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Applied and Environmental Microbiology, April 2000, p. 1321-1327, Vol. 66, No. 4
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Degradation of Pectins with Different Degrees of Esterification
by Bacteroides thetaiotaomicron Isolated from Human
Gut Flora
Gerhard
Dongowski,*
Angelika
Lorenz, and
Horst
Anger
German Institute of Human Nutrition
Potsdam-Rehbrücke, D-14558 Bergholz-Rehbrücke, Germany
Received 5 October 1999/Accepted 8 January 2000
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ABSTRACT |
A complete human fecal flora and cultures of defined species
obtained from fecal flora were investigated in vitro to determine their
ability to ferment the dietary fiber pectin. Bacteroides thetaiotaomicron was tested as a pectin-degrading microorganism alone and in coculture with Escherichia coli.
Macromolecular pectins with different degrees of esterification
were used as substrates in microbial degradation studies. The levels
of oligogalacturonic acids formed in batch cultures were estimated
during a 24- or 48-h incubation period by using
high-performance thin-layer chromatography and
high-performance anion-exchange chromatography. The spectrum and
the amount of unsaturated oligogalacturonic acids formed as intermediate products of pectin fermentation changed permanently in the
culture media during incubation with the complete fecal flora. After
24 h, no oligogalacturonic acids were detected. The pectin-degrading activities of pure cultures of B. thetaiotaomicron were lower than the pectin-degrading activity of
a complete fecal flora. Cocultures of B. thetaiotaomicron
and E. coli exhibited intermediate levels of degradation
activity. In pure cultures of E. coli no pectin-degrading
activity was found. Additionally, the rate of pectin degradation was
affected by the degree of esterification of the substrate. Saturated
oligogalacturonic acids were not found during pectin fermentation. The
disappearance of oligogalacturonic acids in the later stages of
fermentation with both the complete fecal flora and B. thetaiotaomicron was accompanied by increased formation of
short-chain fatty acids.
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INTRODUCTION |
In human nutrition, pectin is one of
the most important sources of dietary fiber. It is present in
vegetables and fruits as a component of the plant cell wall. Pectin
consists mainly of long linear chains of 
-1,4-glycoside-linked
D-galacturonic acid (homogalacturonan; "smooth"
regions) which are partially esterified with methanol. In addition,
branched and complex pectic substances are present in the cell wall
(rhamnogalacturonans I and II; "hairy" regions) (45).
Like other types of dietary fiber, pectin is not depolymerized by
endogenous gastrointestinal enzymes during passage through the stomach
and the small intestine. A number of physiological effects of pectin or
pectin-containing diets have been described; these effects include
decreasing serum cholesterol levels (17), increasing fecal
excretion of steroids (24), interacting with metal ions
(25), and interacting with bile acids in vitro
(11). These effects depend on the macromolecular state of pectin.
In the colon, pectin is fermented more or less completely by the
microflora, as shown previously (8, 9, 18, 28, 48). However,
there have been only a few studies in which the intermediate steps in
pectin degradation by gastrointestinal microorganisms have been
examined. On the other hand, the end products of bacterial fermentation
of pectin are well known; a spectrum of short-chain fatty acids (SCFA)
and different gases (CO2, H2, H2S,
CH4) are formed. In some studies it was shown that feeding
rats pectin can decrease the number of colon tumors (36).
Likewise, it was found that the SCFA butyrate may inhibit the growth of
different colon cancer lines (4, 27, 44) or may decrease the
total number of tumors induced by 1,2-dimethylhydrazine in rats
(33). However, the results of other studies did not support
these conclusions (26).
In some cases, physiological effects of pectin are independent of the
polymer state but dependent on the intermediate cleavage products.
