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Beneficial effects of Lactobacillus casei strain Shirota on academic stress-induced sleep disturbance in healthy adults: A double-blind, randomised, placebo-controlled trial

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The present study examined whether Lactobacillus casei strain Shirota (LcS) improves sleep quality under psychological stress. A double-blind, placebo-controlled trial was conducted in healthy 4th year medical students exposed to academic examination stress. The trial was repeated over two consecutive years in different groups of students, and the data were pooled. For 8 weeks prior to and 3 weeks after a national standardised examination, a total of 48 and 46 subjects received a daily dose of 100 ml of LcS-fermented milk or non-fermented placebo milk, respectively. Study measures included subjective anxiety, overnight single-channel electroencephalography (EEG) recordings, and the Oguri-Shirakawa-Azumi (OSA) sleep inventory scores of subjective sleep quality. Total OSA scores were significantly lower than baseline on the day before the exam and recovered after the exam, indicating a stress-induced decline in sleep quality. There was a significant positive effect of LcS treatment on OSA factors for sleepiness on rising and sleep length. Sleep latency measured by EEG lengthened as the exam approached in the placebo group but was significantly suppressed in the LcS group. The percentage of stage 3 non-REM (N3) sleep decreased in the placebo group as the exam approached, whereas it was maintained in the LcS group throughout the trial. Delta power during the first sleep cycle, measured as an index of sleep intensity, increased as the exam approached in the LcS group and was significantly higher than in the placebo group. These findings suggest that daily consumption of LcS may help to maintain sleep quality during a period of increasing stress. The observed retention of N3 sleep and increased delta power in the LcS group may have contributed to higher perceived sleep satisfaction.
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Beneficial Microbes, 2017; 8(2): 153-162 Wageningen Academic
Publishers
ISSN 1876-2883 print, ISSN 1876-2891 online, DOI 10.3920/BM2016.0150 153
1. Introduction
Accumulating evidence indicates that intestinal
microorganisms influence the brain function and
psychological state of the host through what has come to
be known as the gut-brain-microbiota axis (Bested et al.,
2013; Cryan and Dinan, 2012; Schmidt, 2015). Based on this
concept, probiotics such as lactobacilli and bifidobacteria
have been investigated for their mental health benefits.
Animal studies have shown that probiotics interact with
the neural and endocrine systems to reduce hypothalamus-
pituitary-adrenal (HPA) reactivity to stress or to improve
anxiety- and depression-related behaviours under stressful
conditions (Ait-Belgnaoui et al., 2014; Bravo et al., 2011;
Sudo et al., 2004).
Exposure to psychological stress causes various symptoms
related to mental distress, one of which is sleep disturbance.
Psychological stress perceived by the brain triggers the
release of corticotropin-releasing factor (CRF) from the
hypothalamic paraventricular nucleus, activating both the
HPA axis and the sympathetic nervous system. Studies
Beneficial effects of Lactobacillus casei strain Shirota on academic stress-induced
sleep disturbance in healthy adults: a double-blind, randomised, placebo-controlled trial
M. Takada
1#*
, K. Nishida
2#
, Y. Gondo
1
, H. Kikuchi-Hayakawa
1
, H. Ishikawa
1
, K. Suda
1
, M. Kawai
1
, R. Hoshi
3
,
Y. Kuwano2, K. Miyazaki1 and K. Rokutan2
1Yakult Central Institute, 5-11 Izumi, Kunitachi, Tokyo 186-8650, Japan; 2Department of Pathophysiology, Institute of Biomedical
Sciences, Tokushima University Graduate School, 3-18-15 Kuramoto, Tokushima, Tokushima 770-8503, Japan;
3
Faculty of Research
and Development, Yakult Honsha Co., Ltd., 1-1-19 Higashi-Shimbashi, Minato, Tokyo 105-8660, Japan; mai-takada@yakult.co.jp;
#These authors contributed equally to this work
Received: 21 August 2016 / Accepted: 16 December 2016
© 2017 Wageningen Academic Publishers
RESEARCH ARTICLE
Abstract
The present study examined whether Lactobacillus casei strain Shirota (LcS) improves sleep quality under psychological
stress. A double-blind, placebo-controlled trial was conducted in healthy 4
th
year medical students exposed to
academic examination stress. The trial was repeated over two consecutive years in different groups of students,
and the data were pooled. For 8 weeks prior to and 3 weeks after a national standardised examination, a total of 48
and 46 subjects received a daily dose of 100 ml of LcS-fermented milk or non-fermented placebo milk, respectively.
