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ORIGINAL ARTICLE—ALIMENTARY TRACT
Efficacy of double-coated probiotics for irritable bowel syndrome:
a randomized double-blind controlled trial
Kyungsun Han
1
•Jinghwa Wang
1
•Jae-Gu Seo
2
•Hojun Kim
1
Received: 22 December 2015 / Accepted: 1 May 2016
ÓJapanese Society of Gastroenterology 2016
Abstract
Background The purpose of this study was to elucidate the
effects of a dual-coated probiotic supplement (Duolac
Care) on symptoms of diarrhea-predominant irrita-
ble bowel syndrome in a randomized double-blind clinical
trial.
Methods Fifty subjects with diarrhea-predominant irrita-
ble bowel syndrome were randomly assigned to either the
non-coating group or the dual-coating group in order to
receive two capsules per day of multi-species probiotics
containing 5 billion bacteria per capsule for 4 weeks. Data
from an adequate relief questionnaire were used in
assessment of primary outcome. Daily records of stool
frequencies and the Bristol stool scale, a weekly symptom
diary using 100-mm visual analog scale, and Beck
depression inventories were collected. Blood tests includ-
ing blood cell counts, interleukin-10, tumor necrosis factor-
alpha and inducible nitric oxide synthase, and regulatory T
cells—CD4 ?CD25
high
T cells, CD4 ?LAP ?T cells
and CD25
high
?LAP ?T cells—were analyzed before
and after the study. The shift of gut microbiota was
investigated using a quantitative real-time polymerase
chain reaction assay.
Results Responses to the adequate relief questionnaire
indicated significant improvement in overall discomfort in
the dual-coating group and the ratio of normal stools to
hard or watery stools had a better effect from dual-coated
probiotics compared to non-coated probiotics. This may be
due to a shift of intestinal microbiota, as our correlation
analysis showed significant negative correlation between
Bifidobacterium and urgency of defecation.
Conclusions Our result implies that dual-coating layers of
probiotic supplement can be a candidate for treatment of
diarrhea-predominant irritable bowel syndrome.
Keywords Probiotics Irritable bowel syndrome
Dual-coating technology
Introduction
Irritable bowel syndrome (IBS) is a common gastroin-
testinal disorder that can affect patients’ quality of life,
although it is not a life-threatening disease [1]. The rep-
resentative symptoms include chronic or recurrent
abdominal pain and abdominal discomfort originating from
a functional disorder of bowel movement that cannot be
explained by any structural abnormalities. The prevalence
of IBS in Southeast Asia is 7 %, while 10 % of the United
States populations suffer from IBS symptoms [2]. The
etiological cause of IBS cannot be explained by a single
mechanism. It is thought to be associated with various
factors including disturbed brain-gut axis functions, low-
grade inflammation, infection, intestinal hypersensitivity,
abnormal colonic fermentation, and psychological disor-
ders [3–5]. Intestinal microbiota is another important factor
affecting the intestinal function and inflammation observed
in IBS. Studies have shown that small intestinal bacterial
Electronic supplementary material The online version of this
article (doi:10.1007/s00535-016-1224-y) contains supplementary
material, which is available to authorized users.
&Hojun Kim
kimklar@dongguk.ac.kr
1
Department of Rehabilitation Medicine of Korean Medicine,
Dongguk University Ilsan Hospital, 814 Siksa-dong, Goyang,
Gyeonggi-do, Republic of Korea
2
R&D Center, Cell Biotech Co. Ltd., Gimpo 415-871,
Republic of Korea
123
J Gastroenterol
DOI 10.1007/s00535-016-1224-y
overgrowth is frequently observed in IBS patients [6].
Numerous studies of gut microbiota and dysbiosis reported
a diminished population of Lactobacillius and Bifidobac-
teria in IBS patients while the presence of Veillonella,
Clostridium coccoides subgroup, Streptococci, and Co-
liforms increased [7–9]. Therefore, the therapeutic target of
IBS can focus on normalizing the intestinal microbiota.
Use of probiotics in the treatment of IBS was reported to
be strongly effective in a number of trials [2,10–12]. These
studies suggested the beneficial effects of probiotics by
way of improving immune response, enhancing intestinal
permeability, and altering colonic fermentation [13,14].
