FEMS MicrobiologyEcology95 (1987) 163-171
Published by Elsevier
163
FEC 00118
Polysaccharide breakdown by mixed populations
of human faecal bacteria
H . N . Englyst, S. H a y a n d G . T . M a c f a r l a n e *
M.R. C. Dunn Chmcal Nutrmon Centre, Cambridge CB2 I QL, U K.
Received 5 December1986
Revision received 3 March 1987
Accepted 5 March 1987
Key words: Gut microflora; Polysaccharide fermentation; Volatile fatty acid; Faecal bacterium
1. S U M M A R Y
Measurements of polysaccharide-degrading activity in different fractions of human faeces showed
that bacterial polysaccharidases and glycosidases
were primarily associated with the washed bacterial
fractions. Amylase, pectinase and xylanase were
the major polysaccharide-hydrolysing enzymes
detected, whilst a-L-arabinofuranosidase, /3-Dxylosidase, /3-D-galactosidase and /3-D-glucosidase
were the most active glycosidases. Starch and 3
non-starch polysaccharides (NSP; pectin, xylan
and arabinogalactan) were fermented by mixed
populations of human faecal bacteria in batch
culture. Detailed carbohydrate analysis demonstrated that starch and pectin were the most rapidly
degraded substrates and that arabinogalactan and
the relatively insoluble polysaccharide xylan were
broken down more slowly. Free sugars and oligosaccharides did not accumulate in culture media
with any polysaccharide tested. Time-course measurements of polysaccharide remaining in the
batch culture fermentations showed that the
Correspondence to: G.T. Macfarlane, M.R.C. Dunn Clinical
Nutrition Centre, 100 Tennis Court Road, Cambridge CB2
1QL, U.K.
arabinose side chains of pectin, xylan and
arabinogalactan were co-utilised with the backbone sugars. In these cultures, polysaccharide-degrading activity was mainly cell-associated, but
extracellular polysaccharidase activity increased as
the fermentations progressed. Molar ratios of
acetate, propionate and butyrate produced in these
experiments were dependent upon the polysaccharide substrate tested. Molar ratios of acetate,
propionate and butyrate in the starch, arabinogalactan, xylan and pectin fermentations were
5 0 : 2 2 : 2 9 , 5 0 : 4 2 : 8 , 8 2 : 1 5 : 3 , and 8 4 : 1 4 : 2 , respectively. The presence of starch did not inhibit
the breakdown of arabinogalactan, xylan or pectin
by faecal bacterial, providing evidence that multicomponent substrate utilisation occurs when complex populations of faecal bacteria are provided
with mixed polysaccharide substrates.
2. I N T R O D U C T I O N
Plant polysaccharides can be separated into
starch and the cell-wall and related polysaccharides conveniently called NSP. We have previously shown that all of the dietary NSP and a
0168-6496/87/$03.50 © 1987 Federation of European MicrobiologicalSocxeties
164
considerable amount of starch escapes digestion in
the human small intestine [1-3] and there is now a
substantial body of evidence which shows that
these carbohydrates are fermented in the colon
[4-6]. The majority of the strictly anaerobic
bacteria in the large gut are saccharolytic and
derive their energy from the fermentation of these
carbohydrates, which results in the production of
the volatile fatty acids (VFA) acetate, propionate
and butyrate, the gases CO 2 and H 2 and, in some
individuals, CH 4 [7].
The importance of the large gut fermentation in
human physiology may be considerable, since VFA
are efficiently absorbed from the colon and their
energy value can represent a substantial proportion of that obtained by the host, had the carbohydrate been absorbed in the small bowel [8].
VFA also stimulate salt and water absorption and
are i m p o r t a n t components of intermediary
metabolism in the colonic epithelium, where
butyrate is the principal fuel [9] and in the fiver,
where propionate may influence gluconeogenesis
and cholesterologenesis [10,11]. Carbohydrate fermentation by the intestinal microflora can therefore be regarded as an important process controlling many aspects of large bowel function in man
and may, through VFA, be a contributory factor
in the control of hepatic and peripheral tissue
metabolism.
