(1) Population aging is continuing globally, making the elderly highly susceptible to infection and cancer due to declining immune function. Their gut microbiome also becomes dysregulated with age. (2) Probiotics like Lactobacillus casei Shirota have been shown to improve the altered gut microbiome of the elderly by increasing beneficial bacteria and decreasing pathogenic bacteria. (3) Administration of L. casei Shirota to the elderly provides health benefits like reducing the incidence of infectious diseases, decreasing cancer risk, and improving bowel function and general well-being.

1. Probiotics in Health – Emerging Opportunities
Dec 3-4, 2016, Chennai
Probiotics and the Elderly Microbiome
- Is it ever too late to change? -
Masanobu Nanno
Yakult Central Institute, Tokyo, Japan

2. By J.O.
Today’s topics
1. Health problems in the elderly
– Is microbiome related? ー
2. Probiotics are promising agent
to improve the altered gut
microbiome.
3. Probiotics are beneficial for
health management in the
elderly .

3. Demographics in India and Japan
0
20
40
60
80
100
1950 2010 2025 2050 2100
%
India
<20 20～64 65～74 >75
0
20
40
60
80
100
1950 2010 2025 2050 2100
%
Japan
<20 20～64 65～74 >75
Data from Statistical Handbook of Japan 2016
The proportion of elderly is gradually increasing in India and Japan!
Age Age

4. Data from Ministry of Health, Labor and Welfare (1996)
Incidence(No./105population)
Mortality
Incidence
Mortality
Age
Effect of age on incidence and mortality
of influenza infection in Japan

5. Data from National Cancer Centre Japan (2015)
Effect of age on cancer incidence in Japan
0
500
1000
1500
2000
2500
3000
3500
4000
4500
40-44 45-49 50-54 55-59 60-64 65-69 70-74 75-79 80-84 >85
Incidence
(No.ofpatients/105population)
Male Female
Age

6. Immune functions in the elderly
 Innate immunity:
Neutrophils: reduction of capabilities to migrate and uptake opsonized
particles and pathogens
Monocytes: reduction of ROS production and phagocytosis, and
dysregulation in the release of cytokines
Natural killer cells: defective capacity to secrete chemokines and
reduction of cytotoxic potential
 Acquired immunity:
Dendritic cells: reduction of mobilization and impairment of cytokine
release
T lymphocytes: reduction in the frequency of naïve T cells and lower
capacity to induce antigen-specific T cell responses
accumulation of memory T cells
B lymphocytes: decrease in the number and change in the relative
frequencies of different B cell subsets
Susceptibility to infection and cancer development in elderly
Pinti M et al. Eur J Immunol. 46:2286-2301 (2016)

7. Health problems in the elderly
 Pathogenic infection
 Cancer
 Allergies – food allergy, pollen allergy
 Autoimmune diseases – rheumatoid arthritis
 Metabolic syndrome – obesity, diabetes, cardiovascular diseases
 Mental diseases – depression, dementia
Gut microbiome
Food
Living
environment
Stress
Drugs

8. Aging and gut microbiome
of healthy subjects in Japan
2
4
6
8
10
D1-D3 D7-M1 M3-M6 Y3-19 Y20-59 Y60-79 >Y80
No.ofbacteria(Log10cells/goffeces)
Age
Total bacteria
C. coccoides group
C. leptum subgroup
B. fragilis group
Prevotella
Bifidobacterium
Enterobacteriaceae
Lactobacillus
Staphylococcus
Nomoto K et al. Intestinal Microbiota. 29:9-18 (2015)

9. Gut microbiome in the elderly
Claesson MJ et al. Nature. 488:178-184 (2012)
Gut microbiome in the elderly:
 different from the core microbiome in younger
adults
 higher Firmicutes in subgroup (1) and higher
Bacteroidetes in subgroup (4)
 most diverse in low fat/high fiber group to which
the majority of subgroup (1) belongs
 higher inflammation markers (TNF-a, IL-6, IL-8,
CRP) in subgroup (4)
Subjects: (1) the community-dwelling elderly (n=83)
(2) the elderly attending an out-patient day hospital (n=20)
(3) the elderly in short-term rehabilitation hospital care (n=15)
(4) the elderly in long-term residential care (n=60)
(5) younger adults (n=13)

10. Summary (1)
• Population aging is continuing in
many countries of the world.
• The elderly is highly susceptible to
infection and carcinogenesis due to
decline of immune functions.
• Gut microbiome is dysregulated in
the elderly, the extent of which is
dependent on their lifestyle.
By J.O.

11. Definition of probiotics
Live microorganisms which, when
administered in adequate amounts,
confer a health benefit on the host
(FAO/WHO, 2001)
Is Lactobacillus casei Shirota
(LcS) regarded as probiotics ?

