J.B. PrajapatiPrincipal & Dean

SMC College of Dairy Science, Anand Agricultural University,

Anand–388 110 (Gujarat)

jbprajapati@aau.in

Probiotics for Geriatric Population

JBP-YIMPSF-080315 1

Global ageing…..

2000-2030: Adults worldwide >65 years of age to double from 420 million to 973 million

JBP-YIMPSF-080315 2

The greying of India…

Improvement in health care

Improved living

standards

improvement in

socioeconomic status

JBP-YIMPSF-080315 3

Consequences of ageing…

Loss of physiological functions

Loss of physical and metal faculties, weakness of health

Increased vulnerability to diseases

Chronic, disabling and multiple health problems

Great discomfort to elderly

Distress to family

Clinical, social and economical problems

Martinez et al, 2014; National Academy of Sciences, 2012JBP-YIMPSF-080315 4

Age related changes in the organ system…..1

Organ system Effects of aging

Body composition Progressive reduction in total body water and leanbody massIncrease in body fat

Cardiac andperipheral vascularsystem

Heart changes (stiffening, reduced muscle strength)Reduction in the intrinsic heart rate, Atherosclerosis and loss of elasticity of vessel walls

Musculoskeletal Loss of muscle tissueOsteoarthritisOsteoporosis

Central nervoussystem

Increased sensitivityDecreased blood flowDecline in receptors and pathways (fewer brain cellsand connections)

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Age related changes in the organ system….2

Organ system Effects of aging

Gastrointestinal Decreased secretion of hydrochloric acid and pepsinDysfunction in GI motilityDecreased GI blood flowReduction in liver volume and blood flow

Immune system Decreased immunity to diseasesGreater susceptibility to infections

Respiratory Vital capacity may decline with age, Increased rigidity of chest wallReduced thorax muscle strength and endurance

Sensory Visual impairment, thickening and yellowing of thelens of the eyeHearing impairment,Decline in the ability to taste and smell

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Diseases and clinical conditions associated with ageing

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Ageing associated with multiple illnesses and physical ailments and majority of these chronic conditions defy cure

Hence…..Need of the hour..

Encouraging healthy ageing through diet which can also overcome malnutrition

a preventive or alternative therapy which is milder with less of adverse effects

JBP-YIMPSF-080315 8

MICROBES IN HEALTHY AGEING

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Combating putrefaction in the gut by hygiene, diet and biologicals

Gut microbiota : major player in health and disease

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Elderly gut microbiota different than that of younger adults

high inter-individual variation in microbiota composition in elderly

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Bacteriodetes

Bacteriodetes

Firmucutes

Firmucutes

The solution…

Improve microbial balance of the intestinal tract of elderly

Providing healthier nutrition

Developing elderly specific functional foods

JBP-YIMPSF-080315 12

MOST RELEVANT TOOLS TO MODIFY GUT MICROBIOTA

Probiotics

Reddy et al, 2011JBP-YIMPSF-080315

13

Probiotics in geriatric health-emerging evidences Probiotics have been recommended by WHO as an adjunctive therapy for

nutritional deficiency (Kurniawan and Simadibrata, 2011)

The potential role of probiotics on the gut-brain axis is an emerging study area particularly important in relation to neurodegerative diseases of elderly (Martinez, et al, 2014).

Results of some previous studies……..

JBP-YIMPSF-080315 14

Selected studies in elderly showing effect of probiotics on gut microbiota and the immune system.

Strain Product Assay design1;n2

Age(years)

Effect3 Reference

Bifidobacteriumlactis HN019

Dehydrated sachets mixed with low fat milk

Xover;30 Median 69

↑T helper cell (CD4), activated T lymphocytes (CD 25) and NK cells ↑ ex vivo phagocytic capacity of mononuclear and polymorphonucleaer phagocytes and the tumoricidal activity of NK

Gill et al., 2001

Lactobacillus casei (Shirota)

Probiotic dairy product

DBRPC;124 61±7.3 Improved the mood of those whose mood was initially poorNo improvement of frequency of defecation

Benton et al., 2006

JBP-YIMPSF-080315 15

B. lactis HN019 Mixture with skim milk

DBRPC;4 groups of 20

>60 ↑bifidobacteria, lactobacilli and enterococci↓Enterobacteria

Ahmed et al., 2007

Lactobacillus delbrueckii subsp. bulgaricus B481

Capsule with dehydrated probiotics

DBRPC;61 >85 ↑NK cells↑immune risl parameters↓proinflammatory cytokine IL-B↑ antimicrobial peptide β defensins02(hBD-2)

Moro Garcia et al., 2012

BifidobacteriumlongumBB536

Enteral tube feeding

DBRPC;hospitalized 45

81.7±8.1

↑Bifidobacterium ↑ IgA after influenza vaccination (A/H1N1, A/H3N2 and B)↑ NK cell activity(in subjects with lower NK cell activity)