Study measures included subjective anxiety, overnight single-channel electroencephalography (EEG) recordings,
and the Oguri-Shirakawa-Azumi (OSA) sleep inventory scores of subjective sleep quality. Total OSA scores were
significantly lower than baseline on the day before the exam and recovered after the exam, indicating a stress-induced
decline in sleep quality. There was a significant positive effect of LcS treatment on OSA factors for sleepiness on
rising and sleep length. Sleep latency measured by EEG lengthened as the exam approached in the placebo group
but was significantly suppressed in the LcS group. The percentage of stage 3 non-REM (N3) sleep decreased in the
placebo group as the exam approached, whereas it was maintained in the LcS group throughout the trial. Delta
power during the first sleep cycle, measured as an index of sleep intensity, increased as the exam approached in the
LcS group and was significantly higher than in the placebo group. These findings suggest that daily consumption
of LcS may help to maintain sleep quality during a period of increasing stress. The observed retention of N3 sleep
and increased delta power in the LcS group may have contributed to higher perceived sleep satisfaction.
Keywords: probiotics, psychological stress, sleep quality
OPEN ACCESS
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M. Takada et al.
154 Beneficial Microbes 8(2)
have shown that the CRF-initiated stress responses can
contribute to a decline in sleep quality (Ehlers et al., 1997;
Opp, 1995).
Previous studies have suggested that heat-killed
Lactobacillus brevis amplifies diurnal sleep rhythms in mice
(Miyazaki et al., 2014) and that Lactobacillus helveticus-
fermented milk improves sleep in healthy elderly subjects,
as evaluated by both wrist actigraphy and questionnaires
(Yamamura et al., 2009). Meanwhile, in recent years, we
have been working with an academic stress model to
investigate the beneficial effects of Lactobacillus casei strain
Shirota (LcS) on stress responsiveness in healthy medical
students. Our results have shown that daily consumption of
LcS prevents the onset of abdominal dysfunctions and cold
symptoms in healthy subjects exposed to academic stress,
attenuates a stress-induced rise in salivary cortisol (Kato-
Kataoka et al., 2016a; Takada et al., 2016), and preserves
the diversity of gut microbiota (Kato-Kataoka et al., 2016b).
However, we did not look closely into sleep quality in this
model, and our only previous assessment of sleep using a
questionnaire revealed neither time-dependent changes nor
intergroup differences in chronic sleep-related parameters
(Kato-Kataoka et al., 2016a). Still, tension and stress are
major predictors of poor sleep quality in college students
(Lund et al., 2010). Because the stress-relieving effects of
LcS have been shown previously, we hypothesised that
LcS would also improve sleep during stressful situations.
Therefore, we conducted a double-blind, randomised,
placebo-controlled trial to examine in more detail whether
LcS reverses the stress-induced decline in sleep quality in
our academic stress model. Acute changes in sleep quality
were assessed using subjective questionnaires together with
electroencephalographic (EEG) recordings, which provide
the most reliable and direct measure of sleep architecture.
2. Materials and methods
Test beverages
L. casei strain Shirota YIT 9029 was obtained from the
Culture Collection Research Laboratory of Yakult Central
Institute, Tokyo, Japan. Milk fermented with LcS (1.0×10
9
cfu/ml) and placebo milk not containing LcS were used as
test beverages. Lactic acid was added to the placebo milk
to match the flavour with the LcS-fermented milk. The
placebo and the fermented milk had the same appearance,
flavour, pH, and nutritional content. Test beverages were
distributed and stored below 10°C.
Subjects
Subjects were recruited from healthy 4
th
-grade medical
students at Tokushima University, Japan, who were
preparing for an authorised national qualification
(computer-based) examination for promotion to the next
grade. Students were excluded if they (1) were over 30 years
old; (2) had been diagnosed with physical or mental illness;
(3) were on medication; (4) smoked; or (5) had a milk or
other food allergy. During the trial, subjects were instructed
to avoid all other probiotic and prebiotic products, including
yoghurt and lactic acid bacteria beverages. The trial was
conducted in accordance with the guidelines laid down in
the Declaration of Helsinki and all procedures involving
human subjects were approved by the Institutional Review
Board of Tokushima University Hospital, Tokushima,
Japan. Written informed consent was obtained from all
participants prior to enrolment.
Experimental design
A double-blind, placebo-controlled, parallel-group trial
was conducted during the fall/winter semester. Based on
previously published studies demonstrating positive effects
of certain amino acids on sleep quality in healthy subjects
(Ito et al., 2014; Miyake et al., 2014), we estimated that
around 50 subjects per treatment group would be necessary
to draw firm conclusions. Since the number of subjects
possible to recruit in one trial was limited, the trial was
repeated over two consecutive years in different groups of
students and the data were pooled. The study protocol was
registered with the University Hospital Medical Information
Network (UMIN 000015295, UMIN 000019116).
