nutrients
Article
Bifidobacterium breve CCFM1025 Improves Sleep Quality via
Regulating the Activity of the HPA Axis: A Randomized
Clinical Trial
Yuming Lan 1,2,3,† , Junjie Lu 4,† , Guohong Qiao 5 , Xuhua Mao 4 , Jianxin Zhao 1,2,3,6 , Gang Wang 1,2,3,6 ,
Peijun Tian 1,2,3, * and Wei Chen 1,2,3
Citation: Lan, Y.; Lu, J.; Qiao, G.;
Mao, X.; Zhao, J.; Wang, G.; Tian, P.;
Chen, W. Bifidobacterium breve
CCFM1025 Improves Sleep Quality
via Regulating the Activity of the
HPA Axis: A Randomized Clinical
Trial. Nutrients 2023, 15, 4700.
https://doi.org/10.3390/nu15214700
Academic Editors: Jaime Uribarri
and Rosa Casas
Received: 18 October 2023
Revised: 1 November 2023
Accepted: 3 November 2023
Published: 6 November 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Nutrients 2023, 15, 4700. https://doi.org/10.3390/nu15214700
1 State Key Laboratory of Food Science and Resources, Jiangnan University, Wuxi 214122, China;
6210113187@stu.jiangnan.edu.cn (Y.L.); zhaojianxin@jiangnan.edu.cn (J.Z.);
wanggang@jiangnan.edu.cn (G.W.); chenwei66@jiangnan.edu.cn (W.C.)
2 School of Food Science and Technology, Jiangnan University, Wuxi 214122, China
3 National Engineering Research Center for Functional Food, Jiangnan University, Wuxi 214122, China
4 Department of Critical Care Medicine, Yixing People’s Hospital Affiliated Jiangsu University,
Yixing 214200, China; staff806@yxph.com (J.L.); staff1291@yxph.com (X.M.)
5 Department of Clinical Laboratory, Yixing People’s Hospital Affiliated Jiangsu University,
Yixing 214200, China; staff1000@yxph.com
6 (Yangzhou) Institute of Food Biotechnology, Jiangnan University, Yangzhou 225004, China
* Correspondence: pjtian@jiangnan.edu.cn; Tel.: +86-510-8591-2155
† These authors contribute equally to this work and should be considered co-first authors.
Abstract: Psychobiotics, a newly identified category of probiotics primarily targeting the gut–brain
axis, exhibit tremendous potential in improving sleep quality. In this study, the clinical trial was
registered in advance (identifier: NO. ChiCTR2300067806). Forty participants who were diagnosed
with stress-induced insomnia were chosen and randomly divided into two groups: one received
CCFM1025 at a dose of 5 × 109 CFU (n = 20), while the other was administered a placebo (n = 20),
over a period of four weeks. The results revealed that compared to the placebo group (pre: M = 10.10,
SD = 2.292; post: M = 8.650, SD = 2.793; pre vs. post: F (1, 38) = 15.41, p = 0.4316), the CCFM1025-
treated group exhibited a significant decrease in Pittsburgh Sleep Quality Index (PSQI) scores from
baseline (pre: M = 11.60, SD = 3.169; post: M = 7.750, SD = 3.697, F (1, 38) = 15.41, p = 0.0007).
Furthermore, the administration of CCFM1025 was associated with a more pronounced reduction in
stress marker concentrations. This effect could potentially be linked to changes in serum metabolites
induced by the probiotic treatment, notably daidzein. In conclusion, B. breve CCFM1025 demonstrates
promise as a psychobiotic strain for enhancing sleep quality.
Keywords: probiotics; sleep; insomnia; stress; HPA axis; daidzein
1. Introduction
Natural sleep, constituting about one-third of an individual’s lifespan, plays a vital
role in various aspects of human health, including neurodevelopment, learning, memory,
emotional regulation, and cardiovascular and metabolic functions, as well as the clearance
of cytotoxins [1]. However, numerous factors, both physiological and environmental, can
disrupt sleep patterns, resulting in insufficient and poor-quality sleep. These factors en-
compass conditions like insomnia, obstructive sleep apnea, circadian rhythm disorders,
and even occupational stress [2]. The consequences of chronic sleep deprivation are far-
reaching, contributing to inflammation, metabolic dysfunction, cognitive impairment, and
an increased risk of mental illnesses [3,4]. In the long term, inadequate sleep has been asso-
ciated with the development of serious health conditions such as diabetes, hypertension,
cardiovascular disease, stroke, coronary heart disease, obesity, and depression [5]. Insomnia
https://www.mdpi.com/journal/nutrients
Nutrients 2023, 15, 4700 2 of 12

