ISSN 1062-3590, Biology Bulletin, 2023. © Pleiades Publishing, Inc., 2023.

ANIMAL AND HUMAN PHYSIOLOGY

Bacillus amyloliquefaciens-Inoculated GABA-Rich Rice Upregulate

Neuropeptide Y to Relieve Psychological Stress through Mediations
of GABAB Receptor and Vagus Nerves
Ching-Ju Huanga, Enkhbat Zayabaatara, Sung-Min Wanga, Sunita Kesharib, Wei-Hao Pengc,
Hsiu-Ni Kungd, and Yu-Hsiang Leea, e, *
a
Department of Biomedical Sciences and Engineering, National Central University, Taoyuan City, Taiwan R.O.C.
b
Department of Life Sciences, National Central University, Taoyuan City, Taiwan R.O.C.
c
School of Medicine, National Tsing Hua University, Hsinchu City, Taiwan R.O.C.
d Department of Anatomy and Cell Biology, College of Medicine, National Taiwan University, Taipei City, Taiwan R.O.C.
e Department of Chemical and Materials Engineering, National Central University, Taoyuan City, Taiwan R.O.C.

*e-mail: yuhsianl@ncu.edu.tw
Received June 26, 2022; revised September 19, 2022; accepted October 27, 2022

Abstract—Alteration of neuropeptide Y (NPY) in serum may cause sleep disturbance, circadian rhythm dis-
order, and/or even emotional illness. γ-Aminobutyric acid (GABA) is an inhibitory neurotransmitter and has
been widely used for reduction of depression, anxiety, and/or psychological stress since it can raise the total
or parasympathetic nerve activities in the mammalian central nervous system. However, drawbacks including
short half-life in vivo and low absorption efficiency make synthetic GABA less usability in drug formulation,
while it is more effective to uptake GABA through long term/regular daily diet. In this study, we successfully
produced GABA-enriched rice by inoculating grain seeds of Taiwanese rice with Bacillus amyloliquefaciens
(B. amyloliquefaciens) during germination, and demonstrated that the B. amyloliquefaciens-inoculated rice
(BAR) can grow with a normal root/sprout length but significantly increased germination ratio compared to
the rice without B. amyloliquefaciens treatment. Based on the animal study in association with the hole board
test, our data showed that the level of serum NPY as well as the number of head-dips of the mice treated with
the BAR for 8 days significantly enhanced 2 folds (P < 0.01) compared to the mice without GABA, but such
BAR-induced efficacies dramatically vanished as their gastrointestinal GABAB receptor and/or vagus nerves
were removed through use of GABA receptor antagonist or vagotomy, respectively. These results manifested
the potential of the BAR on anxiolytic applications and unveiled likely mediators for GABA-induced anxio-
lytic activity.

Keywords: B. amyloliquefaciens, γ-aminobutyric acid, neuropeptide Y, vagus nerves, GABA receptor, anxiety,
vagotomy
DOI: 10.1134/S1062359022700054

INTRODUCTION can increase the GABA production of a plant through

Plant microbiome is one of the key determinants
for plant growth (Berendsen et al., 2012). Over the last
decade, increasing numbers of studies have evidenced
that plant growth-promoting rhizobacteria (PGPR)
can be effectively used to increase plant health and
yields by producing the plant growth-regulating chem-
icals such as indole acetic acid (IAA), acetoin, 2,3-
butanediol, and/or metabolites such as phenylacetic
acid (PAA) and γ-aminobutyric acid (GABA)
(Taghavi et al., 2010; Gupta et al., 2021). Bacillus spe-
cies is one of the major PGPRs and has been known to
be able to promote plant growth by providing second-
ary metabolites (Reva et al., 2004; Valenzuela-Soto
et al., 2010). For example, Bacillus amyloliquefaciens
(B. amyloliquefaciens), a gram-positive soil bacteria,
conversion of L-glutamate and thus it is frequently
thought as a beneficial microbe for plant protection
and/or healthy food production (Wang et al., 2019).
GABA, a non-protein amino, is an inhibitory neu-
rotransmitter and has been widely used for reduction
of depression, anxiety and/or psychological stress
since it can raise the total or parasympathetic nerve
activities in mammalian central nervous system (Maz-
zoli and Pessione, 2016; Yoto et al., 2012; Abdou et al.,
2006). GABA can be synthesized through α-decar-
boxylation reaction of L-glutamate in the presence of
glutamate decarboxylase (GAD) (Ramesh et al.,
2015), however, drawbacks including short half-life
in vivo and low absorption efficiency make synthetic
GABA less utilization in drug formulation compared

