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MICROBIAL ECOLOGY IN HEALTH AND DISEASE
VOL.
7: 247-256 (1994)
Effects of Gluconic Acid on Human Faecal Bacteria
T. ASANO*t, K . YUASAt, K. KUNUGITAt, T. TERAJIt AND T. MITSUOKAt
?Chemical Products Research Laboratories, Fujisawa Pharmaceutical Co., Ltd, 5-2-3, Tokodai, Tsukuba, Ibaraki,
300-26 and $Department o j Food Hygiene, Nippon Veterinary and Animal Science University, 1-7-1, Kyonan-cho,
Musashino-shi, Tokyo, 180, Jupan
Received 22 April 1994; revised 8 July 1994
Gluconate was fermented selectively by the Bijidohacterium adolescentis group and some species of other genera,
including Clostridium clostridiiforme, C. innocuum, Propionibacterium acnes, Megasphaera elsdenii, Enterococcus
jaecium and Klebsiella pneumoniae; however it was not utilised by most other bacteria including the Bacteroidaceae.
No other organic acid salts were utilised by B. adolescentis. These salts weakly inhibited the growth of C. perjringens
in vitro, as did gluconate. The absorption rate of gluconate from the ligated small intestinal loop in rats was 19.9 per
cent under conditions when 100 per cent of glucose was absorbed. The effects of ingestion of gluconate on human
faecal bacteria was studied in ten healthy adult males. They ingested 9 g/d or 3 g/d of glucono-6-lactone (anhydride
of gluconic acid). With the 9 g/d ingestion, the number of bifidobacteria significantly increased (P<0.001), whereas
C. perfringens decreased and Enterobacteriaceae remained constant. The concentrations of bifidobacteria also
increased ( W 0 . 0 5 ) following 3 g/d ingestion.
KEY
WORDS-Gluconic
acid; Gluconate; Absorption; Faecal flora; Bifidobacteria.
INTRODUCTION
In the human intestinal microbiota useful bacteria
coexist with harmful bacteria. It is assumed that
harmful bacteria such as Clostridium perjringens
produce putrefactive products or toxins, and may
contribute to diarrhoea, constipation, cancer,
hypertension and/or ageing. On the other hand,
useful bacteria such as bifidobacteria are thought
to inhibit the growth of harmful bacteria and
stimulate immune function, so they are helpful for
maintenance of human health.'
Since the possible beneficial role of bifidobacteria became known, they have been used as
dietary supplements or as starter cultures for
yogurt and other cultured milk products in many
areas of the world including Japan, Scandinavia
and Europe. In Japan, bifidobacteria growthpromoting substances, such as oligosaccharides,
are utilised in various food products as dietary
supplements to promote the multiplication of
bifidobacteria in the human intestine.
Various indigestible oligosaccharides such
as fructoolig~saccharides,~
isomaltooligosaccha*Author to whom correspondence should be addressed
CCC 0891-060X/94/050247-10
0 1994 by John Wiley & Sons, Ltd.
rides,6 soybean oligosaccharides,2 galactooligosaccharides' and xylooligosaccharides" have been
developed as food materials which promote the
growth of bifidobacteria. There is no report on
organic acids which promote the growth of
bifidobacteria. We have directed our attention to
gluconic acid, and have studied its effect on the
growth of bifidobacteria.
Gluconic acid is an oxide of glucose, and its
anhydride is glucono-8-lactone. Gluconic acid
forms gluconate salts with various cations such as
sodium, calcium, potassium and zinc. These gluconic acid salts are widely utilised in various food
products as acids, coagulant and mineral supplement. Gluconic acid exists naturally in rice, honey,
wine (60450 p.p.m.), vinegar, beer (40-60 p.p.m.)
and grape juice (60-380 p.p.m.). On an industrial
scale, gluconic acid is produced from starch by
fermentation.
There are few reports on the effect of gluconic
acid on the growth of bacteria or its absorption
in animals. In the present study, we investigated
the effect of gluconate on the growth of various
intestinal bacteria in vitro, the absorption of
gluconate in rats in situ and the effect of ingestion
of glucono-&lactone on human faecal bacteria.
248
MATERIALS AND METHODS
Utilisation by intestinal bacteria in vitro
A total of 88 strains of intestinal bacteria was
used in fermentation tests. The carbohydrates
tested were sodium gluconate (Fujisawa Pharmaceutical Co., Ltd), fructooligosaccharides (Wako
Pure Chemical Industries Ltd) and glucose
(Nacalai Tesque, Inc.).
