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Probiotics – From Bench to CommunityMar 7-8, 2015, Delhi
New Aspects of Probiotics:A Good Supporter for Assisting Symbiosis
Masanobu NannoYakult Central Institute, Japan
By J.O.
Today’s topics
1. Lactobacillus casei Shirota is regarded as probiotics.
2. L. casei Shirota is efficient for prevention of cancer development in humans and animals.
3. L. casei Shirota opens new possible area for application.
Unbalanced diet
Antibiotics
Aging
Infection
Dysbiosis of gut
microbiota
Severe stress
Diarrhea
Allergy
Irritable bowel
syndrome
Cancer
Gut microbiota and disease
Recovery of well-
balanced gut microbiota
Inflammatory bowel diseaseInflammatory bowel disease
Diarrhea
Allergy
Irritable bowel
syndrome
Cancer
Probiotics
Cancer incidence and mortality in Japan and India
GLOBOCAN 2012: Estimated Cancer Incidence, Mortality and Prevalence Worldwide in 2012 [WHO]
Japan India
Breast cancer
Tsugane S et al, Cancer Causes Control, 1:189-193 (1990)
Life style and cancer incidence
Western life style may enhance the cancer development ?
Cancer incidence was surveyed in the Japanese immigrants who moved to and resided in foreign countries.
日本人 ブラジルの
日本人
ハワイの日本人
0
10
20
30Colorectal cancer
in Japan in Sao Paulo in Hawaii日本人 ブラジル
の日本人
ハワイの日本人
0
10
20
30
40
50Breast cancer
in Japan in Sao Paulo in HawaiiNo.
of
pati
en
ts/1
05 f
em
ale
s
Comparison of gut microbiota between colorectal cancer (CRC) patients and healthy
controls
Chen W et al, PLoS ONE, 7:e39743 (2012)
Mucosa-associatedbacteria
Lumen bacteria
CRC patientsHealthy controls
CRC patientsHealthy controls
2 wks 1 mo
Fecal samples
During ingestion
LcS-containing fermented milk (1 bottle/ day)
Beforeingestion
2 mo 3 mo 4 mo 5 mo 6 mo
Analysis: microbiota, organic acid concentrations and pH in feces
Subjects : 42 inpatients (82±10 years) in Sosen hospital, Japan
Type of trial : open trial to compare the pre- and post-intake of
probiotics (4 x 1010 L. casei Shirota [LcS]/80ml bottle)
Effect of probiotics in the elderly
Bian L et al, Int J Probiotics Prebiotics, 6:123-132 (2011)
Effect of probiotics on the gut microbiota
*, P<0.05; **, P<0.01 ND : Not detected.
Log
10 b
act
eri
al n
um
ber/
g f
ece
s
Bifidobacterium L. casei ShirotaLactobacillus
3456789
10
3456789
1011
ND3456789
10 * * *** ***
beforeafter ingestion (mo)
1 3 6 beforeafter ingestion (mo)
1 3 6 beforeafter ingestion (mo)
1 3 6
C. perfringens
345678
*
71(%) 48 60 61
Staphylococcus10
4
6
89
7
5 62(%) 70 51 65
**
Pseudomonas8
2
4
67
5
3 33 29 16
*
45(%)
beforeafter ingestion (mo)
1 3 6 beforeafter ingestion (mo)
1 3 6 beforeafter ingestion (mo)
1 3 6
*, P<0.05; **, P<0.01
Effect of probiotics on the gut environment
Total organic acids
*
Acetic acid pH
** **
µm
ol/g
fece
s
020
406080
100120
140160
020
406080
100120
140160
5
6
7
8* * p=0.06
beforeafter ingestion (mo)
1 3 6before
after ingestion (mo)
1 3 6before
after ingestion (mo)
1 3 6
Lactobacillus casei Shirota (LcS)・ Survives in the gut lumen and is recovered
alive from the feces.・ Normalizes the altered gut microbiota and
improves the bowel movement.・ Decreases the production of noxious
substances in the gut lumen and allows their clearance.
Lactobacillus casei Shirota is probiotics !
