Efficacy of Indian Probiotic Cultures - ILSI...

34
Efficacy of Indian Probiotic Cultures Efficacy of Indian Probiotic Cultures Sunita Grover, Ph.D Molecular Biology Unit Dairy Microbiology Division Dairy Microbiology Division National Dairy Research Institute

Transcript of Efficacy of Indian Probiotic Cultures - ILSI...

Page 1: Efficacy of Indian Probiotic Cultures - ILSI Indiailsi-india.org/conference-on-feb20-2009/session1/Dr. Sunita Grover.pdf · Quantification of BSH activity by modified ninhydrin assay

Efficacy of Indian Probiotic CulturesEfficacy of Indian Probiotic Cultures

Sunita Grover, Ph.DMolecular Biology Unit

Dairy Microbiology DivisionDairy Microbiology DivisionNational Dairy Research Institute

Page 2: Efficacy of Indian Probiotic Cultures - ILSI Indiailsi-india.org/conference-on-feb20-2009/session1/Dr. Sunita Grover.pdf · Quantification of BSH activity by modified ninhydrin assay

Outline of presentationOutline of presentation

• Probiotics – entry in Indiay• Status of probiotics at global level• Need for Indigenous probiotics

R l f t ti l bi k f i • Role of potential biomarkers for screening and selection of potential strains

• Initiative for Developing Indigenous p g gProbiotics of Indian origin at NDRI, Karnal (DBT)

• Desired Interventions from Indian Desired Interventions from Indian Perspective

• Conclusion

20.2.2009 2ILSI-India

Page 3: Efficacy of Indian Probiotic Cultures - ILSI Indiailsi-india.org/conference-on-feb20-2009/session1/Dr. Sunita Grover.pdf · Quantification of BSH activity by modified ninhydrin assay

Probiotics – the New Nutraceutical Stars d kenter in Indian Market

• An old concept, with a new attitude • Probiotic based functional foods a strong • Probiotic based functional foods – a strong

global market • Japan, Europe - the major playersJapan, Europe the major players• Dairy based probiotic foods / drinks (>

50%)

• Probiotics gaining a foothold in India

20.2.2009 3ILSI-India

Page 4: Efficacy of Indian Probiotic Cultures - ILSI Indiailsi-india.org/conference-on-feb20-2009/session1/Dr. Sunita Grover.pdf · Quantification of BSH activity by modified ninhydrin assay

Status of probiotic cultures at Global levelStatus of probiotic cultures at Global level

• L. rhamnosus GG (Valio)L i Shi (Y k l )• L. casei Shirota (Yakult)

• L. plantarum 299v (Probi AB)• L. johnsonii La7 (Nestle) • L reuteri (BioGaia)• L. reuteri (BioGaia)• L. acidophilus NCFM (Nestle)• L. casei strain DN-114001 (Danisco)• B animalis DN 173010 (Danisco)B. animalis DN 173010 (Danisco)• L. rhamnosus 271 (Probi AB)• L. casei (Chr Hansen)• L. acidophilus La1p• VSL#3 (4 Lactobacillus spp.; 4 Bifidobacterium spp. and one

streptococcus) (CD Pharma)

20.2.2009 4ILSI-India

Page 5: Efficacy of Indian Probiotic Cultures - ILSI Indiailsi-india.org/conference-on-feb20-2009/session1/Dr. Sunita Grover.pdf · Quantification of BSH activity by modified ninhydrin assay

Efficacy / Prospects of probiotic strainsEfficacy / Prospects of probiotic strains• Widely studied strains world wide

S i tifi ll bi ti• Scientifically proven probiotics • Clinical trials conducted for their efficacy• However, results are debatable

• Several factors are known to influence this benefit

Timing of probiotic delivery– Prophylactic, empiric or therapeutic

Specific strain of bacteria– Various secreted components can modulate activity

Quantity of bacteriaMethod of administrationHost factorsHost factors

20.2.2009 5ILSI-India

Page 6: Efficacy of Indian Probiotic Cultures - ILSI Indiailsi-india.org/conference-on-feb20-2009/session1/Dr. Sunita Grover.pdf · Quantification of BSH activity by modified ninhydrin assay

Major Issues for use in Indian population j f p p

• Data in Indian population – lacking ?• Poor adhesion /colonization and short

transit period in the Indian gut due to diff r nt ut c l nd f d h bitsdifferent gut ecology and food habits

• Conditioning effect – critical for acclimatization and optimal functionality acclimatization and optimal functionality

• Non-availability of established / Novel Indian probiotic strains with specific health Indian probiotic strains with specific health functions to demonstrate their efficacy

20.2.2009 6ILSI-India

Page 7: Efficacy of Indian Probiotic Cultures - ILSI Indiailsi-india.org/conference-on-feb20-2009/session1/Dr. Sunita Grover.pdf · Quantification of BSH activity by modified ninhydrin assay