Akatsuet al., 2012

Lactobacillus plantarum CECT 7315 and 7316

Capsule with dehydrated probiotics

DBRPC;nursing home 60

65-85 ↑ response to influenza vaccination (↑ influenza specific IgA and IgG)

Bosch et al., 2012

JBP-YIMPSF-080315 16

Bifidobacteriuminfantis CCUG 52486, B. longumSp 0713, Lactobacillus rhamnosus GG and Lactobacillus casei (Shirota)

Fermemented milk

In vitro PBMC; 16

65-76 Each single probiotic enhanced NK activity with B. infantis effect was influenced by ageing In the youngest ↑Bifidobacterium , ↑IFN-ϒ(not by LGG)↑IL 6 production in the older and B. infantis was the most anti inflammatory

You and Yaqoub, 2012

B. longum BB536

Enteral tube feeding

DBRPC (two trials);Hospitalized B3/123

65-102 Regularised bowel movements with lower input. No other differences

Kondo et al., 2013

B. longum Bar 33 and Lactobacillus helveticus Bar 13

biscuits DBRPC;32

71-88 ↓ opportunistic pathogens Clostridium cluster XI, Clostridium difficle. Clostridium perfringes, Enterococcus faecium and Campylobacter

Rampelliet al., 2013

1Type of Intervention Assay; Xover=cross over assay; DBRPC=double blind randomized placebo control2n=number of subjects3NK=natural killer cells; Il=ineterleukin A/G; IFN-ϒ=interferon-gamma

JBP-YIMPSF-080315 17

Selected studies in elderly showing the effect of synbiotics on gut microbiota and the immune system

Treatment Product Assay design2;n3

Age(years)

Effect4 References

B. bifidum BB-02, B. lactisBL01 and inulin

6 g chicory inulin (Rafilose) plus capsule with capsules with dehydrated probiotics per day

DBRPC;18 >62 ↑bifidobacteriaand lactobacilli

Bartosch et al., 2005

B. longum2C (DSM 14579) and 46 (DSM 14583) vs. B. animalis BB12 and oatmeal

Fermented oat meal

DBRPC;169 Avg 84-3

↑Bifidobacterium species in all samples correlated to↓ TNF –α and IL-10

Ouwehand et al., 2008

JBP-YIMPSF-080315 18

B. longum 46 and B. longum2C and oatmeal

fermented oat meal

DBRPC;66

84±8 ↑Bifidobacteriumcalanufatum, B. bifidumand Bifidobacteriumbreve

Lahtinen et al., 2009

L. rhamnosus GG and FOS

250 g/d commercial yoghurt with LGG and 2.4% scFOS (Actilight)

DBRPC; 12 women constipation, nursing home

76-90 No increase of bifidobacteria↓presence of LGG in faeces of elderly froup than in younger adults↑evaluation number in the elderly, probably due to the presence of FOS

Granata et al., 2013

JBP-YIMPSF-080315 19

Work done at AAU

JBP-YIMPSF-080315 20

Metagenomic and Clinical investigation of synbiotic fermented dairy product containing probiotic

Lactobacillus helveticus MTCC 5463 in geriatric volunteers

21

PARTNERS: Dairy Microbiology Department, Anand Agricultural University, Anand

PI : Dr JB Prajapati, Professor, Dairy MicrobiologyCo-PI : Dr Vijendra Mishra, Associate Prof., Dairy Microbiology Mrs Suja Senan, Asst. Prof., Dairy Microbiology Mrs Sreeja V. Asst. Prof., Dairy Microbiology

Animal Biotechnology Department, Anand Agricultural University, AnandCo-PI : Dr CG Joshi, Prof., Animal Biotechnology

HM Patel Centre for Medical Care & EducationPramukh Swami Medical College, Karamsad-Gujarat

Co-PI : Dr Himanshu Pandya, Prof., Medicine Dr Sunil Trivedi, Prof of Microbiology Dr Uday S Singh, Prof of Community Medicine Dr Rupal Patel, Asst. Prof., Microbiology Dr Manisha Gohel, Asst. Prof. Community Medicine

Dr Ajay Pathak, Statistical Expert

Vidya Dairy, AnandCo-PI : Dr HK Desai, Managing Director

JBP-YIMPSF-080315

22

Objectives

Selection of functional synbiotic dairy product

To conduct feeding trial in old age subjects and validate health benefits

Study changes in composition and biochemical activities of gastrointestinal microflora

Metagenomic analysis of gut microflora composition and metabolic pathways.

To manufacture product on pilot scale and survey its acceptability

Aim of the study

to clinically investigate the influence of an indigenous probiotic culture, fed through well accepted synbiotic dairy food, in geriatric volunteers and carry out metagenomic analysis for gut microflora.