The test medium was PYF broth, PY broth4
containing 4 per cent (vlv) Fildes solution,' with
0.5 per cent of each carbohydrate. The control
medium lacked the added test carbohydrate. Tubes
containing 3 ml of the test medium were inoculated
with 30 pl of the test organisms which were precultured in GAM broth (Nissui Pharmaceutical Co.,
Ltd). The inoculated tubes were incubated at 37°C
for 7 d anaerobically by the Anaero-Pak method
(Mitsubishi Gas Chemical Co., Inc.). After incubation, the optical density (OD) at 660 nm and pH
of the medium were measured.
The efficiency of carbohydrate utilisation by
each bacterial strain was evaluated quantitatively
by calculating the difference in OD between the
test medium and the control medium. The difference in OD was scored in the following manner:
- , <0.099; f , 0.100-0.199; +, 0.200-0.399; + +,
0.400-0-599; + + +, B0.600; v, variable (strains
may be either + or -).
Effect on bacterial growth in vitro
The bacterial strains used in this test were
B. adoIescentis ATCCl5703 and C. perfringens
GKK 16. The organic acids tested were D-gluconic
acid (Fujisawa), D-glucuronic acid (Nacalai), citric
acid (Wako), DL-tartaric acid (Nacalai), m-malic
acid (Wako), maleic acid (Nacalai), fumaric acid
(Wako), m-lactic acid (Nacalai) and acetic acid
(Nacalai). Fructooligosaccharides (Wako) and isomaltooligosaccharides (Wako) were aslo included
as the representative of bifidobacteria growthpromoting substances.
The organic acid salt solution was prepared in a
concentration of 5 per cent (wtIvo1.) neutralised by
the addition of 6 N sodium hydroxide. The basal
medium was Semisolid GAM without dextrose
(Nissui). The test medium was prepared by the
elimination of agar from the basal medium and
with an added 10 per cent (vol./vol.) of each
organic acid salt solution to 0.5 per cent final
concentration. Tubes containing 5ml of the test
medium were inoculated with 50 p1 of 100-fold
dilutions of the test bacteria which were
T. ASANO ET AL.
precultured in GAM broth. The inoculated tubes
were incubated at 37°C for 12 h anaerobically by
the Anaero-Pak method. The OD at 660nm of
the cultured test medium was measured relative
to uninoculated blank medium containing each
organic acid.
The effect of each sample on the growth of B.
adolescentis or C. perfringens was evaluated quantitatively by the percentage difference of the OD in
the test medium and the control medium.
Intestinal absorption in rats in situ
Twelve 7 wk-old male Wistar rats (Charles
River Japan Inc.) weighing approximately 280 g
were divided into four groups of three rats each.
The test solution was either 100mM of sodium
gluconate or 100mM of glucose in saline (9g
sodium chlorideI1).
The rats were anaesthetised with ether after
fasting for 24 h. A central longitudinal incision
was made into the abdominal wall and the small
intestine was exposed. A small intestinal loop
about 10 cm in length was made by ligation with a
silk suture in the portion of either upper intestine
(jejunum) or lower intestine (ileum). 0.5 ml of the
test solution was injected into the ligated loop and
the whole small intestine was put back into the
abdominal cavity. At 30 min after injection, the
rats were killed and the ligated loop (test loop) was
cut off. The contents in the loop were flushed out
with 20ml of saline. At this time, a control loop
was made of the intestinal portion next to the test
loop and as soon as 0.5 ml of the test solution was
injected into the loop, the contents were flushed
out with 20 ml of saline.
The residual gluconate or glucose in the ligated
loop was measured in the contents. Measurement
of gluconate was carried out by Food Analysis
(F-Kit) D-Gluconic acidb-Glucono-6-lactone
(Boehringer Mannheim), and glucose was by
Glucose-CII-Test-Wako (Wako). The absorption
rate of the sample from the ligated loop was
calculated from the amount of the residual sample
in the test loop (T) and that in the control loop (C)
according to the following formula:
Absorption rate (%)=(1 - TIC) x 100
Efect on human faecal bacteria
The test sample used in the human volunteer
tests was glucono-6-lactone (Fujisawa) powder
which reverted to gluconic acid in solution. Two
volunteer studies were carried out independently
GLUCONATE AND FAECAL BACTERIA
with a dose of 9 g/d and 3 g/d respectively. The
subjects were ten healthy male volunteers in each
test. The ages of the subjects ranged from 25 to
50 yr in the 9 gld ingestion study, and from 26 to
44 yr in the 3 gld study. They had stopped ingesting medicines and antibacterial agents 1 mth prior
to and during the test period. However, they could
consume food without restrictions other than
foods with abundant live cultures, and were otherwise asked to maintain their normal diet throughout the test. These studies were performed in
accordance with the Helsinki Declaration as
updated in Tokyo, 1975.