Thus, it has been shown that oligogalacturonic acids (oligoGalA) bind
heavy metal ions, especially lead, with high efficiency. These acids
seem to play an important role in the mechanism which results in
increased excretion of lead into urine after pectin is eaten or
pectin-rich diets are used (13, 51). Such an effect is
directly related to the chemical nature, the amount, and the stability
of cleavage products formed in the gut. Therefore, besides the end
products of fermentation, the intermediate products of bacterial pectin
degradation may have physiological importance.
In a previous pilot study, it was shown that pectin is fragmented into
a spectrum of oligoGalA that are intermediate products during
incubation in vitro with the human fecal flora. Mixtures of unsaturated
di-, tri-, and tetragalacturonic acids were the end products of pectate
lyase activity in the cultures examined. Later, these compounds
disappeared as a result of further fermentation by the gastrointestinal
microflora. Low-methoxyl pectins were depolymerized and fermented
faster than high-methoxyl pectins. Furthermore, it was found that a
mixture of unsaturated oligoGalA prepared from pectic acid by using
pectate lyase from Erwinia carotovora was completely
fermented by human fecal flora in vitro (14).
In this study, the time course of pectin degradation, the transient
formation of oligoGalA, and the conversion of these acids to SCFA
catalyzed by human fecal flora, pure cultures of Bacteroides thetaiotaomicron, or a defined coculture containing B. thetaiotaomicron and Escherichia coli isolated from
human feces were investigated in relation to the degree of
esterification of pectin.
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MATERIALS AND METHODS |
Pectin preparations.
In all experiments,
high-molecular-weight citrus pectin preparations with different degrees
of esterification were used. Pectins B and C were commercial low- and
high-methoxyl citrus pectin preparations without additives produced by
Copenhagen Pectin A/S (Lille Skensved, Denmark). These preparations
were purified further by extraction with acidified 60% ethanol. Very
highly esterified pectin D was prepared by treating pectin C with
methanol-concentrated H2SO4 at 4°C
(14). Pectin A (pectic acid) was prepared by alkaline deesterification of pectin C (11). The pectin preparations
used contained no acetyl or amide groups.
Pectin analysis.
The galacturonan
("anhydro"-galacturonic acid) contents of the pectin preparations
and fractions were determined colorimetrically by the
m-hydroxydiphenyl method (7). The methyl ester
group contents were determined by the chromotropic acid method
(6). Intrinsic viscosity was determined by using an
Ubbelohde viscosimeter at 25.0°C and pH 6.0 in 0.155 M NaCl
(high-methoxyl pectins) or in 0.05 M NaCl-0.005 M sodium oxalate
(low-methoxyl pectins). The relationship between intrinsic viscosity
and the average molecular weight of pectins is described by the
Mark-Houwink equation (2).
Bacterial strains and culture conditions.
An inoculum was
prepared from fresh feces collected from a healthy female volunteer who
ingested a normal Western diet, had no digestive diseases, and had not
taken antibiotics for the previous 6 months. The feces were collected
anaerobically. For batch cultures, 5-g portions of fresh human feces
were incubated in 150-ml portions of nutritive medium in 200-ml bottles
without aeration at 37°C in a water bath. The medium used for the
batch cultures contained 0.25 or 0.5% galacturonan in 0.067 M
Sörensen phosphate buffer (pH 7.8) (medium A) or in
Sörensen phosphate buffer enriched with 1% pancreatic
peptone (Merck, Darmstadt, Germany) (medium B). The pectin-containing
medium was sterilized by filtration with a Sartobran PH minicartridge
(pore size, 0.45 µm; Sartorius, Göttingen, Germany).
B. thetaiotaomicron and E. coli were identified
as pectin-degrading microorganisms obtained from human feces by using
the modified method of Jensen and Canale-Parola (22) and
blood agar plates containing pectin; the organisms were isolated and
rinsed by repeated plating on selective agar plates, as described below for enumeration of viable cells. Organisms were identified by the VITEK
automatic identification method (BioMerieux, Nürtlingen, Germany). The numbers of viable cells in batch cultures were determined by using 0.5-ml samples for serial dilution and subsequent plating on
Columbia blood agar plates (BioMerieux) that were incubated aerobically and anaerobically. Organisms were differentiated on the
following selective media: aerobically incubated Endo agar (Merck) for
E. coli and coliforms, aerobically incubated MRS agar (Merck) for members of the Lactobacillus group, and
anaerobically incubated neomycin-blood agar for B. thetaiotaomicron and members of the gram-negative anaerobic group.
