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Bifidobacterium longum 1714™Strain Modulates Brain
Activity of Healthy Volunteers During Social Stress
Huiying Wang, PhD
1
,
2
,
3
, Christoph Braun, PhD
2
,
4
, Eileen F. Murphy, PhD
5
and Paul Enck, PhD
1
OBJECTIVES: Accumulating evidence indicates that the gut microbiota communicates with the central nervous
system, possibly through neural, endocrine, and immune pathways, and influences brain function.
B. longum 1714™has previously been shown to attenuate cortisol output and stress responses in
healthy subjects exposed to an acute stressor. However, the ability of B. longum 1714™to modulate
brain function in humans is unclear.
METHODS: In a randomized, double-blinded, placebo-controlled trial, the effects of B. longum 1714™on neural
responses to social stress, induced by the “Cyberball game,”a standardized social stress paradigm,
were studied. Forty healthy volunteers received either B. longum 1714™or placebo for 4 weeks at
a dose of 1 310
9
cfu/d. Brain activity was measured using magnetoencephalography and health status
using the 36-item short-form health survey.
RESULTS: B. longum 1714™altered resting-state neural oscillations, with an increase in theta band power in the
frontal and cingulate cortex (P<0.05) and a decrease in beta-3 band in the hippocampus, fusiform, and
temporal cortex (P<0.05), both of which were associated with subjective vitality changes. All groups
showed increased social stress after a 4-week intervention without an effect at behavioral level due to
small sample numbers. However, only B. longum 1714™altered neural oscillation after social stress,
with increased theta and alpha band power in the frontal and cingulate cortex (P<0.05) and
supramarginal gyrus (P<0.05).
DISCUSSION: B. longum 1714™modulated resting neural activity that correlated with enhanced vitality and reduced
mental fatigue. Furthermore, B. longum 1714™modulated neural responses during social stress,
which may be involved in the activation of brain coping centers to counter-regulate negative emotions.
SUPPLEMENTARY MATERIAL accompanies this paper at http://links.lww.com/AJG/A163
Am J Gastroenterol 2019;00:1–11. https://doi.org/10.14309/ajg.0000000000000203
INTRODUCTION
The existence of bidirectional communication between the gut
and brain has long been established. Recently, a body of work has
suggested that the commensal gut microbiota plays an important
role in modulating the gut-brain axis, possibly through immune,
endocrine, and neural system pathways (1). This has led to in-
creased interest in modulating the gut microbiota to target central
nervous system (CNS) functions and improve human behavior
especially in areas such as stress, mood, anxiety, and cognition.
Probiotics may represent a safe and effective way of targeting CNS
function. Indeed, an abundance of preclinical studies have shown
that probiotics acting through the gut-brain axis can affect brain
development, function, and behavior (2). However, the trans-
lation of promising preclinical signals into human subjects with
certain probiotic strains has proven challenging (3). Therefore,
there is a need for more detailed mechanistic insights into the
interaction between the gut microbiota and brain function in
humans.
One probiotic strain, Bifidobacterium longu m 1714™(Zenflore),
has proven effective in modulating CNS functions in animals and
humans. B. longum 1714™has been shown to reduce stress-related
behaviors in preclinical studies (4,5) and improve stress responses
and cognitive function in healthy volunteers (6). However, the
role of B. longum 1714™in modulating brain function in humans
is unclear. Clinical studies with probiotics using neuroimaging
methods have started to emerge and provide insight into mod-
ulations of CNS functions in healthy volunteers (7–9) and in
patients with irritable bowel syndrome (IBS) (10). The first study
to demonstrate neural effects of probiotics in humans used
functional MRI and showed that brain activations to emotional
1
Department of Psychosomatic Medicine and Psychotherapy, University of T ¨
ubingen, T ¨
ubingen, Germany;
2
MEG Center, University Hospital T ¨
ubingen, T ¨
ubingen,
Germany;
3
Graduate Training Center of Neuroscience, IMPRS for Cognitive and Systems Neuroscience, T ¨
ubingen, Germany;
4
CIMeC, Center for Mind/Bra in
Sciences, University of Trento, Trento, Italy;
5
Alimentary He alth Gr oup, Co rk Airport Business Park, Cork, Ireland. Correspondence: Paul Enck. E-mail:
paul.enck@uni-tuebingen.de.
Received August 12, 2018; accepted January 25, 2019; published online April 17, 2019
Copyright © 2019 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of The American College of Gastroenterology The American Journal of GASTROENTEROLOGY
ARTICLE 1
FUNCTIONAL GI DISORDERS
faces were altered by a 4-week consumption of a fermented dairy
drink in comparison to placebo in healthy subjects (7). A more
recent study in healthy volunteers showed positive correlations
between psychometric measures and brain activity (8) as well as
altered functional connectivity (9) in response to emotional
processing after a 4-week intervention with a multi-strain pro-
biotic. Of interest, treatment of patients with IBS using B. longum™
NCC3001 for 6 weeks resulted in an improvement in depressions
scores and reduction in limbic activity to negative emotional
stimuli, but had no effect on gut symptoms (10). Notably, these
previous studies used picture-viewing tasks during brain re-
cording that poorly reflect behavioral changes found in animal
models, which are commonly used to screen the effects of pro-
biotics on stress and emotions. Furthermore, both studies in
healthy volunteers used multi-strain probiotic product making it
difficult to identify the bacterial strain responsible for the ob-
served effects and the pathways involved in strain-specificeffects.