Why Need for Indigenous probiotics ?W y N f g p

• Better colonization / adhesion potentialL t it ti• Longer transit time

• Health promoting functions of probiotics – highly strain and host specific p

• High incidences of diarrhoeal diseases and other gastric disorders in Infants and immuno-compromisedcompromised

• Probiotics as potential Immuno-modulators –Healthy gutsI di bi ti t i i ht b b tt it d t• Indian probiotic strains might be better suited to Indian gut due to longer transit time, colonization / biofilm formations and conditioning effect

20.2.2009 7ILSI-India

Page 8: Efficacy of Indian Probiotic Cultures - ILSI Indiailsi-india.org/conference-on-feb20-2009/session1/Dr. Sunita Grover.pdf · Quantification of BSH activity by modified ninhydrin assay

Initiative for Developing Indigenous Probiotics f di i i N l ( T)of Indian origin at NDRI, Karnal (DBT)

• More than one hundred cultures of indigenousLactobacilli of human origin isolated and studied forth i bi ti tt ib t d l i ti t ti ltheir probiotic attributes and colonization potentials

• Tested for a battery of in vitro tests for culture short li ti

ylisting

• Selection of promising cultures based on strong p g gcolonization and in vitro probiotic attributes

20.2.2009 8ILSI-India

Page 9: Efficacy of Indian Probiotic Cultures - ILSI Indiailsi-india.org/conference-on-feb20-2009/session1/Dr. Sunita Grover.pdf · Quantification of BSH activity by modified ninhydrin assay

Selection of promising cultures based on in vitro probiotic attributesprobiotic attributes

pH 1.5 pH 2.0p

Bile 1.5% Bile 2.0%

20.2.2009 9ILSI-India

Page 10: Efficacy of Indian Probiotic Cultures - ILSI Indiailsi-india.org/conference-on-feb20-2009/session1/Dr. Sunita Grover.pdf · Quantification of BSH activity by modified ninhydrin assay

Contd…..Contd…..

Pepsin Lysozyme

2

4

6

8

10

cell

coun

t (lo

g cf

u/m

l)

0

0h 8.585 8.301 8.9201 8.7447 8.5965 8.672 8.2218

1h 8.0412 8.4471 8.7626 8.231 8.2304 7.4471 8.284

Lp 5276

Lp9 Lp72 Lp75 Lp77 Lp91 Ldch4

Cell surface hydrophobicity Anti-oxidant

20.2.2009 10ILSI-India

Page 11: Efficacy of Indian Probiotic Cultures - ILSI Indiailsi-india.org/conference-on-feb20-2009/session1/Dr. Sunita Grover.pdf · Quantification of BSH activity by modified ninhydrin assay

Adhesion of Lactobacilli (109 cfu/ml for 2 hrs) to human adenocarcinomal cell linesadenocarcinomal cell lines

Caco2 cell line:

HT-29 cell line:

L. plantarum (Lp91)L. plantarum (Lp5276)L. plantarum (LpVS)

HT 29 cell lineL. plantarum (Lp91)

L. plantarum (LpVS)L. plantarum (Lp5276)p ( p )

20.2.2009 11ILSI-India

Page 12: Efficacy of Indian Probiotic Cultures - ILSI Indiailsi-india.org/conference-on-feb20-2009/session1/Dr. Sunita Grover.pdf · Quantification of BSH activity by modified ninhydrin assay

Role of Biomarkers for selection of probioticsRole of Biomarkers for selection of probiotics

• Selection of a proven probiotic strain extremely crucial for application in clinical trials and product developmentdevelopment

• Potential biomarkers need to be developed for screening and selection of probiotic strainsscreening and selection of probiotic strains

Page 13: Efficacy of Indian Probiotic Cultures - ILSI Indiailsi-india.org/conference-on-feb20-2009/session1/Dr. Sunita Grover.pdf · Quantification of BSH activity by modified ninhydrin assay

Expression of bacterial and host genes during transition in GIT of human that can be used as potential biomarkers for

functionality↑ MUC 2↑ INF

functionality

Probiotics ↑ atp operon↑ groEL

↑ INF- α↑ IL-10↑ IL-4 ↑ bsh gene

↑ h 70↑ groEL↑ groES↑ dnaK↑ hsp70

Surface proteins of bacteria↑ MapA

↑ IL-6↑ Cox1↑ hsp-72

↑ hsp70

↑ urvA↑ dps↑ Msr

↑ p↑ Mub↑ CnBp↑ Fbp↑ EF TuStomach

↑ hsp 72

↓ TNF-α↓ IFN-γ

Human epithelial cells

Small intestine↑ EF-Tu↑ dlt operon↑ eps operon

Stomach ↓ IFN γ↓ IL-1β↓ IL-2↓ IL-8

Digestive system

↓↓ Cox2

20.2.2009 13ILSI-India

Page 14: Efficacy of Indian Probiotic Cultures - ILSI Indiailsi-india.org/conference-on-feb20-2009/session1/Dr. Sunita Grover.pdf · Quantification of BSH activity by modified ninhydrin assay

Quantification of BSH activity by modified ninhydrin assay

5

6

tivity

m

l)

P<0.01

3

4

5

drol

ase

act

n/10

8 cfu

/m

0

1

2

2 1 0 7 8 1 0 5 1 9ile sa

lt H

yd(µ

M/m

in

Lp-82

Lj-169

3

LA1La-2

91La-1

95La-1

6Lp-1

1Lp-1

0Lp-2

7Lp-7

8Lp-9

1Lp-90Lp-75Lp-21Lf-

69Bi

Isolate No.

