Anti-hyperlipidemic effect of soybean extract fermented by Bacillus subtilis MORI in db/db mice.

Abstract

The purpose of this study was to investigate the anti-hyperlipidemic effect of soy bean extract solution fermented by Bacillus subtilis MORI (BTD-1E) in obese db/db mice. Eight-week-old male db/db mice were administered 33.3 mg/kg BTD-1E solution orally once a day for four weeks. The BTD-1E group showed significantly lower body weight compared with the db control group (P<0.05). The BTD-1E group showed significantly lower serum total cholesterol and LDL cholesterol levels compared with the db control group, respectively (P<0.05, P<0.01). The BTD-1E group showed significantly decreased liver weight relative to final body weight compared with the db control group (P<0.01). After four weeks of BTD-1E administration, lipid droplets in the liver were apparently decreased in the BTD-1E group compared to the db control group. In summary, our results suggest that BTD-1E has an anti-hyperlipidemic effect in the obese mouse model.

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Anti-hyperlipidemic effect of soybean extract fermented by
Bacillus subtilis MORI in db/db mice
YoonYi Nam1, Harry Jung2, Sankarapandian Karuppasamy2, Jae-Yeon Lee3, Kyung-Don Kang3,
Kyo-Yeol Hwang3, Su-Il Seong3,4, Jun-Gyo Suh1,2*
1Institute of Natural Medicine, Hallym University, Chuncheon, Korea
2Department of Medical Genetics, College of Medicine, Hallym University, Chuncheon, Korea
3R&D Center for Life Science, Biotopia Co., Ltd., Chuncheon, Korea
4Department of Life Science, University of Suwon, Hwaseong, Korea
The purpose of this study was to investigate the anti-hyperlipidemic effect of soy bean extract solution
fermented by Bacillus subtilis MORI (BTD-1E) in obese db/db mice. Eight-week-old male db/db mice were
administered 33.3 mg/kg BTD-1E solution orally once a day for four weeks. The BTD-1E group showed
significantly lower body weight compared with the db control group (P<0.05). The BTD-1E group showed
significantly lower serum total cholesterol and LDL cholesterol levels compared with the db control group,
respectively (P<0.05, P<0.01). The BTD-1E group showed significantly decreased liver weight relative to
final body weight compared with the db control group (P<0.01). After four weeks of BTD-1E
administration, lipid droplets in the liver were apparently decreased in the BTD-1E group compared to
the db control group. In summary, our results suggest that BTD-1E has an anti-hyperlipidemic effect in
the obese mouse model.
Key words: Fermented soybean extract, Bacillus subtilis MORI, 1-deoxynojirimycin, lipid-lowering effect, anti-
obesity
Received 10 May 2012; Revised version received 8 June 2012; Accepted 11 June 2012
For the past decades, obesity has become a serious
worldwide health problem largely due to high caloric
intake and insufficient exercise [1]. Physiologically obesity
is a disorder of energy balance and is fundamentally
considered as a disorder of lipid metabolism [2]. It has
been reported that obesity has a large effect on
lipoprotein metabolism, leading to elevated levels of
low-density lipoprotein cholesterol (LDL-C) and total
cholesterol (T-CHOL) [3,4]. Many health risks such as
heart disease, type 2 diabetes mellitus (T2DM), stroke,
hypertension, osteoarthritis, fatty liver disease, and some
forms of cancer are observed to be associate with excess
weight [5,6]. Life-insurance data and epidemiological
studies confirm that increasing degrees of overweight
and obesity are important predictors of mortality [6,7].
According to a recent report from the World Health
Organization (WHO), it was estimated that 45% of
Korean men and 54% of women were overweight in
2005, and the percentages are expected to increase to
66% and 67%, respectively by 2015 [8]. Although diet
control and lifestyle changes remain the first steps in
obesity management, lifestyle modification (alone) as a
treatment for obesity is widely regarded as ineffective
[9]. Thus, the use of anti-obesity drugs is potentially
important as a supplement to lifestyle modification [10].
At present, several medications are available for
obesity treatment: Orlistat, a gastric and pancreatic lipase
inhibitor [11]; Sibutramine, a centrally acting monoamine-
reuptake inhibitor [12]; Rimonabant, the first endo-
cannabinoid (CB1)-receptor blocker [13]. Despite the
Lab Anim Res 2012: 28(2), 123-129
http://dx.doi.org/10.5625/lar.2012.28.2.123
*Corresponding author: Jun-Gyo Suh, Department of Medical Genetics, College of Medicine, Hallym University, 1 Hallymdaehakgil,