disorder (ID) is particularly noteworthy, being the most prevalent sleep disorder and rank-
ing as the second most common neuropsychiatric disorder [6]. Recent research conducted
by Li et al. [7] showed the extensive prevalence of insomnia symptoms within the Chinese
population, with rates as high as 39.1%. Numerous prior studies have also highlighted the
close relationship between insufficient sleep and elevated levels of job-related stress [8].
The gut–brain axis has garnered increasing attention in the realms of neurology, psy-
chiatry, neurodevelopment, and neurodegenerative diseases due to its profound biological
and physiological implications [9]. Gut microbes mediate brain function and host behaviors
either directly or indirectly, including immune pathways (immune cells and cytokines),
endocrine pathways (HPA axis), and neural pathways (neurotransmitters, neuroactive
metabolites, vagus nerve, enteric nervous system, and spinal nerve) [10]. In fact, several
studies have indicated that treatments using psychobiotics may decrease stress and improve
sleep quality. For example, Lactobacillus gasseri CP2305 can alleviate stress and improve
sleep in young adults by regulating gut microbiota composition and reducing salivary
cortisol and other stress indicators [11,12]. Using miniature polysomnography (PSG), Ho
et al. discovered that a four-week intervention with probiotic Lactobacillus plantarum PS128
could affect sleep architecture and improve the quality of deep sleep [13]. These findings
revealed the therapeutic potential of probiotics in stress management and the alleviation of
stress-related symptoms, such as insomnia.
Bifidobacterium breve CCFM1025, as a potential psychoprobiotic strain, has a positive
regulatory effect on depression, Alzheimer’s disease, and other mental diseases [14,15]. The
underlying mechanisms may involve the alleviation of HPA axis hyperactivity, suppression
of the inflammatory response, modulation of the serotonergic system, and alteration of
the gut microbiota composition [16]. In addition, CCFM1025 was found to colonize in the
human gut [17]. In our prior research, we observed that Bifidobacterium breve CCFM1025 ef-
fectively alleviated anxiety and improved sleep quality in participants with depression [18].
To gain deeper insights, animal studies were conducted, showing that CCFM1025 influ-
enced the production of key metabolites involved in regulating sleep function through the
modulation of gut microbiota [19]. This modulation, in turn, mitigated circadian rhythm
disturbances induced by sleep deprivation in mice. Building on these significant findings,
we initiated a clinical trial to investigate the impact of CCFM1025 intervention on sleep
quality in individuals suffering from insomnia. In contrast to previous approaches, our
study specifically focused on evaluating the probiotic’s effect on participants’ hyperactivity
of the HPA axis.

2. Materials and Methods

2.1. Ethical Approval
This trial was approved by Yixing People’s Hospital (Yixing, China, Approval Code:
Lunshen2022Ke188). The registration number of the Chinese Clinical Trial Registry was
ChiCTR2300067806.

2.2. Sample Size Determination

The software G*Power3.1 was used to analyze prior power. We estimated the effect
size from a published study that improved sleep quality by comparing the mean difference
of the Pittsburgh Sleep Quality Index (PSQI) between the control groups and treated
groups [20]. In order to attain a statistical power of 0.8 for a t-test analysis, it is necessary to
have groups consisting of a minimum of eighteen participants, resulting in an effect size of
d = 0.847 at a significance level of α = 0.05 (See Supplemental Figure S1 for details). We
recruited 20 volunteers per group.

2.3. Study Design

This study was a placebo-controlled, randomized controlled trial (RCT). The CON-
SORT flow diagram is shown in Figure 1. Participants with an age range of 18–65 years were
divided into healthy control groups and sleep disorder groups using the PSQI score before
 2.3. Study Design
This study was a placebo-controlled, randomized controlled trial (RCT). The CON
SORT flow diagram is shown in Figure 1. Participants with an age range of 18–65 yea
Nutrients 2023, 15, 4700 were divided into healthy control groups and sleep disorder groups using the PSQI sco
3 of 12
before enrollment. The sleep disorder group was then divided into placebo an
CCFM1025-treated groups. Participants were blinded and received the intervention pro
enrollment.
uct The sleep administered
independently, disorder group wasby anthen divided into
authorized placebo and CCFM1025-treated
investigator, following a 1:1 allocatio
groups. Participants were blinded and received the intervention
ratio determined by a randomization code [21]. Samples of the healthy product independently,
control group we
administered by an authorized investigator, following a 1:1 allocation ratio determined by a
only taken at baseline, whereas samples of the sleep disorder group were taken at baselin
randomization code [21]. Samples of the healthy control group were only taken at baseline,
and aftersamples
whereas intervention. All of
of the sleep the samples
disorder weretaken
group were collected at theand
at baseline hospital under the guidan
after intervention.
of
Allanother authorized
of the samples investigator.
were collected With a PSQI
at the hospital under score > 7, participants
the guidance with the followin
of another authorized
symptoms
investigator. With a PSQI score > 7, participants with the following symptoms such as with i
such as difficulty falling asleep and waking up early were diagnosed
difficultyParticipants
somnia. falling asleepwho
and waking up early
take sleep aids,were
work diagnosed with insomnia.
night shifts, Participants
have an irregular lifestyle,
who take sleep aids, work night shifts, have an irregular lifestyle, or
have a history of alcohol dependence, pregnant or lactating women, and men or wome have a history of
alcohol dependence, pregnant or lactating women, and men or women who are preparing
who are preparing for pregnancy (childbearing) were excluded. As a result, 60 partic
for pregnancy (childbearing) were excluded. As a result, 60 participants completed the
pants completed the trial (health control group, n = 20, female ratio 50%; placebo, n = 2
trial (health control group, n = 20, female ratio 50%; placebo, n = 20, female ratio 70%;
female
CCFM1025,ration70%;
= 20, CCFM1025, n = 20,
female ratio 60%). Thefemale ratio 60%).
demographic The demographic
characteristics are presentedcharacteristi
in
are presented in Table 1. (The demographic information of all participants
Table 1. (The demographic information of all participants including the healthy control including th
healthy
group and control group
the sleep and group
disorder the sleep disorder
is shown group is shown
in Supplemental in Supplemental Table S1
Table S1).