Bile salt hydrolase activity of lactobacilli cultures was quantified against different conjugated bile salts.

GCA hydrolase activity. GDCA hydrolase activityy y y yTCA hydrolase activity. TDCA hydrolase activity.

Page 15: Efficacy of Indian Probiotic Cultures - ILSI Indiailsi-india.org/conference-on-feb20-2009/session1/Dr. Sunita Grover.pdf · Quantification of BSH activity by modified ninhydrin assay

Anti-Hypercholesterolemic effect of Lp-91

Duration of treatment:- 21 days

Lp91 Lp21

TC 17.2 9.1

TG 27.3 18.9

140)

Total Cholesterol Triglyceride HDL LDL

TG 27.3 18.9

LDL 35.5 13.5

HDL 47.8 14.4

80100120140

le (m

g/dL Diet without Lp-91P<0.01

0204060

Lip

id pr

ofi

ND Ctrl HD Ctrl HD + 91 HD 91 HD Cap91 HD 21

Treatment groups

ND N l Di t HD H h l t l i di t HD 91 H h l t l i di t ith t LND, Normal Diet; HD, Hypercholesterolemic diet; HD+91, Hypercholesterolemic diet without Lp-91; HD91, Hypercholesterolemic diet containing noncapsulated Lp-91; HDCap91,Hypercholesterolemic diet mixed with Microencapsulated Lp-91; HD21,Hypercholesterolemic dietmixed with Lp-21 and HD Shirota, Hypercholesterolemic diet mixed with L. casei (Shirota).

Page 16: Efficacy of Indian Probiotic Cultures - ILSI Indiailsi-india.org/conference-on-feb20-2009/session1/Dr. Sunita Grover.pdf · Quantification of BSH activity by modified ninhydrin assay

Transposon mediated insertional inactivation of bsh gene of L. plantarum 77 strain.

ATGTGTACTGCCATAACTTATCAATCTTATAATAATTACTTCGGTAGAAATTTCGATTATGAAATTTCATACAATGAAATGGTTACGATTACGCCTAGAAAATATCCACTAGTATTTCGTAAGGTGGAGAACTTAGATCACCATTATGCAATAATTGGAATTACTGCTGATGTAGAAAGCTATCCACTTTACTACGATGCGATG

CAAAAACTAAACTTGGTTAATATTAATTTTAGTGAACAATTACCATTATCACCGCTACATTGGTTGGTTGCTGATAAACAGGAATCGATAGTTATTGAAAGTGATAAAGAAGGACTAAAAATTTACGACAATCCAGTAGGTGTGTTAACAAACAATCCTAATTTTGACTACCAATTATTTAATTTGAACAACTATCGTGCCTTATCAAATAGCACACCCCAAAATAGTTTTTCGGAAAAAGTGGATTTAGATAGTTATAGTAGAGGAATGGGCGGACTAGGATTACCTGGAGACTTGTCCTCAATGTCTAGA

GGCAGATTGCAAGAAATAACTTGAACGAATTTAATGACGTAAATTAAGCAAGAAGATCTCGATTGGGGTATGCCAATTAAGACATTTAAGTGGCCGTGAATTCAAATACCAATTAATTTGAACCAATTGGTGATCACTTAGTTCTTCAATGGCTTGGCCTTTGGGAATAAAGCGCCGCAAAACTCGGTTACGGTTCTCATTACTGCCTCTTTCATGCGGTGAATAGGCAGGGGCAAAATAAACCTGTGTACCAGTTCGCCGTTCAATTGTCTGATAGGTAGCGAACTCTTTACCATGATCTACGGTAAGCGTCTTGAG

1057 bp254 bp 619 bp5’- -3’

TGTAGAAAGCTATCCACTTTACTACGATGCGATGAATGAAAAAGGCTTGTGTATTGTGGGATTAAATTTTGCAGGTTATGCTGA

TAGAGGAATGGGCGGACTAGGATTACCTGGAGACTTGTCCTCAATGTCTAGATTTGTCAGAGCCGCTTTTACTAAATTAAACTCGTTGCCGATGCAGACAGAGAGTGGCAGTGTTAGTCAGTTTTTCCATATACTAGGGTCTGTAGAACAACAAAAAGGGCTATGTGAAGTTACTGACGGAAAGTACGAATATACAATCTATTCTTCTTGTCGTGATATGAACAAGGGAGTTTATTACTATAGAACTTATGACAATAGTCAAATTAACAGTGTCAATTTAAACCATGAGCACTTGGATACGACTGAATTAATTTCTTATCCATTACGATCAGAAGCACAATACTATGCAGTTTAACTAA