Chuncheon, Gangwon 200-702, Korea
Tel: +82-33-248-2692; Fax: +82-33-248-2696; E-mail: jgsuh@hallym.ac.kr
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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anti-obesity effects, some critical adverse effects of these
medications such as gastro-intestinal effects [14],
increases in blood pressure and pulse rate [15], and
increased incidence of mood-related disorder are
reported [16].
1-deoxynojirimycin (DNJ) is known to be a strong
intestinal α-glucosidase inhibitor (AGI) [17]. It has been
reported that DNJ is a constituent part of mulberry
leaves, dietary mulberry, and is also produced by several
Bacillus and Streptomyces species isolated from soil
[18,19]. DNJ potently inhibits α-glucosidase in the small
intestine, binding to the active center of α-glucosidase
[20]. It has been reported that DNJ reduces atherogenicity
of LDL-C in patients with impaired glucose tolerance
[21]. Cheik et al. have shown that soy product fermented
by Enterococcus faecium and Lactobacillus Jugurti
significantly decreases the lipogenesis rate and increases
in lipolysis rate in tissue adipocytes and liver in rats with
hypercholestrolemic diet [22]. The preventive effect of
DNJ and AGI on T2DM has been extensively studied
[23-24].
Recently, a paper has reported that a new species
isolated from Bacillus subtilis MORI produces both AGI
and DNJ in high quantities, and is present in traditionally
fermented Korean soybean [25]. The DNJ content and
AGI activity of Bacillus subtilis MORI-fermented soybean
extract (BTD-1) were approximately 5-fold higher than
those of mulberry leaves [26,27]. Our previous study
showed the glucose-lowering effect of BTD-1 in db/db
mice. However, the hypolipidemic effect of BTD-1 has
not been completely studied.
The present study was thus designed to investigate the
lipid-lowering effect of water extract of BTD-1 (BTD-
1E) in db/db mice.
Materials and Methods
Animals
Twelve male db/db mice (five weeks old, Central Lab.
Animal, Inc., Seoul, Korea) were used after a three
weeks acclimatization period. The mice were divided
into two groups of six. The db control group was
administered with 0.9% saline only, whereas the BTD-
1E group was administered with 33.3 mg/kg of BTD-1E
solution orally once a day for four weeks. The animals
were maintained in the experimental animal center at
Hallym University, Korea. The facility was maintained
at 22±2oC, in 55±10% relative humidity, and an
artificial 12 h light and 12 h dark cycle was maintained.
A normal rodent pellet diet (Jeiljedang, Seoul, Korea)
and water were supplied ad libitum. This animal study
was conducted in accordance with the guidelines and the
approval of the Institutional Animal Care and Use
Committee of Hallym University (Hallym-2010-74).
Preparation of the BTD-1E
BTD-1E was supplied by Biotopia Co. (Chuncheon,
Korea). The 5% (w/v) defatted soybean meal was
fermented for five days at 37oC by Bacillus subtilis
MORI (KCCM-10450P), which had been isolated from
chungkookjang, a Korean traditional food. BTD-1
powder was made into a spray by drying the culture
supernatant and the excipient so that they were 1:1 (v/v).
Twenty grams of BTD-1 powder was well mixed with
100 mL distilled water. The mixture was extracted for
1 hr at 60oC in a waterbath. Then the reactant was
centrifuged at 10,000 g for 10 min. The supernatant of
BTD-1E was used in this animal study. The DNJ
component of BTD-1 powder is 0.944±0.04% (w/w).
The α-glucosidase inhibitor unit of BTD-1 was 593,200
GIU/g and the 50% inhibitory concentration (IC50) was
1.7µg/mL. The dosage of the active ingredients in BTD-
1E was considered with the differences between human
(60 kg adult) and mouse metabolic in mind. The dosage
of oral administration of BTD-1E was 2,000 GIU/kg
(mouse body weight) of GIU and the DNJ concentration
was 0.37 mg/kg.
Measurement of body weight and diet intake
Throughout the four week experimentation period,
body weight and food intake were measured weekly.
Serum lipids analyses
Blood was collected from the orbital plexus after an
overnight fast (15 h), weekly. We measured T-CHOL,
LDL-C, HDL cholesterol (HDL-C), and triglyceride
(TG). For these measurements we used a biochemical
analyzer (Kornelab20XT, Thermo, Espoo, Finland) with
commercial reagents from Thermo Fisher Scientific
(Espoo, Finland).
Organ weight
Organ weights, including that of the liver, kidney,
testis, spleen, and epiderminal fats (EPDF) were carefully
separated and weighed. The relative organ weight (%)
was calculated based on final body weight.