12. *, P<0.05: vs. before ingestion
**, P<0.01：vs. before ingestion
a, P<0.01：vs. 3 months after ingestion
b, P<0.01：vs. 6 months after ingestion
Wang C et al. Annal Nutr Metabol. 67:257-266 (2015)
Improvement of gut microbiome
by L. casei Shirota in the children
Bacterialcount
(Log10cells/goffeces)
Bifidobacterium Enterobacteriaceae S. aureus C. perfringens
* ** b
Ingestion
period (mo)
0 121 3 6
11
10
9
8
7
6
5
* **
b
10
9
8
7
6
5
4
Ingestion
period (mo)
0 121 3 6
** *
a, b6
5
4
3
Ingestion
period (mo)
0 121 3 6
6
5
4
3
2
Ingestion
period (mo)
0 121 3 6
*
44
(%)
b
35 35 7 63

13. Improvement of gut microbiome
by L. casei Shirota in the elderly
Bacterialcount(Log10cells/goffeces)
Bifidobacterium L. casei ShirotaLactobacillus
3
4
5
6
7
8
9
10
3
4
5
6
7
8
9
10
11
ND
3
4
5
6
7
8
9
10 * * *** ***
before
after ingestion (mo)
1 3 6
before
after ingestion (mo)
1 3 6
before
after ingestion (mo)
1 3 6
C. perfringens
3
4
5
6
7
8
*
71
(%) 48 60 61
Staphylococcus
10
4
6
8
9
7
5 62
(%) 70 51 65
**
Pseudomonas
8
2
4
6
7
5
3
33 29 16
*
45
(%)
before
after ingestion (mo)
1 3 6
before
after ingestion (mo)
1 3 6
before
after ingestion (mo)
1 3 6
Bian L et al. Int J Prob Preb. 6:123-132 (2011)
*, P<0.05; **,P<0.01; ND, Not detected.

14. Improvement of clinical symptoms
by L. casei Shirota in the elderly
DiarrheaConstipation
Attack of fever
(>37℃)
Feverishduration(days/week)
0
2.0
0.5
1.0
1.5
**
*
*
before
after ingestion
(mo)
1 3 6
Constipation(No./week)
0
0.2
0.8
0.6
0.4
*
before
after ingestion
(mo)
1 3 6
Diarrhea(No./week)
0
0.1
0.2
0.3
0.4
*
before
after ingestion
(mo)
1 3 6
Bian L et al. Int J Prob Preb. 6:123-132 (2011)
*, P<0.05; **,P<0.01, vs. before ingestion

15. Improvement by L. casei Shirota
of gut discomfort in the elderly
Japan study (Mean age 85.9, Intervention 1 year; Sekita Y et al. 2015)
0
5
10
15
20
25
30
35
Before ingestion After ingestion
No.permonth
Evacuation
0
5
10
15
20
25
Before ingestion After ingestion
No.peryear
Suppository
0
2
4
6
8
10
12
14
Before ingestion After ingestion
Daysperyear
Physical condition
x1.21
x0.64 x0.63
Netherlands study (Mean age 83.8, Intervention 6 weeks; van den Nieuwhoer M et al. 2015)
0
1
2
3
4
5
6
Before ingestion After ingestion
No.perweek
Evacuation
0
2
4
6
8
10
12
14
Before ingestion After ingestion
%
Constipation
0
5
10
15
20
25
30
35
40
45
Before ingestion After ingestion
%
Diarrhea
x1.04
x0.41
x0.85

16. 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 !

17. Van Puyenbroeck K et al.
(2012)
Fujita R et al.
(2013)
Belgium study
Age: >65 years
Intervention: 6 months
Japan study
Age: Mean 83 years
Intervention: 7 months
Improvement by L. casei Shirota
of infectious diseases in the elderly
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
0 1 2 3 4 5
OddsRatio
Risk of RTI
0
1
2
3
4
5
6
Placebo LcS-drink
Days
Duration of URTI
Japan study
Age: Mean 85 years
Intervention: 6 months
0
1
2
3
Before After 1
month
After 3
months
After 6
months
Days
Duration with fever
Placebo LcS-drink
Nagata S et al.
(2016)
Time after ingestion (month)

18. Asahara T et al. J Appl Microbiol. 110:163-173 (2010)
Survival rate (%)
Days after infection
with Salmonella
0
20
40
60
80
100
0 3 15 216 9 12 18
Infected
Infected ＋ killed LcS
Infected ＋ live LcS
P<0.05
pH
6.4
6.6
6.8
7.0
7.4
7.2
P<0.01
Not infected Infected
Infected ＋ live LcS Infected ＋ killed LcS
Organic acid
mmol/gofcecalcontents
0
20
40
60
80
100
P<0.01
Anti-infectious mechanism of L. casei Shirota
- Production of short chain fatty acids -

19. Anti-infectious action of L. casei Shirota
- Enhancement of natural killer activity -
Hori T et al. Clin Diag Lab Immunol. 9:105-108 (2002)
Protocol:
BALB/c mice at 15 months old ⇒ LcS feeding for 4 months ⇒ IFV infection
NK activity assay
0
2
4
6
8
10
Control LcS
**
NK activity of splenocytes
0
1
2
Virustiter(10n
)
*
3
Control LcS
IFV titer in nasal washing
*, P<0.05; **,P<0.01