ATTGTCTGATAGGTAGCGAACTCTTTACCATGATCTACGGTAAGCGTCTTGAGCTTGTCTTGAAGTTGACTAGCTAGTTCAAGTACGGCTTGAGTCATGGACTGACTGTCGCGACCATGGAGCCGTTTACAATTGTCAGGCGACTCTTACGCTCCACAAAAGTAGCCACAGCTTGACCTTTACGTTTACCAGAAAGTACGGTATCAGCTTCAAAGTGGCCGAATTCCTGGCGAGTTTCGACTTTATGAGGCCGCTCCTCAATGGAGCGGCCGTGATTGAACGTACCACGCTTTTCTTTAGCACGATGACGACGAATCCCATGATCAGGCAAATCGGGCAACTGTATATCAAGCCATCCTTGATCAATCCAGTTATAGACCGTCTTGTAGGCAATCCCCACCACATGGGCAACTTGTTCAGGGGACCACTTTTGGACTTGAATCTTTTCCTCGATCAAGTGTTTAAGGTTTTTAGTGACCACTTTTGGACTTGAATCTTTTCCTCGATCAAGTGTTTAAGGTTTTTAGTGAGCGAAGACTTCCGCCCCCGTTGACTAACCTTTCGTTCAAAGTCAGTTTGCGCTAGCCCAGCCTGGTACTCACTATTTAGCCCGTGAAGTTCGTTAAAGATGGTGGTCTTACTAAAGCCTAAGTAGTTAGCGATGTATCGCAAGGAACGTCCTTCATTATGTAAGCGTGTCAATGACAACACGGTTCTGGAATGAAAAAATAGTGGTGCTCATCAAGGTCCTTCCTTTCTAATGGAGTGGGTGGTAACATCATTTAAGGACCTTGATTGGGTTTTTCTGGGCCCCTTAAATGTTCAACTTAAATTTTACAATCTGC

873 bp GGCAGATTGCAAGAAATAACTTGAACGAATTTAATGACGTAAATTAAGCAAGAAGATCTCGATTGGG

1057 bp873 bp

ATGTGTACTGCCATAACTTATCAATCTTATAATAATTACTTCGGTAGAAATTTCGATTATGAAATTTCATACAATGAAATGGTTACGATTACGCCTAGAAAATATCCACTAGTATTTCGTAAGGTGGAGAACTTAGATCACCATTATGCAATAATTGGAATTACTGCTGATGTAGAAAGCTATCCACTTTACTACGATGCGATGAATGAAAAAGGCTTGTGTATTGTGGGATTAAATTTTGCAGGTTATGCTGACAAAAACTAAACTTGGTTAATATTAATTTTAGTGAACAATTACCATTATCACCGCTACATTGGTTGGTTGCTGATAAACAGGAATCGATAGTTATTGAAAGTGATAAAGAAGGACTAAAAATTTACGACAATCCAGTAGGTGTGTTAACAAACAATCCTAATTTTGACTACCAATTATTTAATTTGAACAACTATCGTGCCTTATCAAATAGCAC

GTATGCCAATTAAGACATTTAAGTGGCCGTGAATTCAAATACCAATTAATTTGAACCAATTGGTGATCACTTAGTTCTTCAATGGCTTGGCCTTTGGGAATAAAGCGCCGCAAAACTCGGTTACGGTTCTCATTACTGCCTCTTTCATGCGGTGAATAGGCAGGGGCAAAATAAACCTGTGTACCAGTTCGCCGTTCAATTGTCTGATAGGTAGCGAACTCTTTACCATGATCTACGGTAAGCGTCTTGAGCTTGTCTTGAAGTTGACTAGCTAGTTCAAGTACGGCTTGAGTCATGGACTGACTGTCGCGACCATGGAGCCGTTTACAATTGTCAGGCGACTCTTACGCTCCACAAAAGTAGCCACAGCTTGACCTTTACGTTTACCAGAAAGTACGGTATCAGCTTCAAAGTGGCCGAATTCCTGGCGAGTTTCGACTTTATGAGGCCGCTCCTCAATGGAGCGGCCGTGATTGAACGTACCACGCTTTTCTTTAGCACGATGACGACGAATCCCATGATCAGGCAAATCGGGC

ACCCCAAAATAGTTTTTCGGAAAAAGTGGATTTAGATAGTTATAGTAGAGGAATGGGCGGACTAGGATTACCTGGAGACTTGTCCTCAATGTCTAGATTTGTCAGAGCCGCTTTTACTAAATTAAACTCGTTGCCGATGCAGACAGAGAGTGGCAGTGTTAGTCAGTTTTTCCATATACTAGGGTCTGTAGAACAACAAAAAGGGCTATGTGAAGTTACTGACGGAAAGTACGAATATACAATCTATTCTTCTTGTCGTGATATGAACAAGGGAGTTTATTACTATAGAACTTATGACAATAGTCAAATTAACAGTGTCAATTTAAACCATGAGCACTTGGATACGACTGAATTAATTTCTTATCCATTACGATCAGAAGCACAATACTATGCAGTTTAACTAA