1
2 CHING-JU HUANG et al.
to natural GABA (Rashmi et al., 2018). Furthermore,
although a variety of GABA supplements have been
offered in the market nowadays, the intake of GABA
remains short in people due to different dietary habits
and/or non-sustained uptake in daily life (Boonstra
et al., 2015). Rice is the second-most consumed cereal
grain in many regions of the world and could be enzy-
matically modified to enhance or adjust nutrient con-
tents such as GABA (Patil and Khan, 2011). In fact, it
has been demonstrated that GABA-enriched rice can
be generated by various methods such as GAD trans-
fection, fermentation, and germination (Patil and
Khan, 2011; Jannoey et al., 2010), however, the
approach by incorporation with microbial inoculation
is relatively less reported as yet.
GABA may alternatively regulate psychological
stress through induction of neuropeptide Y (NPY)
which is known as an endogenous anxiolytic mediator
(Briguglio et al., 2018). In fact, it has been demon-
strated that the NPY located within the nervous sys-
tem can influence the activity of the gastrointestinal
microbiota as well as their interactions with the gut-
brain axis (Holzer and Farzi, 2014). In the brain, the
NPY Y1 and Y2 receptors, as well as its Y4 and Y5 sub-
types are involved in multiple brain functions such as
regulating feeding behaviors, energy homeostasis, anxi-
ety and memory processing, and stress coping (Vezzani
et al., 1999). Moreover, the NPY can protect against dis-
tinct behavioral disturbances caused by peripheral
immune challenge, ameliorate the acute sickness
response, and prevent long-term depression (Holzer
et al., 2012; Thorsell et al., 2006; Breit et al., 2018).
Vagus nerve is frequently considered as the molec-
ular signaling pathway between digestive system and
brain. It is the tenth cranial nerve extending from the
brainstem to the abdomen, by which the signal deliv-
ery is mediated through activation of hypothalamic
pituitary adrenal (HPA) axis (Howland, 2014). Com-
bined neural (vagus nerves) and hormonal (HPA axis)
communications allow brain to influence the activities
of gastrointestinal cells including immune cells, epi-
thelial cells, and enteric neurons (Mayer et al., 2014).
Although the effectiveness of NPY on physiology
have been widely recognized, how the (1) GABA
receptor and (2) vagus nerves in the gastrointestinal
system regulate NPY expression remain uncertain
nowadays. In this study, first we sought to pro-
duce/cultivate GABA-rich B. amyloliquefaciens-inoc-
ulated Taiwanese rice named BAR, followed by inves-
tigating its effectiveness and the possible mediators on
anxiolytic applications stepwise through analyses of
the serum NPY expression and head-dips behaviors of
mice after treatment.
MATERIALS AND METHODS
Bacterial culture. B. amyloliquefaciens utilized in
the study were self-isolated from the human skin and
identified by 16S ribosomal RNA sequencing method
after culture in agar plate for three days. The B. amylo-
liquefaciens were routinely cultured in reinforced clos-
tridial medium under aerobic conditions at 37°C. The
bacteria was quantified by using an UV–Vis spectro-
photometer (V-650, JASCO, Easton, MD) set at
λabs = 600 nm. Sub-cultivation of the bacteria was per-
formed by 1 : 100 dilution as the value of the optical
density (OD600) was ≥1.0.
BAR production, cultivation and characterization.
The Taiwanese rice (Taiken 9) was cultivated using the
Murashige and Skoog (MS) agar (10 seeds per dish)
with pH 5.75 in the presence and absence of 105 colony
forming unit (CFU) of B. amyloliquefaciens. The cul-
ture conditions were set by 8-/16-h of light/dark cycle
under 57 ± 2% of relative humidity at 25 ± 2°C. The
root and sprout lengths of each germinated seed were
measured every 24 h for eight days. The germination
ratio (GR) of each group was calculated using the
equation (Ruan et al., 2002):
NG
GR = × 100%, (1)
N0
where N0 is the total number of seed used in the exper-
iment. NG represents the number of germinated seed at
the 8th day. To quantify the GABA content in each
group, two germinated grains from each plate were
harvested at the 8th day and were immediately sub-
jected to grinding by using a mortar and pestle under
liquid nitrogen. The quantity of GABA in each ground
rice was measured by spectrophotometry (V-650,
JASCO) set at λ = 630 nm as reported previously
(Karladee and Suriyong, 2012).
Ethical statement for animal study. In this study,
total 96 Institute of Cancer Research (ICR) mice
(CD-1® IGS Mouse, Crl:CD1 (ICR), 8–9 weeks,
female, BioLASCO Taiwan Co., Ltd., Taipei, Taiwan
R.O.C) weighing between 25–35 g were employed to
build up the in vivo BAR-assay model. Each experi-
mental mouse was reared in a single cage and fed with
standard diet and tap water ad libitum in the room,
where the housing conditions were set at room tem-
perature (25 ± 2°C) with a light/dark cycle of 12/12 h.
To evaluate and manage the conditions of detention,
the behaviors of the mice, including defensiveness,
appearance changes, abnormal vocalizations, activity,
and appetite were consistently monitored throughout
the time course.
Vagotomy. In this study, the vagotomy was per-
formed to prepare mice without vagus nerves. After
anesthetized via isoflurane inhalation (3–5% isoflu-
rane accompanied with 1.5 L/min of oxygen), the
abdomen of each experimental mouse was shaved and
sterilized, and then a 2-cm incision through the epi-
dermal tissue and peritoneum was made, by which the
ventral and dorsal branches of the vagus nerves on the
organs can be clearly identified through microscopy.
All the major components of vagus nerves including
BIOLOGY BULLETIN 2023