Both trials consisted of a screening period followed
by a 2-week pre-intervention period and an 11-week
intervention period. The national examination took place
at the end of the 8th week of the intervention. Subjects
were randomly divided into two groups with balanced
distribution of the following parameters evaluated during
the screening period: age, sex, body mass index, health
habits assessed by the Health Practice Index (Morimoto,
2000), psychological well-being assessed by the General
Health Questionnaire (GHQ) score (Goldberg and Hillier,
1979), personality traits assessed by the NEO-Five Factor
Inventory score (NEO-FFI) (Costa and McCrae, 1989),
anxiety levels assessed by the State-Trait Anxiety Index
(STAI) (Nakazato and Mizuguchi, 1982; Spielberger et
al., 1970), recent sleep quality assessed by the Pittsburgh
Sleep Quality Index (PSQI) (Buysse et al., 1989), and sleep
efficiency determined by EEG measurement as described
below. SAS Preclinical Package version 5.0 (SAS Institute
Japan, Tokyo, Japan) was used for complete randomisation
with balancing variables. The two groups were assigned
by a team member not involved with data collection or
analysis to ingest either 100 ml of the LcS-fermented milk
or placebo milk once a day throughout the intervention
period. Treatment was concealed from the other team
members until the set of subjects to be included in the
analysis was fixed. The study schedule is shown in Figure 1.
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Lactobacillus casei Shirota prevents stress-induced sleep disturbance
Beneficial Microbes 8(2) 155
STAI state anxiety level was evaluated at screening, at 8
weeks before the exam (baseline), at 2 weeks before the exam
(6 weeks into the intervention), on the day before the exam,
immediately after the exam on the same day, and at 2 weeks
after the exam. Sleep EEG data were recorded for three
consecutive nights during the screening period, during the
second week of the pre-intervention period (baseline) and
during the 6th, 8th, 9th, and 11th weeks of the intervention
period. On the morning after EEG recording, sleep quality
was assessed using the Oguri-Shirakawa-Azumi (OSA)
sleep inventory (MA version) (Yamamoto et al., 1999). The
OSA sleep inventory consists of 16 items measured by a
four-point rating scale. These items were classified into
five factors of sleep quality: sleepiness on rising, initiation
and maintenance of sleep, dreaming, recovery from fatigue,
and sleep length. Scores were expressed as corrected (Zc)
scores, with higher scores indicating better sleep quality.
OSA scores from three consecutive mornings were averaged
to analyse the changes in sleep quality in subjects exposed
to examination stress.
Measurement and analysis of sleep EEG
All subjects underwent overnight EEG monitoring
using a single-channel EEG (SleepScope™; SleepWell
Co., Osaka, Japan) that has been used in a number of
previous sleep studies (Fujiwara et al., 2015; Yoda et al.,
2015). The instrument is a palm-sized, single-channel,
portable electroencephalograph with two self-adhesive
electrodes, one to be placed on the forehead and another to
be placed behind the ear. On the week of EEG evaluation,
subjects wore the SleepScope on Tuesday, Wednesday, and
Thursday nights during their time in bed. Data collected
on Wednesdays were used for analysis, unless the EEG
measurement was not successful on the Wednesday, in
which case data from Thursday were used. Extraction
of information from the raw EEG data was entrusted to
SleepWell Co., Ltd. The following indicators of sleep quality
were obtained for each subject: sleep latency, total sleep
time, sleep efficiency, percentage of wake after sleep onset
(WASO), percentage of stage 3 non-REM (N3) sleep, and
delta power during the first sleep cycle. Scoring of sleep
stages was performed according to the AASM 2007 manual
(Iber et al., 2007), in which each 30 s epoch of recording
was classified into five stages: wakefulness, REM sleep, and
non-REM sleep stages N1, N2, and N3. Sleep stage was
labelled as N3 when 20% or more of an epoch consisted
of waves with a frequency of 0.5-2.0 Hz and peak-to-peak
amplitude of >75 μV. Onset of sleep was defined by the
first occurrence of 5 min of continuous non-REM sleep.
Total sleep time was calculated as the total time spent in
REM and non-REM sleep. WASO was calculated as the
total time scored as wakefulness between the initial sleep
onset and final sleep offset. Sleep efficiency was given as
the percentage of total sleep time out of the total time in
bed. As a marker of deep sleep, spectral power in the delta
frequency range (0.5-2.0 Hz) was determined. Because sleep
time and number of sleep cycles vary among individuals,
delta power during only the first sleep cycle was compared.