The subjects ingested glucono-6-lactone three
times daily in one-third portions of the appointed
dose after a meal for two consecutive weeks.
Fresh faecal specimens were collected once a week
six times in total. The control specimens were
collected 1 wk before and just before starting
ingestion. The specimens during ingestion were
collected 1 wk and 2 wk after starting ingestion,
and further control specimens were collected 1 wk
and 2 wk after stopping ingestion. Faecal specimens collected from each subject were immediately
refrigerated, and the faecal bacteria were analysed
within 3 h.
The method of Mitsuoka et a[.*,’ was used for
faecal microbial analysis. The results were expressed as log,, of the number of bacteria per gram
wet weight of faecal material. The paired t test and
chi-square test were used for statistical analysis of
the results.
RESULTS
Utilisation of gluconate by various intestinal
bacteria in vitro
The results of fermentation tests in vitro are
shown in Table 1. Among members of the genus
Bijidobacterium, gluconate was utilised selectively
by the B. udolescentis group such as B. adolescentis, B. pseudocutenulaturn and B. catenulatum, but
was not utilised by other bifidobacteria including
B. longum and B. b$dum. In the genus LactobacilEus, gluconate was weakly utilised by L. casri and
L. fermentum. In addition, gluconate was utilised
by facultative bacteria including Enterococcus
faecium and Klebsiella pneumoniae and anaerobic
species including Clostridium clostridiiforme, C.
innocuum, Propionibacterium acnes and Megusphaera elsdenii. However, gluconate was not
utilised by the major part of intestinal bacteria
249
including Bacteroidaceae which are among the
predominant bacteria.
Eflects of orgunic acids and carbohydrates on the
growth of B. adolescentis and C. perfringens in
vitro
The results of the bacterial growth tests in vitro
are shown in Table 2. The growth of B. adolescen(is was promoted by gluconate. The maximal
effects were observed at concentrations of 0.5 per
cent and 1 per cent of gluconate, and a similar
phenomenon was noted with glucose. Other organic acids either had little effect on, or suppressed
the growth of, B. adolescentis, especially maleate
which strongly inhibited growth. On the other
hand, the growth of C. perfringens was weakly
suppressed by gluconate in a concentrationdependent manner. Other organic acids similarly
suppressed, and the inhibitory effects of citrate and
maleate were particularly strong.
Intestinal absorption of gluconate in rats in situ
The results of the absorption tests are shown in
Table 3. In the upper portion of the small intestine,
only 19.9 per cent of injected gluconate was
absorbed from the ligated loop under conditions
when 100 per cent of glucose was absorbed. Similarly in the lower portion of the intestine, the
absorption rate of gluconate was 11.6 per cent
compared with 49.3 per cent for glucose.
EfSrct of glucono-d-lactone ingestion on human
fuecal bacteria
Faecal bacteria results for the 9 gld of glucono&-lactoneingestion test are shown in Table 4.The
changes in bacterial concentrations of the representative intestinal bacteria in the 9 g/d and 3 gld
ingestion tests are shown in Figure 1. Bacteroidaceae, bifidobacteria and eubacteria were
selected to represent the predominant bacteria,
and C. perfringens and Enterobacteriaceae were
selected as representatives of potentially harmful
bacteria. In the 9 g/d ingestion test (Figure lA), the
concentration of bifidobacteria significantly increased (P<O.OO1) during ingestion, whereas C.
perfringens decreased in both levels and frequency
of occurrence. The number of Enterobacteriaceae,
which utilised gluconate in vitro, did not increase
during ingestion. In the 3 g/d ingestion test also
(Figure 1 B), the concentration of bifidobacteria
significantly increased (P<0-05) during ingestion,
250
T. ASANO ET AL.
Table 1. Utilisation of gluconate by various intestinal bacteria
~
Bacterial species
Bifidobacterium
B. adolescentis
B. pseudocatenulatum
B. catenulatum
B. angulatum
B. dentium
B. longum
B. bijidum
B. breve
B. infantis
B. animalis
Lactobacillus
L. acidophilus
L. salivarius
L. gasseri
L. casei
L. fermentum
Eubacterium
E. aerofaciens
E. limosum
E. lentum
E. nitritogenes
Bacteroides
B. fragilis
B. distasonis
B. vulgatus
B. thetaiotaomicron
B. ovatus
B. uniformis
B. melaninogenicus
Fusobacterium
F. necrophorum
l? nucleatum
F. varium
Clostridium
C perfringens
C. dificile
C. paraputrificum
C. butyricum
C. clostridioiforme
C. ramosum
C. innocuum
C. sordellii
C. sporogenes
C. bifementans
C. botulinum
Propionibacterium
P. acnes
Peptostreptococcus
P. magnus
P. anaerobius
P. asaccharolyticus
~
~
~
~~~~~~
Strains (no.)