Emerging studies have supported probiotics in IBS treat-
ment, however, the effectiveness of numerous probiotics is
not consistent and comparisons between probiotics are
lacking. To ameliorate the poor survival of probiotic bac-
teria during digestion, dual-coating technology during the
manufacturing process was patented [15,16]. By coating
bacteria cells with peptides followed by a dual polysac-
charide matrix, probiotic product can survive pH gradient
along the gastrointestinal tract and its bile acid conditions
[17]. A randomized, double-blind controlled study com-
paring non-coated probiotics and double-coated probiotics
in elderly persons with functional constipation showed
improvements in the symptoms as well as significantly
increased fecal bacteria, particularly in the double-coated
probiotics group [18]. However, the clinical evidence of
dual-coating technology is not sufficient. Therefore, we
conducted a randomized, double-blind controlled study to
confirm the effects of dual-coated probiotics on IBS-related
symptoms, immunological parameters from blood samples,
and fecal microbial changes.
Subjects and methods
Study design and subjects
The study protocol was approved by the Institutional
Review Board of Dongguk University Ilsan Hospital (ap-
proval no 2014-05). The study protocol was registered in
CRiS (Clinical Research Information Service, Serial
number: KCT0001226). Participants with chronic diarrhea
and abdominal discomfort were recruited at Dongguk
University Ilsan Oriental Hospital. Subjects were recruited
by advertisements in local newspapers and by posters.
Male and female subjects between ages 19–65 who met the
criteria of diarrhea-dominant Rome III criteria were
included in this study. Among 56 participants, 50 subjects
with abdominal discomfort and diarrhea with no organic
lesion diagnosed within 2 years, and concurrently, who met
the Rome III Criteria were included. Written consent was
acquired prior to the study.
Patients who had undergone abdominal surgery except
appendectomy, Caesarian section, tubal ligation, laparo-
scopic cholecystectomy, hysterectomy, and abdominal wall
hernia repair were excluded. Patients with other organic
symptoms related to the gastrointestinal tract (ex. cholan-
gitis, pancreatitis, enteritis, ulcer, bleeding, cancer),
patients who exhibited alarming symptoms such as dra-
matic reduction of weight, black colored stool, dysphagia,
who took over-the-counter medication within 3 days ahead
of study enrollment, who took antibiotics, oriental medi-
cine, or probiotics within 4 weeks, and subjects who were
pregnant or had any chance of pregnancy (patient with
child bearing potential due to not taking contraception were
required to have a negative pregnancy urine test within
48 h of participation) were excluded from this study.
Patients who did not agree to the written consent were also
excluded.
Study treatments
This was a 4-week double-blind placebo-controlled clinical
study randomizing two parallel groups of 25 subjects.
Randomization codes were established by a statistician
running SAS (version 9.1 or after), and kept confidential
until data collection was completed. Participants were
randomly allocated to either the non-coated probiotics
group or the dual-coated probiotics group and completed a
1-week run-in period followed by a 4-week intervention
period. During the intervention, subjects took the probi-
otics supplement named Duolac Care, twice a day, one
capsule at a time (contains 5 billion bacteria per 2 cap-
sules =5910
9
viable cells/strain) 2 h after meals.
Duolac Care (Cell Biotech Co. Ltd., Gimpo, Korea) is a
probiotic mixture containing multiple species of viable
bacterial genus, Lactobacillus acidophilus,L. plantarum,
L. rhamnosus, Bifidobacterium breve, B. lactis, B. longum,
and Streptococcus thermophilus, and the bacteria are
doubly coated with peptides and polysaccharides to protect
the probiotic microorganisms from destruction in the gas-
trointestinal tract. Probiotics for the comparison group
containing the same bacterial genus but left un-coated were
packaged in the same capsules.
Anthropometrical measurements and blood tests
Anthropometry was performed before and after the study.
Blood pressure (BP) and pulse rate (PR) were measured
using an automatic digital sphygmomanometer. Body
weight (BW) and height were measured using an automatic
scale (G-tech, Uijeongbu, Korea), wearing a hospital gown
(the nearest 0.1 kg and 0.5 cm, respectively). Body mass
index (BMI) was calculated by weight in kilograms divided
by height in meters squared. Body composition, including
J Gastroenterol
123
lean body mass, fat mass, fat percentage, body water, and
waist-to-hip ratio were obtained using the InBody 3.0
device (InBody 3.0, Biospace, Seoul, Korea). This Bio-
electrical Impedance Analyzer measures total body water
and fat free mass by making use of the segmental resistance
through its eight tactile electrodes.