Statistics
All data are shown as means ± standard error of the
mean. All statistical analyses were conducted using the
SAS software (SAS Institute, Cary, NC, USA). P-values
of less than 0.05 were considered statistically significant.
Background characteristics at screening were analysed by
Student’s t-test between groups. STAI scores and OSA total
scores were analysed by Dunnett’s multiple comparison
test for within-group comparisons and Bonferroni adjusted
t-test for between-group comparisons. OSA sub-scores
were analysed for significant effects by repeated measures
ANCOVA with group and time point as factors and
baseline as a covariate, followed by Bonferroni adjusted
t-test between groups at each time point using changes in
scores from baseline. Changes in EEG data were analysed
for significant effects using two-way ANOVA followed by
Bonferroni adjusted t-test. Sleep efficiency of 70% or less,
sleep latency of 90 min or longer, REM sleep latency of 30
min or less, and delta power when REM sleep latency was
30 min or less were excluded from the analysis because such
deviation from the normal range, based on sleep medicine,
suggested that EEG data were not measured accurately or
were not representative of the subjects’ usual sleep pattern.
Allocation
2 weeks before
exam
Intervention period: 11 weeks
9-11 weeks1-8 weeks
1 day
before
exam
Immediately
after exam
2 weeks
after
exam
8 weeks before
exam (baseline)
Screening
period
Pre-intervention
period: 2 weeks
Academic
examination
Figure 1. Study schedule. The measurement taken immediately after the exam was on the same day as the exam.
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M. Takada et al.
156 Beneficial Microbes 8(2)
3. Results
Subjects and compliance
Of the 124 participants who consented to participate in the
study (61 in the first year and 63 in the next year), 25 subjects
were excluded for meeting at least one of the five exclusion
criteria listed in the Materials and Methods section, and 1
subject was excluded for obtaining exceedingly high STAI
trait anxiety, NEO-FFI neuroticism, and GHQ scores. The
remaining subjects were randomly allocated to either the
LcS or placebo group. One subject dropped out before
starting the intervention, and three subjects were excluded
from the analysis for having a major life event (death of a
close relative) during the study, for ingesting a probiotic
formulation other than the provided test beverage, or for
not answering the questionnaires properly. Ultimately, 94
subjects, 48 in the LcS group and 46 in the placebo group,
were included in the data analysis. Participant flow diagram
is shown in Figure 2.
Before pooling data from the two trials, background
characteristics of the participants of the first and the second
year were compared by Student’s t-test (data not shown).
All parameters were similar between the two trials except
for the ‘openness’ score of NEO-FFI (P=0.014), which was
considered to be not an obstacle to pooling data since
openness is a personality trait and not an indicator of
health status. Also, STAI scores and OSA total scores on
the day before the academic exam were similar between the
placebo groups of the first and the second year compared
by Student’s t-test (data not shown), indicating that the
subjects responded similarly to academic stress in both
years.
The combined background characteristics of the 94 eligible
subjects from the two trials are shown in Table 1. These
parameters assessed at screening did not differ between
treatment groups. Self-reported compliance with the test
beverages was high, with a mean compliance rate of 97.0% in
the placebo group and 97.5% in the LcS group. Blinding was
confirmed at the end of the study by asking the participants
to guess which treatment they received. No harmful events
related to test beverage intake were reported.
Changes in stress parameters
The STAI state anxiety scores of both groups increased
significantly 2 weeks before the exam (6 weeks into the
intervention, both P<0.01 versus baseline) and the day
before the exam (both P<0.01 versus baseline; Figure 3).
The observed elevation in STAI state scores supports the
premise that students display maximum stress responses
on the day before an examination, demonstrating that
academic stress was indeed imposed on the subjects in
the present model.
Assessed for eligibility (n=124)
Dropped out (n=1)
Excluded
- Not meeting inclusion criteria (n=25)
- Other reasons (n=1)
Randomised (n=98)
Allocated to placebo
group (n=48)
Received allocated
intervention (n=48)
Analysed (n=46)
Excluded
- Major life event (n=1)
- Not answering questionnaires
properly (n=1)
Allocated to LcS
group (n=50)
Received allocated
intervention (n=49)
Analysed (n=48)
Excluded
- Ingesting other
probiotics (n=1)
Figure 2. Participant flow, showing combined number of subjects across two trials.