Therefore, further investigations are required using targeting
approaches and different stress paradigms.
In this study, we explored the effect of B. longum 1714™on
brain function in response to social stress in a randomized,
double-blinded, placebo-controlled trial, using the neuroimaging
method magnetoencephalography (MEG). Social stress was in-
duced by social exclusion/rejection (11) through a standardized
paradigm, called “Cyberball Game”(CBG) (12). According to
a recent review summarizing previous neuroimaging studies on
the CBG, regions of the prefrontal cortex (PFC), anterior cingu-
late cortex (ACC), and temporal cortex are involved in neural
processing of this type of social stressor (13). Indeed, social stress
during the CBG is consistently associated with altered neural
oscillations in certain brain areas, such as alpha band power in
frontalcortex and theta band powerin the ACC and insula (14–17).
In the current trial, we hypothesized that the probiotic strain
B. longum 1714™alters resting-state brain activity and neuro-
physiological responses to CBG-induced social stress. We as-
sumed that during the CBG, stress-related neural oscillations of
the theta and alpha band power would be changed after probiotic
treatment. The results confirmed that B. longum 1714™modu-
lated neural responses during social stress, suggesting counter-
regulation of negative emotions.
METHODS
Participants
Healthy adults were recruited to the study. Criteria for inclusion
were (i) man or woman aged 18–50 years; (ii) nonsmoker for at
least 3 months; (iii) a body mass index of 18–30; (iv) no chronic
allergies; (v) willing to discontinue their normal consumption
of probiotics and probiotic-containing foods or potentially
immune-enhancing dietary supplements; (vi) receiving no
immune-suppressing intervention and not having any immu-
nosuppressive illness within the past year; (vii) receiving no
antibiotic therapy within the past 2 months; (viii) having no
chronic psychiatric or gastrointestinal disorder; and (ix) having
no nonremovable metal parts in the body. Informed consent was
obtained from all participants before joining the study. Partic-
ipants were screened for IBS and psychiatric disorders using the
Rome III criteria (18) and the Patient Health Questionnaire (19).
During the intervention period, participants were instructed to
avoid consumption of food containing probiotics/prebiotics or
potentially immune-enhancing dietary supplements. This was
supported by providing them with a list of “prohibited”foods
(see Information 1, Supplementary Digital Content 1, http://
links.lww.com/AJG/A163). Demographic and baseline psycho-
logical information was also recorded. The protocol was ap-
proved by the Ethics Board of the University of T ¨
ubingen Medical
School (No. 503/2015BO1, as of August 26, 2015) and registered
at ClinicalTrials.gov (identifier No. NCT02793193).
Design
A randomized, double-blinded, parallel-group design was used.
After screening, participants were randomly allocated to the
probiotic or placebo group for 4 weeks. At baseline (visit 1) and 1
day after completion of the intervention period (visit 2), meas-
urements of resting-state MEG, MEG during CBG, and related
questionnaires to measure the level of distress were performed.
Structural MR images were acquired on a different day, regardless
of the intervention schedule. To record participants health and
quality of life status, participants completed the 36-item short-
form health survey (SF36) (20) at the beginning of each of the 2
visits. The SF36 includes 8 subscales: physical functioning, bodily
pain, role limitations due to physical health problems, role limi-
tations due to personal or emotional problems, emotional well-
being, social functioning, energy/vitality, and general health
perceptions. The randomization scheme was only unblinded after
completion of the experiment and complete data evaluation.
Materials
The probiotic and placebo preparations were provided by
Alimentary Health Group, Cork, Ireland. Participants were ran-
domized to receive sachets containing either probiotic (1 310
9
CFU of B. longum 1714™in 2 g of maltodextrin) or placebo (2 g of
maltodextrin). Probiotic and placebo were identically packaged.
Participants were instructed to consume one sachet every
morning by mixing the content into 50 mL of water. Both subjects
and investigators were blinded to the regime administered.
CBG
The CBG was used to study the effects of social stress and ex-
clusion as outlined previously by Wang et al. (21). Briefly, during
the CBG, participants were asked to play a virtual ball-tossing
game with 2 other virtual players programmed. The CBG involves
2 conditions, the so-called inclusion and exclusion. Figure 1
presents a single trial from 1 player throwing the ball to another
(Figure 1) (see Information 2, Supplementary Digital Content 1,
http://links.lww.com/AJG/A163 for more detail).
CBG questionnaires
After the CBG exclusion or inclusion periods, participants
completed 3 questionnaires to assess the level of acute distress.
