Nutrition & Food Science

Effects of konjac glucomannan hydrolysates on the gut microflora of mice

Abdulmnem A. Elamir Richard F. Tester Farage H. Al-Ghazzewi Hakim Y. Kaal Amna A. Ghalbon Najat A.
Elmegrahai John R. Piggott
Article information:
To cite this document:
Abdulmnem A. Elamir Richard F. Tester Farage H. Al-Ghazzewi Hakim Y. Kaal Amna A. Ghalbon Najat A.
Elmegrahai John R. Piggott, (2008),"Effects of konjac glucomannan hydrolysates on the gut microflora of
mice", Nutrition & Food Science, Vol. 38 Iss 5 pp. 422 - 429
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NFS
38,5
Effects of konjac glucomannan
hydrolysates on the gut
microflora of mice
422 Abdulmnem A. Elamir
Faculty of Agriculture, University of Al-Fateh, Tripoli, Libya
Richard F. Tester and Farage H. Al-Ghazzewi
Glycologic Ltd, c/o Glasgow Caledonian University, Glasgow, UK
Hakim Y. Kaal
Faculty of Agriculture, University of Al-Fateh, Tripoli, Libya
Amna A. Ghalbon and Najat A. Elmegrahai
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Faculty of Pharmacy, University of Al-Fateh, Tripoli, Libya, and

John R. Piggott
Strathclyde Institute of Pharmacy and Biomedical Sciences, Royal College,
University of Strathclyde, Glasgow, UK
Abstract
Purpose – The aim of this study is to determine the effects of depolymerised mannans and
specifically konjac glucomannan hydrolysates (GMH) on the colonic microflora of mice. Blood glucose
and cholesterol were also measured.
Design/methodology/approach – Two groups (n ¼ 20) of 12-week old Wister mice were used for a
period of 14 weeks. One group (treatment group) were fed diets containing 5 per cent konjac GMH
dissolved in drinking water in addition to the control (group) standard diet. Faecal microflora, feed
consumption, body weight, blood glucose and cholesterol were determined.
Findings – The GMH promoted the growth of anaerobes and lactobacilli in the treatment group
where this was statistically, highly significant (P < 0.001). Also, the hydrolysate was able to reduce
highly significantly (P < 0.001) faecal Clostridium perfringens and Escherichia coli counts. A
significant increase in average daily feed consumption (P < 0.05) and weekly body weight
(P < 0.001) was found for the treatment group. The mean ± SD (mmol/l) of blood glucose and
cholesterol was lower in the treatment group.
Originality/value – In addition to modulating the gut microflora, GMH seems to lower the blood
glucose and cholesterol in mice. Although this needs to be verified by further studies, GMH could
also be a candidate for possible treatment of subjects with high cholesterol and for diabetics.
Keywords Food products, Bacteria
Paper type Research paper

