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Effects of Enteric Capsules Containing Bifidobacteria and Lactic

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					   Effects of Enteric Capsules Containing Bifidobacteria and Lactic Acid
               Bacteria on the Fecal Properties in Healthy Volunteers

               Mamiko KOHNO, * Tomoe YOSHINO, Youichi MATSUURA,
                         Masanori ASADA and Yuzo KAWAHARA

               * Research & Development Division, Morishita Jintan Co., Ltd.


Abstract       The effects of oral administration of enteric capsules containing
Bifidobacterium longum JBL01, Lactobacillus gasseri JLG01 and Enterococcus faecium
JEF01 in Bifina R on fecal properties were examined in healthy volunteers (32 men and
62 women, average age 35.4 years). A significant increase in the defecation frequency
was observed in the constipated volunteer group whose initial defecation frequency was
five or less times per week.     In addition, the defecation quantity was increased in the
constipated volunteer group. Also, the increase in the defecation quantity was observed
in the not-constipated volunteer group whose initial defecation frequency was more than
five times per week.    An improvement in the fecal shape, a change in the fecal color from
dark to light color were observed after the administration in all groups.        These results
demonstrated that an intake of Bifina R was effective to improve intestinal properties.
Key words: Bifidobacterium longum; lactic acid bacteria; seamless enteric capsule; fecal
properties


      Bifidobacteria are a group of bacteria most predominant in the intestinal bacterial
flora (9), and oral intake of these bacteria is reported, together with lactic acid bacteria, to
improve the balance of intestinal bacterial flora (3, 9) and exert various effects on body
function including control of intestinal function and enhancement of immunoreactivity (4,
10, 11, 15).
      Bacteria and yeasts that, upon being orally ingested in viable conditions, improve
the balance of intestinal microbial flora thereby acting beneficially on health are called
probiotics (9). Bifidobacteria and lactic acid bacteria which actively multiply in the
lower small intestine and the large intestine (area covering cecum and ascending colon)

Received on January 6, 2004
* 1-1-30 Tamatsukuri, Chuo-ku, Osaka 540-8566, Japan



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are none other than probiotics. However, since these bacteria are very susceptible to the
effect of acid, viable cell number of orally ingested bacteria will be reduced substantially
by the action of gastric acid (pH 1.2) before they reach the site of action (8).
      Acid-resistant seamless capsules containing Bifidobacterium longum JBL01,
Lactobacillus gasseri JLG01, and Enterococcus faecium JEF01 have been developed that
allow these bacteria after oral intake to reach the site at high cell densities with reduced
exposure to gastric acid (1, 5). We conducted a study in which test capsule samples
containing these bacteria were orally administered to healthy volunteers to assess the
effect on stool frequency, stool properties, and feeling after bowel movement.


                                    Materials and methods
1. Materials: Test samples used were: 0.2-gram seamless capsules containing
Bifidobacterium longum JBL01 (2.0  109CFU), 0.1-gram seamless capsules containing
Lactobacillus gasseri JLG01 and Enterococcus faecium JEF01 (5.0  108 CFU each), and
1.1-g packet bifidobacteria preparation (trade name: Bifina R) containing 0.29 g
oligosaccharides (lactulose and raffinose). Other ingredients in the test samples were
erythritol and citric acid.    Each of the test samples was added to 900 ml of Solution I (pH
1.2, artificial solution equivalent to gastric juice) described in the Japanese Pharmacopeia,
14th edition, and the mixture was stirred for 2 hr, after which the survival rate of
bifidobacteria and lactic acid bacteria in capsules was determined.     The survival rate was
71% with bifidobacteria and 80% with lactic acid bacteria (Fig. 1). Since the survival
rate decreased below 0.0001% in 30 min when either of the bacteria in powder form was
treated in the same manner, these capsules, when orally ingested, are expected to be less
subject to gastric acid (pH 1.2) and protect these bacteria from being decimated before
reaching the site of action.