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Lactobacillus casei Shirota prevents stress-induced sleep disturbance
Beneficial Microbes 8(2) 157
Effects of Lactobacillus casei Shirota on sleep scores
As shown in Figure 4A, sleep quality estimated by total
OSA scores was gradually impaired as the exam approached
in both groups. The score significantly decreased on the
day before the exam (P<0.01 versus pre-intervention) and
Table 1. Subjects’ backgrounds at screening.1,2
Placebo Lactobacillus casei
Shirota
Male/female 28/18 27/21
Age (yr) 22.6±0.2 22.8±0.2
Body mass index (kg/m2) 20.5±0.3 20.7±0.3
GHQ 4.4±0.5 4.7±0.5
Health Practice Index 4.9±0.2 5.0±0.2
NEO-FFI
Neuroticism 24.2±1.2 25.4±1.3
Extraversion 24.9±1.0 25.2±0.9
Openness 28.3±0.9 29.4±1.0
Agreeableness 29.7±0.8 31.6±0.7
Conscientiousness 27.0±1.0 25.5±0.9
STAI trait anxiety 45.1±1.4 45.1±1.4
STAI state anxiety 39.1±1.3 38.6±1.2
PSQI 4.6±0.3 4.8±0.4
Sleep efficiency (%) 91.4±0.9 91.4±0.8
1
GHQ = General Health Questionnaire; NEO-FFI = NEO-Five Factor
Inventory; STAI = state-trait anxiety inventory; PSQI = Pittsburgh Sleep
Quality Index.
2
Values are means ± standard error of the mean. No significant differences
were observed between groups (t-test).
**
**
**
**
*
20
25
30
35
40
45
50
55
60
Placebo LcS
STAI state anxiety score
2 weeks after
exam
Immediately
after exam
1 day before
exam
2 weeks
before exam
Before
intervention
(baseline)
Figure 3. STAI state anxiety scores before and during the
intervention. Mean values with standard error of the mean;
* P<0.05, ** P<0.01 versus baseline (Dunnett’s test).
A
B
C
**
**
60
70
80
90
100
110
120
Pre 6 8 9 11
Weeks of intervention
Placebo LcS
**
*
60
70
80
90
100
110
120
Pre 6 8 9 11
Weeks of intervention
*
*
**
60
70
80
90
100
110
120
Pre 6 8 9 11
Weeks of intervention
OSA sleep inventory
total score (pooled)
OSA sleep inventory
total score (trial 1)
OSA sleep inventory
total score (trial 2)
Figure 4. Total scores of the OSA sleep inventory, showing (A)
pooled data of the two trials and individual data of the (B) first
trial and (C) second trial. The black arrow indicates the time
of the academic exam. Mean values with standard error of the
mean; * P<0.05, ** P<0.01 versus baseline (Dunnett’s test).
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M. Takada et al.
158 Beneficial Microbes 8(2)
recovered to baseline after the exam (Figure 4A). This
stress-induced sleep disturbance was reproducible even
when the data of the trials were analysed individually
(Figures 4B and C). No difference was observed between
the placebo and LcS groups regarding total OSA scores.
We subsequently compared sleep quality between the two
groups in more detail using the five subdivided factors of
OSA (sleepiness on rising, initiation and maintenance of
sleep, dreaming, recovery from fatigue, and sleep length).
The LcS group showed higher (better) overall scores of
sleepiness on rising (Figure 5A) and sleep length (Figure 5E).
Thus, daily intake of LcS significantly relieved sleepiness on
rising (P<0.05 versus placebo; Figure 5A) and significantly
increased sleep length (P<0.01 versus placebo; Figure 5E).
#
-6
-4
-2
0
2
4
6 BA
C
E
D
Pre 6 8 9 11
Weeks of intervention
Placebo LcS
-6
-4
-2
0
2
4
6
Pre 6 8 9 11
Weeks of intervention
-6
-4
-2
0
2
4
6
Pre 6 8 9 11
Weeks of intervention
-6
-4
-2
0
2
4
6
Pre 6 8 9 11
Weeks of intervention
-6
-4
-2
0
2
4
6
Pre 6 8 9 11
Weeks of intervention
ΔOSA sleep inventory
Factor V: sleep length
ΔOSA sleep inventory
Factor III: dreaming
ΔOSA sleep inventory
Factor I: sleepiness on rising
ΔOSA sleep inventory
Factor IV: recovery from fatigue
ΔOSA sleep inventory
Factor II: initiation and
maintenance of sleep
Figure 5. Changes in the scores of the five factors of OSA: (A) sleepiness on rising, (B) initiation and maintenance of sleep, (C)
dreaming, (D) recovery from fatigue, and (E) sleep length. The black arrow indicates the time of the academic exam. Mean values
with standard error of the mean; P<0.05, P<0.01, significant effect of interaction (repeated measures ANCOVA); # P<0.05 versus
placebo (Bonferroni adjusted t-test).
