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MICROBIAL ECOLOGY IN HEALTH AND DISEASE

VOL.

8: 259-266 (1995)

Effect of Dietary Alginate on the Faecal Microbiota


and Faecal Metabolic Activity in Humans
A. TERADA*t, H. HARAt and T. MITSUOKA?
TDepartment of Food Hygiene, Nippon Veterinary and Animal Science University, 1-7-1 Kyonan-cho,
Musashino-shi, Tokyo 180, Japan

Received 18 April 1995; accepted 8 August 1995

The effects of alginate consumption on the faecal microbiota and faecal metabolic activity were studied in eight
healthy male volunteers who consumed a mixed free choice diet (control diet) for 1 wk before and after alginate
consumption, and the diet with additional 10 g alginate once a day for 2 wks. During alginate consumption, the
levels of bifidobacteria increased significantly (P<0.05), while the levels of Enterobacteriaceae and the frequency of
occurrence of lecithinase-negative clostridia showed tendencies to decrease. No detectable change occurred in the
levels or the incidence of other microorganisms throughout the experimental period. Faecal sulphide, phenol,
p-cresol, and indole were significantly decreased (P<0.05) during alginate consumption. After 2 wks of alginate
consumption, faecal concentrations of ammonia and skatole were significantly reduced (P<0.05),whereas the values
of acetic and propionic acids were significantly increased (PcO.05). The water content and weight of the faeces were
slightly increased to some extent during the consumption. The odour of the faeces decreased markedly during
alginate consumption.
KEY WORDS:

ammonia; alginate; intestinal microbiota; metabolic products; sulphide.

INTRODUCTION
Alginates are widely distributed in the cell wall and
cytoplasmic membrane of sea vegetables, l 1 such
as Ecklonia cava, E. maxima, Eisenia bicyclis,
Laminaria, Lessonia, Macrocystis, and Ascophyllum nodosum etc., and extracted from sea
vegetables with sodium carbonate. These are composed of a polymer with p-1, 4-D-mannuronate
and a- 1, 4-L-guluronate.
Alginates are used as emulsifier and stabiliser
in foods, medicine, cosmetics, feeds and various
industries, and suppress intestinal absorption of
heavy metals,31have an antitumour e f f e ~ t ,lower
'~
cholester01'~~~
and lower blood p re~ su re.~ '
Pratt et uI.*~ found that seaweed extract had
antibacterial activity in vitro against various organreported that Staphytocoisms. Burkholder et
ccus aureus is more susceptible to the inhibitory
products of algae than were Escherichia coli or
Candida albicans. Akiyama et
have showed a
significant increase in the growth of bifidobacteria
in vitro by the addition of depolymerised alginate.
*Author to whom correspondence should be addressed.
CCC 0891-060)<195/060259-08
0 1995 by John Wiley & Sons, Ltd

In this paper, we describe the effects of dietary


alginate on the faecal microbiota and faecal metabolic activity, weight, pH, and water content,
which are related to health and also to faecal
odour.
MATERIALS AND METHODS
Composition of sodium alginate
The depolymerisation of sodium alginate
(Kibun Food Chemipha Co. Ltd) was by alkaline
extraction from seaweeds which were a 2: 1 mixture
of Lessonia fravicans and L. nigrescens. The
viscosity and the molecular weight of the alginate
were 3.1 CP (5 per cent, 20C) and 30 000, respectively. The ratio of p-1, 4-D-mannuranate to a-1,
4-L-guluronate of alginate was 0.6.
Subjects and diet
The eight subjects were chosen from healthy
male volunteer students (aged 18-23yr) of the
Department of Food Hygiene, Nippon Veterinary
and Animal Science University. An experimental

A, TERADA ET AL.