Possible application of probiotics
1. gut-associated diseases 1) constipation 2) infection of bacteria and virus 3) irritable bowel syndrome 4) inflammatory bowel diseases2. urinary organ-associated diseases3. cancer4. allergy5. autoimmune diseases6. cardiovascular diseases7. stress-induced abnormalities
Protocol of epidemiological study
Control
Sequence Activity
VKRVVKLLKG
YPWLK
3.452.93
2.92・・・
・・・
RRVTKYRVDE
RMHLH
0.490.49
0.48
Sequence Activity
VKRVVKLLKG
YPWLK
3.452.93
2.92・・・
・・・
RRVTKYRVDE
RMHLH
0.490.49
0.48
Sequence Activity
VKRVVKLLKG
YPWLK
3.452.93
2.92・・・
・・・
RRVTKYRVDE
RMHLH
0.490.49
0.48
Sequence Activity
VKRVVKLLKG
YPWLK
3.452.93
2.92・・・
・・・
RRVTKYRVDE
RMHLH
0.490.49
0.48
Sequence Activity
VKRVVKLLKG
YPWLK
3.452.93
2.92・・・
・・・
RRVTKYRVDE
RMHLH
0.490.49
0.48
Sequence Activity
VKRVVKLLKG
YPWLK
3.452.93
2.92・・・
・・・
RRVTKYRVDE
RMHLH
0.490.49
0.48
Sequence Activity
VKRVVKLLKG
YPWLK
3.452.93
2.92・・・
・・・
RRVTKYRVDE
RMHLH
0.490.49
0.48
Sequence Activity
VKRVVKLLKG
YPWLK
3.452.93
2.92・・・
・・・
RRVTKYRVDE
RMHLH
0.490.49
0.48
Sequence Activity
VKRVVKLLKG
YPWLK
3.452.93
2.92・・・
・・・
RRVTKYRVDE
RMHLH
0.490.49
0.48
Sequence Activity
VKRVVKLLKG
YPWLK
3.452.93
2.92・・・
・・・
RRVTKYRVDE
RMHLH
0.490.49
0.48
Sequence Activity
VKRVVKLLKG
YPWLK
3.452.93
2.92・・・
・・・
RRVTKYRVDE
RMHLH
0.490.49
0.48
Sequence Activity
VKRVVKLLKG
YPWLK
3.452.93
2.92・・・
・・・
RRVTKYRVDE
RMHLH
0.490.49
0.48
Sequence Activity
VKRVVKLLKG
YPWLK
3.452.93
2.92・・・
・・・
RRVTKYRVDE
RMHLH
0.490.49
0.48
Sequence Activity
VKRVVKLLKG
YPWLK
3.452.93
2.92・・・
・・・
RRVTKYRVDE
RMHLH
0.490.49
0.48
Sequence Activity
VKRVVKLLKG
YPWLK
3.452.93
2.92・・・
・・・
RRVTKYRVDE
RMHLH
0.490.49
0.48
Sequence Activity
VKRVVKLLKG
YPWLK
3.452.93
2.92・・・
・・・
RRVTKYRVDE
RMHLH
0.490.49
0.48
Now
Sequence Activity
VKRVVKLLKG
YPWLK
3.452.93
2.92・・・
・・・
RRVTKYRVDE
RMHLH
0.490.49
0.48
Sequence Activity
VKRVVKLLKG
YPWLK
3.452.93
2.92・・・
・・・
RRVTKYRVDE
RMHLH
0.490.49
0.48
Sequence Activity
VKRVVKLLKG
YPWLK
3.452.93
2.92・・・
・・・
RRVTKYRVDE
RMHLH
0.490.49
0.48
Sequence Activity
VKRVVKLLKG
YPWLK
3.452.93
2.92・・・
・・・
RRVTKYRVDE
RMHLH
0.490.49
0.48
Sequence Activity
VKRVVKLLKG
YPWLK
3.452.93
2.92・・・
・・・RRVTKYRVDE
RMHLH
0.490.49
0.48
Sequence Activity
VKRVVKLLKG
YPWLK
3.452.93
2.92・・・
・・・RRVTKYRVDE
RMHLH
0.490.49
0.48
Sequence Activity
VKRVVKLLKG
YPWLK
3.452.93
2.92・・・
・・・RRVTKYRVDE
RMHLH
0.490.49
0.48
Sequence Activity
VKRVVKLLKG
YPWLK
3.452.93
2.92・・・
・・・RRVTKYRVDE
RMHLH
0.490.49
0.48
Sequence Activity
VKRVVKLLKG
YPWLK
3.452.93
2.92・・・
・・・RRVTKYRVDE
RMHLH
0.490.49
0.48
Sequence Activity
VKRVVKLLKG
YPWLK
3.452.93
2.92・・・
・・・RRVTKYRVDE
RMHLH
0.490.49
0.48
Sequence Activity
VKRVVKLLKG
YPWLK
3.452.93
2.92・・・
・・・RRVTKYRVDE
RMHLH