JBP-YIMPSF-080315

Selection of synbiotic product

0

2

4

6

8

10

0day 7day 14day 21day 28day

Honey

Carrot

Oat

Musali

Storage period in days

Overall acceptability of synbiotic lassi products during storage at 5 � 2 � c

Se

nso

ry sc

ore

8.5

9

9.5

10

10.5

11

0 7 14 21 28

Honey

Carrot

Oat

Musali

Storage period in days

Lo

g

cfu/m

l

Changes in lactobacilli count (log cfu/ml) of synbiotic lassiproducts during storage at 5 � 2 � c

Changes in lactobacilli count of synbiotic products during storage at 5 ± 2 °c

Overall acceptability of synbiotic lassi during storage at 5 ± 2 °c

JBP-YIMPSF-080315 23

Methodology

24

Representation of the study design and sample collection

JBP-YIMPSF-080315

Intervention- probiotic fermented milk supplemented with honey

Design of study and subjects flow

25JBP-YIMPSF-080315

Data collection….. The changes in composition and biochemical activities of gastrointestinal

microflora were studied on the basis of their population in faecal sampes and facecal enzyme activity.

Collection of blood for Haematological, lipid, and immunological parameters.

Metagenomic analysis of gut microflora composition was studied by using semi conductor based amplicon sequencing on Ion Torrent PGM sequencer.

The metagenomic data obtained were analysed using MG-RAST and QIIME followed by STAMP statistical analysis.

The product was prepared on pilot scale at Vidya Dairy and survey of its acceptability in the geriatrics is conducted.

26JBP-YIMPSF-080315

Quantification of gut flora by traditional plating

JBP-YIMPSF-080315 27

T0 T30 P0 P308.00

8.20

8.40

8.60

8.80

9.00

9.20

9.40

8.47

9.19 9.179.10

Series1

Changes in Lactobacilli count

Quantification of MTCC 5463 by RT-PCR method

a standard curve for the strain Lb. helveticus MTCC 5463 is obtained. The number of cells of lactobacilli in the target samples was determined by comparing the Ct values obtained to the standard curve.

28

1 2 3 4 5 6 70

5

10

15

20

25

30

35

f(x) = − 3.44038138809637 x + 36.9876775955033R² = 0.994054720282937

Standard curve for lactobacilli

Log no. cells/ml

Thre

shol

d cy

cle

(Ct)

JBP-YIMPSF-080315

Levels of L. helveticusMTCC 5463 in faecaes of the elderly volunteers

The strain was not detected in any of the subjects in Group A or B before active test feeding

A statistically significant increase in the fecal amounts of strain confirmed the ability of the strain to colonize the human gut when delivered in a fermented drink.

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T0T30

P0P30

0

1

2

3

4

5

6

7

8

0

7.77751367776294

5.938915834661446.15106445963725

7.91826054724741

Group A

Group B

Time period (30days interval)

Lo

g G

ene

Co

pie

s/g

of

feac

al m

atte

r

T0=Before probiotic FeedingT30= After probiotic FeedingP0= Before placebo feedingP30= After placebo feeding

Group A-Started with test product, Group B – started with placebo product.

JBP-YIMPSF-080315

Feacalβ-glucuronidase activity

The mean β-glucuronidase activity was reduced in test group from 1.40 to 0.73 (Microgram/min/mg of protein) while in case of placebo group, no effect on enzyme activity was observed.

Enzyme β-glucuronidase activity in the faeces of all subjects in the probiotic group, were highly significant (p = 0.00029) while in case of placebo group it showed non-significant difference (p= 0.4082).

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T0 T30 P0 P300.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

1.41

0.73

1.461.52

Time period (30 day interval)

Mic

rog

ram

/min

/mg

ofp

rote

in

T0=Before probiotic Feeding, T30= After probiotic FeedingP0= Before placebo feeding, P30= After placebo feeding

JBP-YIMPSF-080315

Effect on serum calcium and creatinine

Characteristics

Probiotic group Placebo

N T0 T30P-value N P0 P30

P-value

Heamoglobin (13-17 g/dl) 58

12.41±1.52

12.40±1.39

0.89 6512.29±1.

7112.17±1.

710.08

Heamatocrit (36-53%) 57

38.89±3.45

39.57±3.13

0.00 5939.88±3.

4039.61±3.

060.24

Calcium (8.6-10.2mg/dl)

628.45±0.6

19.36±0.4

5<0.001 69

9.56±0.68

8.65±0.76

<0.001

31

Group comparison of hematological parameters and calcium

Serum calcium level was significantly improved in probiotic Lassi Group (p<0.001).

