137

Full Paper Bioscience Microflora Vol. 25 (4), 137146, 2006

The Effects of a Synbiotic Fermented Milk Beverage
Containing Lactobacillus casei Strain Shirota and
Transgalactosylated Oligosaccharides on Defecation
Frequency, Intestinal Microflora, Organic Acid
Concentrations, and Putrefactive Metabolites of Sub-Optimal
Health State Volunteers: A Randomized Placebo-Controlled
Cross-Over Study
Terue SHIOIRI
1
, Keiko YAHAGI
1
, Sachie NAKAYAMA
1
, Takashi ASAHARA
2
, Norikatsu YUKI
2
, Koji KAWAKAMI
2
,
Yoshitaku YAMAOKA
3
, Yuko SAKAI
3
, Koji NOMOTO
2
* and Masumi TOTANI
1
1
Tokyo Kasei University, 1-18-1 Kaga, Itabashi-ku, Tokyo 173-8602, Japan
2
Yakult Central Institute for Microbiological Research, 1796 Yaho, Kunitachi, Tokyo 186-8650, Japan
3
Yakult Honsha Co., Ltd., 1-1-9 Higashi-Shinbashi, Minato, Tokyo 105-8660, Japan
Received May 2, 2006; Accepted July 14, 2006
We evaluated the effects of ingestion of a synbiotic fermented milk beverage containing Lactobacillus casei strain
Shirota (LcS) at 3 10
10
and transgalactosylated oligosaccharides (GOS) at 2.5 g per 80 ml (once a day, 2 weeks) on
the defecation frequency in 35 female university students with constipation as well as the defecation frequency,
intestinal microflora, and intestinal environment in elderly persons in whom the intestinal microflora and the levels of
putrefactive metabolites were abnormal in a placebo-controlled double-blind study. In the female students, the
defecation frequency after 1 week of synbiotic fermented milk beverage ingestion was significantly higher than that
after 1 week of placebo ingestion or before ingestion. In the elderly persons, the fecal Bifidobacterium and
Lactobacillus bacterial counts after 1 and 2 weeks of synbiotic fermented milk beverage ingestion were significantly
higher than those after placebo ingestion or before ingestion (p<0.05 and p<0.01, respectively). The fecal lecithinase-
positive Clostridium bacterial count after 1 week of synbiotic fermented milk beverage ingestion and the fecal
Enterobacteriaceae bacterial counts after 1 and 2 weeks of synbiotic fermented milk beverage ingestion were
significantly lower than those after placebo ingestion (p<0.05). LcS at 10
7
CFU per gram of stool was collected during
the ingestion period. The acetic acid levels after 1 and 2 weeks of synbiotic fermented milk beverage ingestion were
significantly higher than those after placebo ingestion (p<0.01). The stool pH values after 1 and 2 weeks of synbiotic
fermented milk beverage ingestion and the ammonia and phenol levels after 2 weeks of synbiotic fermented milk
beverage ingestion were significantly lower than those after placebo ingestion (p<0.05). These results suggest that
ingestion of the synbiotic fermented milk beverage containing LcS and GOS improves the stool quality, intestinal
microflora, and intestinal environment.
Key words: synbiotics; fermented milk beverage; bowel movements; fecal microflora; organic acid, putrefactive
metabolite
INTRODUCTION
In Japan, increases in the consumption of European/
American food and stress have recently elevated the
incidences of intestinal disorders (25). In particular, the
incidence of colorectal cancer has increased more than 2-
fold during the past 10 years. It is indicated that
colorectal cancer will soon comprise the highest
mortality risk among various cancers, as demonstrated in
Europe and the United States, exceeding the incidence of
gastric cancer (29). Furthermore, infectious intestinal
diseases represented by infection with Escherichia coli
O-157 are an important issue (20). Along with a recent
increase in peoples interests in intestine and enteric
bacteria, some types of beneficial bacteria contained in
yogurts, fermented milk, or other fermented foods have
been recognized as the medical entity of probiotics (24).
Recently, a collaborative FAO/WHO working group for
preparing guidelines for evaluating probiotics defined
*Corresponding author. Mailing address: Yakult Central Institute for
Microbiological Research, 1796 Yaho, Kunitachi, Tokyo 186-8650,
Japan. Phone: +81-425-77-8962. FAX: +81-425-77-3020. E-mail: koji-
nomoto@yakult.co.jp
T. SHIOIRI, et al. 138
probi ot i cs as l i ve mi croorgani sms whi ch when
administered in adequate amounts confer a health benefit
on t he host ( 24) . Pr ebi ot i cs r epr es ent ed by
oligosaccharides are a non-digestive food component
that contributes to the hosts health status by promoting
the proliferation of probiotics and beneficial enteric
bacteria or increasing their activity (11). In addition,
when the two components are combined to achieve
synergistic effects they are called synbiotics (10).
Several studies have reported that foods containing
Lactobacillus casei strain Shirota (LcS), known as a
probiotic, or transgalactosylated oligosaccharides
(GOS), a prebiotic, improve the intestinal microflora by
increasing beneficial enteric bacteria and decreasing
harmful bacteria, regulate the intestinal environment by
decreasing the contents of putrefactive metabolites, and
condition bowel movements in humans by increasing the
defecation frequency (14, 1618, 27). However, no
study has reported the bowel movement-conditioning
effects of a synbiotic food containing both LcS/other
types of Lactobacillus and a prebiotic.