Whilst there have been considerable advances
in recent years with respect to the characterisation
of the polysaccharide-degrading enzymes produced by individual species of intestinal bacteria
[12-14], detailed information concerning the
breakdown of polysaccharides by the large gut
microflora is limited. The aims of the present
study therefore were to investigate the rate and
extent of polysaccharide breakdown by mixed cultures of human faecal bacteria and to assess the
amounts and proportion of VFA produced by
fermentation of different polysaccharides.
3. MATERIALS A N D M E T H O D S
3.1. Polysaccharidase and glycosidase activities in
faeces
Fresh faeces from 5 individuals, who had been
eating normal western diets, were suspended in
anaerobic 0.1 M Na-phosphate buffer pH 6.5 to
produce 10% w / v faecal slurries. Each slurry was
then centrifuged to give a washed bacterial cell
fraction, a particulate fraction and a cell-free supernatant fraction, as described by Macfarlane
and Englyst [15]. The particulate fraction was
resuspended in a 0.001% w / v solution of cetyl
trimethylammonium bromide (CTAB) and vigorously agitated on a wrist-action shaker for 30 min
to remove adhering bacteria. The samples were
then centrifuged at 1000 x g for 20 min to remove
the particulate material. The bacterial suspension
was subsequently centrifuged at 20 000 × g for 30
min. The pelleted cells were washed twice with
anaerobic 0.1 M Na-phosphate buffer pH 6.5,
recentrifuged and resuspended in buffer. All
buffers and washing solutions were boiled, cooled
and maintained under a stream of high-purity
nitrogen gas prior to use. For measurement of
polysaccharidase and glycosidase activities,
bacteria from the washed bacterial cell fractions
and bacteria removed from the particulate fraction were disrupted by 2 passages through a French
pressure cell at 16000 psi and the cells were
microscopically checked for disruption. The homogenate was centrifuged at 40 000 x g for 30 min
to remove cell debris. Enzyme assays were then
carried out on the soluble fractions and also on
the cell-free faecal supernatants.
Polysaccharidase activities were measured as
follows: 1 ml of a solution of either Lintners
starch, pectin (citrus), xylan (larchwood) or
arabinogalactan grade I (larchwood) (all 0.2% w / v
in 0.1 M Na-phosphate buffer p H 6.5) was added
to 1 ml of sample. The mixture was then incubated
anaerobically under a headspace of oxygen-free
nitrogen gas at 37 o C for I h. After incubation, the
reducing sugars released were measured using the
dinitrosalicylate reagent [16], with maltose, galacturonic acid, xylose and galactose used as standards. Glycosidase activities were determined by
measuring the release of p-nitrophenol at 410 nm
from a range of p-nitrophenyl sugar derivatives as
described by Berg et al. [17].
3.2. Polysaccharide fermentation by mixed bacterial
populations
Faecal samples were obtained from 2 individu-
165
als and washed faecal bacterial cells were prepared
as previously described. Batch cultures of these
faecal bacteria were grown anaerobically in 1-1itre
fermenters on a mineral salts medium, as described
by Englyst and Macfarlane [18], with either Lintners starch, xylan, arabinogalactan (grade I) or
pectin (all 10 m g . m 1 - 1 ) as sole fermentable
carbon sources. Culture pH was 6.5 and was controlled using a Modular Fermenter pH Controller
(Gallenkamp). Samples (50 ml) were taken at 0, 3,
6, 12, 24 and 48 h for analysis. Portions (45 ml)
were centrifuged at 20000 × g for 30 min. The
resultant cell pellets were washed with the
anaerobic phosphate buffer and recentrifuged. The
washed cell pellets were resuspended in 5 ml phosphate buffer and disrupted in a French pressure
cell as before. Following centrifugation to remove
cell debris, the homogenates, together with cell-free
culture effluents were assayed for polysacchariease and glycosidase activity, as before.
3.3. Mixed polysaccharide fermentation
In the studies in which the effect of starch on
NSP breakdown was investigated, mixed populations of bacteria were grown in 1-1itre fermenters
as described previously. The initial concentrations
of both the starch polysaccharides and the NSP
(arabinogalactan, xylan or pectin) were 5 mg.
m1-1. Parallel incubations were carried out in
fermenters containing only the NSP (5 mg. ml-1).
Samples were taken for polysaccharide analysis at
0, 3, 6, 12 and 24 h.