The means of probiotic as well as placebo groups of volunteers for haemoglobulin and serum creatinine were not significant before and after intervention

JBP-YIMPSF-080315

Effect on the Immunological parameters (TNF-α, IL2, IFN–γ and IgG or IgM levels)

32

Immunological parameters

Placebo group Probiotic group

P value

Subjects with normal or abnormal value* (as defined for each parameter) before dietary supplement(a)

Subjects with Significant benefit observed

(b)

% Correction

(b/a X100)

Subjects with normal or abnormal value* (as defined) before dietary supplement(c)

Subjects with Significant benefit observed

(d)

% Correction

(d/c X100)

TNF-α## 34* 01 2.9% 44* 12 27.2% 0.011

IFN–γ ** 00 00 00 03 03 100% NA

IL 2** 53 02 3.77% 55 11 20% 0.016

IgG** 58 03 5.1% 58 00 00 0.24#

IgM** 58 00 00 58 00 00 NA# Fisher’s Exact Test* Any value reported beyond 25% of the upper/lower limit of the normal range has been taken as abnormal value.##A moderate increase of TNF-α level from normal value to maximum three times than the upper limit of normal range; as well as, a decrease from abnormally high value (more than ten times higher than the upper limit of the normal range) to at least half of the baseline abnormal level.** A moderate increase of levels from sub-optimal or normal level to at least double of the upper limit of normal range

Salient findings

A significant immunomodulatory effect on the TNF-α and IL2 levels for the benefit of the subjects among probiotic group in comparison to placebo group.

There was however no significant beneficiary effect found on IFN–γ, IgG or IgM levels.

33JBP-YIMPSF-080315

Paired t test results for lipid profile parameters in 2 groups of human subjects

34

Characteristics Probiotic group Placebo 

N T0 T30P-value

N P0 P30P-value

Serum cholesterol (130-220mg/dl)  

54 161.67±41.05158.09±

42.630.12 68

174.32±49.99

167.09±43.11

<0.001

triglyceride (upto 170mg/dl) 

60 103.77±49.84104.00±56.4

30.96 69

116.38±71.01

108.58±70.74

0.03

HDL (30-68mg/dl) 

62 46.21±12.46 47.08±13.97 0.24 6949.67±15.9

748.77±12.9

80.34

LDL(100-129mg/dl)  

55 98.48±37.12 92.93±35.79 0.01 5388.93±38.3

784.56±31.1

30.09

VLDL(upto 38mg/dl) 

61 21.63±12.04 21.34±11.97 0.74 6923.28±14.2

021.71±14.1

20.03

TC/HDL(upto 5.0)  

62 3.91±1.22 3.74±1.20 <0.001 69 3.77±1.33 3.65±1.23 0.04

LDL/HDL(upto 3.5)

62 2.37±0.96 2.21±0.91 <0.001 69 2.23±1.01 2.13±0.96 0.04

Metagenomic analysis of gut microflora composition and metabolic pathways

The metagenomic study of faecal microflora of geriatric volunteers revealed that they were dominated by Firmicutes (50%), Acintobacteria (20%) and Proteobacteria (10%).

Changes in the phylum composition after probiotic feeding included a 7% increase in Firmicutes, 1.5 % rise in Actinobacteria and 1.9% increase in Proteobacteria.

Proteobacteria were higher in non responders than in responders.

The STAMP analysis revealed that among responders and non responders the chief genera of Firmicutes that showed significant difference wereLactobacillus, Clostridium, Eubacterium, and Blautia (q< 0.002) while the genera of Proteobacteria included Shigella, Escherichia, Burkholderia and Camphylobacter (q-value<0.002).

35JBP-YIMPSF-080315

36JBP-YIMPSF-080315

JBP-YIMPSF-080315 37

To summarize… Gut microbiota in elderly was shown to be strongly influenced by diet.

Faecal Lactobacilli count increased and their presence also helped in reducing faecal β-glucuronidas activity.

A significant immunomodulatory effect on the TNF-α and IL2 levels for the benefit of the subjects among treated group in comparison to placebo group was observed.

The metagenomic study revealed that the faecal samples of geriatric volunteers were dominated by Firmicutes (50%), Acintobacteria (20%) and Proteobacteria (10%).

Changes in the phylum composition after probiotic feeding included a 7% increase in Firmicutes, 1.5 % rise in Actinobacteria and 1.9% increase in Proteobacteria

JBP-YIMPSF-080315 38

To summarize…

Very few probiotic intervention studies in India.

Knowledge regarding gut microbiome of elderly of different geographical regions of India is required.

Efforts should be put to better understand bacterial shifts in gut microbiome during a probiotic therapy in geriatric populations

JBP-YIMPSF-080315 39

“Let us give the elderly a healthy living and let us all have a healthy ageing”

Thank youJBP-YIMPSF-080315 40