260
Table 1. Mean daily energy and nutrient consumption

Diet ingested

Before
consumption
CN-1"

Alginate consumption

AS- 1

AS-2"

After
consumption
CN-2"

2698 f 175Id
85.6 f 4.3
82.3 f 5.3
749 f 28
11.1 f 1.3
2.0 f 0.3
25.3 f 1.6
27.6 f 1.5

2652 & 186


84.4 f 4.9
83.2 f 5.1
731 f 31
10.6 f 1.2
2.2 f 0.4
24.5 f 1.2
27.1 f 1.6

2624 f 154
83.9 f 4.1
81.0 f 5.7
736 f 25
10.9 f 1.1
2.1 zt 0.3
25.1 k 1.3
26.0 f 1.8

2641 f 136
84.3 f 3.9
83.1 f 6.3
738 zt 23
11.23~1.3
2.0 f 0.2
23.8 f 1.5
26.8 f 2.1

~~

Energy (KcaVday)
Protein (g/day)
Fat (glday)
Calcium (mglday)
Salt @day)
Crude fibre (g/day)
Saturated fatty acid (g/day)
Unsaturated fatty acid (g/day)

"Subjects took an alginate-free drink per day for a week.


bSubjects took a 10 g alginate-supplemented drink per day for a week.
'Subjects took a 10 g alginate-supplemented drink per day for a second week.
dValues expressed as mean f SD.

drink containing 10 g of alginate was consumed


once a day in addition to their normal diet for two
consecutive weeks. The volunteers consumed the
normal free-choice diet for 1 wk before and after
the alginate period. None of the subjects received
medication or foods with abundant viable cultures
for 1 wk prior to and during the experiment.
Nutrient analysis for food records of each dietary
period is summarised in Table 1.
This work was performed in accordance with
the Helsinki Declaration as updated in Tokyo,
1975.
Collections of specimens
Freshly voided faecal specimens were collected
on day 0 (end of control diet period 1) before
alginate consumption (day seven of the normal
free-choice diet), day 14 (first week of alginate diet)
and day 21 (second week of alginate diet) during
the alginate consumption, and day 28 (end of
control diet period 2) after the alginate consumption ended. The specimens were immediately transported at 4C to the laboratory for analysis. The
faecal microbiota, faecal weight, moisture content
and pH value were analysed within 3 h. The remainder of the samples were frozen at - 80C for
later analysis of bacterial metabolites.
Bacteriological analysis
The faecal microbiota was analysed by using the
methods and media of Mitsuoka et a1.22 and
the heat treatment method of Terada et
as

described p r e v i o u ~ l y .The
~ ~ ' results
~~
are expressed
as the log,, of the number of bacteria per gram wet
weight of faecal material.
Analysis of faecal metabolic products
Faecal ammonia was determined by potentiometer ILO-30 (DKK Co. Ltd, Tokyo) with an
ammonia gas sensing electrode 1716 (DKK Co.
Ltd, Tokyo) using the method of Terada et aZ.34
For the measurement of sulphide, a portion of 1 g
of faeces was suspended in 44 ml of distilled water,
mixed thoroughly with 5 ml of S-DIMAB (NaOH
40 g, L-ascorbic acid 10 g, sodium ethylenediaminetetra acetate 9.3 g, and glycerin 500 ml in
1000 ml of distilled water). The faecal sulphide was
measured using a sulphide electrode 7100 (DKK
Co. Ltd, Tokyo). Faecal concentrations of phenols
and indole were examined by gas chromatography
using the method of Y ~ s h i h a r a Faecal
. ~ ~ analysis
of short chain fatty acids (SCFA) were carried out
by the high-performance liquid chromatographic
method of Hara et al. l 6
Measure of faecal properties
Faecal moisture contents were measured using
approximately 1 g samples, weighed before and
after drying in a vacuum oven at 105C for 2 h.
The difference in weight was regarded as the water
content. The pH values were determined by inserting a flat glass electrode (DKK Co. Ltd, Tokyo)
directly into faeces.