3.4. Chemical analyses and chemicals
V F A in cell-free culture effluents were measured by gas-liquid chromatography (GLC) using
methods described by Macfarlane et al. [19].
Carbohydrate analysis was also carried out using
GLC, as described by Englyst and Cummings
[3,20]. Proteins were determined by the method of
Lowry et al. [21].
Lintners starch was purchased from BDH, all
other chemicals were obtained from Sigma.
4. RESULTS A N D DISCUSSION
4.1. Polysaccharidase and glycosidase activities in
faeces
Studies with pure cultures of human faecal
bacteria have shown that complex plant-cell polysaccharides are degraded by a range of hydrolytic
enzymes (polysaccharidases and glycosidases) that
are inducible and are generally cell-associated
[22,23].
Data presented in Table 1 shows that in human
faeces, polysaccharidase and glycosidase activities
were primarily associated with the bacterial fractions. With the exception of starch-degrading activity, the bulk of which is pancreatic in origin
[15], comparatively low levels of polysaccharidedegrading activity were found in faecal water,
confirming that in the large gut polysaccharidehydrolysing enzymes produced by the microflora
are mainly cell-associated.
Comparison of polysaccharidase activity in
bacteria that were growing unattached to particulate material in faeces, with those that had
been removed from particles by surfactant treatment, showed that the highest levels of starch and
pectin hydrolysis (4.25 and 1.09 /~mol reducing
sugar released, h - 1. mg protein- a, respectively)
occurred in the former, whereas xylan- and
arabinogalactan-hydrolysing activities were greatest in bacteria that were particle-associated (1.40
and 0.79 ~tmol reducing sugar released, h - l . m g
protein- 1, respectively).
/3-Galactosidase, a-arabinofuranosidase, /3-xylosidase and /3-glucosidase were the major glycosidases detected in faecal bacteria (Table 1) and in
general, specific activities of these enzymes were
significantly higher in those bacteria that were
particle-associated as compared to bacteria that
were growing unattached to particulate material.
These results may indicate that specific polysaccharide-degrading populations are associated
with particulate material in the large gut, however,
it seems more likely that bacteria growing on the
surface of food particles were simply expressing
enzyme activities that reflected the nature of the
substrate on which they were growing. Williams
and Strachan in a comprehensive study on the
166
Table 1
Polysaccharidase and glycosidase activities in different fractions of h u m a n faeces
Values are the mean of data obtained from 5 individuals a.
Faecal fraction
Cell-free
supernatant
Extract from washed
bacterial cells
Extract from washed
bacterial cells removed
from particulate
material by surfactant
treatment
Polysaccharide substrate b
Starch
Xylan
Pectin
Arabinogalactan
Galactomannan
Carboxymethylcellulose
Amylopectin
48.20-+ 11.34
ND
0.66 -+ 0.15
0.19 -+ 0.05
ND
ND
ND
4.255:
0.82_+
1.09 -+
0.43+
0.06 _+
0.17_+
0.71_+
1.16
0.18
0.29
0.13
0.02
0.12
0.12
3.96+
1.40+
1.14+
0.79+
1.62
0.47
0.32
0.16
0.19_+
0.06
ND
ND
Glycostde substrate c
p-Nitrophenyl
o-Nitrophenyl
p-Nitrophenyl
p-Nitrophenyl
p-Nitrophenyl
p-Nitrophenyl
p-Nitrophenyl
p-Nitrophenyl
p-Nitrophenyl
p-Nitrophenyl
p-Nitrophenyl
a-L-arabinofuranoslde
fl-o-xylopyranoside
a-D-galactopyranoside
fl-D-galactopyranoside
fl-D-fucopyranoside
fl-D-galacturonide
fl-o-glucuronide
a-D-glucopyranoside
fl-D-glucopyranoside
a-D-mannopyranoslde
fl-D-mannopyranoside
65
102
ND
434
129
ND
ND
113
50
78
20
± 34
_+ 69
1598
955
930
2 570
234
4
130
511
1581
16
52
+ 168
_+ 62
_ 73
_+ 35
5:44
+_ 8
a Values_+ SEM.
b Polysaccharidase activity: ~mol reducing sugar r e l e a s e d . h - 1 . m g protein
c Glycosidase activity: nmol p-nitrophenol released, h - 1 mg p r o t e i n - 1.