Nutrition & Food Science
Vol. 38 No. 5, 2008
pp. 422-429
# Emerald Group Publishing Limited
0034-6659
DOI 10.1108/00346650810906930
Introduction
Glucomannan is a polysaccharide that can be extracted from, for example, the
Amorphophallus konjac plant (or group of plants) which belong to the Araceae genus
(Khanna, 2003). Konjac corms are grown in Asia where they have provided a source of
food for many centuries with very interesting physical and nutritional characteristics
(Khanna, 2003; Zhang et al., 2005).
Glucomannans (such as konjac) have a positive effect on health (Al-Ghazzewi et al.,
2007), by for example stimulating selectively the growth of gut-friendly bacteria and
serving as valuable functional foods (Jones, 2002). Like other polysaccharides, the
polymers can be depolymerised with acids and enzymes. A GMH from konjac has been
described elsewhere (Khanna, 2003; Al-Ghazzewi et al., 2007) where it has a molecular
 weight of 1,000-6,000 Daltons compared to the native polysaccharide with a molecular Effects of konjac
weight of >106 Daltons.
The colon of healthy humans contains a diverse bacterial population. The colon is
glucomannan
dominated by strict anaerobes include Bacteroides spp., Clostridium, Bifidobacterium hydrolysates
spp., Atopobium spp., and peptococci. Facultative anaerobes occur less in numbers and
include lactobacilli, enterococci, streptococci and Enterobacteriaceae. Yeasts occur in
much lower numbers (Gibson and Roberfroid, 1995; Rastall, 2004). Among the bacteria,
lactobacilli and bifidobacteria are the most significant organisms in terms of human 423
health (Gibson and Roberfroid, 1995). The growth of these bacteria is stimulated
specifically by prebiotics (Gibson and Roberfroid, 1995; Al-Ghazzewi et al., 2007).
As bifidobacteria and lactobacilli have a positive and beneficial health effect in the
gut (Wong et al., 2006), stimulating these bacteria to grow specifically leads to reduced
intestinal infections by a number of mechanisms (Gibson et al., 2005). These include
lowering the gut pH (by producing acids), excreting natural antibiotics, blocking
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pathogen adhesion to the gut surface and competition for nutrients. A number of
investigators have reported the potential health benefits of consuming prebiotic and/or
probiotic for many diseases. These include decreasing the risk of colon cancer
(Buddington et al., 2002; Pool-Zobel, 2005; Petrovsky, 2006), increasing the resistance to
pathogens, lowering blood ammonia and increasing the stimulation of the immune
response (Manning and Gibson, 2004).
Konjac glucomannans have broader nutritional benefits outside the gut which include
cholesterol lowering (Arvill and Bodin, 1995; Gallaher et al., 2002; Martino et al., 2005),
reducing the risk of chronic constipation (Passaretti et al., 1991; Loening-Baucke et al.,
2004), diabetes management (Chen et al., 2003) cancer prevention (Luo, 1992) and weight
reduction (Walsh et al., 1984). Mannans may prevent fat storage through inhibiting the
intestinal absorption of dietary fat in high fat diets (Takao et al., 2006).
The work discussed in this study was undertaken to determine the effects of
depolymerised mannans and specifically konjac GMH on the colonic microflora of
Wister mice over three months. The specific effects on faecal lactobacilli, Clostridium
perfringens, Escherichia coli and total anaerobes counts were characterised. The serum
glucose and cholesterol were also measured on representative mice from each group.

Materials and methods

Preparation of GMH
Konjac GMH was prepared according to the procedure of Al-Ghazzewi et al. (2007).
Briefly, konjac flour (Luxara 5867, Arthur Branwell & Co. Ltd., Essex) was dispersed
(~10 per cent, w/v) in citrate buffer (200 mM, pH 4.5) or deionised water containing 15 mg/
mL cellulase (C013P, 3,000U/g, Biocatalysts, Pontypridd) within sealed 250 mL Duran
glass screw neck flasks. Samples were incubated in a shaking water bath (30 cycles/min)
at 60  C for 1 h. Following incubation, the samples were then placed in a boiling water
bath for 15 min to inactivate the enzyme. Next, they were centrifuged at 3,000 rpm
(1500g) for 15 min. The supernatant was then freeze-dried to obtain the hydrolysate.

