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

!