N D , Not detected.
distribution
of polysaccharide-degrading
enzymes
in rumen contents similarly found higher specific
activities of these enzymes associated with popula-
_ 522
+ 153
-+ 125
_+520
_+ 76
_+ 3
+ 38
-+ 76
_+474
_+ 9
_+ 23
2782
1986
832
3 231
145
86
44
586
1293
28
22
_+573
_+308
_+ 66
-+ 491
-+ 19
± 47
-+ 18
± 149
_+261
_+ 14
-+ 13
i.
ttons growing
on particulate
fied different functional
material
and
identi-
groups of microorganisms
on the basis of these observations
[24].
Table 2
Description and chemical nature of polysaccharides used in this study
Polysaccharide
Main structural features
Distribution and properties
Pectin
Arabinogalactan
al --, 4 D-galacturonans with rhamnosyl insertions
fll -~ 4 or 1 --* 3-D-galactopyranosides with arabino
side chains
fll -~ 4-D-xylopyranosyl chains with branching (1
3) and with arabino and 4-D-methyl glucurono side
chains
al ---, 4 and ~1 ~ 6 linked glucan
F o u n d in all plant tissues. Mainly water soluble.
F o u n d in most plant tissues. Water soluble.
Xylan
Starch
Occurs in virtually all plant cell walls. Insoluble in
native state but m a n y forms are water-soluble after
extraction.
Major storage polysaccharide m plants.
167
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10
20
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12
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6
A
10
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o.8
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04
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Incubation
14
~"
12
~'=
u~
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10
2
E
08
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=
o6
=~
-04
=~m~ m
, ~
/
"- .~,
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02
-~
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=,
. "''~'~"
0
12
2
.~
o
4
06
O2
1-6
•
48
24
period
(h)
a "~
*
12
Incubation
~
~
~" 0
24
period
48
(h}
Fig. 1. Breakdownof starch by human faecal bacteria grown in
batch culture anaerobically at 37°C. O, Residual starch; &,
total VFA; t,, cell-associatedamylase activity; ©, extracellular
amylase activity.
Fig. 2. Breakdown of arabinogalactan by human faecal bacteria.
O, Galactose polymer; II, arabinose polymer. A, Total VFA; zx,
cell-associated polysaccharidase activity; O, extracellular polysaccharidase activity.
4.2.1. Polysaccharide fermentation
r e l e a s e d . h - l . m g protein-I), but may also be
explained by the fact that the ability to hydrolyse
starch is considerably more widespread amongst
faecal bacteria, than is the ability to break down
other polysaccharides. Approx. 59% of the starch
fermented was recovered as VFA, with molar ratios
of acetate, propionate and butyrate produced being
50 : 22 : 29, respectively (Table 3). Starch-hydrolysing enzymes were initially cell-associated, extracellular activity was detected after 24 h incubation, however, which correlated with a decrease in
cell-associated activity. These results suggest that
to some extent at least, cell-associated polysaccharide-degrading enzymes were being released
from senescent bacteria.
A description of the chemical properties and
occurrence of the polysaccharides used in this
study is given in Table 2. All four polysaccharides
(starch, arabinogalactan, xylan and pectin) were
fermented by mixed populations of faecal bacteria
with production of VFA (Fig. 1-4). Only trace
amounts of free sugars and oligosaccharides were
detected in culture effluents, which would indicate
that in these experiments, the rate of hydrolysis of
the polysaccharides was the rate-limiting step in
their utilisation. Data are presented as the mean
of values obtained from two separate experiments.
4.2.2. Starch fermentation
Starch was the most rapidly fermented polysaccharide (Fig. 1). This could be attributed to the
high levels of cell-associated polysaccharidase present at zero time (1.45 /tmol reducing sugar
4.2.3. Arabinogalactan fermentation
Results presented in Fig. 2 show that the
arabinose side chains and galactose backbone of
168
10
20
'120
10
18
18
• -- °~o
8
~o
16
-i
5E
1
o
=5
2
14
6
6
t-I
\
"..