 BACILLUS AMYLOLIQUEFACIENS-INOCULATED GABA-RICH RICE UPREGULATE...
ventral and dorsal vagal trunks were gently removed by
using a microhook. The mice that went through stom-
ach and esophagus exposure without removal of vagus
nerves were served as the sham-vagotomized control.
All the surgical ICR mice were applied to the experi-
ment after recovery for seven days.
Hole-Board Test (HBT). The HBT was applied to
assess the anxiety-like behaviors of the mice post
treatment as reported previously (Campos et al.,
2013). It is recognized that the rodents may explore the
holes of the board by poking their heads, named head
dipping, and the number of head-dips is inversely pro-
portional to the anxiety state (Bilkei-Gorzó and
Gyertyán, 1996). In this study, the numbers of head-
dips of the mice with various treatments were counted
for continuously 5 min as performed in the previous
studies (Ibironke and Olley, 2014; Haj-Mirzaian et al.,
2019).
Administration and effect of BAR on mice. The
ground rice made by normal Taiwanese rice (Taiken 9)
or BAR post 7-days cultivation was utilized as the
feeding materials. To prepare the provender, 3 mg of
the ground rice (normal rice or BAR) in 5-mL deion-
ized water was vigorously vortex for 20 min, followed
by centrifugation with 8000 rpm in 4°C for 10 min.
The supernatant was collected, filtrated, and stored in
4°C until use.
To examine the efficacy of BAR on NPY expres-
sion in vivo, 12 ICR mice were separately fed with
BAR provender (10-mg GABA/kg), normal rice prov-
ender, commercial GABA (10-mg GABA/kg, Sigma-
Aldrich; St-Louis, MO, USA), or water through oral
administration every 24 h for eight days. The blood of
each experimental mouse was collected before sacri-
fice at day 8, and all the collected serum samples were
immediately subjected to NPY detection using the
immune assay (RayBio® Mouse NPY Enzyme
Immunoassay Kit, RayBiotech, GA, USA) according
to the manufacturer’s instructions. In addition, the
numbers of head-dips of the mice with various treat-
ments were counted for continuous 5 min after treat-
ment for 8 days.
Assessment of the effects of GABAB receptor on
NPY expression and behaviors of BAR-fed mice. 12
ICR mice were separately fed with water or BAR prov-
ender (10-mg GABA/kg) in the presence and absence
of GABAB receptor antagonists CPG46381 (0.5 mg/kg,
Tocris Bioscience, Bristol, UK) every 24 h through
oral administration for eight days. The blood of each
experimental mouse was collected at the 8th day and
all the serum samples were immediately subjected to
serum NPY assay using the RayBio® Mouse NPY
Enzyme Immunoassay Kit (RayBiotech). In addition,
the numbers of head-dips of the mice with various
treatments were counted for continuous 5 min after
treatment for 8 days.
Assessment of the effects of vagus nerves on NPY
expression and behaviors of BAR-fed mice. 12 vagoto-
BIOLOGY BULLETIN 2023
3
mized ICR mice were separately fed with the BAR
provender (10-mg GABA/kg), normal rice provender,
commercial GABA (10-mg GABA/kg), or water every
24 h through oral administration for eight days. 12
sham-vagotomized ICR mice with identical feeding
protocol were served as the comparisons. The blood of
each experimental mouse was collected at the 8th day
and all the serum samples were immediately subjected
to serum NPY assay using the RayBio® Mouse NPY
Enzyme Immunoassay Kit (RayBiotech). In addition,
the numbers of head-dips of the mice with various
treatments were counted for continuous 5 min after
treatment for 8 days.
Statistical analysis. All data were acquired from
≥three independent experiments and were presented
as the mean ± standard error (SE). Statistical analyses
were conducted using the MedCalc software (Version
17.2) in which comparisons for one condition between
two groups were performed by Student’s t-test with
significance levels of *P < 0.05, **P < 0.01, and ***P <
0.001 throughout the study.
RESULTS
Effects of B. amyloliquefaciens on Rice Germination
and GABA Production
Figure 1a shows the result of 16S ribosomal RNA
sequencing of the bacteria isolated from the human
skin. It reveals that their genome was 99.79% identical
to the B. amyloliquefaciens obtained from the ATCC
(ATCC® 23350™), demonstrating that the self-iso-
lated microbe was surely B. amyloliquefaciens. The iso-
lated B. amyloliquefaciens was then applied to rice
inoculation and the cultivation scenarios were photo-
graphically detected after handling for eight days. In
comparison to the group without B. amyloliquefaciens
(Fig. 1b), it can be seen that the inoculated B. amyloliq-
uefaciens formed colonies and those were attached on the
surfaces of the seed and roots as shown in Fig. 1c. The
colonized B. amyloliquefaciens exhibited regular rod
shape according to the SEM shown in Fig. 1d.
The B. amyloliquefaciens did not influence the
lengths of germinated root and sprout as plotted in
Figs. 1e, 1f, respectively. However, the germination
ratio and the GABA content of the BAR significantly
increased 75% (P < 0.001; Fig. 1g) and two folds (P <
0.001; Fig. 1h) compared to the rice without inocula-
tion of B. amyloliquefaciens after cultivation for eight
days, suggesting that B. amyloliquefaciens is highly
potential for use in development of GABA-rich plants
and/or foods.
Effects of BAR on NPY expression and behavioral
changes of mice. To examine the efficacy of the BAR
on anxiolytic application, both serum NPY level and
head-dipping behavior of the mice were measured
after fed with the BAR for eight days. As shown in
Fig. 2a, the mice fed with GABA or BAR exhibited
1.5- (P < 0.01) and 2.1- (P < 0.001) fold higher NPY
4 CHING-JU HUANG et al.