Blood samples were taken twice, before and after the
clinical study. Blood tests, including white blood cell count
(WBC), red blood cell count (RBC), hemoglobin (Hb),
hematocrit (Hct), platelet, neutrophils, lymphocytes,
monocytes, and eosinophils were determined using a Cobas
8000 modular analyzer (Roche, Brandford, CT, USA).
Immunological markers
To determine the ratio of CD4 ?CD25
high
and CD4 ?
CD25 ?LAP ?, fluorescence-activated cell sorting
(FACS) was performed using a Beckman Coulter FC500
series (Beckman Coulter, Fullerton, CA, USA). We treated
blood samples with CD4-FICS (5 ll) and CD25-PC5
(2.5 ll) and cells were fixed with PerFix-nc (Beckman
coulter). During fixation, after addition of the second fix-
ation solution, anti-human LAP-PE (1.25 ll) was applied,
and Mouse IgG1 jIso control–PE (1.25 ll) (eBioscience,
San Diego, CA, USA) was used as control. To study
changes of cytokines (IL-10, TNF-a) and nitric oxide
synthase (iNOS), plasma were collected by centrifugation
(3000 rpm, 15 min) after 2 h of blood collection, and kept
in -80 °C until examination. Enzyme-linked immunosor-
bent assay (ELISA) was performed according to the man-
ufacturer’s protocol. IL-10 High Sensitivity Human ELISA
kit (Abcam, Inc., Cambridge, MA, USA), TNF-alpha
Human SimpleStep ELISA Kit (Abcam), and human
inducible nitric oxide synthase ELISA kit (Cusabio,
Wuhan, China) were used.
Assessments of symptoms
To exclude any significant difference between the two
groups, functional bowel disorder severity index (FBDSI)
was assessed [19]. The FBDSI scoring system is as follows.
FBDSI ¼current pain by visual analog scale 0100ðÞ½
þdiagnosis of chronic functional abdominal pain½
0 if absent and 106 if presentðÞ
þnumber of physicians visits over previous½
6 months 11
Adequate relief (AR) of IBS pain and discomfort was
used to assess the improvement of IBS symptoms in order
to determine the primary outcome. Patients were asked the
following question before the study, 2 weeks after the
intervention, and after 4 weeks of intervention study: ‘‘In
the past seven days have you had adequate relief of your
irritable bowel syndrome pain and discomfort?’’ [20]
Throughout the study, symptom diaries were distributed
prior to the intervention and patients were asked to com-
plete a diary every week to assess the severity of IBS
symptoms. The stool diary data including stool frequency,
consistency, and ease of passage were recorded based on
the Bristol stool scale [21]. Individual IBS symptoms
(abdominal pain, abdominal discomfort, bloating, flatu-
lence, urgency, and mucus in the stool) were also assessed
on a 100-mm visual analog scale (VAS) [22]. The Beck
Depression Inventory (BDI), composed of 21 self-report
questionnaires and used to measure severity of depression,
was also used before and after the investigation to assess
the quality of life of IBS patients [23,24].
Intestinal microbial analysis
Fecal samples collected in sterile containers at the beginning
and end of the study were brought to the laboratory frozen
and stored at -80 °C until analyzed. Bacterial DNA was
isolated from stool samples using the FastDNA SPIN Kit
(MP Biomedicals, Santa Ana, CA). Reverse-transcriptase
polymerase chain reaction (RT-qPCR) analysis was per-
formed using a LightCycler 480 system (Roche, Germany).
Primers were synthesized commercially by Bioneer (Dae-
jeon, Korea), and the specificity of each primer was varied
using DNA isolated from closely and distantly related bac-
teria as a template. Quantitative PCR was performed in a
96-well plate in a final volume of 20 lL. Reaction mixtures
consisted of 1 lL fecal DNA, 0.5 lL primers (10 pmol
each), 10lL SYBR Green I master mix (Roche, Germany),
and 8 lL H2O. PCR amplification was carried out under the
following conditions: pre-incubation at 94 °C for 4 min,
followed by 55 cycles of amplification (denaturation at
94 °C for 15 s, primer annealing at 55 °C for 15 s, and
elongation at 72 °C for 20 s). The melting curve was ana-
lyzed by heating the reaction from 50 to 90 °C with a
temperature transition time of 5 °C/sec. The primers used in
qPCR analysis are shown in Supplementary Table 1.