Plates were incubated anaerobically by using the Anaerocult System
(Merck). Starter cultures of E. coli and B. thetaiotaomicron were preincubated for 18 and 40 h, respectively, in nutrient broth (Difco, Augsburg, Germany) without aeration. Starter cultures (4 ml) containing 5 × 108
cells/ml were used as inocula for the batch cultures in 150-ml portions
of medium B. Samples (4 ml) were taken immediately after inoculation
and then periodically after 2 to 48 h of incubation.
Characterization of pectin degradation.
To determine the
amounts of high-molecular-weight and low-molecular-weight pectin
fractions and oligoGalA in batch cultures, 4-ml samples were mixed
immediately with 1 ml of 0.2 M HCl and 5 ml of ethanol to stop
bacterial growth and enzymatic reactions. After centrifugation
(6,000 × g, 30 min, 4°C), some of the supernatant was used to determine the galacturonan content (low-molecular-weight pectin fraction content. The remaining supernatant was concentrated in
a vacuum, dissolved in 1.5 ml of H2O, centrifuged
(10,000 × g, 30 min, 4°C), and used for
chromatographic determination of the oligoGalA content.
To remove low-molecular-weight substances, the residues (coagulates)
that remained after centrifugation were extracted three
times with 50%
ethanol and once with 96% ethanol with stirring.
During the second
extraction, enzymes were inactivated by heating
the preparation at
85°C for 15 min. After extraction of the residues
with 0.5% EDTA (pH
6.0) and coagulation at pH 2 in 50% ethanol,
the macromolecular pectin
contents of batch cultures were determined
colorimetrically
(
7).
The oligoGalA content and the oligoGalA composition were determined by
using a combination of two chromatographic techniques
with different
detection
methods.
A high-performance thin-layer chromatography (HPTLC) apparatus obtained
from Camag (Muttenz, Switzerland) included a model
III automatic
thin-layer chromatography sampler, an automatic
development chamber, a
dipping device, and a model II thin-layer
chromatography scanner with
CATS evaluation software. Up to 3
µl of a sample was applied to an
activated Silica Gel 60 F
254 plate (Merck). The
chromatograms were developed four times with
n-propanol-water mixtures (7:4.50 to 7:2.75) by using the
following
conditions: run distance, 40 to 80 mm; drying time, 10 min;
heating
time, 1.5 min; and precondition time, 5 min. The delta-4,5
double
bonds of unsaturated oligoGalA that formed were detected at 235
nm. Then the plates were dipped twice for 3 s in a 0.5% solution
of
m-hydroxydiphenyl in acetone, heated for 10 min at
100°C, and
scanned at 525 nm to obtain information concerning the
total amounts
of substances in individual spots (
12).
Additionally, oligoGalA were analyzed by high-performance
anion-exchange chromatography (HPAEC) by using a chromatography
system
obtained from Kontron (Neufahrn, Germany) and equipped
with a UV
(250-nm) chiralyser and a pulsed amperometric detector.
A CarboPac PA1
column (250 by 9 mm) from Dionex (Idstein, Germany)
with a precolumn
and a nonlinear gradient consisting of 40 to
100% 1 M sodium acetate
in 0.15 M NaOH and 60 to 0% 0.15% NaOH
for 50 min (flow rate, 2 ml/min) were
used.
Both chromatographic methods were calibrated by using a mixture of
oligoGalA with degrees of polymerization between 2 and
>7 prepared
from pectic acid by using pectate lyase from
E. carotovora (
12).