(a) (e) (f)

Normal rice
50 50

Sprout length, mm
BAR

Root length, mm
40 40
30 30
(b) 20 20
10 10

0 2 4 6 8 0 2 4 6 8
Days Days

GABA production, mg/g rice

(g) (h)
100

Germination ratio, %
(c) 10 ***
***
80 8
60 6
40 4
20 2
(d) 0 0
Normal BAR Normal BAR
rice rice

Fig. 1. Effect of B. amyloliquefaciens on germination and GABA production of the BAR. (a) Genomic sequence of the self-iso-
lated B. amyloliquefaciens utilized in this study. (b and c) Photomicrographs of the germinated grains cultivated without (b) and
with (c) inoculation of B. amyloliquefaciens. Scale bar = 2 mm. Arrows in (c) denote the bacterial colonies located on the roots
and surrounding MS agar. (d) SEM image of the colonized B. amyloliquefaciens. Scale bar = 2 μm. (e and f) Root (e) and sprout
(f) lengths of the rice cultivated with and without inoculation of B. amyloliquefaciens during eight days. Values are presented as
mean ± SE (n = 3). (g and h) GR (g) and GABA content (h) of the rice cultivated with and without inoculation of B. amyloliq-
uefaciens after eight days. † BA represents B. amyloliquefaciens. Values are the mean ± SE (n = 3). *** P < 0.001.

level compared to the mice treated with normal rice,
while the NPY level in the latter was similar to the
value gained form the group with water (P = NS). In
addition, the number of head dipping was significantly
increased in the BAR-treated mice compared to the
one treated with normal rice as plotted in Fig. 2b. We
surmise that the NPY level in circulation was effec-
tively upregulated by GABA through feeding with
BAR and consequently rendered a low anxiety behav-
ior (i.e., more head-dipping activity) to the mice.
Effects of GABAB Receptor on NPY Expression
and Behavioral Changes of BAR-fed Mice
To identify whether the efficacies of increased NPY
and head-dips in BAR-treated mice were truly
attributed to GABA, the above effects were repeatedly
examined using the mice with and without GABAB
receptor expression. As shown in Fig. 3a, the BAR-fed
mice reproducibly exhibited enhanced NPY expres-
sion in serum compared to those treated with water
(P < 0.01), while such BAR-mediated NPY enhance-
ment was dramatically inhibited after the mice were con-
currently fed with BAR and CPG46381 for eight days.
Similarly, the mice with BAR + CPG46381
showed markedly decreased number of head-dips
compared to the BAR-treated ones without GABAB
receptor antagonist, and the number in the former was
even similar to the result gained from the group with
water alone (P = NS, Fig. 3b). These results manifest
that the GABAB receptor indeed plays a crucial role in
BAR-mediated NPY upregulation and subsequent
behavioral changes.
Effects of Vagus Nerves on NPY Expression
and Behavioral Changes of BAR-fed Mice
The vagus nerve is an essential part of the brain-gut
axis and has been known to be involved in modulation
of inflammation, digestion, and intestinal homeosta-
sis (Breit et al., 2018). Study from Bravo et al. further
demonstrated that the lactobacillus strain can provide
anti-depressant functionality to mice by regulating the
GABAB receptor through vagus nerves, showing that
the vagus nerves play an important role in communi-
cation between gut and brain (Bravo et al., 2011). In
this study, the effects of vagus nerves on BAR-medi-
ated reactions including NPY expression and behav-
ioral changes were examined by using the mice with
vagotomy. As shown in Fig. 4, we found that the BAR-
fed mice with vagotomy exhibited dramatically
decreased NPY level (P < 0.001; Fig. 4a) and head-
dipping number (P < 0.001; Fig. 4b) compared to the
mice with sham surgery under equal feeding protocol,
and both data in the latter group were even similar to
the results gained from the vagotomized mice with
normal rice. These results indicate that vagus nerves
indeed paly an essential role in BAR/GABA-mediated
NPY upregulation and behavioral changes.