AACTGTATATCAAGCCATCCTTGATCAATCCAGTTATAGACCGTCTTGTAGGCAATCCCCACCACATGGGCAACTTGTTCAGGGGACCACTTTTGGACTTGAATCTTTTCCTCGATCAAGTGTTTAAGGTTTTTAGTGAGCGAAGACTTCCGCCCCCGTTGACTAACCTTTCGTTCAAAGTCAGTTTGCGCTAGCCCAGCCTGGTACTCACTATTTAGCCCGTGAAGTTCGTTAAAGATGGTGGTCTTACTAAAGCCTAAGTAGTTAGCGATGTATCGCAAGGAACGTCCTTCATTATGTAAGCGTGTCAATGACAACACGGTTCTGGAATGAAAAAATAGTGGTGCTCATCAAGGTCCTTCCTTTCTAATGGAGTGGGTGGTAACATCATTTAAGGACCTTGATTGGGTTTTTCTGGGCCCCTTAAATGTTCAACTTAAATTTTACAATCTGC

Page 17: Efficacy of Indian Probiotic Cultures - ILSI Indiailsi-india.org/conference-on-feb20-2009/session1/Dr. Sunita Grover.pdf · Quantification of BSH activity by modified ninhydrin assay

20.2.2009 17ILSI-India

Page 18: Efficacy of Indian Probiotic Cultures - ILSI Indiailsi-india.org/conference-on-feb20-2009/session1/Dr. Sunita Grover.pdf · Quantification of BSH activity by modified ninhydrin assay

Cell surface proteins as probiotic markers f lfor colonization

• Mucus Binding Protein (MBP)• Fibronectin Binding Protein (FnBP)Fibronectin Binding Protein (FnBP)• Collagen Binding Protein (CBP)

EF Tu• EF-Tu• LTA

20.2.2009 18ILSI-India

Page 19: Efficacy of Indian Probiotic Cultures - ILSI Indiailsi-india.org/conference-on-feb20-2009/session1/Dr. Sunita Grover.pdf · Quantification of BSH activity by modified ninhydrin assay

M 1 2 3 4 5 6 7 8 9 10M 1 2 3 4 5 6 7 8 9 10M 1 2 3 4 5 6

405 b

M 1 2 3 4 5 6

405 b

250 bp405 bp

200bp100bp

250 bp405 bp

200bp100bp

Fig. Multiplex PCR with primer pairsLBLMA1 /R-161 and MubD1_F87/ MubD1_R0.5

405 bp250 bp200bp

400bp

Fig. Multiplex PCR with primer pairsLBLMA1 /R-161 and MubD1_F87/ MubD1_R0.5

405 bp250 bp200bp

400bp

Fig. Multiplex PCR for L. plantarum with primer pairs LBLMA1 /R-161 and MubD1_F87/ MubD1_R0.5Lanes: M-100 bp DNA ladder; 1-Lp10; 2-Lp20; 3-Lp75; 4-Lp76; 5-Lp77; 6-Lp5276; 7-Lps2; 8-Lp44; 9-Lp45; 10-Lp68

Fig. Multiplex PCR for L. plantarum with primer pairs LBLMA1 /R-161 and MubD1_F87/ MubD1_R0.5Lanes: M-100 bp DNA ladder; 1-Lp10; 2-Lp20; 3-Lp75; 4-Lp76; 5-Lp77; 6-Lp5276; 7-Lps2; 8-Lp44; 9-Lp45; 10-Lp68

Lanes: M-100 bp marker; 1-2- Genus specific PCR product of Lp9,Lp91 3-4- ‘Mub’ specific PCR product with primer MubD1_F87/ MubD1_R0.5 of Lp9,Lp91 5-6-Multiplex PCR with both primer for Lp9,Lp91 .

Lanes: M-100 bp marker; 1-2- Genus specific PCR product of Lp9,Lp91 3-4- ‘Mub’ specific PCR product with primer MubD1_F87/ MubD1_R0.5 of Lp9,Lp91 5-6-Multiplex PCR with both primer for Lp9,Lp91 .