2.    Study subjects and method of sample ingestion:
      Subjects were 114 healthy adult volunteers, who, upon being provided with a
thorough explanation about the details of the study in accordance with the principle of
Helsinki Declaration, gave consent to participate in the study. A total of 94 subjects (32
males, 62 females, mean age 35.4 years) were eligible for analysis; those excluded from
analysis were 16 subjects who did not complete or submit the bowel movement diary, 3



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subjects who used antibiotics, and 1 subject who became ill unrelated to the study during
the study, 20 subjects in all.
      The study duration consisted of 2 weeks of pre-ingestion period, 2 weeks of
ingestion period, and 2 weeks of post-ingestion period, 6 weeks in total.      During the
ingestion period, subjects were asked to take one packet everyday, and record the
condition of bowel movement in the questionnaire sheet.        As a general rule, the test
sample was to be ingested immediately after awaking in the morning, but ingesting at any
time of the day was permitted as long as one packet was ingested per day.      Throughout
the study period, subjects were allowed to take meals as usual, but were asked to refrain
from taking health foods that may affect bowel condition unless they had been taking them
everyday.




3. Questionnaire survey: Subjects were asked to enter in a questionnaire sheet stool
frequency, stool volume, stool properties and feeling after bowel movement every time
they defecated. Specifically, they were to record (1) stool frequency, (2) stool volume,
(3) shape, (4) color, (5) odor, and (6) refreshing feeling after bowel movement.   Subjects
were to record the number of times of defecation per day as stool frequency, and stool
volume expressed in the number of eggs (large size) that corresponded to the volume by
visual estimation.    The stool shape was to be recorded as (i) very hard, (ii) hard, (iii)
banana shape, (iv) soft, (v) muddy, or (vi) watery.   Color was referred to the DIC color
guide (ver. 17) of Dainippon Ink and Chemicals, Inc. and recorded in one of 6 different
numbers expressing the color closest to fecal color: (i) 203 (yellow), (ii) 340 (brownish
yellow), (iii) 238 (yellowish brown), (iv) 304 (brown), (v) 310 (darkish brown), or (vi)


                                            -3-
521 (dark brown). Stool odor was recorded as (i) strong, (ii) unchanged, or (iii) weak,
and refreshing feeling after bowel movement as (i) unrefreshing, (ii) usual, or (iii)
refreshing.


4. Statistical treatment: Stool frequency and stool volume were analyzed by Wilcoxon’s
signed rank sum test, with significance level p<0.05 and p<0.01. The stool shape, color
and odor as well as refreshing feeling after bowel movement were summarized as
incidence rate for each study period, and subjected to 2 test for independence of
distribution.


5. Matters of special note about the analysis of results: 94 subjects eligible for analysis
were divided into two groups: constipated subjects with stool frequency of 5 times or less
a week during the pre-ingestion period and non-constipated subjects with stool frequency
of more than 5 times a week during the same period. Results are compiled separately for
each group. Subjects in non-constipated group were further divided into two groups:
subgroup A with stool frequency of 14 times a week and subgroup B with stool
frequency of <14 times a week during the pre-ingestion period.        Furthermore, subjects
with soft, muddy or watery stool shape on the average during the 2-week pre-ingestion
period were handled as diarrheic subjects, and data on stool shape were compiled for each
of the groups classified as above.


                                           Results
1.    Stool frequency and volume
      Table 1 shows changes in stool frequency and stool volume. The stool frequency
in the constipated group increased significantly both during the ingestion period and
during the post-ingestion period relative to the frequency during the pre-ingestion period
(p<0.01).     In the non-constipated group, the stool frequency decreased significantly both
during the ingestion period and during the post-ingestion period relative to the frequency
during the pre-ingestion period (p<0.05). The frequency also decreased significantly in a
similar manner in subgroup A, a group of patients with particularly high frequency in the
non-constipated group (p<0.05).
      Stool volume did not show significant difference between the constipated group and



                                             -4-
the non-constipated group, but showed a tendency to increase during the ingestion period
relative to the pre-ingestion period.     Since entry of stool volume in the diary was
incomplete in two subjects in the non-constipated group, data obtained from 27 subjects in
the constipated group and from 65 subjects in the non-constipated group were used for the
calculation of mean values and for statistical analysis.