BIOLOGY BULLETIN 2023

 BACILLUS AMYLOLIQUEFACIENS-INOCULATED GABA-RICH RICE UPREGULATE... 5

(a) (a)
*** **
15 15 ***
**

NPY, ng/mL
NPY, ng/mL

10 10

5 5

0 0
Normal BAR H2O GABA H2 O BAR H2O BAR
rice + +
CPG46381 CPG46381
b) (b)
**
** ***
15 15
***
Number of head-dips

Number of head-dips
10 10

5 5

0 0
Normal BAR H2O
rice
Fig. 2. Effects of the BAR on NPY expression and head-
dipping behavior of the mice. (a) The serum NPY levels of
the mice with various treatments at the 8th day. (b) The
numbers of head-dips of the mice with various treatments
at the 8th day. The dose of GABA for mice feeding was set
as 10-mg GABA/kg. Values are the mean ± SE (n = 3).
** P < 0.01, *** P < 0.001. † BA represents B. amyloliquefa-
ciens.
DISCUSSION
In this study, we have successfully isolated B. amy-
loliquefaciens from human skin and demonstrated that
such microorganism can be applied to produce high-
GABA plants (e.g., BAR) through inoculation with
the seed. In addition, our data showed that oral
administration of BAR to mice (10-mg GABA/kg)
may significantly elevate their serum NPY level and
increase head-dipping number after treatment for
eight days. We further demonstrated that both GABAB
receptor and vagus nerves are the potential intermedi-
ators for the BAR-induced NPY upregulation and
anxiolytic behavioral changes through animal assays.
To the best of our knowledge, this is the first report
showing the production of B. amyloliquefaciens-ino-
culated GABA-enriched rice and their potential in
anxiolytic applications.
Based on the similar 16S ribosomal RNA sequence
and SEM morphology compared to the commercial
B. amyloliquefaciens, our self-isolated bacterial species
GABA H2O BAR H2O BAR
+ +
CPG46381 CPG46381
Fig. 3. Effects of GABAB receptor on NPY expression and
head-dipping behavior of the mice. (a) The serum NPY
levels of the mice after fed with the BAR or water in the
presence and absence of CPG46381 for eight days. (b) The
numbers of head-dips of the mice after fed with the BAR
or water in the presence and absence of CPG46381 for
eight days. The dose of GABA provided by the BAR in
mice feeding was set as 10-mg GABA/kg. Values are the
mean ± SE (n = 3). ** P < 0.01. † BA represents B. amylo-
liquefaciens.
was confirmed as the B. amyloliquefaciens. According
to the reports of clinical studies, uptake of GABA
through regular diet using rhizobacteria-induced
GABA-rich plants are generally considered as an effi-
cient, safe, and inexpensive approach for relieving
depression and/or anxiety compared to medicinal
therapy (Briguglio et al., 2018; Hinton et al., 2019). So
far, a variety of microbes such as Fusarium gramin-
earum (Feehily and Karatzas, 2013), Lactobacillus
paracasei (Dhakal et al., 2012), and B. amyloliquefa-
ciens (Suwanmanon and Hsieh, 2014) have been
known to be able to enhance GABA content in plants,
of which bacillus species can usually provide higher
efficiency of GABA production than the others (Jung
et al., 2019). Moreover, bacillus species such as
B. amyloliquefaciens are able to protect germination as
well as growth of the inoculated rice under various abi-
otic stresses and/or phytohormone treatments that

BIOLOGY BULLETIN 2023

6 CHING-JU HUANG et al.

(a) et al., 2015). In fact, there are abundant of neuropils

Sham surgery composed of GABA in the spinal cord and hypothala-
15 Vagotomy mus (Brumovsky et al., 2007), and the NPY is synthe-
*** sized by neurons of the arcuate nucleus and secreted
** from the paraventricular nucleus (Sah et al., 2007).
GABA is expressed in the subpopulation of NPY neu-
NPY, ng/mL

10
rons in the arcuate nucleus and the interactions of
NPY and GABA in the hypothalamus could be con-
trolled by feeding (Sah et al., 2007). On the other
5
hand, effects of GABA are mainly dependent on the
activations of ionotropic (GABAA and GABAC) and
metabotropic (GABAB) receptors while they are rou-
0
H2 O GABA Normal BAR tinely regulated by both excitatory and inhibitory sig-
rice nals in the enteric nervous system. Moreover, previous
studies showed that chronic anxiety- and panic-like
(b)
*** behaviors in mice can be relieved through use of the
15 GABAB receptor allosteric modulator, indicating that
**
the GABAB receptor is a potential target for treating
Number of head-dips