M 1 2 3 4 5 6 7M 1 2 3 4 5 6 7

PCR Assays405 bp

250 bp200bp400bp 405 bp

250 bp200bp400bp

PCR Assays

Fig. Multiplex PCR for L. casei , L. acidophilus and Bifidoacterium bifidum with primer pairs LBLMA1 /R-161 and MubD1_F87/ MubD1_R0.5Lanes: M-100 bp marker; 1- L casei J7; 2- L casei J9; 3- L casei

Fig. Multiplex PCR for L. casei , L. acidophilus and Bifidoacterium bifidum with primer pairs LBLMA1 /R-161 and MubD1_F87/ MubD1_R0.5Lanes: M-100 bp marker; 1- L casei J7; 2- L casei J9; 3- L caseiLanes: M-100 bp marker; 1- L. casei J7; 2- L. casei J9; 3- L. caseiJ13; 4- L. casei 17; 5- L. casei S3; 6- LA1 (standard); 7- BB12 (Bifidobacerium bifidum)

Lanes: M-100 bp marker; 1- L. casei J7; 2- L. casei J9; 3- L. caseiJ13; 4- L. casei 17; 5- L. casei S3; 6- LA1 (standard); 7- BB12 (Bifidobacerium bifidum)

20.2.2009 19ILSI-India

Page 20: Efficacy of Indian Probiotic Cultures - ILSI Indiailsi-india.org/conference-on-feb20-2009/session1/Dr. Sunita Grover.pdf · Quantification of BSH activity by modified ninhydrin assay

M 1 2 3 4 5 6 7M 1 2 3 4 5 6 7

1.6 kb

M 1 2 3 4 5 6 7

1.6 kb

250 bp500bp

1.5kb

M 1 2 3 4 5 6 7

1.6 kb

250 bp500bp

1.5kb

i ‘ ’ C i i i 0 93 /

1kb

i ‘ ’ C i i i 0 93 /

1kb

Fig. Multiplex PCR for Lp9 with primer pairs LBLMA1/R-161 and MubN_F0.93/ MubN_R165

250 bp

Fig. Multiplex PCR for Lp9 with primer pairs LBLMA1/R-161 and MubN_F0.93/ MubN_R165

250 bp

Fig. ‘Mub’ PCR with primer pair mubN_F0.93 / MubN_R1.65Lanes: M-250 bp DNA ladder; 1- L. brevis (standard); 2-Lp9; 3-Lp91; 4-Lp72; 5-Lp-77; 6-Lp10; 7-Lp20

Fig. ‘Mub’ PCR with primer pair mubN_F0.93 / MubN_R1.65Lanes: M-250 bp DNA ladder; 1- L. brevis (standard); 2-Lp9; 3-Lp91; 4-Lp72; 5-Lp-77; 6-Lp10; 7-Lp20

_ _Lanes: M- Supermix DNA ladder; 1-2-(1:0.5 ratio of Genus and ‘Mub’ primers); 3-4- (1:1 ratio of Genus and ‘Mub’ primers); 5-6- (0.5:1 ratio of Genus and ‘Mub’ primers); 7- 1kb DNA ladder (Chromous)

_ _Lanes: M- Supermix DNA ladder; 1-2-(1:0.5 ratio of Genus and ‘Mub’ primers); 3-4- (1:1 ratio of Genus and ‘Mub’ primers); 5-6- (0.5:1 ratio of Genus and ‘Mub’ primers); 7- 1kb DNA ladder (Chromous)

M1 1 2 3 4 5 6 7 8 9 10 11 12 13 M2

1.6 kb1 5kb

M1 1 2 3 4 5 6 7 8 9 10 11 12 13 M2

1.6 kb1 5kbPCR Assays

1.6 kb

250 bp100bp

500bp

1.5kb 1.6 kb

250 bp100bp

500bp

1.5kb

Fig. Multiplex PCR for L. plantarum isolates with primer pairs LBLMA1/ R-161 and MubN_F0.93/ MubN_R165Lanes: M1-500 bp DNA ladder; 1-L. brevis (standard); 2-Lp5276 (standard); 3-Lp9; 4-Lp10; 5-Lp20; 6-Lp21; 7-Lp72; 8-Lp75; 9-Lp76; 10-Lp77; 11-Lp90; 12-Lp91; 13 Lp 122; 14 Lp130; M2 100 bp DNA ladder

Fig. Multiplex PCR for L. plantarum isolates with primer pairs LBLMA1/ R-161 and MubN_F0.93/ MubN_R165Lanes: M1-500 bp DNA ladder; 1-L. brevis (standard); 2-Lp5276 (standard); 3-Lp9; 4-Lp10; 5-Lp20; 6-Lp21; 7-Lp72; 8-Lp75; 9-Lp76; 10-Lp77; 11-Lp90; 12-Lp91; 13 Lp 122; 14 Lp130; M2 100 bp DNA ladderLp91; 13-Lp-122; 14-Lp130; M2-100 bp DNA ladderLp91; 13-Lp-122; 14-Lp130; M2-100 bp DNA ladder

20.2.2009 20ILSI-India

Page 21: Efficacy of Indian Probiotic Cultures - ILSI Indiailsi-india.org/conference-on-feb20-2009/session1/Dr. Sunita Grover.pdf · Quantification of BSH activity by modified ninhydrin assay

Potential Biomarkers for Selection of Probiotics against ifi dispecific diseases

• Probiotics – Highly strain and host specific(Specific health promoting functions)

• Inflammatory diseases, IBD, Crohn’s disease, Ulcerative colitis, constipation etc., p

• Gastro-intestinal disease• Cancers – colon, bladder etc.• Autoimmune diseases Diabetes Rheumatoid arthritis• Autoimmune diseases – Diabetes, Rheumatoid arthritis• Allergies – rhinitis• Respiratory tract diseases – pneumonia etc.• Liver diseases• Hypertension, obesity etc.