In this study, we conducted a placebo-controlled
double-blind study using a synbiotic fermented milk
beverage containing LcS and GOS. The subjects were
female students with constipation and healthy elderly
per sons i n whom t he i nt est i nal mi cr of l or a and
environment were abnormal and we investigated the
bowel movement-conditioning effects of the symbiotic
fermented milk.
MATERIALS AND METHODS
Subjects
The student study: We conducted a screening test for 2
weeks on 90 female university students aged 19 to 22
years, and selected 35 who reported a defecation
frequency of 9 times or less per 2 weeks. We excluded
students with food allergy, those frequently skipping
meals, and those with serious diseases. In addition, we
excluded those periodically taking intestinal drugs or
agents that may influence bowel movement-conditioning
effects.
The elderly person study: The subjects were 20
healthy elderly persons (stool test was conducted 17
persons out of the 20 volunteers) in whom no specific
diet guidance, diet therapy, or diet restriction was applied
or recommended.
The study was conducted according to the Helsinki
declaration (ratified in 1964, revised in 1975, 1983,
1989, 1996, and 2000). After the contents and methods
of the study had been explained to the subjects, their
written informed consent was obtained.
Test diet
We employed two test diets: a synbiotic fermented
milk beverage containing LcS at 3 10
10
and GOS at 2.5
g per 80 ml bottle and a placebo (also 80 ml). The
synbiotic fermented milk beverage was made from liquid
GOS, sugar, skim milk powder, liquid glucose/fructose,
soybean polysaccharides, a flavoring agent, vitamin C,
and vitamin E. The energy, protein, lipid, carbohydrate,
sodium, vitamin C, and vitamin E contents per bottle (80
ml) were 57 kcal, 1.0 g, 0.1 g, 13.6 g, 18 mg, 30 mg, and
3 mg, respectively. The LcS bacterial count at ingestion
was 3 10
10
or more. Placebo was prepared basically
with the same ingredients and nutritional contents and
with the same taste, color, pH and energy (by adjusting
glucose and sugar compositions) as for the synbiotic
fermented milk beverage except that it contained neither
L. casei strain Shirota nor GOS; Acidic taste was
adjusted by addition of lactic acid.
Study schedule and ingestion of the test diets
We conducted a placebo-controlled double-blind
cross-over study (Fig. 1). The study period was 9 weeks:
Non-ingestion Period 1 (2 weeks), Ingestion Period 1 (2
weeks), Non-ingestion Period 2 (3 weeks), and Ingestion
Period 2 (2 weeks). During the ingestion period, the test
diet at 1 bottle per day was given to the subjects. The
subjects were instructed to ingest the test diet at a specific
time every day, and not to change their normal daily
activities such as dietary and exercise habits.
Examination methods
Survey by diary: During the study period, the subjects
recorded a diary regarding the defecation frequency,
defecation hour, stool quantity, stool quality, health
status, presence or absence of test diet ingestion,
ingestion hour, and contents of meals every day by the 24
hr remembering method. Concerning the stool quantity,
a sample for determining the stool quantity (a column
measuring 1.5 cm in diameter and 5 cm in length) was
delivered to the subjects, and they recorded the stool
quantity as the number of sample bottles used (including
the first decimal place). Furthermore, the subjects were
instructed to avoid ingestion of milk products, foods
containing oligosaccharides, and fermented soybeans as
well as excessive ingestion of other milk products during
the study period. However, there was no dietary, alcohol,
or drug restriction. They recorded alcohol or drugs in a
questionnaire. We excluded subjects who did not have
the test diet on 3 or more of 28 days (period of test diet
ingestion)(rate of test diet ingestion: less than 90%), and
analyzed the study results.
EFFECTS OF SYNBIOTIC FERMENTED MILK 139
Stool test
<1> Sample collection and transport
In the elderly person study, stools were collected on
the final day of Non-ingestion Period 1, at the ends of
Weeks 1 and 2 of Ingestion Period 1, on the final day of
Non-ingestion Period 2, and at the ends of Weeks 1 and 2
of Ingestion Period 2 (total: 6 times) for a stool test (Fig.
1). Immediately after collection, stools were stored in a
refrigerator in an6 anaerobic state using an Anaero Pack
Kenki (MITSUBISHI GAS CHEMICAL COMPANY,
INC., Tokyo), and the fecal microflora were investigated
within 24 hr after collection.