depression (Pin and Prézeau, 2007; Ghose et al.,

10
5 dips number, while those efficacies dramatically
0 aforementioned favorable effects of the BAR/GABA
H2 O GABA Normal BAR
rice
Fig. 4. Effects of vagus nerves on GABA-mediated NPY
expression and head-dips behavior of the mice. (a) The
serum NPY levels of the vagotomized or sham surgery-
treated mice after fed with BAR, normal rice, water, or
commercial GABA for eight days. (b) The numbers of
head-dips of the vagotomized or sham surgery-treated
mice after fed with BAR, normal rice, water, or commer-
cial GABA for eight days. The dose of GABA in mice feed-
ing was set as 10-mg GABA/kg. Values are the mean ± SE
(n = 3). ** P < 0.01, *** P < 0.001. † BA represents B. amy-
loliquefaciens.
make it a good choice of rhizobacteria for production
of GABA-rich plants (Tiwari et al., 2017). In this
study, we found that during the growth of BAR,
GABA was first detected in the roots and the GABA
content in the roots was higher than that in the shoots,
conferring it an increased GR as reported in a previous
study (Isaji et al., 2018). We reason that the enhanced
GR (Fig. 1g) and GABA content (Fig. 1h) in BAR
were likely attributed to such B. amyloliquefaciens-
mediated growth regulation as well as protection on
seedlings.
NPY family is one of the most abundant peptides
expressed in the whole gut-brain axis. In the healthy
human subjects, serum NPY level rises in response to
anxiety and/or depression in order to modulate psy-
chological stress (Morgan III et al., 2002; Enman
2011). In this study, we demonstrated that the BAR
was able to provide anxiolytic effects to mice including
upregulated serum level of NPY and increased head-
declined after fed with the GABAB receptor antago-
nists CPG46381 for eight days, denoting that the
were likely attributed to GABAB receptor activity
accordingly.
The central nervous system is indirectly connected
with the gastrointestinal system through enteric ner-
vous system and the vagus nerves, while vagus nerves
can directly transmit gastrointestinal/physiological
messages to brain (Cryan and O’Mahony, 2011;
Thayer and Sternberg, 2009). In this study, our data
showed that the vagotomized mice did not exhibit
increased head-dips behavior even after fed with the
BAR, manifesting that vagus nerves play a key role in
GABA-mediated anxiolytic effect. Furthermore, we
interestingly found that in addition to the behavioral
changes described above, both BAR and commercial
GABA were not able to enhance the NPY level in vag-
otomized mice but were effective on sham groups,
implying that interactions between GABA and NPY
were also potentially modulated by vagus nerves.
Taken together, we hypothesized that the mechanism
of GABA-mediated anxiolytic effect occurred in
BAR-fed mice was carried out through activation of
GABAB receptor located on the vagus nerves due to
uptake of GABA, by which NPY was upregulated and
led to increased head-dips number thereafter. To clar-
ify how the vagus nerves influence the NPY activation
through GABAB signaling, more studies are certainly
needed and efforts are currently in progress.