26 1

ALGINATE A N D FAECAL BACTERIA

Table 2. Effect of alginate consumption on faecal microorganisms of humans


~~

Organism

Before
consumption
Day 0

Total bacteria
Bifidobacteria

10.95 f 0.17"
9.97 f0.26

Bacteroidaceae

10.66 f 0.21
(100)
9.98 f 0.3 1
(100)
9.34 f 0.66
(100)
8.87 f 0.37
(63)
8.73 f 0.61
(38)
6.10 f 0.20
(38)

Eubacteria
Peptococcaceae
Megasphaerae
Curved rods
Veillonellae
Clostridia
Lecithinase-positive

Lactobacilli
Enterobacteriaceae
Pseudomonads
Streptococci
Staphylococci
Bacilli
Yeasts
Moulds
~

After
consumption
Day 28

Day 14

Day 21

10.96 f 0.10
10.27 f0.12*
( 100)
10.69 f 0.12
(100)
9.94 f 0.31
( 100)
9.69 f 0.29
( 100)
9.23 f 0.35
(75)
8.94 f 0.70
(63)
5.63 f 1.89
(38)

10.86 f 0.10
10.22 f 0.21*
(100)
10.62 f 0.10
(100)
9.62 f 0.41
( 100)
9.49 f 0.57
(100)
9.13 f 0.37
(75)
8.80 f 0.50
6.40 f 0.78
(38)

11.03 f 0.13
9.98 f0.18
( 100)
10.85 f 0.14
(100)
9.82 f 0.33
( 100)
9.53 f 0.25
(100)
9.02 f 0.69
(75)
8.94 iz 0.58
(50)
5.90 f 1.16
(38)

3.55 f 0.90
(75)
9.06 f0-55
(38)
7.39 f 1.28
(88)
7.93 f 1.00
( 100)
3.43 f 0.62
(38)
8.11 f 1.54
(100)
2.95 f 0.79

4.18 f 1.17
(75)
9.13 f 0-49

5.39 f 1.83
(88)
9.30 f 0.33

4.81 f 1.21
( 100)
8.98 f 0.62
(88)
6.11 f 1.76
( 100)
8.37 f 0.84
( 100)
3.29 f 0.68
(50)
7.94 f 1.47
( 100)
3.36 f 0.77
(63)
3.49 f 0.92
(38)
2.94 f 1.13
(50)
2.40
(13)

