243

Fundamental Toxicological Sciences (Fundam. Toxicol. Sci.)
Vol.3, No.6, 243-250, 2016

LetterSafety assessment of probiotic bacteria, Bacillus coagulans
strain SANK70258, in rats
Yui Akagawa1, Yasuyuki Ohnishi1, Masatoshi Takaya2 and Yuko Watanabe2

2

1LSI Medience Corporation, 13-4 Uchikanda 1-chome, Chiyoda-ku, Tokyo 101-8517, Japan
Mitsubishi-Kagaku Foods Corporation, 1-1-1 Marunouchi, Chiyoda-ku, Tokyo 100-8251, Japan(Received September 28, 2016; Accepted October 9, 2016)

ABSTRACT — Bacillus coagulans is a lactic acid, spore-forming gram-positive bacteria that has
the potential of probiotic benefits. To evaluate the toxicological profiles of Bacillus coagulans strain
SANK70258 (active element of LACRISTM-S), a 90-day oral gavage dose study was conducted in rats.
The microbe was administered by oral gavage to 6-week old Crl:CD(SD) rats (10 males and 10 females/group) for 90 days at dose levels of 0, 500, 1000, and 2000 mg/kg/day. According to the results, no deaths
occurred in either males or females, and no treatment-related changes were observed in any clinical sign
including a detailed observation with functional observational battery (FOB), functional test, motor activity, body weight, food consumption, ophthalmoscopy, urinalysis, hematology, blood chemistry, organ
weight, necropsy or histopathology. In conclusion, Bacillus coagulans strain SANK70258 had no subchronic toxicity in rats and the no-observed-adverse-effect-level (NOAEL) was judged to be greater than2000 mg (about 1 × 1012 CFU)/kg/day. The microbe is harmless and can be used as a probiotic.
Key words: Probiotic, Bacillus coagulans, 90-day subchronic-toxicity study, Rat, NOAELINTRODUCTION
Probiotics are defined as the viable microorganismsthat exhibit a beneficial effect on the health of the host by
improving its intestinal microbial balance (Schrezenmeirand De Vrese, 2001). Lactic acid bacteria have a long history of safe use in fermented food production as one of
the most beneficial probiotics. The beneficial functionsof lactic acid bacteria include: improving digestion andimmune function (Rundles et al., 2000); managing lactose
intolerance (Granato et al., 2010); degrading cholesterol(Ooi and Liong, 2010); and preventing cancer (Hirayama
and Rafter, 2000). However, general lactic acid bacteria
are very sensitive to heat, drying, acidity of the stomach,and bile acids. On the contrary, Bacillus coagulans stains
are the spore-forming gram-positive bacteria and have theability to overcome these limitations (Keller et al., 2010).
Although traditional Lactobacillus strains have an
excellent history of safe use in the formation of dairy
products and other foods and some have ‘‘generally recognized as safe’’ (GRAS) status, it is essential to conduct
the safety assessment on any new strains with the intent
to be added into foods or used as a dietary supplement(Donohue and Salminen, 1996). Bacillus coagulans strain

SANK70258 was the first successful mass produced by
Daiichi-Sankyo Co., Ltd. (Tokyo, Japan). Its beneficial
effects have been reported (Ara et al., 2002; Iino et al.,1997a, 1997b; Kajimoto et al., 2005) and this strain has
been used for drugs and foods in safety for about 50 years(Mashita et al., 1964; Takaya, 2014); however, reliable data according to recent preclinical guidelines wereinsufficient for this strain. In this study, we carried out a
sub-chronic toxicity study of Bacillus coagulans strain
SANK70258 when administered daily by oral gavage
to rats for 90 days in compliance with Good Laboratory
Practice (GLP).
MATERIALS AND METHODSThis study was conducted in accordance with the procedures indicated by Organization for Economic Cooperation and Development (OECD) Guidelines for the Testing of Chemicals “Repeated Dose 90-day Oral Toxicity
Study in Rodents” (Test No. 408, Adopted by the Council
on 21 September 1998) and carried out by LSI Medience
Corporation (Tokyo, Japan) in compliance with OECD
Principles of Good Laboratory Practice (as revised in1997). This experiment was approved by the Institutional