BIOLOGY BULLETIN 2023

 BACILLUS AMYLOLIQUEFACIENS-INOCULATED GABA-RICH RICE UPREGULATE...
ACKNOWLEDGMENTS
This work was financially supported by the Veterans
General Hospitals and University System of Taiwan Joint
Research Program (VGHUST111-G4-1-1; Y.-H. Lee).
COMPLIANCE WITH ETHICAL STANDARDS
Conflict of interest. The authors declare that they have no
conflicts of interest.
Statement on the welfare of animals. All the animal protocols
including the daily care and operation of laboratory mice were
in accordance with the guidelines approved by the National
Central University (Approval number: NCU-106-016.
Approval Date: December 19, 2017).
REFERENCES
Abdou, A.M., Higashiguchi, S., Horie, K., Kim, M., Hat-
ta, H., and Yokogoshi, H., Relaxation and immunity en-
hancement effects of gamma-aminobutyric acid (GABA)
administration in humans, Biofactors, 2006, vol. 26,
pp. 201–208.
Berendsen, R.L., Pieterse, C.M., and Bakker, P.A., The
rhizosphere microbiome and plant health, Trends Plant
Sci., 2012, vol. 17, pp. 478–486.
Bilkei-Gorzo, A. and Gyertyan, I., Some doubts about the
basic concept of hole-board test, Neurobiology, 1996, vol. 4,
pp. 405–415.
Boonstra, E., de Kleijn, R., Colzato, L.S., Alkemade, A.,
Forstmann, B.U., and Nieuwenhuis, S., Neurotransmitters
as food supplements: the effects of GABA on brain and be-
havior, Front. Psychol., 2015, vol. 6, p. 1520.
Bravo, J.A., Forsythe, P., Chew, MV., Escaravage, E., Savi-
gnac, H.M., Dinan, T.G., Bienenstock, J., and Cryan, J.F.,
Ingestion of Lactobacillus strain regulates emotional behav-
ior and central GABA receptor expression in a mouse via
the vagus nerve, Proc. Natl. Acad. Sci. U. S. A., 2011,
vol. 108, pp. 16050–16055.
Breit, S., Kupferberg, A., Rogler, G., and Hasler, G., Vagus
nerve as modulator of the brain–gut axis in psychiatric and
inflammatory disorders, Front. Psychiatry, 2018, vol. 9,
p. 44.
Briguglio, M., Dell’Osso, B., Panzica, G., Malgaroli, A.,
Banfi, G., Zanaboni, Dina, C., Galentino, R., and Porta, M.,
Dietary neurotransmitters: a narrative review on current
knowledge, Nutrients, 2015, vol. 10, p. 591.
Brumovsky, P., Shi, T.S., Landry, M., Villar, M.J., and
Hökfelt, T., Neuropeptide tyrosine and pain, Trends Phar-
macol. Sci., 2007, vol. 28, pp. 93–102.
Campos, A.C., Fogaça, M.V., Aguiar, D.C., and Guimar-
aes, F.S., Animal models of anxiety disorders and stress,
Braz. J. Psychiatry, 2013, vol. 35, suppl. 2, pp. S101–S11.
Cryan, J.F. and O’Mahony, S.M., The microbiome–gut–
brain axis: from bowel to behavior, Neurogastroenterol. Mo-
til., 2011, vol. 23, pp. 187–192.
Dhakal, R., Bajpai, V.K., and Baek, K.H., Production of
GABA (γ-aminobutyric acid) by microorganisms: a review,
Braz. J. Microbiol., 2012, vol. 43, pp. 1230–1241.
Enman, N.M., Sabban, E.L., McGonigle, P., and Van
Bockstaele, E.J., Targeting the neuropeptide Y system in
BIOLOGY BULLETIN 2023
7
stress-related psychiatric disorders, Neurobiol. Stress, 2015,
vol. 1, pp. 33–43.
Feehily, C. and Karatzas, K.A.G., Role of glutamate me-
tabolism in bacterial responses towards acid and other
stresses, J. Appl. Microbiol., 2013, vol. 114, pp. 11–24.
Ghose, S., Winter, M.K., McCarson, K.E., Tamminga, C.A.,
and Enna, S.J., The GABAB receptor as a target for antide-
pressant drug action, Br. J. Pharmacol., 2011, vol. 162,
pp. 1–17.
Gupta, K., Dubey, N.K., Singh, S.P., Kheni, J.K., Gupta, S.,
and Varshney, A., Plant growth-promoting rhizobacteria
(PGPR): current and future prospects for crop improve-
ment, in Current Trends in Microbial Biotechnology for Sus-
tainable Agriculture, Singapore: Springer, 2021, pp. 203–
226.
Haj-Mirzaian, A., Nikbakhsh, R., Ramezanzadeh, K., Re-
zaee, M., Amini-Khoei, H., Haj-Mirzaian, A., Ghesmati, M.,
Afshari, K., Haddadi, N.S., and Dehpour, A.R., Involve-
ment of opioid system in behavioral despair induced by so-
cial isolation stress in mice, Biomed. Pharmacother., 2019,
vol. 109, pp. 938–944.
Hinton, T., Jelinek, H.F., Viengkhou, V., Johnston, G.A.,
and Matthews, S., Effect of GABA-fortified oolong tea on
reducing stress in a university student cohort, Front. Nutr.,
2019, vol. 6, p. 27.
Holzer, P., Reichmann, F., and Farzi, A., Neuropeptide Y,
peptide YY and pancreatic polypeptide in the gut–brain ax-
is, Neuropeptides, 2012, vol. 46, pp. 261–274.
Holzer, P. and Farzi, A., Neuropeptides and the microbio-
ta–gut–brain axis, Adv. Exp. Med. Biol., 2014, vol. 817,
pp. 195–219.
Howland, R.H., Vagus nerve stimulation, Curr. Behav.
Neurosci. Rep., 2014, vol. 1, pp. 64–73.
Ibironke, G.F. and Olley, S.M., Cholinergic modulation of
restraint stress induced neurobehavioral alterations in mice,
Afr. J. Biomed. Res., 2014, vol. 17, pp. 181–185.
Isaji, S., Yoshinaga, N., Teraishi, M., Ogawa, D., Kato, E.,
Okumoto, Y., Habu, Y., and Mori, N., Biosynthesis and ac-
cumulation of GABA in rice plants treated with acetic acid,