Animals and experimental protocol

Twelve-week old Wister mice were housed in solid-bottom plastic cages with wood
shavings for bedding in a room maintained on an average of 22  C and approximately
50 per cent relative humidity. The room was regulated to provide a 12 h light (08.00-
20.00)/dark cycle. Mice were allowed free access to water (treatment group containing
5 per cent w/v GMH) and food. Experimental work with the animals was performed at
 NFS the University of Al-Fateh in compliance with the appropriate legislation, laws and
committee guidelines of the University of Al-Fateh, Tripoli, Libya. After two weeks
38,5 adaptation, the mice were divided randomly into two groups (n ¼ 20/group) defined as
control and treatment. Both groups were ‘‘fed Rat and Mouse Standard Diet’’ from
‘‘BEEKAY’’ (Bantin and Kingman Ltd., Hull, UK). Food intake and body weights were
taken daily and weekly, respectively, over the 14 weeks. The treatment group were also
fed the konjac GMH dissolved in the drinking water at 5 per cent (w/v) after the
424 adaptation period. Blood glucose and cholesterol were determined on six subjects from
each group using diagnostic kits (Roche ACCU-CHEK, Blood Glucose System,
04422040018, Lewes, UK; Boots Cholesterol Testing Kits, 2106094, Nottingham, UK).
The internal organs for the slaughtered mice were examined for any physiological
differences by veterinary surgeons.

Enumeration of faecal microflora

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Dehydrated media of anaerobe basal agar (CM972, Oxoid, Basingstoke, UK) and
perfringens agar base (CM587, Oxoid, UK) with selective supplement containing
kanamycin sulphate and polymyxin B (SR93, Oxoid, UK) were used to selectively isolate
total anaerobes and C. perfringens, respectively. Plates were incubated (in triplicate) at
37  C in an anaerobic condition for 48 h using the Oxoid Anaero Gen Atmosphere
Generation System AN0025. E. coli and lactobacilli were isolated selectively using eosin
methylene blue agar and De Man, Rogosa and Sharpe (MRS) (CM69, Oxoid, UK)
respectively. All plates were incubated at 37  C for 24 h under aerobic conditions except
for lactobacilli which were incubated in 5 per cent CO2 cabinets for up to 48 h. Faecal
samples were collected on the same day each week where cages were cleaned the day
before to ensure the material was in the cages for <24 h. The faeces were serially diluted
in sterile peptone water then subcultured using the pour plate technique.

Statistical analysis
Bacterial colony counts, rodent weight and feed consumption data were analysed for
statistical variance using Minitab 14 (Minitab Ltd., Coventry, UK). Differences are
considered as statistically significant at P < 0.05.

Results
During the experimental time-course of this study, all mice were healthy. The GMH
promoted the growth of anaerobes and lactobacilli in the faeces of the treatment group
(group fed 5 per cent GMH). Statistically, two-way ANOVA showed a highly significant
(P < 0.001) increase in the number of lactobacilli and anaerobes in the treatment group
compared with the control. In addition, the hydrolysate was able to reduce significantly
(P < 0.001) faecal C. perfringens and E. coli numbers. Figures 1–4 show the number of
colony forming units (CFU/g wet weight faeces) for the control and treatment groups.
The feed for the mice in both groups was weighed daily during the time-course of the
experiment and the amount consumed per animal was calculated. For the control group
this was in the range 3.89-5.15 g per animal with an average of 4.63 g ± 0.31 per animal
per day. For the treatment group, the daily consumption was in the range of 3.73-5.25 g
with an average 4.86 g ± 0.50 per animal per day. Statistical analysis (two-way ANOVA
and paired t-test) of the feed data, taken as daily averages, showed a significant (P < 0.05)
effect of treatment (treated group ate slightly more). The mouse weights during the
course of experiment were also recorded where weekly determined body weight for the
control group was in the range 21.3-26.7 g with an average of 24.7 g ± 1.50. For the
 Effects of konjac
glucomannan
hydrolysates

425

Figure 1.
Number of CFU of
lactobacilli/g faeces from
mice fed standard diets or
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standard diets plus water

containing GMH

Figure 2.
Number of CFU of total
anaerobes/g faeces from
mice fed standard diets or
standard diets plus water
containing GMH

treatment group, the weekly determined body weight was in the range of 26.2-28.9 g with
an average 27.5 g ± 1.35. Two-way ANOVA showed that the treated group had a
significantly (P < 0.001) higher average weight than that of the control group.
At the end of 14 weeks of the experiment, the blood of mice from each group was
taken for glucose and cholesterol testing. The mean blood glucose concentration for the
treatment group (4.45 mmol/L ± 1.23) was lower than that of the control (7.14 mmol/
L ± 1.98). Similarly, the blood cholesterol level for the treatment group (3.88 mmol/
L ± 0) was lower than the control (4.31 mmol/L ± 0.67).