Statistical analyses
Continuous data were calculated as mean with standard
deviation, while categorical data were presented as fre-
quencies. Continuous variables were compared by ttest, and
categorical variables by Wilcoxon’s signed–rank and Mann–
Whitney tests using SPSS program version 16.0 (SPSS Inc.,
Chicago, IL). Correlation analyses between variables were
assessed using Spearman’s correlation test. Pvalues below
0.05 were considered statistically significant.
J Gastroenterol
123
Results
Clinical outcomes
Among 50 patients enrolled in this study, and 46 patients
completed the study with no adverse effect. The study flow
chart is shown in Fig. 1. Two patients withdrew consent,
one patient was lost to follow-up and one patient dropped
out of this study because of protocol violation. No differ-
ence in age, gender, clinical anthropometric values, and
FBDSI (functional bowel disorders severity index) was
observed between the two groups at baseline. No signifi-
cant changes of anthropometric values were observed after
the study, as shown in Table 1.
Table 2shows changes of blood cell counts of each
group. Blood analysis showed no significant changes in
blood cell counts, including white blood cell, red blood
cell, hemoglobin, hematocrit, platelets, neutrophils, lym-
phocytes, monocytes, and eosinophils.
Immunological parameters
Three important markers (iNOS, IL-10, and TNFa)of
inflammation from blood samples collected before and
after the study were analyzed. No significant changes in
these markers were observed in either group because of
individual difference. CD4 ?CD25 high T cells,
CD4 ?LAP ?T cells, and CD25 high ?LAP ?T cells
also showed no significant difference before and after the
study (Supplementary Table 2). There were no significant
changes in group comparison or in pre-post comparison.
Symptoms and bowel function
We used data from the Adequate Relief (AR) questionnaire
for the assessment of primary outcome to estimate the
improvement after the intervention (Fig. 2a). After
2 weeks of intervention, 76 % of subjects in the dual-
coating group and 36 % in the non-coating group said that
they had achieved adequate relief of their symptoms. At the
end of the trial, both groups showed significant improve-
ment of their IBS symptoms—that is, adequate relief was
achieved in 64 % from the non-coating group and 72 %
from the dual-coating group. However, the difference in
the fourth week of the trial was not significant between
groups (pvalue =0.433).
To assess the severity of IBS symptoms, patients were
required to write in a symptom diary on a daily basis, which
included stool frequency per day and a score on the Bristol
stool scale (Figs. 2b, c, 3). Despite individual variations,
overall frequency decreased in both groups, particularly in
the non-coating group. Compared to the baseline, both
groups showed a rapid decrease during the first week of the
trial. However, statistically there were no significant chan-
ges in both groups. There were also no statistical differences
between the two groups in defecation frequency.
Stool shapes improved throughout the investigations, as
shown in Fig. 3a, however, there was no significance
Fig. 1 Study flow chart
J Gastroenterol
123
between the two groups. Stool with soft blobs with clear
cut edges (type 5) and a sausage- or snake-like stool type
(type 4) showed the highest percentage in both groups. To
optimize the improvement of the stool shape, stool types
3–5 were defined as a regular form in which patients did
not have difficulty in defecation and the ratio of normal
stool to watery or hard types of stool was calculated
(Fig. 3b). The portion of normal stool increased signifi-
cantly in the dual-coating group after 3 weeks (Fig. 3c).