1. Flow
Figure 1.
Figure Flowchart.
chart.
The probiotic treatment group received a daily sachet consisting of Bifidobacterium breve
The probiotic
CCFM1025 treatment
in a dose of 109 CFU per group received
day and a daily
maltodextrin, sachet
while consisting
the placebo groupof Bifidobacteriu
received
breve CCFM1025 in a dose of 109 CFU per day and maltodextrin, while the placebo grou
an identical-appearing sachet containing only maltodextrin for four weeks. (Specific
received an identical-appearing
experimental sachet containing
schedule is shown in Supplemental Figure only maltodextrin
S2). The products wereforproduced
four weeks. (Sp
cific experimental
by Shisheng Yisheng,schedule is shown
Co., Ltd. in Supplemental
(Yangzhou, Figurewere
China). Participants S2). The
askedproducts
to avoidwere pr
supplements and food containing probiotics during their participation in
duced by Shisheng Yisheng, Co., Ltd. (Yangzhou, China). Participants were asked the study, with to avo
no other dietary
supplements restrictions.
and Enrollment
food containing and sample
probiotics collection
during their began on 1 January
participation 2023
in the study, wi
and ended on 31 March 2023. Sample collection and data analysis were completed in the
no other dietary restrictions. Enrollment and sample collection began on 1 January 20
laboratory in June 2023.
and ended on 31 March 2023. Sample collection and data analysis were completed in th
laboratory in June 2023.
Nutrients 2023, 15, 4700 4 of 12

Table 1. Summary of Demographic Information.

CCFM1025
Variable Placebo (n = 20) Statistic Value p Value
(n = 20)
Age, Mean ± SD 36.55 ± 11.31 38.95 ± 10.59 0.693 0.493 a
BMI. Mean ± SD 22.59 ± 3.30 22.76 ± 3.05 0.168 0.866 a
Female 14 (70) 12 (60)
Sex, n (%)
Male 6 (30) 8 (40)
0.440 0.507 b

Race, n (%) Chinese, Han nationality 20 (100) 20 (100) N/A N/A

Medication Before
0 (0) 0 (0) N/A N/A
inclusion, n (%)
Primary 1 (5) 0 (0)
Educational status, Secondary 3 (15) 4 (20)
n (%) Higher 10 (50) 13 (65)
2.534 0.469 b
Unknown 6 (30) 3 (15)
Non-drinker 16 (80) 15 (75)
Alcohol use, n (%)
Occasional drinker 4 (20) 5 (25)
0.143 0.705 b

Non-smoker 16 (80) 13 (65)

Smokers, n (%)
Smoker 4 (20) 7 (35)
1.129 0.288 b

n, number of participants; N/A, not available; a Statistical analysis by t-test; b Statistical analysis by chi square test.

2.4. Sleep Disorder Measurements (Major Outcome Measures)

PSQI was used to quantify sleep quality as a major experimental outcome [22]. Higher
total PSQI scores indicate poorer sleep quality. A PSQI score greater than 7 was used as the
cut-off point for whether a participant had a sleep disorder. We recruited participants with
a total PSQI score > 7. Participants were scored during routine examinations. In addition,
the Athens Insomnia Scale (AIS) was used as an auxiliary judgment for sleep quality.

2.5. Salivary Cortisol Measurements

Salivary cortisol level was measured at the start and after a 4-week period to assess
the HPA axis’s activity in relation to chronic stress [23]. Since the secretion of cortisol
peaks around 9 a.m. [24], salivary samples were collected at this time. Saliva samples
from all participants were collected using a commercial saliva tube (Shanghai Zhizhi
Biotechnology Co., Ltd., Shanghai, China) and centrifuged according to the instructions to
remove particulate matter. The quantification of free cortisol was performed using a saliva
cortisol ELISA kit (Shanghai Coibo Bio Technology Co., Ltd., Shanghai, China), according
to the manufacturer’s instructions.

2.6. Plasma Measurements

After saliva collection, 4 mL of fasting peripheral venous blood was taken from
participants. Anticoagulant was added into the blood collection tube, and plasma was
separated by 3000 r/min and stored in a low temperature freezer (−80 ◦ C). The Cortisol
CLIA Microparticles (Autobio Diagnostics Co., Ltd., Zhengzhou, China) and the ACTH
CLIA Microparticles (Autobio Diagnostics Co., Ltd.) were used to detect the levels of
cortisol and adrenocorticotropin (ACTH) in plasma.

2.7. Serum Measurements

The procedure for serum metabolite extraction was described extensively in the study
conducted by Yuan et al. [25]. To conduct the metabolomic analysis, we utilized the
online software MetaboAnalyst 5.0. For further details, the official website of the software
is http://www.metaboanalyst.ca/ (accessed on 15 July 2023). The parameters for the
Ultraperformance Liquid Chromatography–Mass Spectrometry (UPLC-MS) analysis are
provided in Supplemental S1.1.
 Ultraperformance Liquid Chromatography–Mass Spectrometry (UPLC-MS) analysis are
provided in Supplemental S1.1.