Correspondence: Yui Akagawa (E-mail: akagawa.yui@ma.medience.co.jp)
Vol. 3 No. 6

244
Y. Akagawa et al.

Animal Care and Use Committee of LSI Medience Corporation accredited by the Association for Assessment
and Accreditation of Laboratory Animal Care International (AAAALAC International).
Test article
The test microbe, Bacillus coagulans strain SANK70258(Lot No. 141004AA, 5.1 × 1011 CFU/g), which is an active
element of LACRISTM-S, was prepared by MitsubishiKagaku Foods Corporation (Tokyo, Japan). The microbe
is a gram-positive spore-forming rod that is aerobic to microaerophilic in nature. SANK70258 is a purecell mass consisting solely of Bacillus coagulans andlight brown powder having a slightly unique odor. The
microbe was suspended in water for injection (Otsuka
Pharmaceutical Factory, Inc., Tokushima, Japan) to makeconcentrations of 50, 100, and 200 mg/mL once within
4 days. The stability of the test microbe in water for injection was confirmed for 7 days under refrigeration at 50 and
400 mg/mL.
Animals and husbandry
Male and female Crl:CD Sprague Dawley (SD)rats (4 weeks old) were purchased from Charles River
Laboratories Japan, Inc. (Atsugi, Japan). After quarantine
and acclimation for 2 weeks, 40 healthy animals (6 weeks
of age) of each sex were assigned to each group by thestratified-by-weight randomization method on the basisof the body weights (male: 194.7 g to 234.0 g, female:147.7 g to 187.7 g). Animals were housed individually in hanging type stainless steel wire cages with enrichments (alumina balls, floor plates and laboratory bedding[Diamond Twists, Harlan Laboratories, Tokyo, Japan]) inan air-conditioned room (temperature: 19.0°C to 25.0°C,
relative humidity: 25.0% to 75.0%) with artificial lightingfrom 7:00 to 19:00 and ventilation providing all fresh air.
Food (CR-LPF, radiation sterilized: Oriental Yeast Co.,
Ltd., Tokyo, Japan) and water were given ad libitum. The
initial dosing day was designated as Day 1, and the period from Day 1 to Day 7 was designated as Week 1.
Administration
The dosing suspensions were administered by oral
gavage with a disposable syringe attached to a gastrictube for rats. The dose levels were set at 0, 500, 1000,
and 2000 mg/kg/day. Each dose group consisted of
10 animals of each sex. The control animals were dosedwith the vehicle alone. The dose volume was set at 10 mL/kg, and the individual volume was calculated based on themost recent body weight.

Vol. 3 No. 6

Observation and examinations
Clinical signs were observed twice daily (before andabout 1 hr after dosing). Detailed clinical observations
were performed by the functional observational battery(FOB) once before initial dosing and once a week (after
dosing) during the dosing period. Function tests (approach
response, touch response, auditory response, tail pinch
response, and aerial righting reaction, grip strength offorelimb and hindlimb using a digital force gauge) andmotor activity measurement using a motor activity-measuring device (SUPERMEX, Muromachi Kikai Co., Ltd.,Tokyo, Japan) were performed in Week 13. Body weight
and food consumption were measured every 7 days. Ophthalmoscopy was performed before the start of dosing andin Week 13. Urinalysis was performed on 5 males and5 females in each group in Week 13. Fresh urine samples
were collected within 3 hr in the morning (before dosing) without food and water supply. Subsequently, 20-hr
accumulated urine samples were collected while providing food and water. Paper test (Clinitek 500; Siemens
Healthcare Diagnostics K.K, Tokyo, Japan) and sedimenttest were conducted using fresh urine samples. The volume, specific gravity (Uricon-JE; Atago Co., Ltd., Tokyo,
Japan), and electrolytes (sodium [Na], potassium [K], andchlorine [Cl]: TBA-200FR; Toshiba Corporation, Tokyo,
Japan) were examined using the 20-hr urine samples. At
the end of the dosing period, blood samples were collected from the posterior vena cava under anesthesia (intraperitoneal injection of sodium thiopental) after overnight
fasting. A portion of the blood sample was anticoagulated with ethylenediaminetetraacetic acid (EDTA)-2K andanalyzed for hematology parameters by an automatedhematology analyzer (XT-2000iV; Sysmex Corporation,
Hyogo, Japan). A portion of the blood samples was anticoagulated with 3.2 w/v% trisodium citrate solution, andthen plasma samples were obtained for examination ofprothrombin time (PT) and activated partial thromboplastin time (APTT) by an automated coagulation analyzer (CA-510; Sysmex Corporation, Hyogo, Japan).
In addition, serum samples were obtained from the other portion of the blood samples and analyzed for blood
chemical parameters by an auto-analyzer (TBA-200FR,
Toshiba Corporation, Tokyo, Japan). After the blood samples were collected, animals were necropsied. Organs andtissues were removed and fixed 10 vol% phosphate-buffered formalin. The eyeball with the optic nerve and Harderian gland was fixed with Davidson’s solution, and thetestes were fixed with Bouin’s solution before storage in
10 vol% phosphate-buffered formalin. The main organs
were weighed and the relative organ weights (organ tobody weight ratio) were calculated. All organs and tissues

245
Safety assessment of probiotic bacteria in rats

were processed by the standard method to prepare hematoxylin and eosin-stained (HE) specimens, and examined
microscopically.

ever, none of them were considered to be treatment-related because they are often observed in normal rats or they
were already noted before the start of dosing.

Statistical analysis
Numerical data were first analyzed by Bartlett's test
(P < 0.01). When the group variance was homogeneous,Dunnett’s test (P < 0.05, two-tailed) was employed tocompare with the mean value of the control group. Whenthe group variance was heterogeneous, Bartlett’s test was
performed again after logarithmic conversion of the data.
When the variance was homogeneous, Dunnett’s test wasapplied. When the variance was heterogeneous, Steel test
(P < 0.05, two-tailed) was applied to compare with themean value of control group after rank transformation of
the data.

Clinical chemistry
No treatment-related differences were noted in theurinalysis, hematology or blood chemistry tests amonggroups (Tables 2 and 3). Several statistically significant
differences were observed; however, they were within therange of the background data of the test facility or there
was no dose-relationship. They were not concluded to betreatment-related.

RESULTS
Systemic clinical status
There were no deaths or moribund animals or any
treatment-related change noted in any animal according to
the standard observation or detailed observation by FOB,
functional test, or motor activity throughout the experimental period. There were no differences in body weightor food consumption among groups throughout the experimental period (Fig. 1 and Table 1).
Ophthalmoscopy
No treatment-related changes were noted in any animal. Various changes were observed in each group; how-Fig. 1.