The weekly report of the symptom diary was investi-
gated using the VAS scale in six categories consisting of
representative symptoms of IBS: abdominal pain, abdom-
inal discomfort, bloating, flatulence, urgency, and mucus in
stool. Figure 4b shows the difference in values compared
to the beginning of the study. Overall symptoms improved,
however, no significance was observed between the two
groups. Patients of the non-coating group showed signifi-
cant improvement in all six symptoms at the end of the
trial, while subjects from the dual-coating group showed
significant improvement in bloating, flatulence, urgency,
and mucus in stool. Although dual-coated probiotics did
not seem to affect abdominal pain and discomfort,
Table 1 Baseline characteristics of the patients and changes after the trial
Variables Non-coating group (n=23) Dual-coating group (n=23) P
2
value
Before After Pvalue Before After Pvalue
Gender Male (n=11), female (n=12) Male (n=13), female (n=10)
Age (years) 42.5 ±10.07 45.7 ±9.55 0.338
FBDSI 165.0 ±19.59 – 165.8 ±20.52 – 0.884
Body weight(kg) 61.3 ±10.81 61.2 ±10.59 0.537 63.6 ±9.55 63.7 ±9.57 0.399 0.666
BMI (kg/m
2
) 21.8 ±3.14 21.7 ±3.01 0.539 23.0 ±2.61 23.0 ±2.60 0.129 0.648
Waist circumferences (mm) 798.3 ±97.28 802.3 ±94.12 0.271 829.0 ±78.78 791.6 ±171.23 0.794 0.312
Body fat percentage (%) 23.3 ±73.6 23.8 ±7.16 0.183 25.1 ±7.01 25.0 ±7.35 0.559 0.891
Waist–hip ratio 0.860 ±0.051 0.864 ±0.051 0.073 0.890 ±0.048 0.891 ±0.049 0.072 0.56
Skeletal muscle mass (%) 25.9 ±5.70 25.7 ±5.48 0.139 26.4 ±5.44 26.4 ±5.56 0.685 0.903
Body fat mass (kg) 14.3 ±5.56 14.6 ±5.41 0.277 15.9 ±4.81 15.8 ±5.04 0.421 0.919
Total body water (L) 34.6 ±6.94 34.3 ±6.55 0.082 35.1 ±6.56 35.2 ±6.75 0.631 0.736
Systolic BP (mmHg) 112.7 ±13.29 111.3 ±13.58 0.436 115.6 ±13.56 114.8 ±10.28 0.805 0.333
Diastolic BP (mmHg) 73.0 ±11.86 72.7 ±12.86 0.752 75.5 ±13.02 75.0 ±8.80 0.825 0.474
Pulse rate (/min) 76.4 ±11.65 73.2 ±10.73 0.107 71.6 ±7.44 70.6 ±8.03 0.555 0.356
FBDSI score \36 mild illness; 37–110 moderate illness; [111 severe symptoms. Data presented as mean ±SD. Pvalue: pre-post comparison
within each group. P
2
value: comparison between groups. None of the comparisons had a statistically significant Pvalue
FBDSI functional bowel disorder severity index, BMI body mass index, BP blood pressure
Table 2 Blood cell counts before and after the trial
Variables Non-coating group (n=23) Dual-coating group (n=23) p
2
value
Before After Pvalue Before After Pvalue
WBC (910
3
/ll) 5.8 ±2.67 5.8 ±1.81 0.994 5.6 ±0.80 5.9 ±1.45 0.206 0.907
RBC (910
6
/ll) 4.6 ±0.38 4.6 ±0.40 0.641 4.7 ±0.32 4.6 ±0.32 0.159 0.694
Hb (g/dL) 13.8 ±1.49 13.8 ±1.56 0.581 14.4 ±1.15 14.3 ±1.21 0.47 0.248
Hct (%) 41.0 ±3.91 41.1 ±4.06 0.558 42.4 ±3.09 42.0 ±3.39 0.101 0.453
Platelet (910
3
/ll) 234.8 ±77.77 213.8 ±95.99 0.112 219.0 ±36.14 224.2 ±44.57 0.479 0.639
Neutrophil (%) 52.9 ±10.00 52.3 ±10.18 0.543 52.2 ±5.86 52.4 ±9.49 0.844 0.957
Lymphocyte (%) 37.6 ±9.26 37.7 ±9.21 0.951 38.2 ±5.13 38.0 ±7.69 0.901 0.894
Monocyte (%) 6.5 ±1.62 6.5 ±1.74 0.977 6.2 ±1.28 6.5 ±1.54 0.382 0.964
Eosinophil (%) 2.6 ±1.48 2.9 ±1.80 0.118 2.9 ±2.64 2.7 ±2.48 0.155 0.756
Data presented as mean ±SD. Pvalue: pre-post comparison within each group. P
2
value: comparison between groups. None of the comparisons
showed a statistically significant Pvalue
WBC white blood cell, RBC red blood cell, Hb hemoglobin, Hct hematocrit
J Gastroenterol
123
defecation urgency, bloating sense, and mucus in stool
relatively improved compared to the non-coated probiotics
group.
IBS symptoms ultimately damage overall quality of life,
and can also cause depression. To determine how symptom
changes influenced subjects’ quality of life, BDI scores
were surveyed at week 0, 2, and 4. Feeling of depression
decreased significantly in both groups, but comparison
between the groups showed no significance (Fig. 4a).