2.    Stool shape
      Fig. 2 shows changes in stool shape.           Comparison of stool shape during the
pre-ingestion period showed that the incidence of "hard" or "very hard" stool was higher
in the constipated group than in the non-constipated group.     In the constipated group, the
incidence of "hard" or "very hard" stool decreased during the ingestion period compared
with the pre-ingestion period, while the incidence of "banana-shaped" stool increased
significantly (p<0.05).   In the diarrheic group, the incidence of "soft" stool decreased and
the incidence of "banana-shaped" stool increased significantly during the ingestion period
relative to the pre-ingestion period (p<0.01).       The incidence of "banana-shaped" stool
remained at a significantly higher level even during the post-ingestion period (p<0.01).
In the non-constipated group also, the incidence of "soft" stool decreased during the
ingestion period relative to the pre-ingestion period.     Little or no "muddy/watery" stool
was observed throughout the whole study period in any of the groups, with no significant
changes in the incidence of stool of these shapes.




                                             -5-
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3.    Stool color
      Fig. 3 shows changes in stool color.           Comparison of stool color during the
pre-ingestion showed that the incidence of "dark brown, blackish brown or brown" stool
was higher in the constipated subjects than in the non-constipated subjects.          In the
constipated subjects, the incidence of "brown" stool decreased and the incidence of
"yellowish brown or brownish yellow" stool increased during the ingestion period relative
to the pre-ingestion period.    In the non-constipated group, the incidence of "yellowish
brown" stool decreased and the incidence of "brownish yellow or yellow" stool increased
during the ingestion period relative to the pre-ingestion period. No significant difference
was observed in the distribution of incidences among study periods.


4.    Stool odor
      Fig. 4 shows effects on stool odor. In the constipated group, no difference was
observed in stool odor between the pre-ingestion period and the ingestion period.      In the
non-constipated group, the incidence of "strong" odor decreased during the ingestion
period relative to the pre-ingestion period.   In both groups, the incidence of "strong" odor
showed a tendency of increase.          No significant difference was observed in the
distribution of incidences among study periods.


5.    Effect on feeling after defecation
      Fig. 5 shows data on refreshing feeling after bowel movement.       In the constipated
group, no difference was observed in the refreshing feeling between the pre-ingestion
period and the ingestion period, whereas in the non-constipated group, the incidence of
"unrefreshing feeling" decreased during the ingestion period relative to the pre-ingestion
period, and the incidence of "unrefreshing feeling" tended to decrease further during the
post-ingestion period.    No significant difference was observed in the distribution of
incidences among study periods.




                                               -7-
                                        Discussion
     Constipation refers to the condition wherein the bowel movement does not occur
smoothly with stool remaining in the large intestine for a prolonged time.             Stool
frequency varies from person to person; bowel movement frequency of once per 2 to 3
days may not be constipation in people accustomed to such a frequency, while some
people may be constipated even with the bowel movement of once a day, with no
refreshing feeling after bowel movement.     It is therefore difficult to define constipation
based solely on stool frequency (12). Nevertheless, usually we experience regular bowel
movement of once or twice every 24 to 48 hours, and prolonged retention of stool in the
intestine may cause the growth of "bad" bacteria which produce harmful substances
resulting in impairment of health. Against these backgrounds, we investigated the effect
of probiotics capsules on healthy subjects who were classified into constipated group and
non-constipated group based on the stool frequency during the period before test sample
ingestion. Since diarrhea refers to conditions wherein water content of stool is high,
subjects who had "soft, muddy or watery" stool on the average during 2 weeks of the
pre-ingestion period were handled as diarrheic subjects, and changes in stool shape in
these subjects were analyzed.
     In the constipated subjects, stool frequency increased significantly both during the
ingestion period and the post-ingestion period relative to the pre-ingestion period (p<0.01),
indicating that the test sample was particularly effective in promoting bowel movement in
the constipated group.    In the non-constipated group, the stool frequency decreased
significantly both during the ingestion period and the post-ingestion period (p<0.05),