Nutrients 2023, 15, 4700 2.8. Statistical Analysis 5 of 12

Before the commencement of statistical analysis, all clinical data underwent an initial
normality test using the Shapiro–Wilk test. IBM SPSS 22.0 was employed for conducting
2.8. Statistical
the statisticalAnalysis
analyses. To compare the two groups, including the baseline clinical data
Beforethe
between the groups,
commencement
as wellof statistical analysis,
as the score andall clinical data underwent
concentration changesan between
initial the
normality test
CCFM1025 andusing the Shapiro–Wilk
placebo test. IBM SPSS
groups, an unpaired 22.0 was
Student’s employed
t-test for conducting
was utilized. Cohen’s d was
the statistical
used to assess analyses. To size.
the effect compare
For the
thetwo groups,
analysis of including the baseline
the alterations clinical
in scale scoresdata
and sub-
between the groups, as well as the score and concentration changes between the CCFM1025
stance concentrations before and after the intervention in the two groups, a two-way anal-
and placebo groups, an unpaired Student’s t-test was utilized. Cohen’s d was used to
ysis of variance (ANOVA) was conducted, followed by Tukey’s multiple comparisons. The
assess the effect size. For the analysis of the alterations in scale scores and substance
correlation
concentrations between
before the
and compounds was examined
after the intervention in the twousing the Pearson
groups, correlation
a two-way analysis coeffi-
cient. Statistical
of variance (ANOVA)significance was determined
was conducted, followed by
by considering
Tukey’s multiple a p-value of less than
comparisons. The0.05 for
all comparisons. Graphs were generated using Prism 8.0.
correlation between the compounds was examined using the Pearson correlation coefficient.
Statistical significance was determined by considering a p-value of less than 0.05 for all
comparisons.
3. Results Graphs were generated using Prism 8.0.
3.1. Clinical Grouping and Validation of Insomnia
3. Results
Participants
3.1. Clinical Grouping were
anddivided
Validationinto two groups, a sleep disorder group and a healthy con-
of Insomnia
trol group, based on their scores
Participants were divided into two on thegroups,
PSQI (t a(58) = 11.29,
sleep p < 0.0001,
disorder group andd = 2.813; see Figure
a healthy
2A). Additionally,
control group, basedthere existed
on their scoresnotable disparities
on the PSQI (t (58) =in11.29,
the results of the
p < 0.0001, d =AIS between
2.813; see the
two
Figuregroups (t (58) = 10.42,
2A). Additionally, p <existed
there 0.0001, d = 2.598;
notable see Figure
disparities 2B). We
in the results of also measured
the AIS betweenvarious
the two groupsindicators
physiological (t (58) = 10.42, p < groups.
in both 0.0001, dThe= 2.598;
sleepsee Figure 2B).group
disturbance We also measured
exhibited a signifi-
cantly elevated salivary cortisol level in comparison to the healthy controlexhibited
various physiological indicators in both groups. The sleep disturbance group group (t (58) =
a significantly
9.774, elevated
p < 0.0001, salivary
d = 2.631; seecortisol
Figure level
2C).inFurthermore,
comparison to the the healthy
plasma control
cortisolgroup
and ACTH
(t (58) = 9.774, p < 0.0001, d = 2.631; see Figure 2C). Furthermore, the plasma cortisol and
levels in the sleep disorder group were significantly elevated compared to those in the
ACTH levels in the sleep disorder group were significantly elevated compared to those in
healthy control
the healthy controlgroup
group(plasma cortisol:t (58)
(plasma cortisol: t (58) = 6.175,
= 6.175, p < p0.0001,
< 0.0001, d = 1.553;
d = 1.553; see Figure
see Figure 2D; 2D;
plasma ACTH: t (58) = 4.093, p = 0.0001, d = 1.020; see Figure 2E). These
plasma ACTH: t (58) = 4.093, p = 0.0001, d = 1.020; see Figure 2E). These results align with results align with
previous studies[26,27],
previous studies [26,27], confirming
confirming the the reliability
reliability of ourofinsomnia
our insomnia assessment.
assessment.

2. Differences
Figure 2.
Figure Differencesbetween
between thethe
sleep disorder
sleep and and
disorder healthy control
healthy group.group.
control (A) Baseline PSQI score.
(A) Baseline PSQI score.
(B) Baseline AIS score. (C) Baseline salivary cortisol. (D) Baseline plasma cortisol. (E) Baseline
(B) Baseline AIS score. (C) Baseline salivary cortisol. (D) Baseline plasma cortisol. (E) Baseline plasma
ACTH. ***
plasma p < 0.001,
ACTH. *** p**** p < 0.0001
< 0.001, **** in
p <the unpaired
0.0001 in the t-tests.
unpaired t-tests.
14, x FOR PEER REVIEW 6 of 12

Nutrients 2023, 15, 4700 6 of 12

3.2. CCFM1025 Intervention Enhanced the Sleep Quality of Insomnia Patients

In the CCFM1025-treated group, the
3.2. CCFM1025 Intervention participants’
Enhanced the Sleep PSQI
Qualityscore exhibited
of Insomnia a notable
Patients
drop in comparison to the initial baseline measurement (pre vs. post: F (1, 38) = 15.41, p =
In the CCFM1025-treated group, the participants’ PSQI score exhibited a notable
0.0007; Figure 3A). However,
drop there was
in comparison noinitial
to the statistical significance
baseline measurement in the placebo
(pre vs. post:group
F (1,(pre
38) = 15.41,
vs. post: F (1, 38)p==15.41,
0.0007;pFigure
= 0.4316;
3A). Figure
However,3A). Moreover,
there CCFM1025
was no statistical led to in
significance a more sig- group
the placebo
nificant reduction(pre
in the
vs. participants’
post: F (1, 38)PSQI score
= 15.41, p =compared to the
0.4316; Figure placebo
3A). group
Moreover, (placeboled to a
CCFM1025
more significant reduction in the participants’
vs. CCFM1025: t (38) = 2.106, p = 0.0419, d = 0.666; Figure 3A). PSQI score compared to the placebo group
(placebo vs. CCFM1025: t (38) = 2.106, p = 0.0419, d = 0.666; Figure 3A).