Lecithinase-negative

~~~~~~~

During consumption

(50)

2.79 f 0.70
(38)
2.98 f 0.72
(50)

(0)

<

(50)

(50)

6.17 f 1.29
(88)
7.78 f 0.92
(100)
2.95 f 0-23
(50)
7.89 f 1.00
(100)
3.14 f 0.98
(75)
3.40 f 1.06
(63)
3.03 f 0.52
(38)

(0)

(100)

7.06 f 1.94
( 100)
8.72 f 1.15
(100)
3.02 f 0-40
(63)
8.28 f 0.84
( 100)
4.23 f 1.19
(75)
3.34 f 1.51
(63)
3.06 f 0.44
(50)

2.30
(13)

"Bacterial concentrations expressed as mean f SD of log,, number per gram of stool.


bFrequency of occurrence (YO).
Significant difference from the levels before consumption: *P<0.05.

Statistical analysis of data


The paired t-test and chi-square test were used
for statistical analysis of the faecal microbiota. The
paired t-test was used for analysis of bacterial
metabolites, faecal weight, pH values and water
content.

RESULTS
Faecal mkrobiota analysis
The effects of alginate on the faecal microbiota
analysis of eight volunteers are shown in Table 2.
The numbers of bifidobacteria were significantly
increased (P<0.05) during days seven and 14 of

262

A . TERADA ET AL.

10 -

9
Day 0
Day 14
Day 21
Before
During consumption
consumption

Day 28
After
consumption

Figure 1. Change in faecal levels of bifidobacteria on alginate consumption in


eight adults

alginate consumption compared to before consumption. The levels of Enterobacteriaceae and


the detection rate of lecithinase-negative clostridia
showed tendencies to decrease during alginate consumption, but not significantly so. The levels (log,,
8,80 k 0-71 and log,, 8-96 f0-59; mean f SD) of
lecithinase-negative clostridia on day seven and 14
of consumption, except for the value (log,, 9.60)
in one volunteer, were similar to those (log,,
8.98 & 0.62) before consumption. No detectable
changes were observed in these levels and in the
frequency of occurrence of other organisms
throughout the experimental period. The change
of the levels of bifidobacteria for each volunteer at
each dietary period is shown in Figure 1. The levels
of bifidobacteria in one volunteer decreased
throughout the experimental period.
Faecal putrefactive products
A significant decrease (lWO.05) in the amounts
of faecal ammonia and skatole were observed on
day 14 of alginate consumption, and faecal sulphide was significantly decreased (P<0.05) during
alginate consumption in comparison with that
before alginate consumption (Figures 2 and 3).
The concentrations of phenol were significantly
decreased (P<O.Ol) during consumption compared
to before alginate consumption. A significant
decrease of p-cresol and indole were observed on

days seven (P<0.05) and 14 (P<O.Ol) of consumption. Total faecal SCFA, acetic acid and propionic
acid significantly increased (W0.05) on day 14
of consumption (Figure 4). The odour of faeces
was noted to be less offensive during alginate
consumption.
Faecal weight, water content and p H value
Stool weights and water content showed a slight
tendency to increase during alginate consumption compared with those before consumption
(Table 3). No great difference occurred in faecal
pH values throughout the experimental period.
DISCUSSION

The indigenous faecal microbiota and its metabolic activities are thought to be important in
human
In particular, the increase of
bifidobacteria in the intestinal tract is thought to
exert a beneficial effect for the host by production
of short chain fatty acids, lowering the pH in the
bowel, decreasing putrefactive products, inhibiting
the growth of potential pathogens and immunopotentiation.
The intestinal bacteria (such as
bifidobacteria, ruminococci, clostridia and some
bacteroides) are known to play an important role
on the utilisation of dietary
such as plant
cell ~ a l l s ~ (cellulose,
, ~ ~ . ~ hemicellulose,
~
xylan,
'5320,27

263

ALGINATE AND FAECAL BACTERIA

d
v

'z
0

300
W

250

Em 200

v)
Y-

Bc

150

100

-0.
3

3 :0

.-

LI

u
C

P 50

W
0

Day14

Day21
During consumption

Day 0

Before
consumption

Day 28

0 !,
"

After
consumption

Figure 2. Effect of alginate consumption on faecal ammonia and faecal sulphide.


Ammonia and sulphide are expressed as mean f SD of eight adults, respectively.
Significant difference from the values before consumption; *P<O.O5

found a significant increase of bifidobacteria using


depolymerised alginate supplements in in vitro
cultures.
lndole
The reported effects of the consumption of
various dietary fibre on the human faecal microEthylphenol
biota are conflicting. Some worker^^,^,^^ reported
p-Cresot
that the supplementation of bananas, plantains,
............................
.................
rn
wheat bran and corn fibre, which are rich in
dietary fibre, did not alter the human faecal micro6 40
biota. Moore et al.24 also described no significant
difference in the intestinal microbiota of complete
s 20
'Z
vegetarians.
In contrast, Fuchs et al. l3 observed
e
+J
C
that
the
consumption
of wheat bran (5 g/day) led
0
0
u
to an increase in faecal clostridia and streptococci,