0.490.49
0.48
Sequence Activity
VKRVVKLLKG
YPWLK
3.452.93
2.92・・・
・・・RRVTKYRVDE
RMHLH
0.490.49
0.48
Sequence Activity
VKRVVKLLKG
YPWLK
3.452.93
2.92・・・
・・・
RRVTKYRVDE
RMHLH
0.490.49
0.48
Sequence Activity
VKRVVKLLKG
YPWLK
3.452.93
2.92・・・
・・・
RRVTKYRVDE
RMHLH
0.490.49
0.48
Sequence Activity
VKRVVKLLKG
YPWLK
3.452.93
2.92・・・
・・・
RRVTKYRVDE
RMHLH
0.490.49
0.48
Sequence Activity
VKRVVKLLKG
YPWLK
3.452.93
2.92・・・
・・・
RRVTKYRVDE
RMHLH
0.490.49
0.48
Questionnaire
Before
Comparison
Breast cancer patients
Subjects Japanese women (40 to 55 years old) Breast cancer patients: 306 Control: 662
Questionnaire Lifestyle such as diet and exercise habits (from childhood to now)
Analysis Compare the questionnaire responses and explore factors for the prevention of breast cancer
Epidemiological study for the prevention
of breast cancer
lactic acid bacteria
(LaB)
soy isoflavones
Main purpose of this study:
Question: Is the consumption of LaB and soy isoflavones
effective for the prevention of breast cancer?
Amount of soy isoflavones consumed (mg/day)
1.2
1.0
0.8
0.6
0.4
0.2
0
1.00
0.76
0.53 0.48
Rela
tive r
isk o
f b
reast
can
cer
< 18.76 18.76-28.81
28.81-43.75 ≥ 43.75
Effect of soy isoflavone consumption on the risk of breast cancer
Women who consumed more soy isoflavones have a lower risk of breast cancer.
0.65
1.001.2
1.0
0.8
0.6
0.4
0.2
0< 4 times/week ≥ 4 times/week
Rela
tive r
isk o
f b
reast
can
cer
Average frequency of consuming LaB-drinks
Women who consumed more LaB-drinks have a lower risk of breast cancer.
Effect of lactic acid bacteria (LaB)-containing drink consumption on the risk
of breast cancer
Combinational effect of LaB-drinks and soy isoflavones
Lower (< 18.76 mg)
Higher (≥ 43.75 mg )
Amount of soy isoflavones intake (mg/day)
Lower (< 4
times/week )Higher
(≥ 4 times/week )
Frequency of LaB-drink consumption
1.00 0.49
0.50 0.36
Women who consumed both LaB-drinks and soy isoflavones have the lowest risk of breast cancer.
Summary of case-control study
Breast cancer risk is low er in subjects w ho consum ed LaB-drinks by ≥ 4 tim es per w eek com pared w ith those w ho consum ed LaB-drinks by < 4 tim es per w eek.
The greater is the am ount of soy isoflavones consum ed, the low er becom es the risk of breast cancer.
The low est risk is observed in subjects w ho consum ed both LaB-drinks and soy isoflavones.
Breast cancer risk is lower in subjects who consumed LaB-drinks by ≥ 4 times per week compared with those who consumed LaB-drinks by < 4 times per week.
The greater is the amount of soy isoflavones consumed, the lower becomes the risk of breast cancer.
The lowest risk is observed in subjects who consumed both LaB-drinks and soy isoflavones.