Pathological examination
No treatment-related changes were noted in organ
weights (Table 4), necropsy, or histopathology in anygroup. A statistically significant increase in thyroid
weight was observed in males at 2000 mg/kg; however, it was not of toxicological significance because there
were no corresponding abnormalities in the histopathology. Various changes were observed in each group including the control group; however, they were not considered
to be treatment-related because similar changes are often
observed in normal rats and/or there was no clear dosedependency.
DISCUSSION
Many Lactobacillus stains have been afforded GRAS
classification, and are among the most important bacteria in food microbiology and human nutrition due toBody weight changes of male and female rats administered Bacillus coagulans strain SANK70258 by gavage for 90 days.
The data represents means ± standard deviation for each group (n = 10 animals per group).
Vol. 3 No. 6

Vol. 3 No. 6

2000

1000

500

0

2000

1000

(n = 10 animals per group)

Females

0500

Doses (mg/kg/day)
Males

Mean
SD
Mean
SD
Mean
SD
Mean
SD

Mean
SD
Mean
SD
Mean
SD
Mean
SD

Day

20.94
2.01
20.05
1.89
20.64
2.23
20.85
2.15

29.04
2.26
29.24
2.31
28.03
2.37
28.24
2.76

1 to 8

21.70
2.34
21.00
2.59
20.94
2.85
21.78
1.98

31.11
2.47
31.56
3.61
30.06
2.98
30.88
2.39

8 to 15

22.54
2.58
20.87
2.30
21.64
3.19
22.19
2.16

31.45
2.35
31.56
4.21
30.32
2.85
31.71
1.88

15 to 22

22.37
2.16
21.51
2.78
22.37
3.33
22.95
2.85

31.80
2.68
31.64
3.74
30.40
3.24
32.30
1.83

22 to 29

22.83
3.07
21.47
2.41
22.81
3.75
23.61
2.64

32.75
2.55
32.61
3.32
31.17
3.29
32.52
2.80

29 to 36

22.91
2.26
22.26
2.48
22.75
2.70
23.84
1.96

32.17
1.96
32.07
3.52
30.84
2.91
32.74
2.05

36 to 43

23.19
2.54
22.48
2.29
22.27
3.28
23.25
2.32

31.15
1.85
32.06
3.35
30.76
3.00
32.44
2.13

43 to 50

23.25
2.98
21.60
2.17
22.35
3.04
23.17
2.49

31.39
2.04
31.50
2.95
30.63
2.97
32.14
1.82

50 to 57

21.98
2.54
21.64
2.32
22.14
2.78
22.56
2.81

30.76
1.73
31.57
3.42
30.30
2.89
32.00
1.91

57 to 64

21.57
1.75
21.21
2.03
21.24
2.89
22.40
1.99

30.55
1.77
31.54
3.62
29.82
2.56
31.39
1.67

64 to 71

21.73
1.96
20.66
1.92
21.15
2.96
21.86
2.06

30.19
1.65
30.62
3.77
29.40
2.44
31.63
2.13

71 to 78

Table 1. Mean daily food consumption (g/day) of male and female rats administered Bacillus coagulans strain SANK70258 by gavage for 90 days.

21.75
1.60
21.11
2.48
21.19
2.48
22.00
2.11

29.83
1.88
29.88
3.17
29.35
2.79
30.66
1.81

78 to 85

21.21
2.11
20.19
2.45
19.26
2.15
20.98
2.31

28.57
2.09
28.64
3.51
28.49
3.27
29.42
2.51

87 to 90

246
Y. Akagawa et al.

247
Safety assessment of probiotic bacteria in rats
Table 2. Hematology parameters of male (A) and female (B) rats administered Bacillus coagulans strain SANK70258 by gavage for 90 days.
(A) Males
Doses (mg/kg/day)
0
500
1000
9.023 ± 0.302
8.832 ± 0.443
8.780 ± 0.532
Red Blood Cell Count(106/μL)
Hemoglobin conc.
(g/dL)
15.69 ± 0.65
15.73 ± 0.53
15.11 ± 0.78
Hematocrit(%)
45.43 ± 1.89
45.81 ± 1.37
44.34 ± 2.08
MCV
(fL)
50.34 ± 1.17
51.96 ± 2.23
50.54 ± 1.35
MCH
(pg)
17.39 ± 0.38
17.83 ± 0.60
17.22 ± 0.40
MCHC
(g/dL)
34.53 ± 0.31
34.35 ± 0.41
34.07 ± 0.38*
Platelet Count(103/μL)
1113.9 ± 78.3
1119.0 ± 122.7
1110.0 ± 63.6
Reticulocyte(%)
3.718 ± 0.578
3.223 ± 0.707
3.457 ± 0.624
PT(sec)
15.05 ± 3.25
8.93 ± 0.36**
8.78 ± 0.51**
APTT
(sec)
22.23 ± 2.35
16.11 ± 1.91**
16.58 ± 1.32**
White Blood Cell Count(103/μL)
8.331 ± 1.578
8.776 ± 2.063
8.527 ± 1.166
Lymphocyte(%)
76.59 ± 7.44
77.36 ± 6.36
72.51 ± 6.07
Neutrophil(%)
19.19 ± 6.73
17.20 ± 5.79
22.01 ± 6.09
Eosinophil(%)
1.49 ± 0.47
1.56 ± 0.50
1.52 ± 0.78
Basophil(%)
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
3.96 ± 1.22*
Monocyte(%)
2.73 ± 1.00
3.88 ± 0.99*
6.374 ± 1.362
6.791 ± 1.745
6.215 ± 1.173
Lymphocyte(103/μL)
1.607 ± 0.661
1.514 ± 0.748
1.852 ± 0.460
Neutrophil(103/μL)
Eosinophil(103/μL)
0.120 ± 0.028
0.135 ± 0.045
0.126 ± 0.052
Basophil(103/μL)
0.000 ± 0.000
0.000 ± 0.000
0.000 ± 0.000
0.230 ± 0.100
0.336 ± 0.094*
0.334 ± 0.092*
Monocyte(103/μL)(n = 10 animals per group), *: Significantly different from control (P < 0.05) **: Significantly different from control (P < 0.01)
Items