C
Day0
Day 14
Day 21
Day 28
23
Before
During consumption
After
and a decrease in lactobacilli and eubacteria.
consumption
consumption Benno et al.' found that rice fibre decreased
Figure 3. Effect of alginate consumption on faecal putrefactive the concentrations and incidence of clostridia
products. Data are expressed as mean of eight adults. Signifi- in human faeces. A decrease in the frequency of
cant difference from the values before consumption; *P<0.05, occurrence of lecithinase-negative clostridia was
**P<O.OI, ***P<O.OOI
also observed during c h i t o ~ a n and
~ ~ polydextrose" consumption in humans. The increase of
arabinogalactan and pectin), plant gums43 (guar bifidobacteria with dietary fibre consumption in
gum and gum arabic), seaweed polysaccharide the intestines of rat receiving 10 per cent konjac
(alginate), mucins's,26 (salivary mucin and gastric mannan23or 2 per cent hemicellulose isolated from
mucin) and mucopolysaccharides (chondroitin sul- rice bran3 has been observed. Wyatt et
found
phate and heparin). Hidaka et a l l 7 reported that that gum arabic enhanced the growth of
intestinal organisms could use greenpeas fibre BiJidobacterium Iongum in the human intestine. In
in vitro, but not dietary fibre such as sodium the present study, a significant increase in the
alginate, cellulose and carrageenan. Bayliss and faecal concentration of bifidobacteria during algiHouston4 demonstrated that bifidobacteria fer- nate consumption was similar to the findings in
mentated xylan, amylose, amylopectin, arabino- humans,'6J7'32,33and
fed various
galactam, gum arabic and mucin. Akiyama et aL2 oligosaccharides. The tendency to lower faecal

........I.

!!:::::-.!

264

A. TERADA ET AL.

...

Butyric acid
Propionic acid
Acetic acid

........... Formic acid


......
::.:$b;:
......

Day0
Day 14
Day21
Day28
Before
During consumption
After
consumption
consumption

Succinic acid

c]Lactic acid
..

Figure 4. Effect of alginate consumption on faecal short chain fatty acids (SCFA). Data are expressed
as mean of eight adults. Significant difference from the values before consumption; *P<0.05

Table 3. Effect of alginate consumption on human faecal weight, pH and water content
During consumption

Product

Before
consumption
Day 0

Day 14

Day 21

After
consumption
Day 28

Faecal weight (g)


PH
Water content (%)

108.4 z t 24.9
6.6 f 0.4
72.9 f 3.1

135.8 k 39.2
6.5 f 0.5
75.5 & 5.5

127.3 f 31.7
6.6 zt 0.6
76.1 f 4.3

103.6 zk !0.3
6.5 & 0.4
74.1 + 4.3

Data expressed as mean i SD.

concentrations of Enterobacteriaceae and the hemicellulose.30Faecal sulphide formed by deamilower detection rates of lecithinase-negative nation of cysteine and methionine in the intestine
clostridia during alginate consumption is similar decreased significantly during alginate consumpto that seen during lactosucrose16 and ~ h i t o s a n ~tion
~ similarly to that seen during lactosucrose6
consumption, respectively.
consumption. Phenolic and indolic compounds
The principal bacterial end-products in the produced by deamination of tyrosine, phenylhuman intestine are ammonia, sulphide, amines, alanine and tryptophan are detoxified by glucurophenols, the SCFA, fermentation gases and nide or sulphate conjugation in the liver and
energy, which the organisms utilise for rowth and excreted in urine. The decrease of faecal phenol,
the maintenance of cellular function! The end- p-cresol and indole in this study were similar
products of the metabolites produced during the to those seen with o l i g o s a ~ c h a r i d e s and
~~~~
catabolism of proteins are potentially harmful to ~ h i t o s a nconsumption.
~~
the host. Ammonia formed by deamination of
Cummings and MacFarlane8 reported that little
amino-acids, peptides and protein is absorbed change in faecal SCFA occurred with variation in
from the gut and detoxified by urea formation in diet, but that the total SCFA increased in parallel
the liver. In this study, the reduction of faecal with faecal weight. Adding corn bran to a low fibre
ammonia have resulted from either the sup- diet increased total SCFA in proportion of faecal
pression of ammonia production in the intestine, output. In the present study, the concentration
or from enhanced ammonia absorption from of total SCFA, acetic and propionic acids were
the large intestine as seen in humans fed corn increased during alginate consumption as has been

265

ALGINATE AND FAECAL BACTERIA

noted in humans fed c h i t o ~ a na n~d~ a cereal fibre


diet.7 Thus it appears that alginate promotes organic acid production and inhibits the putrefactive
activity of intestinal bacteria.
From these results, it may be concluded that
alginate consumption influences the composition
and the metabolic activity of the intestinal
microbiota.
ACKNOWLEDGEMENTS

11.
12.

13.

The authors would like to thank Kibun Food Chemipha


Co. Ltd, for supplying sodium alginate.

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