Determination of SCFA contents.
The concentration of SCFA
was determined by gas-liquid chromatography by using a Carbowax 20M
column (25 m by 0.32 mm [inside diameter]) attached to a
Hewlett-Packard model 5890A chromatograph equipped with a flame
ionization detector and a split injector. Helium was used as the
carrier gas. The column temperature was maintained at 125°C, and the
injector port and detector temperatures were 200°C.
Isobutyrate (internal standard), perchloric acid, and an NaOH solution
were added to 1 ml of each sample. After freeze-drying,
the material
was homogenized in a mixture containing 100 µl of
5 M formic acid and
400 µl of acetone. A 1-µl sample of the organic
phase was injected
into the gas-liquid
chromatograph.
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RESULTS |
Pectin substrates.
The galacturonan concentrations of the
pectin preparations used were between 59 and 73%. The degrees of
esterification of the substrates were as follows: pectin A, 0%; pectin
B, 34.4%; pectin C, 66.0%; and pectin D, 94.7%. The free and
esterified carboxyl groups in the pectin macromolecules were
distributed in a random (statistical) manner. All of the pectins used
were high-molecular-weight preparations; their intrinsic viscosities were between 270 and 920 ml/g of galacturonan.
Microbiology.
Table 1 shows the
effects of incubation time and degree of esterification of the
substrate on selected groups of intestinal microbes, as indicated by
the number of viable cells under the experimental conditions used. The
viable cell content increased slightly or, in some cases, significantly
for the Bacteroides group. The number of bacteria belonging
to the Enterobacteriaceae increased. The colony counts for
lactobacilli (log 3 to 4) and the total anaerobic cell counts (log 8.5 to 9.2) were nearly constant. Furthermore, the degree of esterification
of pectin did not have a significant effect on the viable cell counts.
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TABLE 1.
Numbers of viable cells of members of the
Enterobacteriaceae and the Bacteroides group
after a 24 h of incubation of fecal flora with pectins with
different degrees of esterification
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After 24 h, the numbers of viable cells were similar for pure
cultures of
E. coli or
B. thetaiotaomicron and
for mixed cultures,
as well as for complete feces cultures (Table
2).
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TABLE 2.
Numbers of viable E. coli and B. thetaiotaomicron cells after 24-h of incubation of different
microbial cultures with pectin B (degree of
esterification, 34.4%)
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The pH of the culture medium decreased from 7.7 at the beginning of
incubation to 7.2 to 6.7 at the end of
incubation.
Pectin degradation with complete fecal flora.
During
incubation of pectin with a complete feces culture in vitro the
following effects were observed. The portion of macromolecular pectin
that was not soluble in 50% ethanol decreased during incubation, whereas the portion of galacturonan in the low-molecular-weight fraction that was soluble in 50% ethanol increased continuously during
the first phase of fermentation. When high-methoxyl pectin C was used
as a substrate, the amount of the low-molecular-weight fraction reached
a maximum value after 10 to 12 h (Fig.
1). Then it decreased almost like the
amount of the macromolecular fraction decreased. After incubation for
24 h, only very small amounts of both pectin fractions were
present in the experimental culture. Generally, the high-methoxyl
pectins were degraded more slowly than the minimally esterified
substrates by the total fecal flora (14).
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FIG. 1.
Concentrations of macromolecular (cross-hatched bars)
and low-molecular-weight (solid bars) pectin fractions during in vitro
fermentation of high-methoxyl pectin C (degree of esterification,
66.0%) with complete human fecal flora (n = 6).
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The changes in oligoGalA composition were measured during incubation by
HPTLC and HPAEC. The multiple development technique
used with HPTLC
improved separation of the individual oligomers
by a
"pseudogradient." HPAEC with combined UV, pulsed amperometric,
and
chirality detection was a suitable technique for determining
oligoGalA
contents. The UV absorption values which were related
to the number of
double bonds in the unsaturated oligoGalA were
affected by the presence
of acetate in the elution buffer. This
effect could be suppressed by
obtaining UV measurements at 250
nm instead of 235 nm. The sensitivity
of detection decreased with
the degree of polymerization of oligomers.