Quantitative changes in intestinal microbiota
The changes in intestinal microbiota are shown in Fig. 5a.
Ingestion of probiotic supplementation resulted in an
increase of total bacteria in both non-coating and dual-
coating groups (from 9.76 to 10.38 in the non-coating group
and 9.98–10.42 in the dual-coating group: numbers pre-
sented as log
10
). The number of intestinal bacteria in both
Gram-positive bacteria (from 6.76 to 7.68 in the non-coating
group, from 7.05 to 7.77 in the dual-coating group) and
Gram-negative bacteria (from 7.89 to 8.02 in the non-
coating group, from 8.20 to 8.26 in the dual-coating group)
both increased, although the shifts were not significant. The
same tendency was observed in the Lactobacillus group and
Bifidobacterium group. Only Bifidobacterium increased
significantly in both groups (from 7.03 to 7.78 in the non-
coating group: pvalue \0.05, from 7.38 to 8.14 in the dual-
coating group: pvalue \0.01). An increase was observed in
the Lactobacillus group after the probiotics supplement,
however, the changes were not significant (from 6.55 to 6.76
in the non-coating group, from 6.76 to 6.92 in the dual-
coating group). To investigate the changes of intestinal
microbiota other than administered probiotic bacteria,
changes of Prevotella,Clostridium coccoides group, and
Veillonella were analyzed. Pre-post comparison showed no
significant difference between groups (Data not shown).
Fig. 2 Changes of irritable bowel syndrome (IBS)-related symptoms
after treatment of either dual-coated probiotics or non-coated
probiotics. aProportions of the AR questionnaire. Subjects were
asked the following question: ‘‘In the past 7 days have you had
adequate relief of your irritable bowel syndrome pain and
discomfort?’’ *Pre-post comparison within each group using Wil-
coxon’s signed–rank test;
comparison between groups using the
Mann–Whitney test. *p\0.05, **p\0.01,
p\0.05, bdifferences
in the number of defecations compared to the baseline. cNumber of
defecation times per day. *pvalue \0.05 compared between groups
J Gastroenterol
123
Correlation analysis between immunological
markers and intestinal microbiota
The correlation between improved symptoms related to
IBS and clinical markers, including blood cell counts,
immunological markers, and intestinal bacteria, was ana-
lyzed (Fig. 5b). Among the six representative symptoms of
IBS, correlation between urgency and clinical parameters
was noted. Urgency showed a negative correlation with
increased numbers of gut microbiota. Significant values
were observed for Bifidobacterium (R=-0.564,
p=0.005) and Gram-positive bacteria (R=-0.443,
p=0.034). In addition, Prevotella showed a negative
correlation with the degree of mucus in stool
(R=-0.0539, p=0.031) while the Clostridium
coccoides group showed a negative correlation with
abdominal pain (R=-0.490, p=0.033).
Discussion
Administration of probiotics for treatment of IBS has been
a promising strategy and a number of investigations have
been conducted using diverse multispecies probiotics. The
current clinical trial was conducted for comparison of the
effectiveness of a probiotic supplement, Duolac Care,
produced with double-coated layers of proteins and
polysaccharide to increase the survival rate of probiotics in
the gastrointestinal tract during digestion. Previous studies
showed significant improvement in smooth evacuation,
Fig. 3 Bristol stool scale. aBristol stool scale changes throughout
the study. There was no significance between the two groups. bRatio
of normal stool (categories 3–5) compared to hard or watery stool
(categories 1, 2, 6, and 7). cPercentage of normal stool (categories
3–5). *pvalue \0.05 compared between groups
J Gastroenterol
123
defecation frequencies, and obstructive sensation during
evacuation using dual-coated probiotics in elderly persons
with functional constipation [18]. The number of fecal
bacteria also increased significantly in both non-coated and
dual-coated probiotics, particularly in use of the latter [18].
In-vivo and in-vitro examination using dual-coated probi-
otics showed that dual-coated probiotics had effective
immunological activity by way of TNFaand NO produc-
tion by macrophages, which inhibits tumor cells or bacteria
and through an inhibitory effect on b-glucosidase activity
and b-glucuronidase activity in rat feces [16]. However,
correlations between IBS symptoms, immunological
markers, and intestinal microbiota are still unclear. At the
same time, this study is the first to investigate correlations
between the shift of gut microbiota, immunological
markers related to IBS, and IBS-related symptoms on the
basis of dual-coating technology.