12
4
-
~"
12
-q
10
4
~
&
2
i
O8
'
-06
04
•
/
0
l
//
¶
~08
m
L'
=-=~a
\
\
j
02
~
\
-06
~
i
1
-°
E
O2
,
12
Incubation
24
period
48
(h)
Fig. 3. Breakdown of xylan by human faecal bacteria. O,
Xylose polymer; i, arabinose polymer. A, Total VFA; z~,
cell-associated polysaccharidase activity; O, extracellularpolysaccharidase activity.
arabinogalactan were simultaneously degraded by
colonic bacteria. About 43% of the polysaccharide
fermented was recovered as VFA. Molar ratios of
acetate (50), propionate (42) and butyrate (8) differed from those that occurred in the starch fermentations, in that more propionate and comparatively less acetate and butyrate were produced
(Table 3). Arabinogalactan-degrading activity was
not detectable at zero time in the washed cells
from either faecal sample but cell-associated activity was found in samples taken at 3 h (Fig. 2). The
rapid appearance of arabinogalactan-degrading
activity would suggest that enzymes were being
induced in bacteria already present in high numbers in the culture, rather than that selection of
arabinogalactan-degrading
p o p u l a t i o n s had
occurred, although it is recognised that this may
have occurred later. These data illustrate the nutritional versatility of colonic bacteria which enables
==
~A
0
~~
0
12
Incubation
48
24
per=od
E
(h)
Fig. 4. Breakdown of pectin by human faecal bacteria. O,
Uronic acid polymer; i, arabinose polymer. A, Total VFA; zx,
cell-associated polysaccharidaseactivity; ©, extracellularpolysaccharidase activity.
organisms to rapidly synthesise inducible catabolic enzyme systems thereby enabling them to
quickly adapt to the presence of new substrates.
4.2.4. Xylan fermentation
The insoluble polysaccharide xylan was slowly
and incompletely broken down by mixed cultures
of gut bacteria (Fig. 3). The arabinose side chains
and xylose backbone of the polysaccharide were
degraded concomitantly, as was the case with
arabinogalactan. In the early stages of xylan
breakdown the arabinose side chains and xylose
backbone sugars were utilized at comparable rates,
but whilst xylose utilisation continued after 12 h
incubation, the rate of arabinose uptake was considerably reduced. Since considerable xylan-degrading activity occurred in cultures at zero time
(Fig. 3) these results suggest that the relative insolubility of xylan may be an important factor
169
Table 3
VFA produced by mixed populations of human faecal bacteria
after 48 h growth on different polysaccharide substrates
Bacteria were grown in batch culture at 3 7 ° C anaerobically.
Molar ratios of VFA are given in parentheses.
Polysacchande
Starch
Arablnogalactan
Xylan
Pectin
VFA produced
( m g / m g polysaccharide utihsed) a
Acetate
Proplonate
Butyrate
Total
0.25 (50)
0.13 (22)
0.21 (29)
0.59
0.19 (50)
0.42 (82)
0.27 (84)
0.20 (42)
0.10 (15)
0.06 (14)
0.04 (8)
0.02 (3)
0.01 (2)
0.43
0.54
0.35
a Results are the mean of data obtained from separate experiments in which faecal samples from 2 different persons were
used.
affecting the rate of hydrolysis of this polysaccharide. Molar ratios of acetate, propionate and
butyrate produced were 82 : 15 : 3, respectively
Table 4
Glycos~dase activities on mixed populations of human faecal
bacteria after 24 h growth on different polysaccharide substrates
Bacteria were grown in batch culture at 37 ° C anaerobically.
Glycosidase
Glycosidase actiwty a
(nmol p-nitrophenol released
• h 1. mg protein- 1)
Starch
culture
a-L-Arabmofuranosldase
fl-D-Xylosidase
a-D-Galactosidase
fl-D-Galactosidase
fl-D-Fucosidase
fl-D-Galacturonidase
fl-D-Glucuromdase
a-D-Glucosidase
fl-D-Glucosidase
a-D-Mannosidase
fl-D-Mannosidase
1 207
1 168
910
1197
128
11
70
1149
1001
ND
58
Arabmogalactan
culture
Xylan
culture
1054
1036
200
1095
128
ND
51
1 020
859
ND
50
963
909
900
947
71
ND
ND
947
769
ND
ND
Pectin
culture
764
788
620
739
129
120
ND
681
572
19
38
a Results are the mean of data obtained from separate experiments in which faecal samples from 2 different persons were
used.