In contrast, pulsed
amperometric detection was closely related to the
reducing end
groups of the oligomers, and the analytical sensitivity
decreased
with chain length. Chirality detection was related to monomer
units in the chain, and except for very low degrees of polymerization,
the response was not related to chain length. A typical HPAEC
chromatogram is shown in Fig.
2.
Determinations of the qualitative
and the quantitative compositions of
oligoGalA were optimized
by using both chromatographic techniques with
different detection
methods.
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FIG. 2.
Determination of oligoGalA contents by HPAEC with pulsed
amperometric (PAD), UV (250-nm), and chirality detection after
incubation of low-methoxyl pectin B with complete human fecal flora
(peaks 2 through 7 correspond to degrees of polymerization of 2 through
7, respectively).
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A broad spectrum of oligoGalA was found in cultures during incubation.
The quantity of oligoGalA also depended on the degree
of esterification
of the substrate. Maximal formation of these
oligomers occurred after
approximately 8 h if low-methoxyl pectin
B was used as the
substrate. No maximum was observed for up to
12 h when
high-methoxyl pectins C and D were fermented. After
24 h,
oligoGalA were absent in the culture inoculated with the
complete
fecal flora (Fig.
3). Saturated oligoGalA
were not formed
as a result of bacterial action.
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FIG. 3.
Unsaturated oligoGalA contents and compositions during
incubation of pectins with different degrees of esterification (DE)
with complete human fecal flora (n = 4).
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The oligoGalA formed must have been depolymerized further to the
unstable monomer, which was rearranged to
4-deoxy-
L-
threo-5-hexoseulose
uronic acid
(
37) by unidentified enzymes. It was not possible
to
detect this monomer chromatographically, perhaps because it
was formed
intracellularly. The end products of fermentation of
pectin are the
SCFA, which are physiologically important metabolites
(
40).
The concentration of SCFA (acetic acid, propionic acid, and butyric
acid, as well as low concentrations of valeric acids)
increased
continuously during incubation (Table
3).
In accordance
with the rate of formation of oligoGalA, the amount of
SCFA produced
was significantly larger when low-methoxyl pectins were
used as
substrates. The lower concentration of SCFA obtained after
24
h when high-methoxyl substrates were used was related to
incomplete
depolymerization of the pectins to monomeric
units (Fig.
1). The
preferred SCFA formed was acetic acid, which
accounted for more
than 75 mol%. The molar concentrations of
propionic acid and butyric
acid were relatively similar for incubation
times up to 12 h.
It was remarkable that significantly more
butyrate than propionate
was found after 24 h of incubation with
all of the pectin substrates
(Table
3). Only very small amounts of
n-valerate and isovalerate
were present. Additionally, the
pH decreased continuously in the
media as a result of formation of
SCFA.
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TABLE 3.
Formation of SCFA and pH values in cultures during
fermentation of pectins with different degrees of esterification
when the complete human fecal flora was used
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Pectin degradation with defined bacterial cultures.
Compared with the complete fecal flora cultures, development of
the different pectin fractions in pure cultures of B. thetaiotaomicron was similar, but the reaction was slower.
This effect was found to be independent of the degree of
esterification of pectin, but it was less pronounced when
high-methoxyl substrates were used (Fig.
4 and 5).
Distinct degradation did not begin before a culture had been incubated
for approximately 6 h. Therefore, maximum formation of oligoGalA
occurred later than it occurred in experiments in which the complete
fecal flora was used. A reduction in the amount of the
low-molecular-weight pectin fraction did not occur in any of these
cultures until after 48 h of incubation.
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FIG. 4.