Unit

2000
8.892 ± 0.394
15.58 ± 0.77
45.52 ± 2.27
51.23 ± 2.41
17.54 ± 0.75
34.23 ± 0.25
1159.7 ± 126.3
3.319 ± 0.509
8.73 ± 0.51**
17.03 ± 0.95**
8.418 ± 2.160
77.15 ± 6.33
18.31 ± 5.98
1.28 ± 0.26
0.00 ± 0.00
3.26 ± 0.63
6.571 ± 2.040
1.466 ± 0.349
0.109 ± 0.042
0.000 ± 0.000
0.272 ± 0.084(B) Females
Items

Unit

0
8.129 ± 0.447
Red Blood Cell Count(106/μL)
Hemoglobin conc.
(g/dL)
15.03 ± 0.41
Hematocrit(%)
43.51 ± 1.22
MCV
(fL)
53.63 ± 2.40
MCH(pg)
18.52 ± 0.74
MCHC
(g/dL)
34.54 ± 0.58
1110.8 ± 147.5
Platelet Count(103/μL)
Reticulocyte(%)
2.925 ± 0.590
PT(sec)
7.69 ± 0.36
APTT
(sec)
15.51 ± 1.61
5.702 ± 3.029
White Blood Cell Count(103/μL)
Lymphocyte(%)
85.12 ± 5.16
Neutrophil(%)
11.91 ± 5.08
Eosinophil(%)
1.33 ± 0.47
Basophil(%)
0.01 ± 0.03
Monocyte(%)
1.63 ± 0.73
4.900 ± 2.748
Lymphocyte(103/μL)
0.627 ± 0.291
Neutrophil(103/μL)
Eosinophil(103/μL)
0.072 ± 0.032
0.001 ± 0.003
Basophil(103/μL)
0.102 ± 0.093
Monocyte(103/μL)(n = 10 animals per group), *: Significantly different from control (P < 0.05)

their contribution to fermented food production or theiruse as probiotics (Donohue and Salminen, 1996). Toxicological studies were conducted on several Bacillus coagulans species, and demonstrated that there are no safety issues (Endres et al., 2009; Keller et al., 2010; Sudha
et al., 2011). The purpose of this study is to assess thepotential sub-chronic toxicity of Bacillus coagulans strain
SANK70258, which is an active element of LACRISTM-S,
and to confirm commercial availability as the probiotic
strain. Bacillus coagulans strain SANK70258 was admin-

Doses (mg/kg/day)
500
1000
8.108 ± 0.405
8.396 ± 0.426
15.02 ± 0.52
15.29 ± 0.66
43.45 ± 1.64
44.58 ± 2.05
53.64 ± 1.27
53.14 ± 2.09
18.53 ± 0.37
18.23 ± 0.63
34.58 ± 0.44
34.30 ± 0.22
1160.4 ± 71.4
1145.9 ± 77.8
3.375 ± 0.420
2.815 ± 0.577
7.89 ± 0.26
7.71 ± 0.25
14.99 ± 2.00
14.94 ± 1.28
5.133 ± 1.515
6.503 ± 1.434
84.39 ± 3.63
82.09 ± 7.65
12.09 ± 3.48
13.86 ± 6.91
1.45 ± 0.53
1.40 ± 0.54
0.00 ± 0.00
0.00 ± 0.00
2.07 ± 0.92
2.65 ± 1.08*
4.335 ± 1.274
5.392 ± 1.531
0.614 ± 0.255
0.860 ± 0.374
0.077 ± 0.040
0.087 ± 0.026
0.000 ± 0.000
0.000 ± 0.000
0.107 ± 0.054
0.164 ± 0.068

2000
8.256 ± 0.443
15.21 ± 0.69
44.26 ± 1.88
53.65 ± 1.57
18.44 ± 0.56
34.38 ± 0.45
1161.2 ± 119.5
3.186 ± 0.673
7.64 ± 0.24
14.93 ± 1.97
5.553 ± 2.246
82.11 ± 5.10
14.11 ± 4.98
1.61 ± 0.67
0.00 ± 0.00
2.17 ± 0.93
4.606 ± 2.038
0.750 ± 0.347
0.083 ± 0.033
0.000 ± 0.000
0.114 ± 0.061

istered by oral gavage at 0, 500, 1000, and 2000 mg/kg/
day to SD rats for 90 days to assess its toxicity according
to OECD guideline in compliance with GLP.
According to the results, no deaths occurred in males or
females, and no treatment-related changes were observedin any examination. Since the test microbe was administered for a period longer than 90 days in this study, it
has no potentials to produce some toxins or induce abnormal fermentation in the intestinal tract causing diarrhea.
In the microbe safety assessment, it is important to evalVol. 3 No. 6