Sodium gluconate
Fruc tooligosaccharides
Glucose
5
++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
++
++
+++
+++
+++
+
1
2
2
2
2
1
1
+++
+++
V
+++
-
+++
+++
+++
+++
-
+++
+++
++
1
1
-
f
f
+
++
++S
++
+++
+++
+++
+
+
+
++
++
-
f
+
+
+
+
6
2
-
1
1
1
1
1
1
1
+++
+
++
+
2
1
1
+
-
+++
++
+++
+++
++
++
+++
++
+
+++
++
++t
f
f
-
25 1
GLUCONATE AND FAECAL BACTERIA
Table 1. Continued
~
~
Bacterial species
Peptostreptococcus
P. prevotii
P.micros
Veillonella
V. parvura
V. alcalescens
Megasphaera
M . elsdenii
Enterococcus
E. faecaiis
E. faecium
Streptococcus
S. pyogenes
S. mutans
Staphylococcus
S. aureus
S. epidermidis
Escherichiu
E. coli
Klebsiella
K. pneumoniae
Proteus
P. mirabilis
~
_
Strains (no.)
_
_
_
Sodium gluconate
1
1
~
~~
~
~
~
Fructooligosaccharides
~
~
_
~
Glucose
f
-
1
1
-
1
++
2
++
+++
1
1
1
1
1
-
-
+++
+
++
1
+
+
1
f
4
Bacterial growth assessed by differences in optical density (OD) at 660 nm between control and test experiments: - , c0.099;
f , 0.1004199; +, 0.200-0.399; + +, 0.400 -0.599; + + +, >0.600; v, variable (strains may be either + or - ).
but the extent of the increase was smaller than that
of the 9 g/d ingestion test.
The changes in relative ration of the numbers of
some dominant bacteria to the total number of
bacteria are shown in Figure 2.The ratios of bifidobacteria in the control periods before starting
ingestion and after stopping ingestion ranged from
17-0 to 24.2 per cent in both 9 g/d and 3 g/d
ingestion tests. In the 9 gid ingestion test, the ratio
of bifidobacteria increased to 45.5 per cent during
ingestion, while the ratio in the 3 g/d ingestion test
increased to 35.1 per cent.
DISCUSSION
In vitro fermentation tests showed that gluconate
was utilised selectively by B. adolescentis group
bacteria including B. adolescentis, B. pseudocatenulatum and B. catenulatum. Other Bijidobacteria
species isolated from the intestine of human adults,
such as B. longum and B. bijidum, did not utilise
gluconate. Yaeshima et al. l 3 identified 56 strains of
bifidobacteria isolated from the faeces of human
adults, and identified them as 31 strains of B.
adolescentis, 17 strains of B. pseudocatenulatum,
four strains of B. catenulatum and four strains of
B. longum. From among the 56 strains, 52 strains
belonged to the B. adolescentis group, and utilised
gluconate, with the exception of some strains of B.
pseudocatenulatum. In the present study, we isolated 51 strains of bifidobacteria from the faeces of
the subjects, and identified them as 47 strains of
the B. adolescentis group and four strains of B.
longum on the basis of carbohydrate fermentation
patterns. These results suggest that the major
Bifidobacteria species within the intestines of
human adults belong to the B. adolescentis group,
which can utilise gluconate. Since gluconate is not
utilised by the numerically dominant intestinal
bacteria, including Bacteroidaceae which are the
most predominant bacteria, it can be efficiently
utilised by bifidobacteria. Although Enterobacteriaceae utilised gluconate in vitro, they did not
increase in concentration in the human volunteer
_
252
T. ASANO ET AL.
Table 2. Effects of organic acid and carbohydrates on the growth of B. adoolescentis and
C. perfringens in vitro
Organic acid or
carbohydrate
(Yo)
Concentration
Control
Fructooligosaccharides
Isomaltooligosaccharides
Glucose
0.5
0.5
0.25
0.5
1
Gluconate
Glucuronate
Citrate
Tartrate
Malate
Maleate
Fumarate
Lactate
Acetate
Data are expressed as ratio
2
0.25
0.5
1
2
0.5
0.5
0.5
B. adolescentis
ATCC15703
C. perfringens
GKKl6
100
152.4
178.9
172.4
197.2
20 1.2
174.8
142.3
172.8
172.8
100
99.6
135.6
161.0
201.3
201.3
194.9
93.6
86.9
74.6
69.5
95.3
0.2
80.1
144.3
87.8
0.5
77.2
97.2
93.1
0.5
0
0.5
63.0
89.4
91.9
0.5
0.5
70.3
36.7
65.3
71.8
69.9
(W,)
of the OD in test medium to the OD in control medium.