Variation in macromolecular (cross-hatched bars) and
low-molecular-weight (solid bars) fractions during in vitro
fermentation of low-methoxyl pectin B with B. thetaiotaomicron (B. theta.), E. coli, and a
mixed culture containing B. thetaiotaomicron and E. coli (n = 6).
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FIG. 5.
Variation in macromolecular (cross-hatched bars) and
low-molecular-weight (solid bars) fractions during in vitro
fermentation of very highly esterified pectin D with B. thetaiotaomicron (B. theta.), E. coli, and a
mixed culture containing B. thetaiotaomicron and E. coli (n = 6).
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In the case of an
E. coli strain, the agar plate test for
pectin degradation activity resulted in a positive reaction on
pectin-blood
agar but no reaction on pectin-Endo agar. Likewise, no
pectin
degradation was observed in batch cultures (medium B) of
E. coli.
However, in cocultures with
B. thetaiotaomicron, the degradation
activity was greater than the
degradation activity in pure
Bacteroides cultures (Fig.
4
and
5). When the mixed bacterial culture was
used, low-methoxyl pectin
was degraded more pronounced than high-methoxyl
pectin. After 48 h
of incubation, a decrease in the oligomer content
was observed if
low-methoxyl pectin was
used.
In pure
B. thetaiotaomicron cultures, a broad spectrum of
oligoGalA was detected (Fig.
6 and
7). This spectrum was similar
to the
spectrum obtained in experiments performed with a complete
fecal flora.
Also, in all cases only unsaturated oligoGalA were
detected.
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FIG. 6.
Unsaturated oligoGalA contents during incubation of
low-methoxyl pectin B with B. thetaiotaomicron (B. theta.), E. coli, and a mixed culture containing
B. thetaiotaomicron and E. coli (n = 4).
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FIG. 7.
Unsaturated oligoGalA contents during incubation of very
highly esterified pectin D with B. thetaiotaomicron
(B. theta.), E. coli, and a mixed culture
containing B. thetaiotaomicron and E. coli
(n = 4).
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The influence of the degree of esterification of pectins on the
formation of oligoGalA suggests that pectate lyase and pectin
esterase
activities occurred in the bacterial cultures from feces.
Although we
could not directly detect pectin esterase activity,
we hypothesized
that this enzyme was involved in pectin degradation,
especially if more
highly esterified pectins were used. The degrees
of esterification of
low- and high-methoxyl substrates were stable
under the conditions used
in the absence of fecal bacteria for
up to 48 h. However, a slight
decrease in the degree of esterification
was observed if the very
highly esterified pectin D was treated
under the same
conditions.
Degradation of oligoGalA to SCFA took place throughout incubation.
Approximately 15 µmol of acetic acid per ml was formed
during the
first 8 h of incubation with pectin acid or low-methoxyl
pectin.
On the other hand, only approximately 10 µmol/ml was formed
when
high-methoxyl or very highly esterified pectin was used.
In the case of
high-methoxyl pectin, the amount of acetic acid
increased continuously
during incubation. Compared with other
substrates used, more acetic
acid was formed between 8 and 12
h. Relatively small amounts of
propionic and butyric acids were
formed continuously with all of the
pectins.
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DISCUSSION |
It is well known that pectin (which is consumed mostly in the form
of fruits and vegetables but also as hydrocolloid in "functional foods," jellies, milk products, etc.) is fermented in the large intestine to SCFA and gases. Decomposition of the polysaccharide pectin
occurs during the following main steps: (i) macromolecular pectin, (ii)
(unsaturated) oligoGalA, (iii) monogalacturonic acid (or its
rearrangement products), and (iv) SCFA (and gases).
Formation of oligoGalA as intermediate products during pectin
degradation by human or animal microflora has not been analyzed systematically previously. Our results show that the velocity of
oligoGalA formation depends on the structure of the pectin used
(especially the degree of esterification), although all pectins are
fermented more or less completely by microflora in vitro and in vivo in
the end. The velocity of formation of the oligoGalA also acts as a
limiting factor for later formation of SCFA.