                                            -8-
presumably because stool frequency decreased to a marked extent in subgroup A (subjects
with stool frequency of 14 times per week). Subjects in subgroup A tended to be
diarrheic and the results suggest that the test sample is also effective for adjusting stool
frequency.
      Stool volume did not show significant difference between the constipated group and
the non-constipated group, but increased during the ingestion period relative to the
pre-ingestion period. Thus, the results suggest the possibility that refreshing feeling may
improve by the increase in stool volume in people who, despite bowel movement of at
least once a day, do not have refreshing feeling after bowel movement due to small stool
volume and are therefore considered to be constipated (12).
      "Banana-shaped" stool is usually thought to be an ideal shape of stool.        In the
present study, the incidence of "banana-shaped" stool increased in all groups during the
ingestion period relative to the pre-ingestion period, and this tendency was particularly
marked in the constipated group and the diarrheic group.      In contrast, the incidence of
"hard or very hard" stool, which was observed at a high incidence rate in the constipated
group, decreased. Also decreased was the incidence of "soft" stool which was observed
at a high incidence rate in the diarrheic group and the non-constipated group. These
results indicate that the test sample is effective not only in improving the stool shape in
constipated people but also in bringing the stool shape close to the ideal shape in the
diarrheic people. Regarding stool color, the incidence of "brownish yellow or yellowish
brown" stool increased in the constipated subjects who showed a high incidence of black
to brown (dark brown, blackish brown, brown) stool during the pre-ingestion period, while
the incidence of "brownish yellow or yellow" stool tended to increase in the
non-constipated group who had a high incidence of yellowish brown stool during the
pre-ingestion period. Thus, stool color tended to become brighter in both groups. Since
stool of brighter color is generally regarded as ideal, the results suggest that the test
sample is also effective in improving the stool color.
      No significant difference was observed in stool odor or refreshing feeling after
bowel movement between groups, while the incidence of "strong" odor and of
"unrefreshing feeling" remained unchanged or tended to decrease during the ingestion
period relative to the pre-ingestion period. These results suggest that stool odor and
refreshing feeling are improved by the ingestion of the test sample.



                                             -9-
      Oral intake of a test sample containing seamless capsules of B. longum JBL01 as the
sole beneficial bacteria has been reported to improve bowel movement in patients with
anus-related diseases (6). Also, effect of oral intake of the same sample was studied in
dialysis patients who are likely to become constipated due to restriction of water intake,
and it was reported that the treatment improved intestinal bacterial flora and suppressed
the formation of putrefactive products (7).      Furthermore, in a study which compared a
test sample containing seamless capsules of B. longum JBL01 only and a test sample
containing seamless capsules of B. longum JBL01 and seamless capsules of L. gasseri
JLG01 and E. faecium JEF01 for the activity to improve intestinal bacterial flora
following oral ingestion, both samples were found to increase the number of bifidobacteria
in feces, but the sample containing the capsules of lactic acid bacteria was more effective
in significantly increasing the number of bifidobacteria (unpublished data). The present
study also demonstrated the activity of the test samples to improve the stool frequency and
stool properties. Since it is reported that intake of lactic acid bacteria increases the
number of bifidobacteria (14), and that peristaltic movement of the intestine is stimulated
by organic acids produced by bifidobacteria and lactic acid bacteria (13), simultaneous
intake of bifidobacteria capsules and lactic acid bacteria capsules is expected to provide a
synergistic effect.
      These results, taken together, raise an expectation that the seamless capsules
containing B. longum JBL01, L. gasseri JLG01 and E. faecium JEF01 will be effective in
controlling the intestinal function in healthy adults.


      In concluding, we express our sincere gratitude to Tomotari Mitsuoka, Emeritus
Professor of the University of Tokyo for his advice and guidance throughout the study.


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