248
Y. Akagawa et al.
Table 3. Blood chemistry parameters of male (A) and female (B) rats administered Bacillus coagulans strain SANK70258 bygavage for 90 days.
(A) Males
Doses (mg/kg/day)
0
500
1000
ASAT
(U/L)
76.3 ± 9.2
79.3 ± 14.6
85.9 ± 15.1
ALAT
(U/L)
25.9 ± 5.6
21.3 ± 5.0
21.8 ± 4.9
Gamma GT
(U/L)
0.9 ± 0.3
0.8 ± 0.4
1.0 ± 0.0
ALP
(U/L)
278.5 ± 30.8
257.7 ± 43.2
247.3 ± 66.6
Total Bilirubin(mg/dL)0.06 ± 0.05
0.08 ± 0.06
0.11 ± 0.06
Total Bile Acid(μmol/L)
22.67 ± 14.20
16.55 ± 6.67
13.39 ± 5.85
Urea Nitrogen(mg/dL)13.07 ± 1.40
13.33 ± 1.93
13.57 ± 1.73
Creatinine(mg/dL)0.28 ± 0.04
0.25 ± 0.05
0.27 ± 0.05
Glucose(mg/dL)149.1 ± 8.0
146.8 ± 10.8
146.4 ± 11.9
Total Cholesterol(mg/dL)76.2 ± 15.7
68.9 ± 19.2
75.1 ± 16.7
Phospholipid(mg/dL)113.2 ± 18.2
108.3 ± 23.7
110.1 ± 19.0
Triglyceride(mg/dL)47.3 ± 17.6
64.6 ± 22.7
49.5 ± 10.8
Total Protein(g/dL)
7.23 ± 0.38
6.98 ± 0.35
6.79 ± 0.29*
A/G Ratio
0.980 ± 0.113
0.986 ± 0.107
0.947 ± 0.067
Albumin(%)
49.37 ± 2.77
49.49 ± 2.68
48.59 ± 1.78
Alpha1 Globulin(%)
20.24 ± 2.80
19.67 ± 2.40
19.46 ± 2.83
Alpha2 Globulin(%)
10.22 ± 1.25
9.73 ± 0.99
10.18 ± 1.39
Beta Globulin(%)
15.21 ± 1.24
15.73 ± 1.04
15.98 ± 0.87
Gamma Globulin(%)
4.96 ± 0.92
5.38 ± 1.23
5.79 ± 1.65
Albumin(g/dL)
3.57 ± 0.21
3.45 ± 0.21
3.30 ± 0.16**
Alpha1 Globulin(g/dL)
1.47 ± 0.24
1.38 ± 0.21
1.33 ± 0.23
Alpha2 Globulin(g/dL)
0.74 ± 0.08
0.68 ± 0.07
0.69 ± 0.07
Beta Globulin(g/dL)
1.10 ± 0.13
1.10 ± 0.09
1.08 ± 0.07
Gamma Globulin(g/dL)
0.36 ± 0.07
0.37 ± 0.08
0.39 ± 0.12Ca
(mg/dL)10.53 ± 0.47
10.38 ± 0.41
10.31 ± 0.24IP
(mg/dL)7.61 ± 0.45
7.61 ± 0.39
7.80 ± 0.53Na(mmol/L)
148.4 ± 1.0
148.1 ± 1.3
147.7 ± 0.8
K(mmol/L)
4.39 ± 0.17
4.40 ± 0.13
4.45 ± 0.23
Cl(mmol/L)
104.3 ± 1.3
105.0 ± 1.1
105.0 ± 1.5(n = 10 animals per group), *: Significantly different from control (P < 0.05) **: Significantly different from control (P < 0.01)
Items

Unit

2000
94.9 ± 23.7*
26.0 ± 6.9
0.9 ± 0.3
259.2 ± 60.8
0.10 ± 0.00
16.70 ± 6.67
13.82 ± 1.30
0.29 ± 0.03
148.1 ± 7.5
70.5 ± 18.6
113.9 ± 23.4
68.0 ± 33.9
7.18 ± 0.27
0.937 ± 0.116
48.22 ± 2.90
19.73 ± 2.35
10.25 ± 1.28
15.75 ± 1.36
6.05 ± 1.10
3.46 ± 0.17
1.42 ± 0.19
0.74 ± 0.09
1.13 ± 0.12
0.44 ± 0.09
10.57 ± 0.28
7.44 ± 0.53
150.2 ± 4.7
4.59 ± 0.34
106.7 ± 3.8(B) Females
Items