<2> Microflora test
Stools were weighed in an anaerobic glove box (CO
2
:
5%, H
2
: 4%, N
2
: 91%), and homogenized. A 0.5 g
sample was mixed with 4.5 ml of anaerobic transport
medium (Lab lemco powder (Becton, Dickinson and
Company, USA), 1% w/v; Bact Agar (Becton, Dickinson
and Company), 0.05% w/v; sodium thioglycollate,
0.075% w/v; glycerin, 10% w/v; KH
2
PO
4
, 0.0225% w/v;
K
2
HPO
4
, 0.0225% w/v; NaCl, 0.045% w/v; (NH
4
)
2
SO
4
,
0.0225% w/v; CaCl
2
, 0.00225% w/v; MgSO
4
, 0.00225%
w/v; Na
2
CO
3
, 0.3% w/v; L-cysteine 1 hydrate (Wako
Pure Chemical Industries, Ltd., Osaka), 0.05% w/v; and
resazurin, 0.0001% w/v; pH 7.4 to 7.6) to prepare a stool
solution (dilution ratio: 10). This solution was serially
diluted with an anaerobic dilution buffer (BACT Agar
(Becton, Dickinson and Company), 0.05% w/v; Tween
80, 0.05% w/v; KH
2
PO
4
, 0.0225% w/v; K
2
HPO
4
,
0.0225% w/v; NaCl, 0.045% w/v; (NH
4
)
2
SO
4
, 0.0225%
w/v; CaCl
2
, 0.00225% w/v; MgSO
4
, 0.00225% w/v;
Na
2
CO
3
, 0. 3% w/v; L-cysteine HCl (Wako Pure
Chemical Industries, Ltd.), 0.05% w/v; and resazurin,
0.0001% w/v) in multiples of 10. Various concentrations
of samples at 0.05 ml were inoculated to various types of
medium. A total of 10 items, i. e., total obligate anaerobe
count, Bacteroidaceae, Bifidobacterium, lecithinase-
positive Clostridium, Lactobacillus, Enterobacteriaceae,
Enterococcus, Staphylococcus, Candida, and LcS, were
exami ned using t he fol lowi ng agar pl at e media,
respectively: M10 (3), NBGT (1), TOS propionic acid
agar (pH 5.7) (Eiken Chemical Co. Ltd., Tokyo), CW
(Nikken BioMedical Laboratory, Inc., Kyoto), modified
LBS (1), DHL (Nissui Pharmaceutical Co., Ltd., Tokyo),
COBA (23), MSE (Nikken BioMedical Laboratory,
Inc. ), Sabouraund dextrose (Ni kken BioMedi cal
Laborat ory, Inc. ), and LLV agar medi um (30).
Anaerobic culture was performed using M10, NBGT,
TOS propionic acid agar (pH 5.7), CW, and modified
LBS agar medium. Using the other media, aerobic
culture was performed. After culture at 37C for a
specific period (1 to 5 days), the number of colonies was
measured. The colonies appearing in various media were
morphologically classified, and Grams staining of the
representative colonies was performed to examine the
bacterial morphology. In the colonies appearing in
COBA and MSE media, a catalase test was conducted,
and Enterococcus and Staphylococcus were confirmed,
respectively. LcS was identified by enzyme-linked
immunosorbent assay (ELISA) with a strain-specific
monoclonal antibody (30). The bacterial count was
expressed as the logarithmic mean per gram of stool
standard deviation. The lower detection limit was 2.3,
and the detection rate was calculated as the number of
positive samples/the number of tested samples.
<3> Measurement of the fecal concentrations of
organic acids
A portion of the homogenized stool was isolated,
weighed, mixed with 0.15 M perchloric acid at a 4-fold
volume, and reacted at 4C overnight. Then, the mixture
was centrifuged at 4C at 15,000 rpm for 10 min, and the
supernatant was filtrated with a 0.45-m membrane filter
Fig. 1. Study design. Female university students: Group A, n=20; Group B, n=20. Elderly
persons: Group A, n=9; Group B, n=8. : Fecal sample (Only the elderly).
T. SHIOIRI, et al. 140
(Mi l l i por e Japan, Tokyo) , and st er i l i zed. The
concentrations of organic acids in this sample were
measured using a Waters high-performance liquid
chr omat ogr aphy ( HPLC) syst em ( Wat er s 432
Conductive Detector, Waters, USA) and a Shodex
Rspack KC-811 column (Showa Denko, Tokyo)(13).
We prepared a standard mixed solution consisting of 1 to
20 mM succinic acid, lactic acid, formic acid, acetic acid,
propionic acid, isobutyric acid, butyric acid, isovaleric
acid, and valeric acid, and calculated the concentrations
of organic acids based on the standard curve.
<4> Stool pH and water content
The stool pH was measured by directly inserting the
glass electrode of a D-51 pH meter (Horiba Seisakusho
Co. Ltd., Tokyo) into the homogenized stool. The stool
water content was calculated as weight differences
between before and after freeze-drying of a portion of the
stool.
<5> Analysis of fecal putrefactive metabolites
We measured the fecal levels of indole, ammonia,
phenol , and p-Cresol. A stool sample weighing
approximately 2.5 g was mixed with 0.1 M phosphate
buffer (PB)(pH 5. 5) at 9 ti mes the st ool weight ,
homogenized with glass beads, and filtrated with gauze.
A 10-fold serial dilution of this sample was used for
measurement. To measure the levels of phenol and p-
Cresol, the sample was mixed with PB at 9 times the
stool weight, and homogenized using a stomacher
(Organo Corporation, Tokyo). The stool suspension was
stored at 30C until measurement. Various putrefactive
metabolites were measured as described below. 1)
Indol e: A col ori ng react i on t est was performed
immediately after the dilutions were prepared. To 1.5 ml
of a coloring solution prepared by dissolving 14.7 g of p-
dimethyl aminobenzaldehyde in a sulfuric acid/alcohol
mixture (52 ml of concentrated sulfuric acid and 948 ml
of 95% ethanol) or a control coloring solution (the
sulfuric acid/alcohol mixture), 0.3 ml of a 70-fold
dilution of a stool sample was added. The mixture was
immediately stirred, reacted at room temperature for 20
min, and centrifuged at 1,390 G for 10 min. The
supernatant was placed on a microplate at 0.2 ml/well,
and the absorbance at 570 nm was determined using a
microplate reader (FLUO star, BMG Lab Technologies).