20. 0
10
20
30
40
Placebo LcS
Incidence(%)
Administration
0.0
0.2
0.4
0.6
0.8
1.0
1.2
＜4 times ＞4 times
Oddsratio
Frequency
0
10
20
30
40
50
None LcS
Incidence(%)
Administration
Toi M et al. (2013)Ishikawa H et al. (2005)Aso Y et al. (1995)
Superficial bladder cancer
(recurrence rate 1 year
after operation)
High grade colon cancer
(crisis rate 4 years
after operation)
Breast cancer
(relative risk)
Prevention by L. casei Shirota
of cancer development

21. Takagi A et al. Carcinogenesis. 22:599-605 (2001)
Cancerdevelopmentrate(%)
0
10
20
30
40
50
1 2 3 4 5 6 7 8 9 10
Development of cancer
cont
LcS
Time after 3-MC administration (wks)
Anti-cancer mechanism of L. casei Shirota
- Enhancement of natural killer activity -
0
4
8
12
16
25 50 100
Lysisoftargetcells(%)
E/T ratio
NK cell activity
C57BL/6 cont
C57BL/6 LcS
Beige cont
Beige LcS
Protocol:
C57BL/6 mice ⇒ 3-MC injection ⇒LcS feeding for 10 weeks ⇒ cancer development
NK activity assay

22. Subjects: 55～74 years old; male n=12, female n=18
Protocol: cross-over experiment
probiotics-drink (1.3 x 1010 L. casei Shirota/130 ml)
or skimmed milk, each 4 weeks intake
Dong H et al, Eur J Nutr, 52:1853-1863 (2013)
Enhancement of natural killer activity
by L. casei Shirota in the elderly
0
10
20
30
0 50 100 150
%ofspecificlysis
E/T ratio
Before
Placebo LcS-drink
0
10
20
30
0 50 100 150
%ofspecificlysis
E/T ratio
After
Placebo LcS-drink
4 weeks
* *, P<0.05

23. Anti-cancer mechanism of L. casei Shirota
- Amelioration of inflammatory response -
Matsumoto S et al. Immunology. 128:e170-e180 (2009)
LcS
LcSDPSPG-I
Control
0 20 40 60 80
IL-6/G3PDH mRNA
Expression of IL-6
P <0.05
P <0.05
0 1 2 3
Tumor count/mice
Tumor count
P <0.01
P <0.05
DSS-induced colitis-
associated colon cancer
Protocol:
BALB/c mice ⇒ DSS treatment ⇒LcS feeding for 20 weeks ⇒ cancer development

24. Modified from nature INSIGHT 16 June 2011
Increased gene expression
(cryptidin, PPARg)
IL-12
LcS
Epithelial cell layer
Treg
Mf
Th17Th1NK
IL-6
Intestinal lumen
Intestinal mucosa
Immunostimulation
Mucous layer
Normal flora
Anti-inflammation
Mf
Maintenance of
homeostasis Immune balance
Short-chain fatty acid
Immuno-modulating mechanism
of L. casei Shirota

25. Summary (2)
• Probiotics improves the gut
microbiota of healthy persons
irrespective of age.
• Probiotics helps the elderly to
maintain the healthy state.
• Probiotics in cooperation with gut
microbiota improves the immune
functions through various pathway.
By J.O.

26. Inflammatory bowel diseases
Yes
Ulcerative
colitis
Infection
Yes
Diarrhea
Cold
Yes
Constipation
Intestinal
discomfort
Predicted effects
Cancer
Yes
Superficial
bladder cancer
Colon cancer
Autoimmune
diseases
Maybe
Arthritis
Intriguing effects
Psychological
stress
Yes
Medical students
Desk workers
Amazing effects
Effectiveness of L. casei Shirota
confirmed in clinical trials

27. Individual difference of gut microbiome
 How can the healthy gut microbiome be
defined?
Regional difference of gut microbiome
 Is the role of gut microbiome common in all
the populations of the world?
Importance of infant gut microbiome to keep
health for all one’s life
 When do we start to ingest probiotics for our
health?
Responder vs non-responder to probiotics
 What determines the efficacy of probiotics?
 Is it necessary to find the individual-specific
probiotics?
Issues to be examined in the future

28. Acknowledgments
 Juntendo University:
Satoru Nagata, Yuichiro Yamashiro,
Kazuyoshi Takeda, Ko Okumura
 The University of Tokyo:
Retsu Fujita, Yasuo Ohashi
 Yakult Central Institute:
Hirokazu Tsuji, Takuya Takahashi,
Takashi Asahara, Koji Nomoto,
Akira Kushiro, Akimitsu Takagi, Takeshi Matsuzaki
Kan Shida, Tetsuji Hori, Satoshi Matsumoto
Thank you for
your attention !

29. Cell Wall Polysaccharide I
Cell Wall Polysaccharide II
Cell Wall
Cell Wall Polysaccharide II
L. casei Shirota D-PSPGI
L. casei Shirota
Cell Wall
Cytoplasm