J. Pestic. Sci., 2018, vol. 43, pp. 214–219.
Jannoey, P., Niamsup, H., Lumyong, S., Suzuki, T., Kata-
yama, T., and Chairote, G., Comparison of gamma-ami-
nobutyric acid production in Thai rice grains, World J. Mi-
crobiol. Biotechnol., 2010, vol. 26, pp. 257–263.
Jung, W.Y., Kim, S.G., Kim, H.K., Huh, S.Y., Kim, D.W.,
Yoon, D.U., Yang, C.H., Kim, H.Y., and Jang, E.Y., The
effect of oral administration of black sticky rice with giant
embryo on brain GABA concentrations, Psychiatry Invest.,
2019, vol. 16, pp. 615–620.
Karladee, D. and Suriyong, S., γ-Aminobutyric acid (GABA)
content in different varieties of brown rice during germination,
Sci. Asia, 2012, vol. 38, pp. 13–17.
Mayer, E.A., Savidge, T., and Shulman, R.J., Brain–gut
microbiome interactions and functional bowel disorders,
Gastroenterology, 2014, vol. 146, pp. 1500–1512.
Mazzoli, R. and Pessione, E., The neuro-endocrinological
role of microbial glutamate and GABA signaling, Front. Mi-
crobiol., 2016, vol. 7, p. 1934.
Morgan, C.A. 3rd, Rasmusson, A.M., Wang, S., Hoyt, G.,
Hauger, R.L., and Hazlett, G., Neuropeptide-Y, cortisol,
and subjective distress in humans exposed to acute stress:
8 CHING-JU HUANG et al.
replication and extension of previous report, Biol. Psychia-
try, 2002, vol. 52, pp. 136–142.
Patil, S.B. and Khan, M., Germinated brown rice as a value
added rice product: a review, J. Food. Sci. Technol., 2011,
vol. 48, pp. 661–667.
Pin, J.P. and Prézeau, L., Allosteric modulators of GABAB
receptors: mechanism of action and therapeutic perspec-
tive, Curr. Neuropharmacol., 2007, vol. 5, pp. 195–201.
Ramesh, S.A., Tyerman, S.D., Xu, B., Bose, J., Kaur, S.,
Conn, V., Domingos, P., Ullah, S., Wege, S., Shabala, S.,
Feijó, J.A., Ryan, P.R., and Gilliham, M., GABA signalling
modulates plant growth by directly regulating the activity of
plant-specific anion transporters, Nat. Commun., 2015,
vol. 6, p. 7879.
Rashmi, D., Zanan, R., John, S., Khandagale, K., and Na-
daf, A., γ-Aminobutyric acid (GABA): biosynthesis, role,
commercial production, and applications, Stud. Nat. Prod.
Chem., 2018, vol. 57, pp. 413–452.
Reva, O.N., Dixelius, C., Meijer, J., and Priest, F.G., Tax-
onomic characterization and plant colonizing abilities of
some bacteria related to Bacillus amyloliquefaciens and Ba-
cillus subtilis, FEMS Microbiol. Ecol., 2004, vol. 48,
pp. 249–259.
Ruan, S., Xue, Q., and Tylkowska, K., Effects of priming on
germination and health of rice (Oryza sativa L.) seeds, Seed
Sci. Technol., 2002, vol. 30, pp. 451–458.
Sah, R., Parker, S.L., Sheriff, S., Eaton, K., Balasubrama-
niam, A., and Sallee, F.R., Interaction of NPY compounds
with the rat glucocorticoid-induced receptor (GIR) reveals
similarity to the NPY-Y2 receptor, Peptides, 2007, vol. 28,
pp. 302–309.
Suwanmanon, K. and Hsieh, P.C., Isolating Bacillus subtilis
and optimizing its fermentative medium for GABA and nat-
tokinase production, CyTA—J. Food, 2014, vol. 12, pp. 282–
290.
Taghavi, S., van der Lelie, D., Hoffman, A., Zhang, Y.B.,
Walla, M.D., Vangronsveld, J., Newman, L., and Monchy, S.,
Genome sequence of the plant growth promoting endo-
phytic bacterium Enterobacter sp. 638, PLoS Genet., 2010,
vol. 6, p. e1000943.
Thayer, J.F. and Sternberg, E.M., Neural concomitants of
immunity—focus on the vagus nerve, Neuroimage, 2009,
vol. 47, pp. 908–910.
Thorsell, A., Slawecki, C.J., El Khoury, A., Mathe, A.A.,
and Ehlers, C.L., The effects of social isolation on neuro-
peptide Y levels, exploratory and anxiety-related behaviors
in rats, Pharmacol. Biochem. Behav., 2006, vol. 83, pp. 28–
34.
Tiwari, S., Prasad, V., Chauhan, P.S., and Lata, C., Bacillus
amyloliquefaciens confers tolerance to various abiotic stress-
es and modulates plant response to phytohormones through
osmoprotection and gene expression regulation in rice,
Front. Plant Sci., 2017, vol. 8, p. 1510.
Valenzuela-Soto, J.H., Estrada-Hernández, M.G., Ibarra-
Laclette. E., and Délano-Frier, J.P., Inoculation of tomato
plants (Solanum lycopersicum) with growth-promoting Ba-
cillus subtilis retards whitefly Bemisia tabaci development,
Planta, 2010, vol. 231, pp. 397–410.
Vezzani, A., Sperk, G., and Colmers, W.F., Neuropeptide Y:
emerging evidence for a functional role in seizure modula-
tion, Trends Neurosci., 1999, vol. 22, pp. 25–30.
Wang, H., Huang, J., Sun, L., Xu, F., Zhang, W., and
Zhan, J., An efficient process for co-production of γ-ami-
nobutyric acid and probiotic Bacillus subtilis cells, Food Sci.
Biotechnol., 2018, vol. 28, pp. 155–163.
Yoto, A., Murao, S., Motoki, M., Yokoyama, Y., Horie, N.,
Takeshima, K., Masuda, K., Kim, M., and Yokogoshi, H.,
Oral intake of γ-aminobutyric acid affects mood and activ-
ities of central nervous system during stressed condition in-
duced by mental tasks, Amino Acids, 2012, vol. 43, pp. 1331–
1337.
BIOLOGY BULLETIN 2023