Unit

0
ASAT
(U/L)
94.2 ± 26.7
ALAT
(U/L)
20.6 ± 6.6
Gamma GT
(U/L)
1.0 ± 0.0
ALP
(U/L)
141.3 ± 37.1
Total Bilirubin(mg/dL)0.09 ± 0.03
Total Bile Acid(μmol/L)
28.90 ± 17.26
Urea Nitrogen(mg/dL)15.76 ± 2.18
Creatinine(mg/dL)
0.33 ± 0.05
Glucose(mg/dL)140.1 ± 23.4
Total Cholesterol(mg/dL)83.2 ± 18.0
Phospholipid(mg/dL)146.3 ± 25.9
Triglyceride(mg/dL)26.4 ± 11.5
Total Protein(g/dL)
7.39 ± 0.60
A/G Ratio
1.284 ± 0.155
Albumin(%)
56.05 ± 2.97
Alpha1 Globulin(%)
15.47 ± 1.76
Alpha2 Globulin(%)
7.91 ± 1.42
Beta Globulin(%)
14.32 ± 1.62
Gamma Globulin(%)
6.25 ± 1.38
Albumin(g/dL)
4.15 ± 0.51
Alpha1 Globulin(g/dL)
1.14 ± 0.14
Alpha2 Globulin(g/dL)
0.58 ± 0.11
Beta Globulin(g/dL)
1.06 ± 0.13
Gamma Globulin(g/dL)
0.46 ± 0.09
Ca
(mg/dL)10.02 ± 0.33IP
(mg/dL)6.51 ± 0.47Na
(mmol/L)
146.2 ± 1.3
K(mmol/L)
4.22 ± 0.26
Cl(mmol/L)
106.4 ± 2.2(n = 10 animals per group), *: Significantly different from control (P < 0.05)

Vol. 3 No. 6

Doses (mg/kg/day)
500
1000
93.3 ± 12.5
103.7 ± 17.6
21.3 ± 4.4
23.9 ± 3.9
1.0 ± 0.0
1.0 ± 0.0
127.8 ± 25.5
109.8 ± 24.7
0.11 ± 0.06
0.15 ± 0.05*
32.96 ± 22.58
31.83 ± 15.54
15.87 ± 2.16
17.05 ± 2.33
0.30 ± 0.00
0.32 ± 0.06
131.4 ± 15.4
128.7 ± 15.6
78.6 ± 15.7
88.1 ± 13.7
138.9 ± 25.6
148.8 ± 17.0
23.3 ± 9.0
23.5 ± 8.3
7.41 ± 0.35
7.55 ± 0.30
1.260 ± 0.141
1.275 ± 0.128
55.59 ± 2.66
55.90 ± 2.52
15.07 ± 2.21
15.73 ± 2.16
8.24 ± 1.49
7.86 ± 1.47
14.23 ± 1.53
13.56 ± 1.15
6.87 ± 1.50
6.95 ± 1.35
4.12 ± 0.32
4.22 ± 0.30
1.12 ± 0.16
1.19 ± 0.16
0.61 ± 0.11
0.59 ± 0.11
1.05 ± 0.13
1.02 ± 0.10
0.51 ± 0.11
0.52 ± 0.09
10.02 ± 0.15
10.13 ± 0.33
6.50 ± 0.57
6.44 ± 0.50
146.2 ± 0.6
145.9 ± 0.7
4.29 ± 0.20
4.46 ± 0.25
107.0 ± 1.1
107.0 ± 0.8

200097.7 ± 16.1
25.4 ± 6.3
1.0 ± 0.0
115.8 ± 29.4
0.12 ± 0.04
26.65 ± 11.34
15.2 ± 1.88
0.30 ± 0.0
138.2 ± 9.9
86.9 ± 13.9
144.1 ± 20.3
21.7 ± 9.4
7.31 ± 0.30
1.175 ± 0.178
53.77 ± 3.56
16.52 ± 1.82
7.63 ± 1.50
14.92 ± 1.79
7.16 ± 1.70
3.94 ± 0.39
1.21 ± 0.14
0.56 ± 0.10
1.09 ± 0.12
0.52 ± 0.12
10.23 ± 0.37
6.58 ± 0.67
146.1 ± 0.9
4.24 ± 0.32
107.1 ± 0.7

249
Safety assessment of probiotic bacteria in rats
Table 4. Absolute and relative organ weights of male (A) and female (B) rats administered Bacillus coagulans strain SANK70258
by gavage for 90 days.
(A) Males
Items

Unit

0
544.40 ± 30.70

Doses (mg/kg/day)
500
1000
560.23 ± 75.58
543.98 ± 53.28

Final Body Weight
Absolute organ weights
Brain
(g)
2.178 ± 0.067
2.203 ± 0.086
2.141 ± 0.085
Pituitary(mg)
13.90 ± 1.81
14.95 ± 1.54
14.72 ± 1.71
Thyroids(mg)31.70 ± 4.31
34.02 ± 4.25
34.00 ± 3.69
Thymus(mg)346.0 ± 72.0
379.3 ± 80.8
336.3 ± 115.5
Submand.GLs(g)0.714 ± 0.054
0.789 ± 0.091
0.767 ± 0.071
Lungs
(g)
1.680 ± 0.147
1.715 ± 0.167
1.674 ± 0.128
Heart
(g)
1.710 ± 0.155
1.588 ± 0.159
1.578 ± 0.151
Liver
(g)
13.396 ± 0.998
13.753 ± 2.485
13.545 ± 1.766
Spleen(g)
0.899 ± 0.112
0.828 ± 0.161
0.853 ± 0.139
Kidneys(g)
3.291 ± 0.120
3.432 ± 0.416
3.420 ± 0.434
Adrenals(mg)58.40 ± 7.53
65.48 ± 11.56
60.84 ± 7.75
Testes(g)
3.678 ± 0.148
3.781 ± 0.283
3.580 ± 0.229
Prostate(g)0.741 ± 0.111
0.821 ± 0.167
0.827 ± 0.181
Epididymides(g)1.435 ± 0.104
1.494 ± 0.159
1.492 ± 0.192
Relative organ weights
Brain
(%)
0.401 ± 0.029
0.398 ± 0.038
0.397 ± 0.035
Pituitary(10-3%)
2.56 ± 0.33
2.68 ± 0.12
2.71 ± 0.20
Thyroids(10-3%)
5.82 ± 0.68
6.12 ± 0.78
6.28 ± 0.66
Thymus(10-3%)
63.30 ± 12.10
67.73 ± 10.90
61.62 ± 19.81
Submand.GLs(%)
0.131 ± 0.009
0.142 ± 0.014
0.142 ± 0.014
Lungs
(%)
0.309 ± 0.020
0.308 ± 0.025
0.309 ± 0.026
Heart
(%)
0.315 ± 0.027
0.286 ± 0.028
0.292 ± 0.032
Liver
(%)
2.462 ± 0.136
2.446 ± 0.190
2.486 ± 0.142
Spleen(%)
0.165 ± 0.018
0.147 ± 0.013
0.156 ± 0.018
Kidneys(%)
0.606 ± 0.033
0.616 ± 0.058
0.628 ± 0.046
Adrenals(10-3%)
10.79 ± 1.85
11.74 ± 1.75
11.29 ± 1.87
Testes
(%)
0.677 ± 0.045
0.685 ± 0.099
0.663 ± 0.065
Prostate(%)
0.137 ± 0.023
0.150 ± 0.039
0.152 ± 0.030
Epididymides(%)
0.264 ± 0.019
0.270 ± 0.038
0.275 ± 0.031(n = 10 animals per group), *: Significantly different from control (P < 0.05) **: Significantly different from control (P < 0.01)