20.2.2009 21ILSI-India

Page 22: Efficacy of Indian Probiotic Cultures - ILSI Indiailsi-india.org/conference-on-feb20-2009/session1/Dr. Sunita Grover.pdf · Quantification of BSH activity by modified ninhydrin assay

Development of DM2 and CVD through id i i fl doxidative-inflammatory cascade

20.2.2009 ILSI-India 32

Page 23: Efficacy of Indian Probiotic Cultures - ILSI Indiailsi-india.org/conference-on-feb20-2009/session1/Dr. Sunita Grover.pdf · Quantification of BSH activity by modified ninhydrin assay

Evaluation of the evidence - Preclinical diStudies

• Cell lines (In vitro)Cell lines (In vitro)– Too simplistic to mimic human systems– Important to screen strain characteristicsImportant to screen strain characteristics– Important to understand mechanisms

• Small experimental animal models (InSmall experimental animal models (In vivo)– Cannot be used for proof of efficacyp y– Excellent for safety

20.2.2009 23ILSI-India

Page 24: Efficacy of Indian Probiotic Cultures - ILSI Indiailsi-india.org/conference-on-feb20-2009/session1/Dr. Sunita Grover.pdf · Quantification of BSH activity by modified ninhydrin assay

Evaluation of the evidence - Clinical StudiesEvaluation of the evidence Clinical Studies

• Human target population RDBPCT : g p pcornerstone of efficacy in human studies

• Phase I, II and III • Preventive / therapeutic• Preventive / therapeutic• Critical Data analysis• Post market surveillance studies

20.2.2009 24ILSI-India

Page 25: Efficacy of Indian Probiotic Cultures - ILSI Indiailsi-india.org/conference-on-feb20-2009/session1/Dr. Sunita Grover.pdf · Quantification of BSH activity by modified ninhydrin assay

Identification of probiotics – a pre-requisite

• Physiological function – highly strain specificPhysiological function highly strain specific • Identification at strain level – very important• False claims / labeling – spurious products• Molecular based identification – more reliable at

genus, species and strain level16s rRNA sequencing– 16s rRNA sequencing

– House keeping genes (rpo, tuf etc.)Lb. casei (13), Lb. paracasei (4), Lb.fermentum (25), Lb. plantarum (26), Lb. delbrueckii (2), Lb.rhamnosus(2), Lb.reuteri(4) and Lb. acidophilus (2)

20.2.2009 25ILSI-India

Page 26: Efficacy of Indian Probiotic Cultures - ILSI Indiailsi-india.org/conference-on-feb20-2009/session1/Dr. Sunita Grover.pdf · Quantification of BSH activity by modified ninhydrin assay

Nucleotide sequence of unique RAPD band of Lb casei with primer OPBB-02

• >OPBB-02 • CACTGGCTGGGAAGGCACCGAGTTGTATGTTCAGCTAGTTCCGG

AAGGCAAATTTGAAGGTGACACGTTAAATCCGTATTTTCTGATCAAAACCGCGGATGAAGCGTTCAGCTTGTGGTCACCGACTGACTGTAAACCGCGGATGAAGCGTTCAGCTTGTGGTCACCGACTGACTGTGACATTCTCGCTGAAGATTGGCAACTTGTTGACGCATGATAGCATTCGATTTTAGCGGCAAAACGGTTGTTGTGACTGGTGCGGCATCGGGAATCGGCGCGGCTCAGGCAGCGGCGTTTACAGCAGCTGGTGCACGGGTTATTGGCGTCGATCTTCAGCCGATGACTGGGCTAGCCGTCACCATTCAGGCAGATGTCAGTAAGGCCGCGACAGCAGAAAACATCGTTCGTGACTATGCGCCCGACATTGTCTGCAACACGGCGGGCATTCTGGATGGTTATCAAGACGTCGCCGCCACTGATCTGGCGGCATTCTGGATGGTTATCAAGACGTCGCCGCCACTGATCTGGCAACCTGGCAGCACATTCTTGATGTTGATCTCACCAGCCAGT

• >probe1• 5’-TGGCAGCACATTCTTGATGTTGATCTCACCAG -3’5 TGGCAGCACATTCTTGATGTTGATCTCACCAG 3

20.2.2009 26ILSI-India

Page 27: Efficacy of Indian Probiotic Cultures - ILSI Indiailsi-india.org/conference-on-feb20-2009/session1/Dr. Sunita Grover.pdf · Quantification of BSH activity by modified ninhydrin assay