We employed the self-report measures of the Need Threat Scale
(NTS), the Mood Questionnaire (MQ), and the Subjective “Ex-
clusion Perception”(SEP) (see Information 3, Supplementary
Digital Content 1, http://links.lww.com/AJG/A163); all these
scales are validated standards for the CBG (12,22). The NTS is
designed to measure the feelings and emotional consequences of
social rejection, and higher scores related to higher distress level.
Its 4 items (rated between 1 and 5 for “weak”to “strong”) com-
prised self-esteem, belonging, meaningful existence, and control,
and combined ratings have been used as a measure of social
distress in previous studies. The MQ is used to assess mood, using
8 questions (are you feeling bad, good, happy, sad, pleasant, an-
gry, friendly, and unfriendly), all rated between 1 and 5. The SEP
The American Journal of GASTROENTEROLOGY VOLUME 00 | MONTH 2019 www.amjgastro.com
FUNCTIONAL GI DISORDERS
Wang et al.2
is used to record participants’feeling of being included/ostracized
by asking them to rate 2 statements (“I was ignored”and “I was
excluded”) between 1 and 5.
MEG recording
Brain magnetic fields were measured with a 275-channel whole-
head MEG (CTF Omega, Port Coquitlam, Canada). Participants
Figure 1. Schematic outline of a trial in the Cyberball Game (CBG).
Figure 2. The CONSORT flow diagram of the clinical trial. Reprinted from Schulz et al. Copyright BMJ (48). All permission requestsfor this image should be
made to the copyright holder.
Copyright © 2019 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of The American College of Gastroenterology The American Journal of GASTROENTEROLOGY
FUNCTIONAL GI DISORDERS
Bifidobacterium longum 1714ÔStrain Modulates Brain Activity 3
were studied in sitting position. During each session, 5 minutes
resting state was recorded before playing the CBG. During the
resting state, participants were instructed to move as little as
possible and to stay alert while keeping their eyes closed. During
the CBG, task instructions were projected onto a screen in front of
the participants using a video projector and a mirror system.
MEG signals were sampled at a rate of 585.94 Hz with an anti-
aliasing filter set to 146.49 Hz.
To overlay the brain activity derived from MEG on anatomical
scans, high-resolution (1 mm, isotropic) T1-weighted structural
MR images were acquired using an MPRAGE sequence with
a Siemens MAGNETOM Trio 3T scanner (Siemens AG, Erlan-
gen, Germany) (12-channel array head coil) for each participant,
but at a separate occasion.
Data analysis
Power calculation. The primary endpoint was change in brain
activity. Based on previously published data (7), we estimated
that—with a power of 0.95 for a 2 32 repeated measure
ANOVA—a minimum sample size of 28 was required to dem-
onstrate an effect size f50.23 at a50.05 in a parallel-group
designed study (see Information 4, Supplementary Digital Con-
tent 1, http://links.lww.com/AJG/A163 for more detail).
Data analysis: MEG
Analysis of the MEG data was performed using MATLAB
(Mathworks, Natick, MA) and the open-source toolboxes Field-
Trip (23). Data from the resting state and during the CBG were
analyzed, respectively, after procedures of preprocessing (trial
segmentation and artifacts rejection)—Time-frequency analysis
(multitaper windowed fast Fourier transform)—Source analysis
(dynamic imaging of cortical sources: DICS)—Source statistics
(cluster-based permutation statistics).
Data analysis: Questionnaires
Data analysis was conducted using SPSS 21 (IBM, Armonk, NY).
To examine whether there was a significant difference in health
Table 1. Demographic and baseline information
B. longum 1714™Placebo Pvalue
Sex
Male N 57N57ns
Female N 513 N 513
Birth delivery
Caesarean section N 52N52ns
Vaginal delivery N 518 N 518
Age 31.00 62.28 33.00 62.83 0.45
BMI 23.00 60.68 22.00 60.55 0.26
SF36
Physical functioning 96.84 61.03 97.63 60.80 0.71
Role limitations due to physical health 100.00 60.00 100.00 60.00 1.00
Role limitations due to emotion problems 100.00 60.00 95.00 65.00 0.81
Energy/vitality 72.25 62.42 75.25 62.94 0.57
Emotional well-being 85.78 61.22 84.42 61.75 0.52
Social functioning 100.00 60.00 94.74 62.20 0.20
Pain 88.75 62.67 89.75 62.80 0.84
General health 82.50 62.31 87.37 62.40 0.11
CBG
NTS
Inclusion 228.30 63.55 236.90 62.28
Exclusion 14.33 64.55 26.90 63.48 P
intervention
50.59
MQ
Inclusion 13.80 61.47 18.03 60.99
Exclusion 2.38 62.25 25.48 62.17 P
intervention
50.21
SEP
Inclusion 24.68 60.28 25.30 60.21
Exclusion 0.15 60.92 2.23 60.61 P
intervention
50.15
BMI, body mass index; CBG, Cyberball Game; MQ, mood questionnaire; ns, not significant; NTS, Need Threat Scale; SEP, subjective exclusion perception; SF36, 36-item
short-form health survey.