2000
577.38 ± 32.34
2.242 ± 0.073
14.34 ± 1.58
38.11 ± 3.96**
341.9 ± 123.8
0.754 ± 0.124
1.730 ± 0.162
1.660 ± 0.163
14.454 ± 1.686
0.901 ± 0.200
3.354 ± 0.232
59.87 ± 9.94
3.686 ± 0.359
0.717 ± 0.184
1.475 ± 0.110
0.389 ± 0.020
2.49 ± 0.27
6.61 ± 0.62*
59.06 ± 20.02
0.131 ± 0.020
0.300 ± 0.026
0.287 ± 0.019
2.499 ± 0.195
0.156 ± 0.031
0.582 ± 0.043
10.40 ± 1.88
0.640 ± 0.065
0.124 ± 0.029
0.256 ± 0.028(B) Females
Items
Final Body Weight
Absolute organ weights
Brain
Pituitary
Thyroids
ThymusSubmand.GLs
Lungs
Heart
Liver
SpleenKidneys
Adrenals
Ovaries
UterusRelative organ weights
Brain
Pituitary
Thyroids
ThymusSubmand.GLs
Lungs
Heart
Liver
SpleenKidneys
Adrenals
Ovaries
Uterus(n = 10 animals per group)0297.16 ± 41.06

Doses (mg/kg/day)
500
1000
297.02 ± 34.00
296.16 ± 39.47

2000
300.74 ± 40.01

(g)
(mg)
(mg)
(mg)
(g)
(g)
(g)
(g)
(g)
(g)
(mg)
(mg)
(g)

2.019 ± 0.102
18.32 ± 2.76
22.84 ± 4.01
280.2 ± 68.4
0.472 ± 0.046
1.239 ± 0.089
0.995 ± 0.099
7.537 ± 1.054
0.517 ± 0.068
1.903 ± 0.226
64.57 ± 9.42
92.23 ± 16.75
0.823 ± 0.338

1.960 ± 0.062
18.70 ± 2.37
23.91 ± 3.13
267.1 ± 36.6
0.482 ± 0.055
1.259 ± 0.078
1.006 ± 0.117
7.542 ± 1.043
0.563 ± 0.090
1.930 ± 0.216
66.19 ± 6.80
84.41 ± 18.41
0.764 ± 0.214

1.975 ± 0.088
18.50 ± 2.26
26.27 ± 4.79
245.7 ± 44.5
0.452 ± 0.040
1.273 ± 0.123
0.975 ± 0.125
7.442 ± 0.939
0.550 ± 0.067
1.908 ± 0.273
62.40 ± 5.09
91.91 ± 16.98
0.566 ± 0.089

1.982 ± 0.088
19.09 ± 1.74
25.04 ± 3.51
293.2 ± 74.4
0.471 ± 0.069
1.259 ± 0.083
1.034 ± 0.092
7.906 ± 0.980
0.565 ± 0.071
1.993 ± 0.126
65.94 ± 10.39
89.78 ± 13.67
0.782 ± 0.367(%)(10-3%)(10-3%)(10-3%)
(%)
(%)
(%)
(%)
(%)
(%)
(10-3%)(10-3%)(%)

0.692 ± 0.106
6.22 ± 0.91
7.78 ± 1.54
94.78 ± 21.29
0.161 ± 0.021
0.422 ± 0.049
0.337 ± 0.027
2.542 ± 0.187
0.175 ± 0.013
0.644 ± 0.059
22.17 ± 4.76
31.49 ± 6.78
0.285 ± 0.133

0.667 ± 0.073
6.31 ± 0.58
8.11 ± 1.17
90.10 ± 9.79
0.163 ± 0.017
0.427 ± 0.034
0.339 ± 0.024
2.538 ± 0.178
0.189 ± 0.018
0.652 ± 0.052
22.52 ± 3.36
28.69 ± 6.21
0.259 ± 0.075