Figure 3. B. breve 0
Figure 3. B. breve CCFM1025′s effectCCFM1025 s effect on sleep (A)
on sleep improvement. improvement.
Alteration (A) Alteration
in the score ofinPSQI.
the score
(B) of PSQI.
The change in rating scores of the PSQI(sleep quality). (C) The change in rating scores of the PSQI of the
(B) The change in rating scores of the PSQI(sleep quality). (C) The change in rating scores
disturbance). (D) The change in rating scores of AIS. * p < 0.05, *** p < 0.001 in Tukey’s
(sleep disturbance).PSQI
(D) (sleep
The change in rating scores of AIS. * p < 0.05, *** p < 0.001 in Tukey’s multiple
multiple comparisons test post two-way ANOVA. # p < 0.05, ## p < 0.01 in the unpaired t-tests. ns:
comparisons test post two-way ANOVA. # p < 0.05, ## p < 0.01 in the unpaired t-tests. ns: not statis-
tically significant. not statistically significant.
CCFM1025 also improved sleep quality by enhancing participants’ subjective sleep
CCFM1025 also improved
quality sleepsleep
and reducing quality by enhancing
disturbance participants’
(sleep quality score: presubjective sleep
vs. post: F (1, 38) =18.19,
quality and reducing sleepFigure
p = 0.004, disturbance
3B; sleep(sleep qualityscore:
disturbance score: pre
pre vs.vs. post:
post: F (1,
F (1, 38)38) =18.19,
= 0.937, p = 0.038,
Figure
p = 0.004, Figure 3B; sleep3C). Conversely,score:
disturbance there was no significant
pre vs. post: F (1,difference
38) = 0.937, in pthe placebo
= 0.038, group (sleep
Figure
3C). Conversely,quality score:no
there was presignificant
vs. post: F (1,
difference in pthe
38) = 18.19, = 0.2557,
placebo Figure 3B; sleep
group disturbance
(sleep quality score:
pre vs. post: F (1, 38) = 0.937, p = 0.5226, Figure 3C).
score: pre vs. post: F (1, 38) = 18.19, p = 0.2557, Figure 3B; sleep disturbance score: pre vs.
In the AIS table, AIS scores significantly decreased before and after CCFM1025 inter-
post: F (1, 38) = 0.937, p = 0.5226, Figure 3C).
vention, while there was no significant difference in the placebo group before and after
In the AIS table, AIS scores
intervention significantly
(pre-placebo decreased Fbefore
vs. post-placebo: (1, 38) and after
= 13.46, p =CCFM1025 inter-
0.1558; pre-CCFM1025 vs.
vention, while there was no significant difference in the placebo
post-CCFM1025: F (1, 38) = 13.46, p = 0.0155; Figure 3D). group before and after
intervention (pre-placebo vs. post-placebo: F (1, 38) = 13.46, p = 0.1558; pre-CCFM1025 vs.
post-CCFM1025:3.3. Effects of CCFM1025 Intervention on Stress Markers in Insomnia Patients
F (1, 38) = 13.46, p = 0.0155; Figure 3D).
Further assessments were conducted by measuring salivary and plasma stress markers.
There was no statistically significant difference seen in the level of salivary cortisol. (pre-
3.3. Effects of CCFM1025 Intervention on Stress Markers in Insomnia Patients
placebo vs. post-placebo: F (1, 38) = 1.67, p = 0.8768; pre-CCFM1025 vs. post-CCFM1025:
Further assessments
F (1, 38) =were
1.67, conducted by measuring
p = 0.0564; Figure salivary
4A). However, it isand plasma stress
noteworthy that themark-
reduction in
ers. There was nosalivary
statistically
cortisolsignificant difference
concentration betweenseen in the
the two levelshowed
groups of salivary cortisol.
statistical significance.
(pre-placebo vs. Specifically, compared
post-placebo: F (1,to 38)
the placebo
= 1.67,group, CCFM1025
p = 0.8768; led to a more significant
pre-CCFM1025 vs. post- reduction
in saliva cortisol concentration (placebo vs. CCFM1025: t (38) = 3.331,
CCFM1025: F (1, 38) = 1.67, p = 0.0564; Figure 4A). However, it is noteworthy that the re- p = 0.0019, d = 2.764;
Figure 4A).
duction in salivary cortisol concentration between the two groups showed statistical sig-
nificance. Specifically, compared to the placebo group, CCFM1025 led to a more signifi-
cant reduction in saliva cortisol concentration (placebo vs. CCFM1025: t (38) = 3.331, p =
0.0019, d = 2.764; Figure 4A).
x FOR PEER REVIEW 7 of 12
Nutrients 2023, 15, 4700 7 of 12