The American Journal of GASTROENTEROLOGY VOLUME 00 | MONTH 2019 www.amjgastro.com
FUNCTIONAL GI DISORDERS
Wang et al.4
status between groups at baseline, scores of SF36 during the first
visit were entered into a nonparametric independent-sample
Mann-Whitney Utest of Intervention as between factor (B. lon-
gum 1714™vs placebo) because parametric assumptions of these
data were violated. To test the intervention-related changes in
participants’health status scored by SF36, changes from before to
after the 4-week intervention were computed by subtracting the
baseline assessment from the corresponding postintervention
values. A nonparametric two-way independent-sample Mann-
Whitney Utest was used to examine the change of SF36 between
Intervention (B. longum 1714™vs placebo).
To examine whether subjective ratings for the CBG were different
between groups at baseline, scores of NTS, MQ, and SEP acquired
during the first visit were entered into an independent ttest with
Intervention as between factor (B. longum 1714™vs placebo). To
control the intervention-related changes in the scores of the NTS,
MQ, and SEP during the CBG, changes after each intervention were
computed for each condition and entered into a 2 32 repeated
measure ANOVA with Intervention as a between-factor (B. longum
1714™vs placebo) 3Condition as a within-factor (exclusion vs
inclusion). Where significant main effects or interaction were ob-
served, pairwise post hoc comparisons were used with a Bonferroni-
adjusted threshold (a50.025). Mean data are reported as M 6s.d.
Correlation between stress measures and quality of life
assessments with MEG data
To investigate the relationship between changes in neural activity and
changes in subjective scores induced by B. longum 1714™, correlation
analysis was performed for both, the change in brain activity in the
resting state and after the CBG. For the resting state, averaged source
power was calculated for the clusters that were significantly different
between both visits. The averaged source power was correlated with
changes in health status (SF36) for each group separately. For the
CBG, for each condition and each intervention, source power within
clusters significantly different between both visits was averaged. The
averaged source power was correlated with changes in the scores of
the NTS, MQ, and SEP separately for each condition and each group,
using Pearson correlations.
RESULTS
Subjects and baseline characteristics
A total of 40 healthy volunteers completed the study. After
screening, 43 subjects were randomized to either B. longum 1714™
or placebo. Three subjects could not be included in the final
analysis because of use of antibiotics during the intervention
period (Figure 2 for detailed trial profile). A total of 40 partic-
ipants were included in the analysis with n 520 per intervention
group. Sex of participants was matched between groups. Age and
body mass index of participants were not significantly different
between groups (Table 1 for details).
Administration of social stress (“CBG”) altered brain activity in
healthy volunteers at baseline
At baseline, stress effects were analyzed across all participants.
No difference between groups at baseline during rest was ob-
served in any frequency band. As expected, source statistics of
Figure 3. Neural activities in different frequency bands during exclusion vs inclusion condition. (a) Theta frequency band (6 Hz): increased power in the
bilateral cerebellum (CBL), right middle and inferior frontal cortex (MFC and IFC), left fusiform cortex (P50.004); decreased power in the left middle frontal
and bilateral superior frontal cortex (SFC) (P50.02). (b) Alpha frequency band (11 Hz): increased power in the bilateral CBL, left fusiform, right inferior
occipital cortex (P50.002); decreased power in the right SFC (P,0.05). (c) Beta-1 frequency band (16 Hz): increased power in the bilateral CBL, left
MFC, left inferior temporal, right middle and superior temporal cortex, and right parahippocampal (P50.004). (d) Beta-2 frequency band (21 Hz):
increased power in the right CBL, left IFC, right inferior and middle temporal cortex (MTC) (P50.002). (e) Beta-3 frequency band (26 Hz): increased power
in the bilateral CBL, right fusiform, left parahippocampal, left MTC (P50.04).
Copyright © 2019 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of The American College of Gastroenterology The American Journal of GASTROENTEROLOGY
FUNCTIONAL GI DISORDERS
Bifidobacterium longum 1714ÔStrain Modulates Brain Activity 5
MEG power in each frequency band showed brain regions that
had significantly different activities during the exclusion
compared with the inclusion condition (Figure 3). The global
NTS (t
37
513.39, P,001) and SEP (t
38
59.99, P,001)
scores were significantly higher, and the MQ scores (t
39
5
29.42, P,001) were lower in the exclusion condition com-
pared with inclusion condition (Table 1). At baseline, there
were no significant differences in SF36 scores or CBG sub-
jective scores (NTS, MQ, and SEP) after the social stress be-
tween groups (Table 1).
B. longum 1714™altered resting-state brain activity measured
by MEG
After a 4-week intervention, comparison between probiotic and
placebo group showed that B. longum 1714™feeding increased
theta band (6 Hz) power (P,0.05; Figure 4a) in the bilateral
inferior, middle, and superior frontal cortex (IFC, MFC, and SFC)
as well as in the bilateral anterior and middle cingulate cortex
(ACC and MCC). Furthermore, feeding B. longum 1714™reduced
beta-2 band (26 Hz) power (P,0.05; Figure 4b) in the bilateral
fusiform gyrus, the bilateral hippocampus, the left inferior and
superior temporal and bilateral middle temporal cortex, and the
left cerebellum.
