.25 ± 0.66
31.5 ± 0.73
33.63 ± 0.83
Mean (g/rat)
32.17 ± 0.47
32.08 ± 0.35
31.78 ± 0.29
32.24 ± 0.36
The data are expressed as means ± standard errors of the means (n = 10 rats/group)
Table 2 Body weight (g) of rats gavaged with water, FL, CAM30 or inulin three times weekly for four consecutive weeks
Measurement
Body weight (g)
Water group
Week 1
Week 2
304.8 ± 7.0
348.4 ± 8.2
Inulin group
304.1 ± 4.4
348.8 ± 5.3
FL group
305.4 ± 4.4
353.5 ± 4.9
CAM30 group
308.54 ± 5.6
354.29 ± 6.1
Week 3
393.2 ± 9.7
392.3 ± 6.5
396.64 ± 5.1
395.6 ± 6.3
Week 4
431.8 ± 10.7
431.1 ± 7.5
435.24 ± 5.8
434.5 ± 6.8
369.53 ± 6.43
369.3 ± 5.7
372.7 ± 5.1
373.24 ± 5.3
Mean (g)
The data are expressed as means ± standard errors of the means (n = 10 rats/group)
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World J Microbiol Biotechnol (2010) 26:1735–1743
gens in the large intestine and the carcinogenic load on the
intestine (Burns and Rowland 2000; Yamamoto et al.
1997). The production of acids by these bacteria also
lowered the pH value of the colon and faeces (Yamamoto
et al. 1997). Moreover, it has been found that modification
of the gut microflora may interfere with the process of
carcinogenesis and this opens up the possibility for dietary
modification of colon cancer risk. Probiotics and prebiotics, which modify the microflora by increasing numbers of
lactobacilli and/or bifidobacteria in the colon, have been a
particular focus of attention in this regard (Burns and
Rowland 2000).
The bifidogenic activity of inulin found in the present
study matches other studies performed in rats and humans.
Many human intervention studies had previously found a
bifidogenic effect of inulin (Bouhnik et al. 2007; Van de
Wiele et al. 2004). This gives a strong indication of the
validity of the results obtained in this study.
The results have provided the first evidence that consumption of FL and CAM30 can beneficially affect faecal
parameters related to colon cancer risk. Under the present
experimental conditions, this study showed that CAM30
and FL can decrease the activity of b-glucuronidase and
increase the activity of b-glucosidase, which could be
perceived as potentially beneficial for the host. It has been
reported that certain bacteria (such as Escherichia coli,
Clostridium perfringens and Bacteroides species) have
high b-glucuronidase activity (Skar et al. 1998). Moreover,
this result may explain the significant increase in the
numbers of bifidobacteria and lactobacilli in the same
caecal samples. Results from some studies with probiotics
have shown that increasing the proportion of the fecal flora
represented by bifidobacteria is associated with lower
activity of reductive enzymes including caecal b-glucuronidase, and inhibition of the development of Azoxymethane (AOM)-induced colon carcinogenesis (Kulkarni and
Reddy 1994; Ling et al. 1994).
The importance of this finding is generated from the fact
that the bacterial enzyme b-glucuronidase is considered to
be one of the enzymes that increases risk for colorectal
cancer (Gorbach and Goldin 1990; Hambly et al. 1997). It
has been suggested that b-glucuronidase is a key enzyme in
the activation of the procarcinogen 1,2-dimethylhydrazine
(DMH) into its toxic carcinogen (Johansson et al. 1997).
Therefore, b-glucuronidase has assumed significance as
colon cancer risk marker and its inhibitors have been
suggested to have anticarcinogenic properties because they
will increase clearance of glucuronidated carcinogens
(Walaszek et al. 1986). Accordingly, lower activity of this
enzyme can be considered beneficial in terms of the risk of
colon cancer (Goldin 1990).
Some studies have shown that certain tumours and some
bacteria have high b-glucuronidase activity (Brady et al.
1741
2000), and that such tumours or bacterial infections may be
treated by means of b-glucuronidase inhibitors which are
toxic to the tumour cell or bacterial cell. Other studies have
shown that the activities of fecal bacterial enzymes such as
b-glucuronidase, azoreductase and nitroreductase increased
in animals and humans consuming a high beef diet which is
associated with a high risk of colon cancer (Gorbach 1982;
Reddy et al. 1977). The results of the present study may
suggest that CAM30 and/or FL extracts are good candidates for decreasing/inhibiting the activity of b-glucuronidase which is involved in colon cancinogenesis.
The increment in the activity of b-glucosidase probably
due to the stimulation of the growth of lactic acid bacteria,
which have high levels of b-glucosidase activity in comparison with the other members of the gut microflora (Saito
et al. 1992). Previous studies have shown that oligosaccharides have the ability to increase the activity of this
enzyme and this has been attributed to the ability of oligosaccharides to stimulate the growth of lactic acid bacteria
(Saito et al. 1992; Rowland and Tanaka 1993). Marteau
et al. (1990) found an increase in faecal b-glucosidase
activity in subjects consuming milk fermented with Lactobacillus acidophilus and Bifidobacterium bifidum. Fuller
and Perdigon (Fuller and Perdigon 2000) reported that
consumption of dairy products with viable L. acidophilus,
which is bile resistant, can cause a significant decline in
some faecal bacterial enzymes including b-glucosidase.
Again, the results of the present study have provided the
first evidence that consumption of FL and CAM30
decreases the caecal pH which can be considered beneficial
in terms of the risk of colon cancer. An association
between high colonic (or stool) pH and increased risk of
colon cancer has been reported, suggesting that colonic
neoplasia might be related to a reduction in the fermentation of dietary polysaccharides to organic acids in the large
bowel (Pietroiusti et al. 1985). A number of potential
mechanisms are suggested. Bile acids, for example, are
degraded more or less efficiently depending on stool pH;
the lower the pH the greater the inhibition of secondary bile
acid formation, which is a key factor in development of
colon cancer (Cummings and Bingham 1987).
Conclusions
This study has identified CAM30 and FL as good prebiotic
agents that can significantly promote the growth of friendly
bacteria such as bifidobacteria and lactobacilli, and lower
the numbers of undesirable bacteria such as bacteroides
and clostridia in the cecum of rats. This study also showed
that CAM30 and FL can decrease the activity of b-glucuronidase and increase the activity of b-glucosidase, which
could be perceived as potentially beneficial for the host.
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These promising results need to be confirmed in human
studies involving both healthy volunteers and subjects with
clinical conditions that have a supposed microbial aetiology (e.g. ulcerative colitis, bowel cancer and irritable
bowel syndrome).
Acknowledgments This study was supported by ‘‘Four Leaf Japan
Co., Ltd.’’ and ‘‘Just the Berries Ltd’’.
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