Toi M et al, Curr Nutr Food Sci, 9:194-200 (2013)
8 times (85 mg/kg of body weight) Analysis
Start
0 1 17 weeksIntake of experimental
diets
Animal experiments for the prevention
of breast cancerExperimental protocol: Sprague-Dawley rats (female, 4 weeks old) were randomized into 4 groups (n=42/group), and PhIP* (chemical carcinogen) was administered orally to all the rats. Rats were given one of experimental diets for 17 weeks, and the incidence and diameter of breast cancer were measured at intervals. *PhIP: 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine
Basal diet AIN-76A (high-fat) Isoflavone (groups B and D) 335 mg/kg of diet LcS (groups C and D) 2 x 1011 CFU/kg of diet
Experimental diets: Group A: basal diet Group B: soy beverage Group C: LcS Group D: soy beverage + LcS
PhIP administration Observation period
3
4 8 12 160
25
50
75 C
an
cer-
beari
ng
rat
(%)
Time after PhIP administration (weeks)
*
*
Effect of soy beverage and LcS on the incidence of breast cancer
LcSbasal soy beverage
soy beverage + LcS
Intake of soy beverage prevented the carcinogenesis in mammary glands induced by PhIP.
Differential effects of soy beverage and LcS
soy beverage → inhibition of carcinogenesisLcS → inhibition of expansion of cancer cells
4 8 12 160
1
2
3
4
Time after PhIP administration
(weeks)
*
*
No. of cancer per rat(multiplicity)
0 2 4 6 80
5
10
15
20
25
*
*
Time after carcinogenesis (weeks)
Diameter of cancer(cancer volume)
LcSbasal soy beverage
soy beverage + LcS
(mm)
Combinatorial effect of soy beverage and LcS
Assess the range of ER-positive cells in the breast cancers in PhIP-administered rats.
Isoflavone can bind estrogen receptor (ER) on cancer cells competitively, followed by their death.
Range of estrogen receptor-positive cells (%)Intake of both soy beverage and LcS reduced the range of ER-positive cells in breast cancer !
highlow
highlow
highlow
Rela
tive r
ati
o (
%)
0
10
20
30
40 basal
0
10
20
30
40 soy beverage
0
10
20
30
40 soy beverage + LcS
Estrogen cannot bind.
Cancer cells stop to multiply and then die.
isoflavone
Kaga C et al, Cancer Sci, 104:1508-1514 (2013)
Summary of animal experiment
Daily intake of soy beverage and LcS prevented the breast cancer developm ent induced by PhIP. It appears that soy beverage inhibited the appearance of cancer cells and LcS suppressed the enlargem ent of cancers. Soy beverage and LcS m ay synergistically prevent the expansion of m alignant cancer cells.
Daily intake of soy beverage and LcS prevented the breast cancer development induced by PhIP. It appears that soy beverage inhibited the appearance of cancer cells and LcS suppressed the enlargement of cancers. Soy beverage and LcS may synergistically prevent the expansion of malignant cancer cells.
Enhancement of NK activity by LcS in tumor-bearing mice
0
10
20
30
40
50
1 2 3 4 5 6 7 8 9 10
controlL. casei Shirota
Beige mice
Incid
en
ce o
f can
cer
(%)
0
10
20
30
40
50
1 2 3 4 5 6 7 8 9 10
Weeks after 3-methylcholanthrene injection
C57BL/6 mice
controlL. casei Shirota
P=0.054
Takagi A et al, Carcinogenesis, 22:599-605 (2001)
Incid
en
ce o
f can
cer
(%)
25 50 1000
4
8
12
16
NK cell activity
C57BL/6 contC57BL/6 LcSBeige contBeige LcS
E/T ratio
% o
f sp
ecifi
c l
ysis
Schedule
DSS (9 times at intervals of 1 wk)
Administration of probiotics or saline (5 days/wk)
1 wk
Saline LcS0
1
2
No
. o
f ca
nce
rs/m
ou
se
Saline LcS0
10
20
30
40
50
60
70
80
Ca
nce
r in
cid
en
ce
(%
)
Matsumoto S et al, Immunology, 128:e170-e180 (2009)
Effect of LcS on the development of colitis-associated cancer in mice
Schedule
DSS (9 times at intervals of 1 wk)
Injection of sgp130Fc (4 times)
1 wk
0.0
0.5
1.0
1.5
2.0
2.5
Controlsgp130Fc(500mg)
sgp130Fc(50mg)
No.