Figure 4. B. effect
breve CCFM1025 0
s effect on stress markers. (A)
Figure 4. B. breve CCFM1025′s on stress markers. (A) Alteration inAlteration in the concentration
the concentration of salivaryof salivary
cortisol. (B) The rating concentration change in the plasma cortisol. (C) The rating concentration change
cortisol. (B) The rating concentration change in the plasma cortisol. (C) The rating concentration
change in the plasma in ACTH.
the plasma# pACTH. p <p0.05,
< 0.05,# ## ## pin
< 0.01 < 0.01
the in the unpaired
unpaired t-tests.
t-tests. ns:ns:
notnotstatistically
statistically significant.
signif-
icant. Similar findings were observed in plasma cortisol levels (pre-placebo vs. post-placebo:
F (1, 38) = 0.088, p = 0.7626; pre-CCFM1025 vs. post-CCFM1025: F (1, 38) = 0.088, p = 0.5058;
Similar findings were observed
concentration reduction:in plasma cortisol
placebo vs. levels (pre-placebo
CCFM1025: t (38) = 2.237, pvs. post-pla-
= 0.0312, d = 0.707;
cebo: F (1, 38) = 0.088,
Figurep =4B).
0.7626; pre-CCFM1025
However, it is importantvs.
to post-CCFM1025:
note that after both FCCFM1025
(1, 38) = 0.088, p = and
intervention
placebo
0.5058; concentration intervention,
reduction: placebo there
vs.were no statistically
CCFM1025: t (38) =significant
2.237, p =differences
0.0312, d =in0.707;
the changes
in plasma ACTH levels (pre-placebo vs. post-placebo: F (1, 38) = 1.66, p = 0.9997; pre-
Figure 4B). However, it is important to note that after both CCFM1025 intervention and
CCFM1025 vs. post-CCFM1025: F (1, 38) = 1.66, p = 0.3141; Figure 4C), and there were no
placebo intervention, there were no statistically significant differences in the changes in
statistically significant differences in the reduction in plasma ACTH content (placebo vs.
plasma ACTH levels (pre-placebo
CCFM1025: vs. post-placebo:
t (38) = 1.935, F (1, Figure
p = 0.0605, d = 0.6616; 38) = 4C).
1.66, p = 0.9997; pre-
CCFM1025 vs. post-CCFM1025: F (1, 38) = 1.66, p = 0.3141; Figure 4C), and there were no
3.4. Metabolomic
statistically significant differences Analysis
in theReveals Daidzein
reduction in as a Biomarker
plasma ACTH in CCFM1025 Intervention
content (placebo vs.
The stability of the metabolomic
CCFM1025: t (38) = 1.935, p = 0.0605, d = 0.6616; Figure 4C). profiles was initially assessed. As illustrated in the
PCA plots (Figure S3), the serum QC samples in both ESI- and ESI+ modes clustered
together. The serum metabolome composition was modeled using partial least squares-
3.4. Metabolomic Analysis Reveals Daidzein as a Biomarker in CCFM1025 Intervention
discriminant analysis, and biomarkers were identified based on their variable importance in
The stability projection
of the metabolomic
(VIP) values. profiles was initially
Several metabolites wereassessed.
identified As illustratedwith
as biomarkers in the
a VIP > 2.0
(VIP values of compounds were shown in Supplemental
PCA plots (Figure S3), the serum QC samples in both ESI- and ESI+ modes clustered to-Table S4). Notably, changes in
gether. The serumspecific human serum
metabolome metabolites
composition waswere observed
modeled before
using and after
partial leastCCFM1025 and placebo
squares-dis-
interventions. In the CCFM1025 intervention group, six biomarkers, including choline,
criminant analysis, and biomarkers were identified based on their variable importance in
gentian violet, oleamide, daidzein, pentadecanoic acid, and acetylcholine, were identified as
projection (VIP) values. Several(Figure
key substances metabolites
5A). Wewere identified
first analyzed theas biomarkers
relationship withthese
between a VIP
six>biomarkers
2.0
(VIP values of compounds weremarkers.
and three stress shownThe in Supplemental Table
results revealed that onlyS4).
oneNotably,
substance,changes
daidzein, in
exhibited
specific human serum metabolites
a significant were with
correlation observed before
the stress and after
markers CCFM1025
(Figure 5C). Daidzeinand also
placebo
served as a
biomarker
interventions. In the CCFM1025with a intervention
VIP > 2 in the group,
placebo six
intervention groupincluding
biomarkers, (Figure 5B). Therefore, we
choline,
chose it for
gentian violet, oleamide, further analysis.
daidzein, pentadecanoic acid, and acetylcholine, were identified
as key substances (Figure 5A). We first analyzed the relationship between these six bi-
omarkers and three stress markers. The results revealed that only one substance, daidzein,
exhibited a significant correlation with the stress markers (Figure 5C). Daidzein also
served as a biomarker with a VIP > 2 in the placebo intervention group (Figure 5B). There-
fore, we chose it for further analysis.
 15, x4700
Nutrients 2023, 14, FOR PEER REVIEW 8 8of
of 12
12

B. breve
Figure 5. B. CCFM10250 s effect
breve CCFM1025′s effect on
on serum
serum metabolites.
metabolites. (A)
(A) Biomarkers
Biomarkers ofof serum
serum metabolites in
the CCFM1025 intervention group. (B) (B) Biomarkers
Biomarkers ofof serum
serum metabolites
metabolites in the
the placebo
placebo intervention
intervention
group.
group. (C) Correlation analysis between biomarkers and stress markers. (D) The relative abundance
(C) Correlation analysis between biomarkers and stress markers. (D) The relative abundance
changes
changes in
in the
the serum
serum daidzein.
daidzein. (E)
(E) The
The correlation
correlation between
between salivary
salivary cortisol
cortisol and
and daidzein.
daidzein. (F)
(F) The
The
correlation
correlation between plasma cortisol and daidzein. (G) The correlation between plasma ACTH and
between plasma cortisol and daidzein. (G) The correlation between plasma ACTH and
daidzein. * p < 0.05 in the unpaired t-tests. ns: not statistically significant.
daidzein. * p < 0.05 in the unpaired t-tests. ns: not statistically significant.