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Lactobacillus casei Shirota prevents stress-induced sleep disturbance
Beneficial Microbes 8(2) 159
The recovery in sleepiness on rising after the examination
was particularly remarkable; the score measured at week 9
of the intervention was significantly higher in the LcS group
than in the placebo group (P<0.05 versus placebo; Figure
5A). Initiation and maintenance of sleep, dreaming, and
recovery from fatigue were not affected by LcS treatment.
Effects of Lactobacillus casei Shirota on sleep EEG
In addition to the OSA questionnaire, EEG monitoring was
used to subjectively and physiologically assess the effect of
LcS on stress-induced sleep disturbance. Changes in sleep
parameters derived from sleep EEG are shown in Figure 6.
Sleep latency tended to increase as the exam approached
in the placebo group, whereas daily LcS intake significantly
suppressed the prolongation of sleep latency (Figure 6A).
Changes in total sleep time (Figure 6B) and sleep efficacy
(Figure 6C) were similar between the two groups. Although
WASO was relatively constant in both groups during the
intervention period, daily intake of LcS significantly affected
the percentage changes in WASO (P<0.05 versus placebo;
Figure 6D). In contrast, LcS intake completely prevented
the reduction in N3 sleep, and this effect was significant
when compared with the placebo group (P<0.01 versus
placebo; Figure 6E). There was a significant difference in
N3 sleep between groups at week 9 of the intervention
(P<0.05 versus placebo; Figure 6E). Moreover, it should
be noted that daily LcS intake increased delta power more
than 20% as the exam approached (Figure 6F). This effect
was significant (P<0.05) and considerable when compared
with the placebo group, whose delta power was unchanged
during the intervention (Figure 6F).
4. Discussion and conclusions
The present double-blind, randomised, placebo-controlled
trial examined whether an LcS-fermented milk beverage
improves the sleep quality of healthy medical students
exposed to academic stress. Consistent with previous
reports (Kamezaki et al., 2012; Kato-Kataoka et al., 2016a),
STAI anxiety scores increased as the exam approached
and recovered after the exam, supporting the suitability
of the present model for assessing the effects of LcS on
academic stress.
Total OSA scores in both groups decreased significantly
from baseline as the exam approached and recovered after
the exam, showing an inverse pattern to that of stress
parameters, which suggested diminished subjective sleep
quality due to academic stress. Among the five different
factors of the inventory, sleepiness on rising, recovery from
fatigue, and sleep length tended to be negatively affected by
stress, showing a dip at week 8 of the intervention. Lower
scores in these factors reflect feeling sleepy or tired on
awakening, indicating that the subjects tended to experience
relatively unsatisfactory sleep near the exam day. The OSA
sleep inventory has been developed to evaluate sleep quality
in healthy subjects, and data from healthy Japanese adults
are accumulating (Kobayashi et al., 1999; Miyake et al.,
2014). In comparison to the PSQI score, a subjective
measure of chronic sleep disturbance that was previously
shown to be unaffected by academic stress (Kato-Kataoka et
al., 2016a), the OSA sleep inventory had greater sensitivity,
reflecting acute changes in sleep quality caused by academic
stress. Thus, OSA was considered to be better suited than
PSQI for the evaluation of sleep quality in healthy medical
students exposed to examination stress.
The total OSA scores of both groups during the pre-
examination period were lower than 99.4, the previously
reported average total OSA score of 284 healthy Japanese
adults (Yamamoto et al., 1999). However, the score of the
LcS group recovered to a level above the standard average
at week 9 of the intervention. LcS treatment also had a
significant effect on sleepiness on rising and sleep length,
with overall scores higher compared with the placebo
group. These findings suggest that LcS prevented the
stress-induced decline in perceived sleep quality and also
enhanced its recovery just after the exam. In support of
these questionnaire results, analysis of the EEG further
revealed that LcS treatment was associated with a higher
percentage of N3 sleep and higher delta power during
the first sleep cycle compared with the placebo group.
There was a higher percentage of N3 sleep at week 9 of the
intervention in the LcS group compared with the placebo,
supporting the quick recovery in the OSA results observed
in the LcS group immediately after the exam. N3, also
known as slow-wave sleep, represents the deepest sleep
stage of non-REM sleep, whereas delta power is an index
of sleep intensity that is associated with deep slow-wave
sleep. Kim and Dimsdale (2007) reported in a systematic
review that slow-wave sleep is often reduced by both daily
life stressors and experimental psychological stressors.