Previously, the fate and fermentation of the dietary fiber pectin
during passage through the gastrointestinal tracts of humans and
animals were investigated in vivo and in vitro. Cummings et al.
(9) observed no increase in fecal excretion of pectin after intake of 36 g of pectin per day for 6 weeks by male volunteers. This was because there was intense bacterial fermentation of pectin in
the colon. Different human intestinal bacteria are known to degrade
pectin; these bacteria include Bacteroides strains,
eubacteria, clostridia, and Bifidobacteria (15, 20, 30, 41,
42). Dekker and Palmer (10) observed that a
Bacteroides strain from human feces contained constitutive
polysaccharidases, especially polygalacturonic acid-degrading
activities. Jensen and Canale-Parola (22, 23) described
Bacteroides sp. strains as pectin-degrading microbes in the
human intestinal flora. When B. thetaiotaomicron was grown
in a pectic acid-containing medium, cell-associated polygalacturonic
acid lyase (EC 4.2.2.2) and hydrolase (EC 3.2.1.15) were present
(29). A Clostridium butyricum-Clostridium
beijerinckii strain isolated from human feces was able to produce
pectate lyase and to decompose pectic acid (31). Recently,
Matsuura (32) identified a pectate lyase of the endotype
which splits pectic acid into unsaturated oligoGalA in human feces
extracts. It is well known that B. thetaiotaomicron can
utilize a wide variety of plant polysaccharides (50). For
instance, Reeves et al. characterized an outer membrane protein which
is essential for utilization of maltooligosaccharides and starch by
this Bacteroides species (38). Tomlin et al.
(49) determined that pectin was completely fermented by
human fecal bacteria within 21 h, that the viscosity of the pectin
was lost, and that the culture pH declined. In some of these studies,
the workers investigated the disappearance of pectin, the enzymes or
microorganisms involved, and/or the formation of SCFA. Sometimes, the
fermentation of pectin-containing complex substrates, like apple fiber,
by human fecal bacteria was studied (19). Recently, Tierny
et al. (47) found pectate lyase, pectin esterase, and
polygalacturonase activities in B. thetaiotaomicron 217 grown on pectin as the sole carbon source. These authors investigated molecular cloning and expression of genes encoding the pectate lyase
and pectin esterase activities in E. coli.
Only limited information is available concerning the variation in
pectin molecules or the dynamics of pectin degradation during bacterial
activity in the colon.
Consistent with the results of in vivo experiments performed with rats
(35), low-methoxyl pectins were fermented in vitro more
efficiently than high-methoxyl pectins were fermented. Therefore, Dongowski and Lorenz concluded that low-methoxyl pectins are the preferred substrates of the pectin-depolymerizing enzymes of the human
microflora (14).
The mechanism of oligoGalA formation from ingested pectin has not been
sufficiently investigated. In the experiments described here,
macromolecular pectin was depolymerized and enzymatically degraded to a
spectrum of unsaturated oligoGalA. The pattern of oligoGalA
formed as intermediate products showed that the key enzyme during this
pectin degradation is pectate lyase. It is well known that
high-methoxyl and very highly esterified pectins are degraded at
decreased rates by pectate lyases (or polygalacturonases) (39). Pectin lyase is the only enzyme which is able to split all pectins independent of the degree of esterification. However, this
enzyme is preferentially present in fungi (39). Distinct chemical deesterification was not detected under the conditions used.
The influence of the degree of esterification of pectin on
fermentation, the formation of oligoGalA, and the decrease in the pH of
the medium indicated that both pectate lyase activity and pectin
esterase activity were present. However, we did not detect pectin
esterase activities in in vitro experiments.