However,
However, itit is
is important
importanttotohighlight
highlightthat
thatthetherelative
relative abundance
abundance of of daidzein
daidzein in se-
in serum
rum varied differently between two groups (Figure 5D). Although
varied differently between two groups (Figure 5D). Although the content of daidzein the content of daidzein
increased
increased inin both
bothgroups
groupsafterafterintervention,
intervention,the therelative
relative abundance
abundance of of daidzein
daidzein in the
in the se-
serum
rum of participants
of participants in theinCCFM1025
the CCFM1025 groupgroup was significantly
was significantly higher higher
after after intervention
intervention than
than
beforebefore intervention
intervention (pre vs.(pre vs. tpost:
post: (19) =t 2.29,
(19) =p = 2.29, p = 0.0337,
0.0337, d = 0.738;d =Figure
0.738;5D).Figure 5D). In
In contrast,
contrast,
there wasthere was no significant
no significant differencedifference
before andbefore and after intervention
after intervention in the placebo in the placebo
group (pre
group (pre vs. post: t (19) = 0.1374, p = 0.8921,
vs. post: t (19) = 0.1374, p 0.8921, d = 0.044; Figure 5D). d = 0.044; Figure 5D).
Further
Further investigation
investigationwas wasconducted
conducted to to
examine
examine thethe
linklink
between
between stress markers
stress and
markers
the
andrelative abundance
the relative abundance of daidzein. The concentrations
of daidzein. The concentrations of stress markers,
of stress including
markers, sali-
including
vary cortisol,
salivary plasma
cortisol, plasma cortisol, andand
cortisol, plasma ACTH,
plasma exhibited
ACTH, negative
exhibited correlations
negative withwith
correlations the
relative abundance
the relative abundance of serum
of serumdaidzein, respectively
daidzein, (salivary
respectively cortisol
(salivary vs. daidzein:
cortisol vs. daidzein:R =
R = −0.41,
−0.41, p < 0.001, Figure
p < 0.001, 5E; 5E;
Figure plasma
plasmacortisol vs.vs.
cortisol daidzein: −0.36,
daidzein:R R= =−0.36, p p< <0.001,
0.001,Figure
Figure5F;5F;
daidzein: R = −0.24, p < 0.05, Figure 5G).
plasma ACTH vs. daidzein: R = −0.24, p < 0.05, Figure 5G).

4. Discussion
4. Discussion
This study
This study delved
delved into
into the
the impact
impact of Bifidobacterium breve
of Bifidobacterium breve CCFM1025
CCFM1025 on on sleep
sleep quality
quality
in individuals with insomnia. After a 4-week intervention, we observed
in individuals with insomnia. After a 4-week intervention, we observed a substantial im- a substantial
improvement
provement in sleep
in sleep quality
quality inCCFM1025
in the the CCFM1025 group,group, as evidenced
as evidenced by a significant
by a significant reduc-
reduction in their PSQI scores in comparison to the placebo group.
tion in their PSQI scores in comparison to the placebo group. This improvement This improvement
encom-
encompassed
passed not onlynot only subjective
subjective sleep quality
sleep quality but alsobutthealso the alleviation
alleviation of sleepof sleep disorders,
disorders, partic-
particularly early waking at night, often associated with abnormal cortisol
ularly early waking at night, often associated with abnormal cortisol secretion (as illus- secretion (as
illustrated in Figure
trated in Figure 3A–C). 3A–C).
Stress has
Stress hasbeen
beenfound
foundtotobebe associated
associated with
with thethe occurrence
occurrence of insomnia
of insomnia episodes,
episodes, be-
being a major contributing factor among the various potential causes of the
ing a major contributing factor among the various potential causes of the sleep disorder. sleep disorder.
When acute
When acute stressors
stressorsare
areencountered,
encountered,the theproduction
productionand andrelease
releaseofof
corticotropin-releasing
corticotropin-releas-
hormone (CRH) and arginine vasopressin (AVP) are increased, which in turn triggers the
ing hormone (CRH) and arginine vasopressin (AVP) are increased, which in turn triggers
Nutrients 2023, 15, 4700 9 of 12