Discussion
The reduction of C. perfringens and E. coli counts in the faeces might be caused by
stimulation of the growth of lactobacilli and anaerobes including bifidobacteria (Al-
Ghazzewi et al., 2007) which produce acid during fermentation and, in turn, inactivates
 NFS
38,5

426

Figure 3.
Number of CFU of C.
perfringens/g faeces from
mice fed standard diets or
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standard diets plus water

containing GMH

Figure 4.
Number of CFU of E. coli/g
faeces from mice fed
standard diets or
standard diets plus water
containing GMH

pathogenic bacteria. These results agree broadly with those of Chen et al. (2005) (whose
work was conducted over a much shorter time period). They found an increase in
bifidobacteria but a decrease in C. perfringens and E. coli in the caecal content of Balb/c
mice. This trend was associated with fermentation of the konjac glucomannan
(Kitamoto et al., 2003). Campbell et al. (1997) reported that caecal bifidobacteria and
total anaerobes were higher in rats fed fructooligosaccharide diets than those fed
control diets. These carbohydrates ferment to form short chain fatty acids (SCFAs) and
a lower pH, with an apparent beneficial impact on gastrointestinal health. It is
established that konjac glucomannan forms SCFAs by intestinal anaerobic bacteria
activity (Matsuura, 1998).
The konjac GMH appears to have unique prebiotic properties which make it
potentially universally valuable for use as a prebiotic in different foods (Al-Ghazzewi
et al., 2007). The addition of mannans to milk replacer diets has been shown to improve
 faecal probiotic profiles and feed intake in calves (Heinrichs et al., 2003). It also Effects of konjac
improves the weight gain in dairy calves (Dvorak and Jacques, 1997). glucomannan
The lower levels of blood glucose and cholesterol in the mice fed the konjac GMH is
not unexpected as a number of studies have reported the ability of dietary fibre to hydrolysates
lower blood glucose and cholesterol (Hundemer et al., 1991; Arjmandi et al., 1992).
However, this does need to be verified by further study.
Konjac glucomannan has been reported to lower blood cholesterol and sugar levels 427
(Zhang et al., 2005). Arvill and Bodin (1995) studied the effect of short-term ingestion of
konjac glucomannan on serum cholesterol in humans and found that glucomannan
treatment lowered the cholesterol level. Glucomannan has also been reported to
decrease plasma total cholesterol and low density lipoprotein cholesterol in
hypercholesterolemic children (Martino et al., 2005). Lowering the serum total
cholesterol and increasing the body weight by konjac consumption have also been
shown for Wister rats (Zhou, 1991). Chen et al. (2003) studied the effect of konjac
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glucomannan on blood lipid and glucose levels in hyperlipidaemic type 2 diabetic

patients. These authors reported that compared with placebos, konjac glucomannan
reduced the plasma cholesterol and glucose levels in diabetic subjects.
In terms of health effects of the GMH on the mice used in this study, when they were
sacrificed and examined by a veterinary surgeon (at the end of the study), no
differences were observed for the organs in situ or subsequent to dissection for either
the control or GMH groups.

Conclusions
In conclusion, it is apparent that consuming GMH can potentially improve gut health
by promoting the growth of beneficial bacteria and suppressing the pathogenic ones. In
addition to modulating the gut microflora, GMH seems to lower the blood glucose and
cholesterol in mice. The GMH appears, therefore, to have highly desirable functional
food and prebiotic properties.

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Corresponding author
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Farage H. Al-Ghazzewi can be contacted at: f.h.alghazzewi@glycologic.co.uk

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