B. longum 1714™–induced change in neural activity correlated
with increased vitality
No significant difference was found in changes of SF36 scores
after intervention between groups. To investigate the re-
lationship between changes in neural activity during resting
state and quality of life, the changes in averaged theta and beta-
2 band power with changes in SF36 scores were correlated. In
both groups, a significant positive correlation was obtained
between changes of the SF36 subscale “Energy/Vitality”with
changes of theta band power (r50.33, P50.04; Figure 5a).
Group-specific correlations revealed that only in the B. longum
1714™group, changes in SF36 score for “Energy/Vitality”
positively correlated with changes in averaged theta band
power (r50.61, P50.007), and negatively correlated with
changes in beta-3 band power during the resting state (r52
0.50, P50.04; Figure 5b).
Figure 4. Difference of neural activity change during resting state comparing
B. longum 1714™vs placebo. (a) After the intervention, an increased theta
band (6 Hz) power was obtained in a cluster including regions of bilateral
inferior frontal cortex, middle frontal cortex, and the bilateral anterior cingu-
late cortex and middle cingulate cortex, comparing B. longum 1714™with
the placebo group (P,0.05). (b) After the intervention, reduced beta-2
band (26 Hz) power was obtained in a cluster, consisting of the bilateral
fusiform gyrus and hippocampus, left inferior temporal cortex and superior
temporal cortex, bilateral middle temporal cortex and left cerebellum, com-
paring B. longum 1714™with the placebo group (P,0.05).
Figure 5. Correlation between neural activity change during resting state and SF36 change. (a) In all groups, a positive correlation was obtained between
changes of SF36 item “Energy/vitality”with changes of theta band power in the cluster (r50.33, P50.04). (b) In only B. longum 1714™group, changes of
SF36 item “Energy/vitality”positively correlated with change of averaged theta band power (r50.61, P50.007), and negatively correlated with change of
beta-3 band power in the activated clusters during the resting state, respectively (r520.50, P50.04). SF36, 36-item short-form health survey.
The American Journal of GASTROENTEROLOGY VOLUME 00 | MONTH 2019 www.amjgastro.com
FUNCTIONAL GI DISORDERS
Wang et al.6
All groups reported enhanced social stress measured by
subjective ratings after a 4-week intervention
Analysis of changes in NTS, MQ, and SEP scores showed significant
main effects of condition on NTS (F(1, 33) 55.91, P50.02) and on
SEP (F(1, 36) 55.61, P50.02) as determined by ANOVA
(Intervention 3Condition). Participants in all groups reported in-
creased scores of NTS (exclusion: M 55.20 62.37; inclusion:
M524.32 61.85) and SEP (exclusion: M 50.94 60.42; inclusion:
M520.30 60.16) in the exclusion compared with inclusion
condition, after 4 weeks of intervention. The changes in MQ scores
(exclusion: M 521.87 61.08;inclusion:M50.30 60.86) showed
no difference after intervention (F(1, 34) 51.72, P50.20)
(Figure6).Therewerenoeffects of intervention on the subjective
scores of these questionnaires (Table 2).
B. longum 1714™–altered brain activity during social stress
measured by MEG
After a 4-week intervention, B. longum 1714™induced changes in
source power in theta (6 Hz) band and alpha band (11 Hz) in
response to the CBG, compared with placebo. B. longum 1714™
increased theta band power in 1 cluster, consisting of the right IFC
and the bilateral MFC and SFC, the left ACC, the bilateral MCC, and
the right supramarginal gyrus in both conditions—inclusion and
exclusion (P50.03; Figure 7a). Furthermore, B. longum 1714™also
increased the alpha band power in the cluster, including regions of
the right IFC, the bilateral MFC and SFC, the bilateral ACC and
MCC, and the right supramarginal gyrus in both conditions (P5
0.04; Figure 7b). No main effects of condition or interaction of
intervention and condition were observed.
B. longum 1714™–induced change in neural activity in social
stress correlated with changes in distress levels
Correlation analysis between changes in neural activities and changes
in subjective ratings during the CBG revealed that only with B. lon-
gum 1714™and only during the exclusion condition, NTS changes
positively, correlated with changes in the theta band power (r50.62,
P50.008) and alpha band power (r50.54, P50.03; Figure 8).
Summaries of frequency bands and neuroanatomical areas
found to be related to B. longum 1714™intervention, and asso-
ciations of changes in neural activity and subjective effects are
provided in Tables 3 and 4, respectively.
Figure 6. Main effects of condition on NTS and SEP. Participants in all groups
reported increased scores of NTS and SEP in the exclusion condition com-
pared with inclusion condition, after a 4-week intervention. MQ, mood ques-
tionnaire; NTS, Need Threat Scale; SEP, subjective exclusion perception.