ND, Not detected.
(Table 3). The relative levels of acetate were considerably higher, and the propionate and butyrate
levels substantially lower than in the starch or
arabinogalactan fermentations.
4. 2.5. Pectin fermentation
The arabinose and uronic acid constituents of
pectin were rapidly utilised by faecal bacteria (Fig.
4). High levels of both cell-associated and extracellular pectinolytic activity were observed in
the early stages of the pectin fermentations. Only
about 35% of the pectin utilised was recovered as
VFA; molar ratios of acetate (84), propionate (14)
and butyrate (2) were almost identical to those
found in the xylan fermentations (Table 3), suggesting that similar bacterial species may possibly
have been involved in both xylan and pectin
breakdown.
4.2.6. Glycosidase activtties
a-Arabinofuranosidase, B-xylosidase, fl-galactosidase and fl-glucosidase were the major glycosidases formed by faecal bacteria, irrespective of
the polysaccharide growth substrate. In general,
however, relative glycosidase activities were greatest in starch-grown cultures, and least in cultures
grown on pectin (Table 4). These particular enzymes were also the predominant glycosidases detected in faeces (Table 1).
4.3. Significance of polysaccharide fermentation in
the large gut
The four polysaccharides studied in this investigation were each broken down at different rates
and to different degrees by faecal bacteria. The
rapid breakdown of starch suggested that, when a
mixture of NSP and starch was available to gut
bacteria, fermentation of NSP might be affected
by the operation of catabolite control mechanisms
[25,26]. The results shown in Fig. 5, however,
demonstrate that mixed cultures of faecal bacteria
fermented pectin, xylan and arabinogalactan concomitantly with starch. Indeed, the presence of
starch appeared to stimulate arabinogalactan (Fig.
5a, b) and xylan (Fig. 5c, d) breakdown, indicating that whilst catabolite control of metabolism
may be important to individual bacterial species
170
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b
n~
2
•
L
"~z~
0t
zx
~
5
4
-i
d
c
"
"~D
3
\
o
1
~A
0
~
z
5
4
x
~
O
~
O
t~
f
e
~,
\,
2
•
0---~-.-...._~_.
:\
00-"
o
•
:
1-'2
2 "4
Incubation
0
12
24
period (h)
Fig. 5 (a-f). Effect of starch on the breakdown of NSP by
mixed populations of faecal bacteria. (a) Arabinogalactan control; (b) arabinogalactan and starch; (c) xylan control; (d)
xylan and starch; (e) pectin control; (f) pectin and starch. O,
Residual starch; A, uronic acxd polymer; II, galactose polymer;
O, xylose polymer; zx, arabinose polymer.
[27], it does not appear to significantly inhibit
polysaccharide breakdown by mixed bacterial
populations. Data in Figs. 1-5 do suggest, however, that a hierarchy of polysaccharide utilisation
may occur in the large gut that may in part be a
result of the physical and chemical characteristics
of the individual polysaccharides. Previous studies
in which the breakdown of mixtures of polysaccharides was investigated in a multichamber continuous culture system support this conclusion
[28].
There are marked differences between starch
and NSP both in their role in the small intestine
and, as shown in the present study, in their fermentation by faecal bacteria. In future studies of
the large gut fermentation therefore it is important
to know the relative amounts of NSP and starch
that are available for fermentation. Different
amounts and proportions of VFA were formed
during the fermentation of different polysaccharides. The breakdown of xylan and pectin was
characterised by the production of large amounts
of acetate. In contrast, less acetate and more propionate and butyrate were formed during the fermentation of arabinogalactan and especially starch
(Table 3).
The high levels of butyrate that are formed
during the fermentation of starch are of special
interest since butyrate has a particularly important
role in the colon. Butyrate is the preferred substrate for energy generation by colonic epithelial
cells [29] and it has also been shown that butyrate
protects these cells against agents that lead to
cellular dedifferentiation [30]. Furthermore,
butyrate is also known to inhibit tumour growth
[311.
Foods containing substrates that when fermented in the large gut, result in large quantities
of butyr~/te being produced, may therefore be particularly beneficial to the host. Previous studies on
digestibility of starch in the small intestine of man
[1-3,32] and results presented in this paper suggest that starch may be such a substrate.
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