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Histopathological analysis by Oil Red O staining in
liver
At the end of the experiment, livers were collected
from mice under anesthesia with diethyl ether after 15 h
of fasting. The livers were fixed in 4% paraformaldehyde
for 24 h. The fixed livers were then transferred to a 20%
sucrose solution for 48 h. Then the livers were treated
with OCT compound for 24h. The tissues were processed
routinely for frozen-sectioned 10 µm in thickness and
stained with Oil Red O solution (O-0625, SIGMA-
ALDRICH, St. Louis, USA). For nucleus staining, the
tissues were stained with hematoxylin and eosin
according to general procedures. Morphology of the
livers was examined under an inverted microscope
(ZEISS, Oberkochen, Germany) and the images were
captured using the Axiovision Rel. 4.7 program.
Statistical analysis
Data are presented as the mean±SDM. To determine
statistical significance, data were analyzed using student’s
t test. A value of P<0.05 was considered to be
statistically significant.
Results
Change of body weight and food consumption
Body weight and food consumption were measured
once a week during the experimental period. BTD-1E
treatment significantly decreased mean body weight in
the mice throughout the experimental period compared
with that of db control group (P<0.05) (Figure 1A).
Average food consumption was slightly higher in the
BTD-1E group compared with the db control group
(Figure 1B). The difference in the food consumption of
the mice treated with BTD-1E was not significant when
compared with that of the db control group.
Serum lipid profiles
The contents of various types of lipid in serum
including T-CHOL, LDL-C, HDL-C, and TG were
analyzed in the db/db mice treated with the BTD-1E
solution. At 3 and 4 weeks, the BTD-1E group showed
significantly lower T-CHOL levels: 74.6 and 64.8% of
that of the db control group, respectively (P<0.05)
(Figure 2A). At 2, 3, and 4 weeks, the BTD-1E group
showed significantly lower LDL-C levels: 63.4, 39.4,
and 64.8% of the db control group, respectively (P<0.05,
P<0.01, P<0.01) (Figure 2B). The HDL-C level was
slightly lower in the BTD-1E group compared with the
db control group (Figure 2C). TG level was slightly
higher in the BTD-1E group compared with the db
control group (Figure 2D). The differences in the HDL-
C and TG levels of the mice treated with BTD-1E were
not significant when compared with the db control
group.
Relative organ weights
Changes of liver, spleen, testis, kidney, and EPDF
Figure 1. Effects of BTD-1E on body weight and diet intake. (A) body weight, and (B) dietary consumption after administration of
BTD-1E in db /db mice. Data represent the means±SD (n=6). *P<0.05 as compared to the db control group.

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weights were measured in relation to final body weight
(Figure 3). The relative final body weights and liver
weights of the BTD-1E group were only 88.1 and 79.7%
of those of the db control group, respectively (P<0.05,
P<0.01) (Figure 3A and 3B). The relative spleen, testis,
and kidney weights of the BTD-1E group were 124,
105.7, and 128.2% of those of the db control group,
respectively (P<0.01) (Figure 3C-3E). There were no
significant differences in EPDF weight among
experimental groups (Figure 3F). However, these results
show the relative values to body weight, all actual
weights of organs in BTD-1E group were lower than
those of the db control group.
Lipid accumulation in liver
There were no lipid droplets in the livers of the
C57BL/6J mice detected by Oil Red O stains (Figure
4A). Large fat droplets were present extensively in the
livers of the db control mice (Figure 4B). The lipid
droplets were remarkably less in the BTD-1E
administered mice compared with the db control mice
(Figure 4C).
Discussion
We evaluated the inhibitory effect on lipid metabolism
of feeding BTD-1E through the analysis of serum lipid
contents and lipid accumulation in the liver in db/db
mice for four weeks. The major findings of our studies
are that BTD-1E decreases body weight due to its effects
on lipid metabolism, as a dietary therapy for obesity.
Overweight and obesity are usually defined using BMI
[28]. Thus, decreases in body weight and body fat are
important and are the primary targets in obesity therapy
[29,30]. The BTD-1E group showed significantly lower
body weight compared with the db control group. This
Figure 2. Effects of BTD-1E on serum lipid profiles. (A) total cholesterol, (B) LDL cholesterol, (C) HDL-cholesterol, and (D)
triglyceride levels. Data represent the means±SD (n=6). *P<0.05 and *P<0.01 as compared to the db control group.

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anti-hyperlipedemic effect was not associated with the
inhibition of diet consumption because the BTD-1E
group showed a slightly higher level of diet consumption
compared with the db control group.
Body weight is usually related with lipid metabolism.
Thus we analyzed the contents of serum lipid after BTD-
1E treatment. In the present study, BTD-1E resulted in
remarkably lower serum T-CHOL and LDL-C levels
compared with the db control group. These results show
that BTD-1E can regulate lipogenesis and lipolysis to
maintain serum T-CHOL and LDL-C levels in the blood
of the mice. It was reported that Monascus-fermented
soybean extract reduced T-CHOL and LDL-C levels in
rats fed a high cholesterol diet [31]. In several studies, a
Figure 3. Effects of BTD-1E on relative organ weight. (A) final body weight, (B) relative liver weight: (liver weight/final body weight)
X 100%, (C) relative spleen weight, (D) relative testis weight, (E) relative kidney weight, and (F) relative epidermal fat weight. Data
represent the means±SD (n=6). *P<0.05 and *P<0.01 as compared to the db control group.