Table 2. Changes in subjective scores of SF36, NTS, MQ, and SEP
B. longum 1714™Placebo Pvalue
SF36
Physical functioning 1.11 60.95 0.53 60.36 0.94
Role limitations due to physical health 0.00 60.00 0.00 60.00 1.00
Role limitations due to emotion problems 0.00 60.00 0.00 60.00 1.00
Energy/vitality 23.42 62.02 23.00 62.03 0.71
Emotional well-being 0.00 61.82 20.84 61.72 0.59
Social functioning 23.13 61.63 4.17 62.34 0.83
Pain 4.88 62.27 5.00 62.11 0.83
General health 20.26 62.31 20.79 61.68 0.95
CBG
NTS
Inclusion 4.11 63.24 4.53 61.79
Exclusion 24.82 63.82 25.58 63.02 P
intervention
50.98
MQ
Inclusion 0.63 61.34 20.03 61.11
Exclusion 20.61 61.74 23.00 61.30 P
intervention
50.60
SEP
Inclusion 20.25 60.24 20.34 60.22
Exclusion 0.53 60.58 1.35 60.60 P
intervention
50.63
CBG, Cyberball Game; MQ, mood questionnaire; NTS, Need Threat Scale; SEP, subjective exclusion perception; SF36, 36-item short-form health survey.
Copyright © 2019 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of The American College of Gastroenterology The American Journal of GASTROENTEROLOGY
FUNCTIONAL GI DISORDERS
Bifidobacterium longum 1714ÔStrain Modulates Brain Activity 7
DISCUSSION
B. longum 1714™has been shown to reduce stress-related
behaviors in preclinical studies (4,5) and improve stress respon-
ses and cognitive function in healthy volunteers (6). The current
functional neuroimaging study showed that B. longum 1714™
significantly altered neural activities in the resting state and in
response to a social stressor in healthy participants after a 4-week
intervention. The alteration in the resting brain activity by B.
longum 1714™was associated with the SF36 scale “Energy/Vi-
tality.”In response to the social stress, the changed neural ac-
tivities occurred only in the probiotic group. These data suggest
that B. longum 1714™may play a role in managing stress
responses by modulating the relevant neural processes.
During the resting state, B. longum 1714™induced an increase
in theta band power in the frontal and cingulate cortex and
a decrease in beta-3 band power in the fusiform cortex, hippo-
campus, temporal cortex, and cerebellum. Although no signifi-
cant change of SF36 was noted after the 4-week probiotic intake,
the increase in theta band and decrease in beta-3 band power
found in the resting state was associated with increase in per-
ceived energy/vitality levels, as assessed by the SF36. Energy/
vitality in the SF36 measures subjective reception of participants—
how full of pep and energetic they felt. Other studieshave shown an
association between increased power in theta band (whole brain,
especially prominent in the frontal regions) on a resting-state EEG
with improved attention and arousal after consumption of glucose
(24,25) whereas decreased theta band power measured by MEG in
cingulate cortex has been correlated with subjective level of fatigue
(26). Of interest, an increase in beta band power has been linked to
mental fatigue (27), andinversely, reduced beta power could index
increased alertness and arousal, accompanied with decreased
anxiety and stress (28,29). These data suggest that B. longum 1714™
altered neural activities in a manner associated with enhanced vi-
tality and reduced mental fatigue.
From an evolutionary perspective, humans are programmed
to be highly sensitive to social exclusion and have developed
monitoring systems to detect cues (30,31). This is necessary for
human survival as social isolation is often a fatal threat (32,33). As
expected, the CBG induced a social stress effect at baseline across
all participants. Social exclusion produced differential neural
oscillations in all frequency bands in various areas, such as frontal,
temporal, and fusiform cortex. These results were similar to
previous EEG and MEG studies, showing modulation of neural
oscillations by social exclusion (14,15,17,21). Previously reported
ACC activations were related to processing of negative emotions
and event appraisal due to the social stress/exclusion, and PFC
activation was related to emotional regulation (13,34–36). Theta
band oscillations in ACC and insula were described as a marker of
social pain in the context of the CBG and was also found during
a cold pressor test and physical pain (37,38). The participants
reported enlarged subjective distress and decreased mood level
during the exclusion condition compared with the inclusion
Figure 8. Correlation between neural activity change during the Cyberball
game and subjective score changes. Only in B. longum 1714™group and
only during the exclusion condition, NTS changes positively correlated with
changes of the theta band power (r50.62, P50.008) and alpha band
power (r50.54, P50.03). NTS, Need Threat Scale.
Figure 7. Difference in neural activity change during the Cyberball game com-
paring B. longum 1714™vs placebo. (a) Theta band showed an increased
power in a cluster, consisting of the right inferior frontal cortex (IFC) and the
bilateral middle frontal cortex (MFC) and superior frontal cortex (SFC), the left
anterior cingulate cortex (ACC), the bilateral middle cingulate cortex (MCC), and
the right supramarginal gyrus (SMG), comparing B. longum 1714 group and the
placebo group in both conditions (P50.03). (b) Alpha band power also showed
an increased power in cluster, including regions of the right IFC, the bilateral MFC
and SFC, the bilateral ACC and MCC, and the right SMG, comparing B. longum
1714™group and the placebo group in both conditions (P50.04). No main
effects of condition or interaction of intervention and condition were observed.