Table 3. Absorption of gluconate from a rat ligated
intestinal loop
Absorption rate (YO)
Small intestine
Gluconate
Glucose
Upper portion
Lower portion
19.9 i 6.0
11.6+ 15.2
100 f 0.0
49.3 7.4
+
Values refer to mean f SD of three rats. 0.5 ml portion of
sample was injected into the ligated intestinal loop and the
amount of residual sample was measured after 30 min.
tests. The reason why they do not increase in vivo is
not clear, but it is possibly a consequence of the
increase in bifidobacteria.
Gluconate, like other organic acids, weakly suppressed growth of C. perfringens in vitro, and it
also decreased the number of C. perfringens in the
volunteer test. However the in vitro effect is so
weak that it may not be the cause of the in vivo
effect. Some oligosaccharides, which could not
suppress the growth of C. perfringens in vitro, also
decreased the number of this bacterium in the
volunteer tests. 11,12 In these cases, the decrease of
C. perfringens may have resulted from the environmental changes in the intestine caused by the
increase of bifidobacteria, and the same reason
could be applied in the case of gluconic acid.
One of the conditions necessary for bdidobacteria growth-promoting factor to be effective is
that it reaches the large intestine where it may be
utilised by bifidobacteria. Therefore, it is necessary
for gluconic acid not to be absorbed from the small
intestine. At first we were afraid that ingested
gluconic acid would be absorbed from the small
intestine, since its structural formula is so similar
to glucose. Unexpectedly, only 20 per cent of
injected gluconate was absorbed from the ligated
intestinal loop of rats under conditions where 100
per cent of glucose was absorbed. This result
suggests that most of the ingested gluconate is not
absorbed from the small intestine and reaches the
large intestine. It is said that some oligosaccharides
which are absorbable from the intestine or are
unstable in gastric juice, need high ingested doses
to gain the effect in vivo. Since gluconic acid does
not have these defects, it could be an efficient
growth-promoting substance for bifidobacteria. In
253
GLUCONATE A N D FAECAL BACTERIA
hhhhhhhhh
0 0 0 0 0 0 0 0 0
2 2 2 = 2 ZcZz
254
T. ASANO ET AL.
8
11.2
v)
aJ
1110.8-
b
Enterobacteriaceae
Total
A
--7
65-
4cd
P
Bifidoba cteri um
9.6-
8
9.49.2
3-
Eubacteri um
00
I
I
1
I
I
2
I
11.2 I
8
d
I
I
I
I
I
1
7-
5
I
Enterobacteriaceae
6-
10.4
10.2
Bifidob$cterium
cd
L
c1
aJ
9.8
DD
0
E
D
-
Eubacterium
9.6
g-4L717J
9.2
-7
0
7
14
21
28
Figure I. Effects of glucono-F-lactone ingestion with the dose of 9 g/day (A) and 3 &day (B) on human faecal bacteria in ten
volunteers. Values are expressed as mean of log,, bacterial concentration from each individual. Frequency of occurrence (YO)is
shown in parentheses. Significant difference form the concentration of day 0: *P<0.05,***P<O.OOI
fact, gluconod-lactone effectively increased
bifidobacteria in the human volunteer tests. T h s
shows that the minimum daily dose of gluconic
acid is less than 3 g, which is sufficiently
comparable to the minimum dose of other oligosaccharide^.^,^
GLUCONATE AND FAECAL BACTERIA
h
x
a-
a
\
M
fm
W
M
n
x
cd
YM
m
W
Q
255
T. ASANO ET AL.
From these results, we conclude that gluconic
acid is utilised effectively by bifidobacteria and
contributes to increasing the number of faecal
bifidobacteria. Furthermore this effect is attained
with a relatively small daily dose of 3 g. Gluconic
acid is already used as a food additive, for example
in acids, as a coagulant and in mineral supplements such as calcium salt. The present study
demonstrates that gluconic acid can be used like
other oligosaccharides as a bifidobacteria growthpromoting substance, and not only as a food
additive.
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