As control indole solutions, 0 to 0.3 mM (ratio: 1.5, 11
concent rat i ons) i ndol e sol ut i ons were prepared
immediatel y before use. 2) Ammonia: A kit for
measuring ammonia (Ammonia Test, Wako Pure
Chemical Industries) was used. 3) Phenol and p-Cresol:
In a glass test tube (TST-SCR, ASAHI TECHNO
GLASS Co., Chiba), 5 ml of a 10-fold stool dilution, 2 ml
of concentrated hydrochloric acid, and 0.1 ml of an
i nt er nal st andar d sol ut i on ( 250 g/ ml phenol
parachlorophenol solution) were placed. The tube was
sealed with a heat-proof screw cap (9998CAPH415-15,
ASAHI TECHNO GLASS Co.), and the mixture was
hydrolyzed at 100C for 60 min in an aluminum block
heater (TAITEC, Saitama). It was cooled at room
temperature, mixed with 4 ml of diethylether, agitated for
1 min for extraction, and centrifuged at 1,650 G for 10
min. The solvent layer (supernatant) at 3 ml was placed
in another test tube, mixed with 3 ml of 0.05 N NaOH/
methanol, and evaporated to dryness using a centrifugal
concentrator (VC-360, TAITEC) and an aspirator (ASP-
13, ASAHI TECHNO GLASS Co.). The pellet was re-
dissolved in 1.0 ml of distilled water, and centrifuged at
12,000 G for 20 min. The supernatant was filtrated
(0.45 m), and used as an HPLC assay sample. As
standard solutions, we used 0.02 to 6 g/ml phenol
solutions and 0.2 to 60 g/ml p-Cresol solutions. These
solutions were assayed by HPLC using a fluorescence
detector (excitation wavelength: 260 nm, measuring
wavelength: 305 nm) and a UV detector (270 nm).
Statistical analysis
Non-ingestion Periods 1 and 2 were established before
Ingestion Periods 1 and 2, respectively. The values
obtained at Week 2 of Non-ingestion Period 1 and at
Week 3 of Non-ingestion Period 2 were analyzed as pre-
ingestion values. The survey parameters in the diary
were analyzed using the weekly collected data. The
results were compared between the synbiotic fermented
milk beverage group and the placebo group before
ingestion and after 1 and 2 weeks of ingestion. In
addition, the pre-ingestion values were compared with
the values after 1 and 2 weeks of ingestion.
The defecation frequency, number of days with bowel
movements, stool quantity, and fecal microflora were
analyzed using non-parametric paired Wilcoxons rank
sum test. The bacterial detection rate was analyzed using
Fishers direct probability test. The stool pH and water
content were analyzed using the paired t-test. We
employed SPSS Ver. 11 software (SPSS Japan Inc.,
Tokyo). p<0.05 was regarded as significant.
RESULTS
Student study
Influence on the defecation frequency, number of days
with bowel movements, and stool quantity: The mean
age, height, body weight, and body mass index (BMI) of
the 35 female students enrolled in the study were 19.4
0.8 years, 158.3 4.2 cm, 51.2 6.6 kg, and 20.6 2.2,
EFFECTS OF SYNBIOTIC FERMENTED MILK 141
respectively.
Before ingestion, the mean defecation frequency per
week was 4.0 1.5 times and 4.4 2.2 times in the
synbiotic fermented milk beverage and placebo groups,
respectively (Table 1). There were no significant
differences in the defecation frequency, number of days
with bowel movements, or stool quantity between the
two groups. The defecation frequency and number of
days with bowel movements after 1 week of synbiotic
fermented milk beverage ingestion were significantly
higher than the values after 1 week of placebo ingestion
(p<0.05, respectively). After 2 weeks of ingestion, there
were no significant differences in any parameters
between the two groups. There were no significant
differences in stool quantity after 1 or 2 weeks of test diet
ingestion between the two groups. The defecation
frequency and number of days with bowel movements
after 1 week of synbiotic fermented milk beverage
ingestion were significantly higher than the values before
ingestion (p<0.05, respectively). However, there were
no significant changes in the placebo group. After 2
weeks of ingestion, there were no marked changes in
these parameters in comparison to the pre-ingestion
values in either group. However, the means of during the
2-week ingestion period in the synbiotic milk beverage
group were significantly higher than the means before
ingestion and after 1 and 2 weeks of placebo ingestion
(data not shown).
Elderly person study
Influence on the defecation frequency, number of days
with bowel movements, and stool quantity: The mean
age, height, body weight, and BMI of the 20 subjects (5
males, 15 females) were 74.4 6.6 years, 156.0 8.2 cm,
58.1 10.8 kg, and 23.8 3.8, respectively.
Before ingestion, the mean defecation frequency per
week was 8 times or more in the synbiotic fermented
milk beverage and placebo groups (Table 2). There were
no significant differences in the defecation frequency,
number of days with bowel movements, or stool quantity
after ingestion of the test diet between the two groups.
After ingestion, there were no marked changes in
comparison to the pre-ingestion values.