of
tum
ors
***
Effect of sgp130Fc
Matsumoto S et al, J Immunol, 184:1543-1551 (2010)
IL-6/sIL-6R trans-signaling in colitis-associated cancer
Intestinalepithelial
cells
Macrophages
Intestinal bacteria
IL-6
sIL-6Ra
IL-6/sIL-6Ra complexDendritic cells
Effect of Lactobacillus strains on IL-6 release
from LPMCs in colitis-suffering mice
Matsumoto S, Clin Exp Immunol, 140:417-426 (2005)
LcS
L. plantarum ATCC14917
L. johnsonii Y50092
L. rhamnosus ATCC53103
1.252.55.0
1.252.55.0
1.252.55.0
1.252.55.0
(μg/mL)
Inhibition of IL-6 release (%)
-100-80 -60 -40 -20 0 20 40 60
Enhancement Inhibition
0 1 2 3
LcS
LcSDPSPG-I
Saline
Tumor number/mouse
0 0.5 1.0 1.5
SOCS3/G3PDH mRNA
0 20 40 60 80
IL-6/G3PDH mRNA
Matsumoto S et al, Immunology, 128:e170-e180 (2009)
Tumor number IL-6 SOCS3
LcS
LcSDPSPG-I
Saline
PSPG-I is critical in the prevention of colitis-associated cancer
*
**
*
*
*
**
Peyer’s
patch
LcS
Amelioration of IL-6-mediated inflammatory responses
Maintenance of symbiotic microbiota
Recovery from dysbiosis
Inflammatory condition
Uptake of LcS?
Disruption of gut barrier
Physiological condition
Possible roles of LcS for gut homeostasisin health and disease
Prevention of inflammatory bowel disease
and colon cancer
Enforcement of immune responses
against cancer and infection
Modulation of IL-10/IL-12-balance
Stimulation of dIgA production
Effect of LcS on glucose response in diet-induced obesity mice
0
100
200
300
400
500
600
0 30 60 90 120
Time after glucose loading (min)
Pla
sm
a g
lucose
(mg
/dl) *
**
HF diet
HF diet + LcS
Increase of plasma glucose level after glucose loading was improved by LcS !
16 wks old
4 weeks
12 wks5 wks
HF diet(60 kcal %
fat)HF diet
+LcS (0.05%)
HF diet
Experimental design
Naito E et al, J Appl Microbiol, 110:650-657 (2011)
Adrenal SNALiver SNA%
ch
an
ge
% c
han
ge
10 min
10
0 s
pik
es/5
sec
Effects of LcS on sympathetic nerve activities
Tanida M et al, J Diabetes Invest, 5:153-161 (2014)
○ placebo ■ LcS 1 x 108 ○ placebo ■ LcS 1 x 108
Effect of LcS on glucose response in high-fat diet-induced obesity mice
insulin
insulin action LcSskeletal muscle
glucose uptake glucose release
liver
inflammatory adipokines
pancreasobesity
sympathetic nerve
adipose tissue
endotoxaemia
Experimentally confirmed beneficial effects of L. casei Shirota
Immunomodulation may be related to beneficial effects !
Allergy
YesOVA-induced
Infection
Yesbacteria
virus
Cancer
Yes3MC-induced
CAC
Autoimmunity
Yesarthritis, SLE,
EAE
YesHF-induced
Metabolic disorder
Stress
Yeswater-
avoidance
Acknowledgments Kyoto University: Masakazu Toi The University of Tokyo: Yasuo Ohashi Juntendo University: Yuichiro Yamashiro, Ko Okumura Kanazawa Medical University: Mamoru Tanida Yakult Central Institute: Chiaki Kaga, Akimitsu Takagi, Kan Shida, Satoshi Matsumoto, Takashi Asahara, Koji Nomoto, Eiichiro Naito, Mitsuhisa Kawai
Thank you for your attention
!