The American Journal of GASTROENTEROLOGY VOLUME 00 | MONTH 2019 www.amjgastro.com
FUNCTIONAL GI DISORDERS
Wang et al.8
condition, consistent to previous studies (12,14,15,17). After the
intervention, as expected, all participants reported higher distress
level during the exclusion condition compared with the inclusion
condition after 4 weeks, regardless of the type of intervention
(probiotic, placebo). However, a clinical effect of B. longum 1714™
on reducing stress was not observed—the study was not powered
to detect differences in subjective stress. Furthermore, as with
other stress paradigms, the repetition of stress may reinforce the
feeling of being excluded and consolidated the memory of the
stressful event making it difficult to detect differences with small
numbers.
Although both placebo and probiotic groups reported
higher subjective distress, changes in neural processing of
social stress were observed only after B. longum 1714™con-
sumption and not placebo. In addition, the correlation be-
tween changes in subjective distress and neural activities was
seen only with B. longum 1714™. Therefore, these data support
the notion that B. longum 1714™may play a role in managing
stress responses by modulating the relevant neural processes;
B. longum 1714™affecting individuals’neurophysiology is
anovelfinding and was previously only reported for behavioral
data in animals and humans (4–6).
However, in comparison to other neurophysiological
studies with the CBG (22,23), our study involved repeated
exposure to the social stress 4 weeks apart. Indeed, similar
changes in theta band activity induced by B. longum 1714™
were observed in the resting-state data (before playing the
CBG) of the current study, which correlated with increased
vitality. These data suggest that the increase in theta and alpha
band power by B. longum 1714™before the stressor may be to
some degree a priming for the stressor and may represent an
upregulation of appraisal processes and coping mechanisms in
the expectation of being exposed to a similar stressful situation
again. Furthermore, lower frontal alpha power has been as-
sociated with anxiety (39,40). The increased alpha oscillation
activity in our study may indicate the inhibition of limbic ac-
tivity and thereby counter-regulate negative emotions and
stress. Taking this argument further, we suggest that in con-
trast to placebo, B. longum 1714™reduced participants’stress
response and enabled them to manage the increased distress
level by upregulating processes appraising stressful events and
downregulating negative emotions.
Other studies, in line with our data, showed neural modu-
lation by different psychobiotic strains, and provided some
clues of the potential mechanisms involved. Preclinical studies
have reported an increase in serotonin and dopamine level, 2
crucial neurotransmitters regulating mood and emotions, in
brain regions of mice such as PFC after interventions with
Lactobacillus plantaraum and Lactobacillus helveticus (41–43).
Another study showed Lactobacillus rhamnosus JB-1 modulated
GABAergic system and reduced stress-related psychiatry-like
behaviors in mice, and some of the effects were mediated by the
vagus nerve (44). However, effects of the L. rhamnosus JB-1 were
not found in a clinical study in healthy volunteers, indicating
possibly the challenge of translating preclinical studies into
clinical relevance (3). The lack of evidence of translating pre-
clinical to clinical studies was also addressed by a recent meta-
analysis on the anxiolytic effect of probiotics (45), suggesting
a need to investigate strain specificity and use functional imag-
ing techniques in clinical studies in alternative to subjective
questionnaires.
In our recent study, antistress effects of rifaximin on social
stress were associated with a reduction of frontal and cingulate
beta band power in the insula, frontal and cingulate gyrus,
after 1-week intervention (21). Although causing a stress re-
duction like B. longum 1714™, rifaximin may act through dif-
ferent pathways—different neural oscillations and brain
regions. Because the peripheral and central mechanisms by
which rifaximin affects stress management are not well un-
derstood, one may speculate on an “eubiotic”effect of this
antibiotic promoting beneficial bacteria such as Bifidobacteria
and Lactobacilli (46,47). Therefore, although effects of probiotics
Table 3. Summarized neuroanatomical areas and frequency
bands of changed neural activities influenced by effect of
condition, intervention and/or interaction of condition and
intervention
Comparison
Frequency
band
Brain
region Hemisphere
P
value
Intervention effect on resting
state: B. longum group vs
placebo group
Theta ↑IFC B ,0.05
MFC B
SFC B
ACC B
MCC B
Beta-3 ↓FFG B ,0.05
HIPP B
ITC L
MTC B
STC L
CBL L
Intervention effect on the CBG
for all conditions: B. longum
group vs placebo group
Theta ↑IFC R 0.03
MFC B
SFC B
ACC L
MCC B
SMG R
Alpha ↑IFC R 0.04
MFC B
SFC B
ACC B
MCC B
SMG R
ACC, anterior cingulate cortex; B, bilateral; CBG, Cyberball Game; CBL, cerebellum;
FFG, fusiform gyrus; HIPP, hippocampus; IFC, inferior frontal cortex; ITC, inferior
temporal cortex; L, left; MCC, middle cingulate cortex; MFC, middle frontal cortex;
MTC, middle temporal cortex; R, right; SFC, superior frontal cortex; SMG,
supramarginal gyrus; STC, superior temporal cortex.