Infl uence on fecal mi crofl ora: There was no
significant difference in the fecal microflora before
ingestion between the synbiotic fermented milk beverage
and pl acebo gr oups ( Tabl e 3) . However , t he
Bifidobacterium and Lactobacillus bacterial counts after
1 week of ingestion in the synbiotic fermented milk
beverage group were significantly higher than those in
the placebo group (p<0.05 and p<0.01, respectively).
These values were also significantly higher than the pre-
ingestion values (p<0.05 and p<0.01, respectively). In
addition, these counts after 2 weeks of synbiotic
fermented milk beverage ingestion significantly differed
from those in the placebo group or the pre-ingestion
values. In the synbiotic fermented milk beverage group,
the Bifidobacterium bacterial count further increased
after 2 weeks of ingestion compared to that after 1 week
of ingestion. After 1 and 2 weeks of ingestion, ingested
LcS was collected at bacterial counts of 7.2 0.8 and 7.4
1.9 (Log) per gram of stool, respectively. After 1 and 2
weeks of synbiotic fermented milk beverage ingestion,
the Lactobacillus bacterial counts were 7.8 0.7,
significantly higher than the collected LcS levels
(p<0.05, respectively). In the synbiotic fermented milk
beverage group, the lecithinase-positive Clostridium and
Enterobacteriaceae bacterial counts after 1 week of
ingestion were significantly lower than those in the
placebo group (p<0.05, respectively). After 2 weeks of
ingestion, the Enterobacteriaceae bacterial count in this
group remained lower than that in the placebo group
(p<0.05). There were no significant differences in the
total obligate anaerobe count or Bacteroidaceae,
Enterococcus, Staphylococcus, or Candida counts
between the two groups.
Influence on fecal levels of organic acids: Before
ingestion of the test diet, there were no significant
differences in the fecal levels of organic acids between
the synbiotic fermented milk beverage and placebo
groups. After 1 week of ingestion, the total organic acid,
acetic acid, and butyric acid levels in the synbiotic
fermented milk beverage group were significantly higher
than those in the placebo group (p<0.05, p<0.01, and
p<0.05, respectively)(Table 4). After 2 weeks of
ingestion, the fecal level of acetic acid in the synbiotic
fermented milk beverage group remained higher than
that in the placebo group (p<0.01). Simultaneously, the
fecal level of succinic acid was lower than that in the
placebo group (p=0.06).
Influence on the stool pH and water content: The stool
pH values after 1 and 2 weeks of ingestion in the
synbi ot i c ferment ed mi l k beverage group were
significantly lower than those in the placebo group
(p<0. 05, respecti vely)(Table 5). There were no
significant differences in the water content between the
two groups (Table 5).
Influence on fecal putrefactive metabolites: There
were no significant differences in the fecal levels of
putrefactive metabolites before ingestion or after 1 week
of ingestion between the synbiotic fermented milk
beverage and placebo groups (Table 5). However, after 2
T. SHIOIRI, et al. 142
weeks of ingestion, the fecal levels of ammonia and
phenol in the synbiotic fermented milk beverage group
were significantly lower than the values in the placebo
group (p<0.05, respectively). There were no significant
differences in the fecal levels of indole or p-Cresol.
DISCUSSION
Deguchi et al. conducted a study with a GOS-
containing beverage in females (age: 17 to 29 years) and
males (age: 19 to 45 years) with mild constipation, and
reported that the defecation frequency and number of
days with bowel movements in the group taking 5 g of
GOS were significantly higher than those in the placebo
group, but there were no significant changes in the group
taking 2.5 g of GOS (6). In this study, we gave a
synbiotic fermented milk product containing LcS and
GOS to female university students with mild constipation
(age: 19 to 22 years), and the defecation frequency and
number of days with bowel movements after 1 week of
ingestion in the synbiotic fermented milk beverage group
were higher than those in the placebo group, suggesting
that this product markedly improved the defecation
frequency via synbiotic coordination between LcS and
GOS, because the GOS content of this product was 2.5 g.
However, after 2 weeks of ingestion, there was no
improvement in the defecation frequency, possibly
because continuous ingestion of oligosaccharides
promoted proliferation of Bifidobacterium in the large
intestine, which rapidly metabolizes oligosaccharides,
restricting large intestine osmotic pressure. Essentially,
a low molecular weight GOS-related increase in large
intestine osmotic pressure and the promotion of
peristalsis related to organic acids produced via the
f er ment at i on of Lact obaci l l us and i nt r i ns i c
Bi f i dobact eri um may be i nvol ved i n t he act i on
mechanism by which synbiotic fermented milk improves
defecation problems (21). Recently, Matsumoto et al.
investigated the intestine-conditioning effects of a
Table 1. Effect of synbiotic fermented milk beverage containing L. casei strain Shirota and transgalactosylated oligosaccharides on
the number of bowel movements, number of days with bowel movements, and stool quantity of female university students
Before ingestion Week 1 of ingestion Week 2 of ingestion
Item Synbiotic fermented Synbiotic fermented Synbiotic fermented
Placebo Placebo Placebo
milk beverage milk beverage milk beverage
Number of bowel movements 4.0 1.5 4.4 2.2 5.0 2.1
b, cc
4.1 1.7 4.1 1.7 4.6 2.0
(times/weeks)
Number of days with 3.6 1.3 3.8 1.3 4.1 1.1
b, c
3.5 1.5 3.7 1.6 3.9 1.4
bowel movements
(days/weeks)
Stool quantity
a
5.5 2.1 5.4 2.4 5.9 2.8 5.4 2.7 5.4 2.7 5.3 2.6
(/week)
The results are expressed as the mean SD (n=35).
a
Stool quantity was expressed as the number of samples to the first decimal
place, using a standard sample size (1.5 cm 5 cm).
b
p<0.05: Significant difference between the synbiotic fermented milk beverage and the placebo (Wilcoxon signed-rank test).
c
p<0.05,
cc
p<0.01: Significant difference between before intake and week 1 of intake (Wilcoxon signed-rank test).