The primary outcome of the current study was patients’
feelings regarding improvement of their IBS symptoms,
determined using the AR questionnaire. Significant
improvement in the symptoms was observed in both
groups. In particular, the dual-coating group showed rapid
improvement after 2 weeks and satisfaction persisted until
the end of the study. A similar tendency was observed in
Fig. 4 Changes of IBS-related symptoms after treatment of either
dual-coated or non-coated probiotics. aBeck depression inventory
(BDI) scale changes. Higher BDI scores indicate more severe
symptoms of depression.Pvalue \0.05 compared to week 0. There
was no significance between groups. bChanges of representative
symptoms of IBS presented in a 100-mm visual analog scale (VAS)
compared to week 0. All symptoms showed a significant decrease
(pvalue \0.05) in both groups compared to week 0. No significant
differences were found between groups
J Gastroenterol
123
stool frequencies. Frequencies of defecation had decreased
during the first two weeks of intervention in both non-
coating and dual-coating groups. Form of stools classified
using the Bristol stool scale improved rapidly in both
groups during the first 2 weeks of probiotics administra-
tion. The improvement progressed in the dual-coating
group and the ratio of normal stool compared to hard or
watery stool showed the greatest disparity in the third week
of intervention. Although the clinical parameters including
body compositions and blood cell counts did not show
significant differences after the study, relief of IBS-related
symptoms is a meaningful outcome. Stress and psycho-
logical aspects greatly contribute to IBS symptoms [25]
and an IBS cohort study found that there was a 40 % higher
probability of depression in IBS patients compared to non-
IBS patients [26]. Our result regarding BDI scores, which
reflect severity of depression, indicated that both groups,
particularly the dual-coating group, showed improved
quality of life. However, as the subjects involved in this
trial did not have severe depression, remarkable changes
were not found in both groups. BDI score 0–9 may be
regarded as normal while 10–20 may be considered as mild
depression [27]. In this clinical trial, none of the subjects
had BDI scores greater than 20. Nevertheless, our result is
meaningful in the way that IBS symptoms affect patients’
quality of life and in that depressed emotion related to IBS
significantly improved after probiotic supplementation.
In assessment of IBS symptoms by a weekly diary using
100 mm VAS, a significant decrease was observed in all
six representative symptoms—abdominal pain, abdominal
discomfort, bloating, flatulence, urgency, and mucus in
stool—in both groups. The dual-coating group showed
rapid improvement in bloating, urgency, and mucus in
stool, while abdominal pain and abdominal discomfort
Fig. 5 a Shift of intestinal microbiota after the intervention. Pvalue:
pre-post comparison within each group. There were no significant
differences between two groups. *pvalue \0.05, **pvalue \0.01.
bCorrelation analysis between IBS-related symptoms and clinical
outcomes. Asterisk p value \0.05
J Gastroenterol
123
showed better efficacy in the non-coating group. Previous
studies using probiotics for treatment of bowel disorders
have shown effectiveness in abdominal symptoms, partic-
ularly in bloating and flatulence [28,29]. Contrasting
results were reported in a study conducted with a probiotic
supplement for IBS patients, which showed decreased
duration of abdominal pain and distension intensity but did
not alter pressure or volume thresholds to reach the sen-
sation of distension or urgency for defecation [30].
Overall, the results proposed a question of why subjects
of the dual-coating group showed significant improvement
in AR questionnaire and BDI scores, despite the insignifi-
cancy in abdominal pain and discomfort findings, which
are generally considered as representative symptoms in
IBS. Recent guidelines for the clinical evaluation of IBS
have recommended critical symptoms for entry criteria of
IBS patients differentially according to subgroups. For
constipation-predominant IBS patients, stool frequency and
abdominal pain intensity are critical symptoms while stool
consistency using the Bristol stool scale and abdominal
pain intensity are considered essential for evaluation of
diarrhea-predominant IBS patients [31,32]. Our results
from diarrhea-predominant subjects correspond well to the
guideline that emphasizes stool shape more than defecation
frequency in diarrhea-predominant IBS patients. Symp-
toms related to stool shape such as mucus in stool and
bloating sensation, might have led to the more satisfactory
results in the dual-coating group despite overall improve-
ments in the non-coating group.
There are various aspects regarding the mechanisms of
probiotics in improvement of IBS symptoms, however, a
review study noted that the probiotic supplements can
potentially benefit IBS symptoms related to behavior and
mood by way of modulation of the gut-brain axis [33].