0.675 ± 0.069
6.31 ± 0.91
9.01 ± 2.19
82.97 ± 10.04
0.154 ± 0.015
0.433 ± 0.034
0.330 ± 0.021
2.518 ± 0.140
0.188 ± 0.030
0.645 ± 0.051
21.39 ± 3.19
31.14 ± 4.79
0.194 ± 0.038

0.668 ± 0.083
6.41 ± 0.76
8.40 ± 1.27
96.86 ± 16.56
0.158 ± 0.022
0.423 ± 0.041
0.346 ± 0.021
2.633 ± 0.103
0.189 ± 0.024
0.671 ± 0.078
22.12 ± 3.55
30.14 ± 5.01
0.271 ± 0.146

Unit

Vol. 3 No. 6

250
Y. Akagawa et al.

uate whether the microbe has a potential to infect mammals. In this study, there were no increases in white blood
cell count (WBC), neutrophil, or eosinophil, or any histopathologic changes indicative of inflammation in any
organ/tissue including the digestive tract at the maximum
dose level, suggesting that the test microbe did not infectrats.
The no-observed-adverse-effect-level (NOAEL) of
Bacillus coagulans strain SANK70258 was judged to begreater than 2000 mg/kg/day in males and females. The
dose level is equivalent to 1 × 1012 CFU/kg/day. It corresponds to 7 × 1013 CFU/human/day when the body weightin adult is regarded as 70 kg. Considering the expecteddaily intake of about 108 to 109 CFU in human, the test
microbe is harmless and available as a probiotic.
Conflict of interest---- The authors declare that there isno conflict of interest.
REFERENCES
Ara, K., Meguro, S., Hase, T., Tokimitsu, I., Otsuji, K., Kawai, S.,
Ito, S. and Iino, H. (2002): Effect of spore-bearing lactic acidforming bacteria (Bacillus coagulans SANK 70258) administration on the intestinal environment, defecation frequency, fecal
characteristics and dermal characteristics in humans and rats.
Microb. Ecol. Health Dis., 80, 581-583.
Donohue, D.C. and Salminen, S. (1996): Safety of probiotic bacteria. Asia Pacific J. Clin. Nutr., 5, 25-28.
Endres, J.R., Clewell, A., Jade, K.A., Farber, T., Hauswirth, J. and
Schauss, A.G. (2009): Safety assessment of a proprietary preparation of a novel probiotic, Bacillus coagulans, as a food ingredient. Food Chem. Toxicol., 47, 1231-1238.
Granato, D., Branco, G.F., Nazzaro, F., Cruz, A.G. and Fariaet,
J.A.F. (2010): Functional foods and nondairy probiotic fooddevelopment: trends, concepts, and products. CRFSFS, 9, 292-

Vol. 3 No. 6302.
Hirayama, K. and Rafter, J. (2000): The role of probiotic bacteria incancer prevention. Microbes Infec., 2, 681-686.
Iino, H., Ito, M., Abe, S. and Sugaura, T. (1997a): The effects ofspore-forming lactic acid bacteria (Bacillus coagulans) in
improving human intestinal bacterial flora. J. Clin. Exp. Med.,17, 3299-3302.
Iino, H., Ito, M., Abe, S. and Sugaura, T. (1997b): The effects ofspore-forming lactic acid bacteria (Bacillus coagulans) in
improving the properties of feces. J. Clin. Exp. Med., 17, 33033308.
Kajimoto, O., Suwa, M., Takahashi, Y., Uchida, N., Hatayama, M.,
Tamura, M., Nakagawa, S. and Kajimoto, Y. (2005): Yuuhoushisei nyuusankin(Bacillus coagulans SANK70258) no sugikafunsyou ni taisuru yuuyousei no kentou Study of usefulness of sporeforming lactic acid bacteria (Bacillus coagulans SANK70258) tocedar pollen allergy. J. New Rem. Clin., 54, 1505-1524.
Keller, D., Farmer, S., McCartney, A. and Gibson, G. (2010): Bacillus coagulans as a probiotic. Food Sci. Technol. Bulletin., 7,103-109.
Mashita, H., Shimizu, K., Ogasawara, M. and Nakashima, H.
(1964): Nakayamashi yuuhoushisei nyuusankin ni kansuru
kisoteki rinsyouteki kenkyuu Basic clinical study on Nakayama
spore-forming lactic acid bacteria. J. New Rem. Clin., 13, 977982.
Ooi, L.G. and Liong, M.T. (2010): Cholesterol-Lowering Effects of
Probiotics and Prebiotics: A Review of in vivo and in vitro Findings. Int. J. Mol. Sci., 11, 2499-2522.
Rundles, S.C., Ahrné, S., Bengmark, S., Liang, R.J., Marshall, F.,
Metakis, L., Califano, C., Dunn, A.M., Grassey, C., Hinds, G.,
and Cervia, J. (2000). Probiotics and immune response. AJG.,
95, S22-S25.
Schrezenmeir, J. and De Vrese, M. (2001). Probiotics, prebiotics,
and synbiotics—approaching a definition. Am. J. Clin. Nutr., 73,
361S-364S.
Sudha, R.M., Sunita, M. and Sekhar, B.M. (2011). Safety studies
of Bacillus coagulans Unique IS-2 in rats: Morphological, Biochemical and clinical evaluations. Int. J. Probiotics Prebiotics, 6,43-48.
Takaya, M. (2014). Bacillus coagulans. Bio. Clinica., 29, 47-51.