Table 2. Effect of synbiotic fermented milk beverage containing L. casei strain Shirota and transgalactosylated oligosaccharides on
the number of bowel movements, number of days with bowel movements, and stool quantity of the elderly persons
Before ingestion Week 1 of ingestion Week 2 of ingestion
Item Synbiotic fermented Synbiotic fermented Synbiotic fermented
Placebo Placebo Placebo
milk beverage milk beverage milk beverage
Number of bowel movements 8.7 4.3 8.6 3.3 8.7 4.1 9.2 3.7 9.2 4.2 9.2 4.3
(times/weeks)
Number of days with 5.9 1.5 6.4 1.1 6.3 1.0 6.3 1.2 6.3 0.9 6.3 1.1
bowel movements
(days/weeks)
Stool quantity
a
5.8 2.9 5.1 2.7 5.3 2.5 5.1 2.4 5.2 2.6 5.0 2.4
(/week)
The results are expressed as the mean SD (n=17).
a
The stool quantity was expressed as the number of samples to the first
decimal place, using a standard sample size (1.5 cm 5 cm).
EFFECTS OF SYNBIOTIC FERMENTED MILK 143
fermented milk beverage containing L. casei strain
Shirota at 4 10
10
bacteria, and reported that the
defecation frequency after 1 and 2 weeks of ingestion
was significantly higher than that before ingestion.
However, in their study, ingestion of the beverage
decreased the fecal levels of organi c aci ds (17).
Ingestion of fermented milk may promote the absorption
of organic acids in the intestinal tract. In the future,
whether changes in the intestinal levels of organic acids
influence constipation alleviation should be further
investigated.
Mitsuoka et al. investigated fecal microflora in 72
healthy elderly persons aged 65 to 86 years, and
compared it with that in 29 healthy adults aged 20 to 64
years. In the elderly persons, the Bifidobacterium
bacterial count and detection rate were significantly
lower, and the lecithinase-positive Clostridium bacterial
count and detection rate were significantly higher (19).
I n t hat s t udy, a pr e- i nges t i on t es t conf i r med
abnormalities in the intestinal flora and environment in
the elderly persons leading a normal healthy daily life
with a mean age of 74 years; the Bifidobacterium
bacterial count was lower, the detection rates for
lecithinase-positive Clostridium and Candida were
higher, and the ammonia and p-Cresol levels were higher
than the reference values for healthy adults. In the
Table 3. Effect of synbiotic fermented milk beverage containing L. casei strain Shirota and transgalactosylated oligosaccharides on
fecal microflora in the elderly persons
Before ingestion Week 1 of ingestion Week 2 of ingestion
Organisms Synbiotic fermented Synbiotic fermented Synbiotic fermented
Placebo Placebo Placebo
milk beverage milk beverage milk beverage
Total anaerobes 10.2 0.3(17/17) 10.2 0.4(17/17) 10.3 0.2 (17/17) 10.3 0.3 (17/17) 10.3 0.4 (17/17) 10.4 0.2 (17/17)
Bacteroidaceae 9.6 0.4(17/17) 9.5 0.5(17/17) 9.6 0.4 (17/17) 9.6 0.4 (17/17) 9.6 0.5 (17/17) 9.6 0.4 (17/17)
Bifidobacterium 8.3 1.2(17/17) 8.3 1.5(17/17) 8.9 1.1
b, c
(17/17) 8.3 1.4 (17/17) 9.3 0.8
b, d
(17/17) 8.2 1.7 (17/17)
Clostridium (L+)
a
4.1 1.6(11/17) 4.2 1.4(10/17) 3.5 1.3
b
(9/17) 4.9 0.9 (12/17) 3.6 1.2
e
(10/17) 4.4 1.1 (12/17)
Lactobacillus 7.0 0.4(17/17) 6.7 1.6(17/17) 7.8 0.7
bb, cc
(17/17) 6.7 1.4 (17/17) 7.8 0.7
bb, dd
(17/17) 6.8 1.4 (17/17)
Enterobacteriaceae 6.9 1.5(17/17) 7.2 1.2(17/17) 6.6 1.1
b
(17/17) 7.3 0.9 (17/17) 6.7 1.0
b
(17/17) 7.4 1.1 (17/17)
Enterococcus 7.2 1.1(17/17) 6.9 1.5(17/17) 6.9 1.2 (17/17) 6.8 1.2 (17/17) 7.0 1.3 (17/17) 7.3 1.3 (17/17)
Staphylococcus 3.6 0.9(10/17) 3.4 0.9 (9/17) 3.2 0.8 (11/17) 3.5 0.9 (7/17) 3.3 0.7 (12/17) 4.1 0.9 (7/17)
Candida 4.3 1.2(12/17) 4.2 1.2(12/17) 4.1 1.1 (12/17) 4.1 0.9 (11/17) 4.2 1.2 (13/17) 4.2 1.0 (11/17)
L. casei strain Shirota ND (0/17) ND (0/17) 7.2 0.8 (17/17) ND (0/17) 7.4 1.9 (17/17) ND (0/17)
The results are expressed as the mean Log
10
c.f.u. and S.D. per gram of feces. The results in parentheses are the number of samples in
which the bacteria were detected/the number of samples examined. ND: Not detected (<2.3 Log
10
c.f.u. and S.D. per g).