Copyright © 2019 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of The American College of Gastroenterology The American Journal of GASTROENTEROLOGY
FUNCTIONAL GI DISORDERS
Bifidobacterium longum 1714ÔStrain Modulates Brain Activity 9
on CNS functions appear to be strain-specific (4), results of
existing studies help to understand the possible pathways of the
strain we used in the current trial. More studies are required to
investigate the effects of different strains on diverse brain activi-
ties in response to stress in order to obtain a detailed picture of the
effects of probiotics on brain functioning.
CONCLUSION
B. longum 1714™influenced resting neural activities associated
with enhanced vitality and stress-related neural responses,
which may be involved in the counter-regulation of negative
emotions. Our results further support the role of B. longum
1714™in reducing stress responses and provides new evidence
that this probiotic affects brain function through modulating
neural oscillations in certain brain regions. The understanding
of neural oscillations induced by stress and their modulation
by probiotics is still at its beginning. Studies that examine the
mechanisms by which probiotics modulate the gut-brain axis
and compare their effects on other CNS functions with other
interventions are warranted. To fully understand the effects of
B. longum 1714™on brain function and human behavior, it is
necessary to consider the effects of the strain on other CNS
functions such as pain sensitivity, mood, and memory in both
healthy controls and in patients with psychiatric (depression,
anxiety), neurologic (neurodegenerative), and gastrointestinal
disorders such as IBS.
ACKNOWLEDGMENTS
We thank Dr. Gerald Clarke, Prof. John Cryan, and Prof. Ted
Dinan at the APC Microbiome Institute, University College Cork,
Ireland for support on training and advice on the probiotic
selection.
1714™, 1714-Serenitas™, and Zenflore™are trademarks of Alimentary
Health.
CONFLICTS OF INTEREST
Guarantor of the article: Paul Enck, PhD.
Specific author contributions: P.E.: inception of the study and
publication of the work. H.W.: contributed to the design of the study,
data collection and analysis, drafting of the manuscript, and critical
revisions of the manuscript. C.B., E.M., and P.E.: contributed to the
design of the study, data analysis, and critical revisions of the
manuscript. All authors approved the final version of the manuscript.
1714, 1714-serenitas and Zenflore are trademarks of Alimentary
Health.
Financial support: The research leading to these results has received
funding from the People Programme of the European Union’s
Seventh Framework Programme under REA grant agreement no.
607652 (NeuroGut).
Potential competing interests: E.M. is the Technical Director at
Alimentary Health Group. Alimentary Health provided the
placebo and probiotic containing the B. longum 1714™strain (also
known as 1714-Serenitas), but had no further influence on data
collection and data evaluation. P.E. is a consultant for Alimentary
Health. The other authors declare no potential conflicts of
interest.
Table 4. Summarized correlations of changes of averaged power in activated areas with changes of subjective results
Groups Subjective item
Resting-state MEG Functional MEG during CBG
Theta band
power change
Beta-3 band
power change
Theta band
power change
Alpha band
power change
Both groups SF36-energy/vitality r50.33
P50.04
———
B. longum group SF36-energy/vitality r50.61
P50.007
r520.50
P50.04
——
NTS r50.62
P50.008
r50.54
P50.03
The changes in neural activity during resting state was correlated with changes of SF36, and the changes in the neural activity during CBG was correlated with changes of
the NTS, respectively, for each of the 2 conditions of exclusion and inclusion.
CBG, Cyberball Game; MEG, magnetoencephalography; NTS, Need Threat Scale; SF36, 36-item short-form health survey.
Study Highlights
WHAT IS KNOWN
3Accumulating evidence indicates that the gut microbiota
communicates with the CNS, possibly through neural,
endocrine, and immune pathways, and influences brain
function.
3Effects of probiotics on the CNS functions are strain specific.
3B. longum 1714™has been shown to reduce stress-related
behaviors in preclinical studies and improve stress responses
and cognitive function in healthy volunteers.
WHAT IS NEW HERE
3B. longum 1714™altered brain activity of healthy
volunteers during social stress as measured by
neuroimaging method MEG with high temporal resolution
and fine spatial resolution.
3B. longum 1714™influenced resting neural activities
associated with enhanced vitality and stress-related neural
responses, which may be involved in the counter-regulation of
negative emotions.
3Our results further support the role of B. longum 1714™in
reducing stress responses in humans and provides new
evidence that this probiotic strain affects brain function
through modulating neural oscillations in brain regions
involved in emotional regulation.
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FUNCTIONAL GI DISORDERS
Wang et al.10
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FUNCTIONAL GI DISORDERS
Bifidobacterium longum 1714ÔStrain Modulates Brain Activity 11