However, by far, the studies were conducted using certain
bacterial strains such as Bifidobacterium longum,Lacto-
bacillus helveticus, and Bifidbacterium infantis [34,35].
Thus, other genuses such as Veillonella,Clostridium coc-
coides group, and Prevotella were analyzed in the attempt
to determine whether probiotic supplements can cause a
shift of other bacterial composition. However, no signifi-
cant change was observed in this study. Based on these
findings, we analyzed the correlation between IBS symp-
toms and clinical parameters including gut microbiota and
immunological markers. Unlike previous studies, urgency
showed a significant association with the shift of intestinal
bacteria rather than bloating or flatulence. Bifidobacterium,
one of the major genera of Gram-positive bacteria, showed
a negative association with urgency of defecation. Further
investigation to determine the association between urgency
and the shift of gut microbiota is needed. In one study,
administration of Bifidobacterium infantis in a malted milk
drink for IBS patients led to improvement of overall
symptoms along with normalizing IL-10/IL-12 ratio and
pro-inflammatory cytokine Th-1, suggesting that probiotic
supplementation can improve IBS symptoms by way of an
immune modulating effect [36]. However, in the current
study, no significant changes were observed in immuno-
logical markers IL-10, TNFa, and iNOS or in regulatory T
cells related to suppressive function on the immune func-
tion after probiotic supplements. This may be due to the
short duration of intervention time or small sample size,
and severity of symptoms. In another interesting correla-
tion study, the genus Prevotella showed negative correla-
tion with the amount of mucus in stool. Prevotella is
known to express mucin-desulfating glycosidases that can
degrade heavily sulfated mucins in the intestinal tract,
which acts as a gut barrier and can cause abdominal pain
with rapid degradation of intestinal mucus [37]. Mucin-
type O-glycans are known to have a major role in mucin
homeostasis influenced by specific members of the bacte-
rial community such as Bacteroides, Prevotella, and Acti-
nobacteria muciniphila [38]. Our results showed negative
correlation between mucus in stool and the amount of
Prevotella in IBS patients fragmentarily, which implies
that probiotic supplements may be helpful in regulatory
functions of the gut barrier such as the mucus layer.
The limitation of this study is that in IBS treatment,
determining the placebo effect is difficult. Approximately
40 % of IBS patients are affected by the placebo effect
and may vary from 0 to 91.7 % in IBS treatment, which
may be due to frequent visits to the hospital and the
duration of the trial [2,39]. Thus, many strategies to
lessen the placebo effect during clinical trials related to
IBS have been proposed [40,41]. Although the validity is
not clear, a longer run-in period of more than 3 weeks can
bring about a lower placebo response [41]. In addition, a
long duration of the study period more than 3–6 months
can lessen the placebo effect based on the integrated plots
made from clinical studies [42]. According to the 27
randomized controlled trials with IBS patients, placebo
response maximally increased during 6–8 weeks and
diminished after 6 months [42]. Taken together, a suffi-
cient run-in period and long duration of study might have
brought a better result in the dual-coating group compared
to the non-coating group.
In summary, the current study is a randomized, double-
blinded study using a multispecies probiotic supplement
(Duolac Care) in diarrhea-predominant IBS patients. Our
hypothesis that dual-coated probiotics can exert a benefi-
cial effect in IBS-related immunological markers and gut
microbiota failed to show a significant effect, which may
be due to a strong placebo effect of non-coated probiotics.
Since various clinical studies using probiotic supplements
had shown ameliorating effect in IBS, it is relatively well
recognized that even probiotics without dual-coating
J Gastroenterol
123
technology can improve symptoms of IBS patients [22,43,
44]. However, to some extent, dual-coated probiotic sup-
plements showed a satisfactory effect on improvement of
the stool morphology and IBS symptoms including bloat-
ing, urgency and mucus in the stool of diarrhea-predomi-
nant IBS patients. This may be due to a shift of beneficial
intestinal microbiota, as our correlation analysis showed
significant negative correlation between Bifidobacterium
and urgency of defecation.
Acknowledgments This study was supported financially by Cell
Biotech.
Compliance with ethical standards
Conflict of interest H Kim received funds from CellBiotech. JG Seo
is an employee of CellBiotech, which does not affect the result or
interpretation of the study. Other authors do not have any conflict of
interest.
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