a
Lecithinase
positive Clostridium.
b
p<0.05,
bb
p<0.01: Significant difference between the synbiotic fermented milk beverage and the placebo
(Wilcoxon signed-rank test).
c
p<0.05,
cc
p<0.01: Significant difference between before intake and week 1 of intake (Wilcoxon signed-rank test).
d
p<0.05,
dd
p<0.01, Significant difference between before intake and week 2 of intake (Wilcoxon signed-rank test).
e
p=0.09, Difference between the synbiotic fermented milk and the placebo (Wilcoxon signed-rank test).
Table 4. Effect of synbiotic fermented milk beverage containing L. casei strain Shirota and transgalactosylated oligosaccharides on the
concentration of organic acids in the elderly persons
Before ingestion Week 1 of ingestion Week 2 of ingestion
Item Synbiotic fermented Synbiotic fermented Synbiotic fermented
Placebo Placebo Placebo
milk beverage milk beverage milk beverage
Total organic acids 125 37 (17/17) 110 38 (17/17) 129 37
a
(17/17) 101 35 (17/17) 114 17
b
(17/17) 95 27 (17/17)
Succinic acid 5.9 10.7 (13/17) 6.5 10.7 (12/17) 1.7 1.4 (14/17) 3.4 5.0(15/17) 2.1 1.9
b
(11/17) 3.9 5.3 (12/17)
Lactic acid 0 (0/17) 2.7 (2/17) 3.0 1.1 (4/17) 3.3 1.4 (3/17) 2.2 1.2 (6/17) 2.7 2.2 (5/17)
Formic acid 1.1 0.3 (5/17) 1.8 2.6 (9/17) 1.1 0.4 (5/17) 1.1 1.0 (5/17) 3.4 5.7 (4/17) 0.9 0.5 (7/17)
Acetic acid 67 24 (17/17) 60.6 22.6 (17/17) 72 18
aa
(17/17) 51 17 (17/17) 68 14
aa
(17/17) 49 16 (17/17)
Propionic acid 22 7 (17/17) 22.5 11.4 (17/17) 24 10 (17/17) 23 14 (17/17) 20 6 (17/17) 19 7 (17/17)
iso-Butyric acid 2.4 0.9 (15/17) 1.8 1.1 (14/17) 2.1 1.0 (15/17) 2.1 1.0(15/17) 1.7 0.9 (16/17) 1.8 1.0 (17/17)
Butyric acid 23 12 (17/17) 15.7 8.1 (17/17) 24 16
a
(17/17) 16 7 (17/17) 17 4 (17/17) 16 5 (17/17)
iso-Valeric acid 4.1 2.3 (15/17) 3.7 2.6 (11/17) 3.3 2.6 (15/17) 3.3 2.5(15/17) 3.4 1.6 (16/17) 3.4 2.1 (17/17)
Valeric acid 2.5 0.8 (13/17) 2.4 1.6 (12/17) 2.4 1.2 (12/17) 2.2 0.6(13/17) 2.5 0.9 (13/17) 2.9 1.1 (13/17)
The results are expressed as the mean mol and S.D. per gram of feces.
The results in parentheses are the numbers of samples in which the organic acids were detected/the number of samples examined.
a
p<0.05,
aa
p<0.01: Significant difference between the synbiotic fermented milk beverage and the placebo (Wilcoxon signed-rank test).
b
p=0.06: Difference between the synbiotic fermented milk beverage and the placebo (Wilcoxon signed-rank test).
T. SHIOIRI, et al. 144
elderly person study, no subject showed constipation
prior to ingestion of the test diet. However, in future
studies, the correlation between disordered intestinal
flora/environment and constipation should be examined
in a larger number of elderly persons.
In the elderly person study, the stool Bifidobacterium
bacteria count after 1 week of ingestion in the synbiotic
fermented milk beverage group was significantly higher
than that in the placebo group, and further increased after
2 weeks of ingestion. In the synbiotic fermented milk
beverage group, the counts of harmful bacteria such as
lecithinase-positive Clostridium and Enterobacteriaceae
were significantly lower than the values in the placebo
group. In addition, the stool pH value was significantly
lower, and the level of acetic acid was significantly
higher. The GOS contained in this synbiotic fermented
milk beverage product is a Bifidobacterium-selective
growt h fact or (12), and t he mai n met abol i t e of
Bifidobacterium is acetic acid. Acetic acid has potent
bact er i ci dal act i vi t y agai ns t pat hogeni c
Enterobacteriaceae (4, 8, 9, 22), and several studies have
suggested that the bactericidal actions of acetic acid are
achieved in a non-dissociation state (4, 7). Based on
these findings, Bifidobacterium proliferating in the
intestinal tract may reduce the intestinal pH via the
production of a large amount of acetic acid, inhibiting
harmful bacteria such as Enterobacteriaceae. Ingestion
of the synbiotic fermented milk beverage significantly
increased the intestinal intrinsic Lactobacillus bacterial
count in comparison to the pre-ingestion or control
values. A study reported that ingestion of a beverage
containing 5 g of GOS significantly increased the
intestinal intrinsic Lactobacillus bacterial count in
comparison to the pre-ingestion value (18). Another
study indicated that several strains of Lactobacillus
isolated in the human intestine, including L. casei,
metabolized GOS (12). Therefore, synbiotic fermented
milk beverage GOS may have increased intrinsic
Lactobacillus; however, the mechanism should be
investigated in a future study.