Consistent with this report, the placebo group in our
present study tended to show a decreased percentage of
N3 sleep as the exam approached. In contrast, N3 sleep was
not impaired in the LcS group throughout the intervention
period. Delta power was also higher in the LcS group,
implying that the LcS group spent more time in deep non-
REM sleep. It has been reported that subjectively good
sleep is related to sleep efficiency and amount of slow-wave
sleep (Keklund and Akerstedt, 1997), so retention of N3
sleep and an increased delta power in the LcS group may
have contributed to a higher perceived sleep satisfaction
in the OSA questionnaire. Interestingly, there was an
inconsistency between the subjective and objective lengths
of sleep. The change in the OSA factor for sleep length
revealed a significant effect of LcS, with the LcS group
scoring higher than the placebo group, whereas no significant
effect of LcS was observed for the change in total sleep time
from baseline as measured by EEG. This suggests that the
http://www.wageningenacademic.com/doi/pdf/10.3920/BM2016.0150 - Friday, April 28, 2017 6:18:07 PM - IP Address:27.140.136.17
M. Takada et al.
160 Beneficial Microbes 8(2)
observed positive effects of LcS on sleep relied on depth of
sleep rather than the actual amount of sleep.
Sleep latency tended to lengthen as the exam approached
in the placebo group, in agreement with a previous study
reporting that medical students under academic stress
undergo longer sleep latencies and worse subjective sleep
quality than in the non-examination period (Jernelov et
al., 2009). The increased sleep latency was suppressed in
the LcS group just before and after the exam, indicating a
#
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-10
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35
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-2.0
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BA
C
E
D
Weeks of intervention Weeks of intervention
Weeks of intervention Weeks of intervention
Weeks of intervention Weeks of intervention
Δ N3 sleep (%) Δ sleep efficiency (%) Δ sleep latency (min)
Δ wake after sleep onset (%)
F
Δ delta power during
first sleep cycle (%)
Δ total sleep time (min)
Figure 6. Changes in EEG-derived sleep parameters: (A) sleep latency, (B) total sleep time, (C) sleep efficiency, (D) percentage of
wake after sleep onset, (E) percentage of N3 sleep, and (F) percent change in delta power during the first sleep cycle. The black
arrow indicates the time of the academic exam. Mean values with standard error of the mean; P<0.05, P<0.01, significant effect
of interaction (two-way ANOVA); # P <0.05 versus placebo (Bonferroni adjusted t-test).
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Lactobacillus casei Shirota prevents stress-induced sleep disturbance
Beneficial Microbes 8(2) 161
potentially positive effect of LcS on sleep initiation. On the
other hand, the change in WASO from baseline was higher
in the LcS group than in the placebo group. However, the
percentage changes in WASO were <1%, which may not
significantly affect sleep quality.
At present, there is not enough evidence to explain how
LcS improved sleep. Nonetheless, it is widely accepted
that stress-induced activation of the HPA axis and/or the
sympathetic nervous system negatively impact sleep, with
evidence that CRF-initiated stress responses contribute
to a decline in sleep quality (Ehlers et al., 1997; Opp,
1995) and that poor sleep is correlated with exaggerated
cortisol reactivity (Hori et al., 2011). LcS has previously
been reported to suppress the stress-induced increase in
glucocorticoids in both a human academic stress model
and rat water-avoidance stress model (Takada et al., 2016).
LcS also suppresses CRF-induced sympathetic activation
in rats (Tanida et al., 2016). These observations led us to
speculate that LcS improves sleep by suppressing stress-
induced HPA and/or sympathetic activation.
In conclusion, the present findings suggest that daily
consumption of LcS may help to maintain the perceived
quality of sleep during a period of increasing stress by
preventing a decrease in the percentage of N3 sleep and
increasing the delta power. As far as we know, this is
the first report to clearly show the beneficial effects of
probiotics on sleep using both subjective questionnaires
and objective EEG measures. Because of the complexity
of the interactions between probiotics and the host, the
complete mechanisms of action of LcS remain unknown.
Further studies are needed to clarify the mechanisms
underlying the effects of LcS in maintaining sleep quality
under examination stress conditions.
Acknowledgements
We thank Akito Kato-Kataoka for his previous work on
the subject, which led to our present study. We also thank
Osamu Watanabe and Tomoki Igarashi for supporting the
collaborative research and Kaori Kashiwagi and Masaki
Yoshida for extracting information from the raw EEG data.