release of ACTH from the anterior pituitary. ACTH then stimulates the synthesis and
release of cortisol from the adrenal cortex [28]. An overexcited and hyperactive HPA axis
can lead to wakefulness, while regulating the HPA axis can be instrumental in enhancing
sleep quality and, consequently, treating insomnia [29].
Insomnia’s onset and progression are closely linked to the dysfunction of the HPA
axis [30]. Recent studies have highlighted the relationship between changes in HPA axis
function and sleep disorders. For instance, individuals with poor sleep exhibit increased
24-h urine cortisol secretion [31], and there is a positive correlation between total waking
time and 24-h urine cortisol secretion in individuals with mild insomnia [32]. Adolescents
with insomnia show elevated plasma cortisol levels from 9 pm to 1 am [33]. These findings
underscore the interplay between alterations in HPA axis function and sleep disturbances.
In alignment with prior research on probiotics, our study did not uncover significant
differences in the levels of stress-related hormones in saliva and blood before and after the
intervention [12,34]. However, it is noteworthy that, in comparison to the placebo group,
there was a more substantial reduction in participants’ salivary and plasma cortisol levels
after the intervention with CCFM1025 (Figure 4A,B). This suggests that CCFM1025 may
possess a regulatory effect on the HPA axis, and this effect might become more pronounced
with extended intervention periods. This hypothesis is consistent with a recent meta-
analysis, which demonstrated that participants receiving probiotic therapy for eight weeks
or longer showed the greater improvements in mean PSQI score and sleep efficiency [35].
The intervention with CCFM1025 had an impact on the serum metabolites of partici-
pants. In our study, we identified a key substance, daidzein, which displayed differential
expression in the serum before and after the experimental intervention in both the probiotic
and placebo groups (Figure 5A,B). Daidzein, classified as a phytoestrogen isoflavonoid
belonging to the category of nonsteroidal estrogens, boasts a diverse array of pharmacolog-
ical effects [36]. Notably, daidzein has been implicated in the regulation of the endocrine
system [37] and functions as an antioxidant [38]. Moreover, it exerts neuroprotective effects
and exerts a beneficial impact on neurological disorders through multiple mechanisms.,
such as shielding nerve cells from death under hypoxic conditions by activating gamma
receptors [39]. It enhances memory by upregulating the expression of brain-derived neu-
rotrophic factor and stimulating the cholinergic system. It can also improve cognitive
function through the activation of the p-CREB/BDNF pathway [40,41]. In primary cultured
nerve cells, daidzein has demonstrated significant protection against beta-amyloid-induced
neurotoxicity and glutamate excitotoxicity. Recent studies have also illuminated daidzein’s
influence on the HPA axis. Chronic or sub-chronic daidzein administration has been found
to alleviate elevated serum levels of stress-related hormones, namely, cortisol and ACTH,
in both depressed and stressed model rats [42]. In our experiment, daidzein is an important
serum biomarker. Following intervention with CCFM1025, there was a significant increase
in the relative abundance of daidzein in serum (p < 0.05, Figure 5D), indicating that the
administration of CCFM1025 can, to some extent, influence the levels of daidzein in the
serum. Our correlation analysis further unveiled a negative relationship between stress
indicators and the relative abundance of serum daidzein (Figure 5C–E), suggesting that
changes in serum daidzein levels due to CCFM1025 intervention may influence HPA axis
activity, enhance awakening behavior, and consequently improve sleep quality. However,
the precise mechanisms underlying this process necessitate further exploration.
Our study has several limitations that warrant attention and should be addressed in
future research endeavors. Firstly, it is essential to increase the number of participants in
the second round of human trials, taking into account potential sex differences within the
treatment group. This step will enhance the robustness and generalizability of our findings.
Secondly, our study did not implement strict dietary controls for participants, with the
exception of antibiotic use. Consequently, we were unable to determine whether diet di-
rectly influenced serum metabolite levels. Given that the gut microbiome is predominantly
influenced by diet, it is possible that the specific impact of probiotics on the gut microbial
profile remains unclear. To obtain more meaningful insights, future investigations should
Nutrients 2023, 15, 4700 10 of 12

consider implementing rigorous dietary controls and employing shotgun metagenomics

analysis. Undoubtedly, alterations in our living environment, work, social schedules, and
biological elements can influence sleep to some extent [43]. Nevertheless, our study primar-
ily concentrated on the connection between the independent application of probiotics and
sleep quality, without addressing other potential influencing variables. In future research,
these variables should be considered. Additionally, our study collected cortisol samples
at a specific time, around 9:00 am, since it can represent the basal cortisol level [44]. To
obtain a comprehensive understanding of serum cortisol levels, it is worthwhile to consider
collecting samples at different times, as suggested in a recent study.

5. Conclusions
In line with the anticipated outcomes based on preclinical research, Bifidobacterium breve
CCFM1025 emerges as a promising candidate among psychobiotic strains for enhancing
sleep quality. The mechanisms underlying this effect may be linked to the influence of
serum metabolites. Our study contributes fundamental insights into the potential use of
psychobiotics to enhance sleep quality. However, to fully establish the therapeutic efficacy
of CCFM1025 and delve deeper into the underlying mechanisms, further investigations are
warranted. These future studies should encompass larger-scale clinical trials with varying
durations of follow-up. Such endeavors will allow for a more comprehensive assessment
of CCFM1025’s therapeutic potential and provide a detailed exploration of the mechanisms
responsible for its beneficial effects on sleep quality.

Supplementary Materials: The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/nu15214700/s1, Figure S1: Power analysis; Figure S2: Experimental
schedule; Figure S3: Quality control assessment. Table S1: Demographic characteristics of all
participants; Table S2: Statistical summary of PSQI and AIS; Table S3: Statistical summary of stress
markers. Table S4: The VIP value of compounds.
Author Contributions: Conceptualization, P.T. and J.L.; methodology, Y.L., G.Q. and X.M.; soft-
ware, Y.L.; validation, G.W. and J.Z.; data curation, J.Z.; writing—original draft preparation, Y.L.;
writing—review and editing, P.T. and J.L.; visualization, Y.L., X.M. and G.Q.; supervision, W.C.;
project administration, G.W.; funding acquisition, P.T. and G.W. All authors have read and agreed to
the published version of the manuscript.
Funding: This work was supported by the National Natural Science Foundation of China (No.
32201988, 31972052), the Natural Science Foundation of Jiangsu Province (BK20210456), China
National Postdoctoral Program for Innovative Talents (BX2021114), the Fundamental Research Funds
for the Central Universities (JUSRP123047, JUSRP22006), and the Program of Collaborative Innovation
Centre of Food Safety and Quality Control in Jiangsu Province.
Institutional Review Board Statement: The study was approved by Yixing People’s Hospital (Ap-
proval Code: Lunshen2022Ke188, Date: 24 April 2023).
Informed Consent Statement: Informed consent was obtained from all subjects participating in
the study.
Data Availability Statement: The data presented in this study are available upon request from the
corresponding author.
Conflicts of Interest: The authors report no conflict of interest.

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