Matsumoto et al. measured the fecal levels of
putrefactive metabolites in 22 healthy adults (21 males, 1
female) with a mean age of 39 10 years, and reported
that the indole, ammonia, phenol, and p-Cresol levels
were 1.8 0.7 mol/g, 297 203 g/g, 3.2 3.2 g/g,
and 29 41 g/g, respectively (18). In the elderly person
study, the fecal indole and phenol levels before ingestion
of the test diet were similar to the reference values for
healthy adults. However, the ammonia and p-Cresol
levels were markedly higher (synbiotic fermented milk
beverage group: 675 352, 78 55, placebo group: 597
442, 72 68, respectively). It is known that intestinal
ammonia and p-Cresol are produced via the enteric
bacteria-related decomposition of amino acids (2, 15).
The intestinal ammonia and p-Cresol levels increase with
a high-protein diet-related increase in the substrates
utilized by enteric bacteria (5). Therefore, in the elderly
subjects, an age-related reduction of digestive tract
function such as digestion/absorption and peristalsis may
have allowed a massive influx of the substrates into the
large intestine, increasing the ammonia and p-Cresol
levels. In the synbiotic fermented milk beverage group,
the fecal ammonia and phenol levels were significantly
lower than the values in the placebo group. The
production of ammonia and phenol is inhibited at a low
pH (26, 28). Therefore, concerning the mechanism by
which ingestion of synbiotic fermented milk beverage
inhibits the fecal production of putrefactive metabolites,
an increase in the intestinal organic acid (acetic acid)
level may reduce ammonia/phenol-producing bacteria
Table 5. Effect of synbiotic fermented milk beverage containing L. casei strain Shirota and transgalactosylated oligosaccharides on fecal
pH, water content, and putrefactive metabolites in the elderly persons
Before ingestion Week 1 of ingestion Week 2 of ingestion
Organisms Synbiotic fermented Synbiotic fermented Synbiotic fermented
Placebo Placebo Placebo
milk beverage milk beverage milk beverage
pH 6.7 0.3 (17/17) 6.8 0.4 (17/17) 6.7 0.3
a
(17/17) 7.1 0.5 (17/17) 6.9 0.3
a
(17/17) 7.1 0.3 (17/17)
Water content (%) 73.0 6.9 (17/17) 75.5 10.6 (17/17) 71.3 12.4 (17/17) 71.6 10.0 (17/17) 73.2 7.9 (17/17) 72.2 9.4 (17/17)
Indole 1.7 1.0 (17/17) 1.8 2.4 (17/17) 1.3 0.7 (17/17) 1.3 0.9 (17/17) 1.7 1.0 (17/17) 1.4 0.8 (17/17)
Ammonia 675 352 (17/17) 597 442 (17/17) 525 302 (17/17) 719 612 (17/17) 496 288
b
(17/17) 620 351 (17/17)
Phenol 4.5 7.0 (14/17) 3.6 5.5 (13/17) 3.9 7.2 (16/17) 5.4 11.6 (15/17) 3.0 4.0
b
(15/17) 10.3 14.1 (13/17)
p-Cresol 78 55 (15/17) 72 68 (15/17) 66 67 (17/17) 87 74 (16/17) 72 41 (15/17) 83 69 (16/17)
The results are expressed as the mean and S.D. in mol/g feces for indole, in g/g feces for ammonia, Phenol and p-Cresol. The
results in the parentheses are the number of samples in which the putrefactive metabolites were detected/the number of samples
examined.
a
p<0.05: Significant difference between the synbiotic fermented milk beverage and the placebo (Students paired t-test).
b
p<0.05: Significant difference between the synbiotic fermented milk beverage and the placebo (Wilcoxon signed-rank test).
EFFECTS OF SYNBIOTIC FERMENTED MILK 145
such as Ent er obact er i aceae, Cl ost ri di um, and
Staphylococcus, and restrict an elevation of pH, as
observed in the placebo group, inhibiting the production
of putrefactive metabolites.
In this study, ingestion of the synbiotic fermented milk
product containing LcS and GOS improved defecation
probl ems i n the student s wi th mi ld consti pat ion
(increases in the defecation frequency and number of
days with bowel movements) and the intestinal flora and
environment in the elderly persons (an increase in
beneficial bacteria, a decrease in harmful bacteria, and
decreases in the levels of putrefactive metabolites). Our
st udy i s t he fi rst report i n whi ch t he i nt est i ne-
conditioning actions of symbiotic beverage, such as
improvement in defecation frequency, an increase in
beneficial bacteria, a decrease in harmful bacteria, and
improvement in the intestinal environment, were
demonstrated when compared with the placebo beverage
in the same ingestion test. In future studies, the roles of
LcS and GOS in synbiotic fermented milk-related
improvement in the stool quality and intestinal flora/
environment should be analyzed.
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