Conflict of interest
Clinical trials were performed based on a collaboration
between Yakult Central Institute of Yakult Honsha Co.,
Ltd and Tokushima University with the sponsorship and
provision of test beverages from Yakult Honsha Co., Ltd.
The sponsor provided support in the form of salaries for
authors MT, YG, HKH, HI, KS, MK, RH, and KM, but did
not have any additional role in study design, data collection
and interpretation, decision to publish, or manuscript
preparation. All remaining authors declare no potential
conflicts of interest with respect to the authorship and
publication of this article.
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... Takada and colleagues measured sleep quality in a group of healthy students who received Lacticaseibacillus casei Shirota (Table 1) (Takada et al., 2017). The students were subjected to stress as they were all studying for national standardised examination. ...
... Sleep latency prolonged in both groups when the national standardised examination was approaching, but sleep latency prolongation was less in the intervention group. EEG measurements also showed that, as the examination approached, the time spent in NREM stage 3 (NREM3) sleep was reduced in the placebo group but maintained in the intervention group (Table 1) (Takada et al., 2017). A study by researchers at Tokushima University did not find changes in sleep quality after the consumption of L. casei Shirota ( (Buysse et al., 1989). ...
... As described previously in this review, L. casei has been associated with improved sleep quality by Takada and colleagues. Their trial did, however, not find any differences in anxiety test scores between the intervention and control group (Table 2) (Takada et al., 2017). Yet, another trial, also in students who were subjected to academic stress, demonstrated that L. casei significantly reduced salivary cortisol levels in comparison with the control group (Table 2) (Kato-Kataoka et al., 2016). ...
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This pilot study investigated the effects of the probiotic Lactobacillus casei strain Shirota (LcS) on psychological, physiological, and physical stress responses in medical students undertaking an authorised nationwide examination for promotion. In a double-blind, placebo-controlled trial, 24 and 23 healthy medical students consumed a fermented milk containing LcS and a placebo milk, respectively, once a day for 8 weeks until the day before the examination. Psychophysical state, salivary cortisol, faecal serotonin, and plasma L-tryptophan were analysed on 5 different sampling days (8 weeks before, 2 weeks before, 1 day before, immediately after, and 2 weeks after the examination). Physical symptoms were also recorded in a diary by subjects during the intervention period for 8 weeks. In association with a significant elevation of anxiety at 1 day before the examination, salivary cortisol and plasma L-tryptophan levels were significantly increased in only the placebo group (P<0.05). Two weeks after the examination, the LcS group had significantly higher faecal serotonin levels (P<0.05) than the placebo group. Moreover, the rate of subjects experiencing common abdominal and cold symptoms and total number of days experiencing these physical symptoms per subject were significantly lower in the LcS group than in the placebo group during the pre-examination period at 5-6 weeks (each P<0.05) and 7-8 weeks (each P<0.01) during the intervention period. Our results suggest that the daily consumption of fermented milk containing LcS may exert beneficial effects preventing the onset of physical symptoms in healthy subjects exposed to stressful situations.
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Objective: Poor sleep quality is an independent predictor of cardiovascular events. However, little is known about the association between glycemic control and objective sleep architecture and its influence on arteriosclerosis in patients with type-2 diabetes mellitus (DM). The present study examined the association of objective sleep architecture with both glycemic control and arteriosclerosis in type-2 DM patients. Design: Cross-sectional study in vascular laboratory. Methods: The subjects were 63 type-2 DM inpatients (M/F, 32/31; age, 57.5±13.1) without taking any sleeping promoting drug and chronic kidney disease. We examined objective sleep architecture by single-channel electroencephalography and arteriosclerosis by carotid-artery intima-media thickness (CA-IMT). Results: HbA1c was associated significantly in a negative manner with REM sleep latency (interval between sleep-onset and the first REM period) (β=-0.280, p=0.033), but not with other measurements of sleep quality. REM sleep latency associated significantly in a positive manner with log delta power (the marker of deep sleep) during that period (β=0.544, p=0.001). In the model including variables univariately correlated with CA-IMT (REM sleep latency, age, DM duration, systolic blood pressure, and HbA1c) as independent variables, REM sleep latency (β=-0.232, p=0.038), but not HbA1c were significantly associated with CA-IMT. When log delta power was included in place of REM sleep latency, log delta power (β=-0.257, p=0.023) emerged as a significant factor associated with CA-IMT. Conclusions: In type-2 DM patients, poor glycemic control was independently associated with poor quality of sleep as represented by decrease of REM sleep latency which might be responsible for increased CA-IMT, a relevant marker for arterial wall thickening.
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