There are at least two processes that occur side by side,
depolymerization of galacturonan macromolecules and decomposition of
the oligomers formed to the end products of fermentation, SCFA and
gases. The results of depolymerization are increases in the oligoGalA
content and the presence of relatively high concentrations of these
oligomers in batch cultures after 4 to 6 h. Later, the level of
oligoGalA formation decreases in connection with a decrease in the
pectin content of the culture media under the conditions which we used.
Formation of SCFA starts immediately after oligoGalA appear, but the
reaction speed seems to be lower than the reaction speed of the pectin
degradation process.
The effect of pectin on excretion of heavy metals, such as lead, via
kidneys has been discussed previously (13, 51). OligoGalA were described as the active substances. Anger et al. (3)
found that 9 to 45% of oligoGalA directly injected into the ceca of rats were recovered in the urine within 16 h.
Based on our results obtained in vitro, we propose the following
hypothesis. The pectin content in a continuous-flow culture (like the
gut) may not decrease for a long time. Therefore, formation of
oligoGalA may continue with a high yield over a relatively long period.
With this background, the results suggest the possibility that some of
the bioactive molecules formed from ingested pectin may be absorbed in
the colon.
The increasing numbers of E. coli cells in feces cultures
and positive reactions on blood agar plates indicate that these microbes may participate in pectin degradation. This hypothesis is not
supported by the results obtained with pure cultures of E. coli. On the other hand, the pectin-degrading activity was greater
in mixed cultures containing E. coli and B. thetaiotaomicron than in pure Bacteroides cultures.
This indicates that E. coli may participate in
degradation of pectin.
There are two possible ways to interpret these results: (i)
E. coli is able to promote the degrading activity of
B. thetaiotaomicron (e.g., by deesterification) without
affecting the viable cells in the culture; or (ii) E. coli has an inducible pectate lyase which requires external
stimulation or support. Further investigations may answer the questions
raised here.
Although some of the oligoGalA formed from pectin in the colon may be
absorbed (3), most of them are fully degraded to the main
fermentation end products of dietary fiber, gases and SCFA, which are
detected in feces. The SCFA butyrate plays an important physiological
role (46). It is the major energy source for colonic
epithelial cells (40) and acts as a regulator in the cell
cycle. The effects of butyrate on normal and neoplastic cells may be
different or opposite (21). Butyrate may have a role in
preventing certain types of colitis (43). However, the hypothesis that butyrate protects against colon cancer was not supported by all of the studies performed (26). Barry et al. (5) described an interlaboratory study in which it was found that pectin was almost completely fermented (97.4%) within 24 h
in vitro. Compared with cellulose, sugar beet fiber, soybean fiber, or
maize bran, greater total SCFA production (67.7 mmol/g after 24 h), a molar ratio of acetic acid to propionic acid to butyric acid of
74.4:8.9:16.9, and a decrease in pH of 0.93 U were estimated. Other
authors found between 2 and 17% butyrate in the SCFA during
fermentation of pectin by human fecal bacteria in vitro (1, 16,
34).
In conclusion, we found that distinct amounts of unsaturated oligoGalA
are formed as metabolites during pectin fermentation. The rate of the
enzymatic reactions is influenced by both molecular parameters of the
substrate, such as the degree of esterification, and the synergistic
effects of bacteria. Later formation of oligoGalA due to the use of
highly esterified pectins as substrates resulted in later formation of
SCFA. Therefore, it seems to be possible to extend the region of
intense formation of SCFA into the distal parts of the colon by using
dietary fibers (like pectin) with special structural parameters.
| |
ACKNOWLEDGMENTS |
We thank Ingrid Vogel and Horst Maischack for their skillful
technical assistance.
This study was supported by the German Federal Ministry for Education,
Science, Research and Technology.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: German Institute
of Human Nutrition Potsdam-Rehbrücke, Department of Food Science and Preventive Nutrition, D-14558 Bergholz-Rehbrücke, Germany. Phone: 49 33200 88268. Fax: 49 33200 88444. E-mail:
dongo{at}www.dife.de.
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Abstract
Introduction