Probiotics : Few Questions which remain l dunresolved

Selection of probiotic culture – what should be the criteria to define the most ideal probiotic strain for a specific disease

• Monostrain vs multistrain ?• Will cell free extracts work ?• Quantity and quality of probiotic needed for desired effect ?• How best to assess the activity / viability ?How best to assess the activity / viability ?• Which Probiotics remain viable in GI tract ?• How long do they remain in the gut to be effective?• The best target site in pathogenesis of a particular disease for

interventionsinterventions• Cell lines / cell culture and Animal models • Lack of correlation (In vitro versus in vivo assays) e.g. in vitro

evaluation of adhesion using cell lines do not account for complex and changing mucus lining and changing mucus lining

• Probiotic safety ?• Clinical trials on human subjects for validating health benefits

20.2.2009 27ILSI-India

Page 28: Efficacy of Indian Probiotic Cultures - ILSI Indiailsi-india.org/conference-on-feb20-2009/session1/Dr. Sunita Grover.pdf · Quantification of BSH activity by modified ninhydrin assay

Points to ponderPoints to ponder• Probiotic definition or selection criteria should include:

Abilit t t i– Ability to generate an immune response– In vitro assays to show that probiotics activate immune cells

• Does in vitro screening based on acid and bile predict in vivosurvival capacity (other stresses like oxidative, nutrientp y ( ,limitation, antimicrobial components etc. might influence?)

• Selection of probiotics on the basis of Adhesion potential using in vitro assays is being debated ? Can not be extrapolated to GIT (in vivo) due to(in vivo) due to – Host defense systems– Competition for nutrients and space with commensals– Mucosal shedding– Peristaltic flow that continuously washes GIT

• Hence, extrapolation of these findings to clinical applications requires caution

20.2.2009 ILSI-india 36

Page 29: Efficacy of Indian Probiotic Cultures - ILSI Indiailsi-india.org/conference-on-feb20-2009/session1/Dr. Sunita Grover.pdf · Quantification of BSH activity by modified ninhydrin assay

Current problems with “probiotics” in IndiaCurrent problems with probiotics in India• Extravagant claims without research

– Specific species and strain effectsSpec c spec es a d st a e ects• Lack of good manufacturing practices

– Quality assurance– Label vs content– Viability of bacterial species

• Validated biomarkers for assessing function and activityy

• Identification needs to be done using molecular tools

16S rRNA16S rRNAFISH

• No specific guidelines currently– In IndiaIn India

20.2.2009 29ILSI-India

Page 30: Efficacy of Indian Probiotic Cultures - ILSI Indiailsi-india.org/conference-on-feb20-2009/session1/Dr. Sunita Grover.pdf · Quantification of BSH activity by modified ninhydrin assay

Desired Interventions from Indian iPerspective

• A comprehensive database on Indian probiotic cultures, their diversityand novel physiological functions for human health well being andand novel physiological functions for human health, well being anddisease mitigation need to be generated as a National priority

• Indian gut – a rich habitat of diversified microbiota (gut ecology - foodhabits and regional and anthropological differences) can be exploredhabits and regional and anthropological differences) – can be exploredby metagenomic approaches

• High probability of biodiversity amongst probiotic strains fromdifferent gut niches at Genomics and Proteomics leveldifferent gut niches at Genomics and Proteomics level

• Initiatives for mining the complete genome of promising Indian strainsand the functional genes associated with novel physiological function

• Search for Novel probiotic strains (Lactobacilli and Bifidobacteria)with specific physiological functions in the gut using comparativegenomics, Transcriptomics, Proteomics approaches etc.

20.2.2009 30ILSI-India

Page 31: Efficacy of Indian Probiotic Cultures - ILSI Indiailsi-india.org/conference-on-feb20-2009/session1/Dr. Sunita Grover.pdf · Quantification of BSH activity by modified ninhydrin assay

Contd…Contd…• Identification of potential Biomarkers for evaluating functionality of

probiotics because greatest challenge concerns functionality ofprobiotics to strengthen health claims probiotics confer on hostprobiotics to strengthen health claims probiotics confer on hosthealth

• Development of consortia of proven strains for ethnic dairy basedproduct development (Dahi / Lassi) for boosting mucosal immunityproduct development (Dahi / Lassi) for boosting mucosal immunity

• Biosafety evaluation of selected probiotics and appropriatevalidation of health claims through clinical trials in target humansubjectssubjects

• Efforts should be made to conduct clinical trials in Indian populationfor comprehensive evaluation of the efficacy of the Indian probioticcultures against diarrhoeal as well as life style diseases vis a viscultures against diarrhoeal as well as life style diseases vis a visestablished western cultures to prove their efficacy

20.2.2009 31ILSI-India

Page 32: Efficacy of Indian Probiotic Cultures - ILSI Indiailsi-india.org/conference-on-feb20-2009/session1/Dr. Sunita Grover.pdf · Quantification of BSH activity by modified ninhydrin assay

Time for a paradigm shiftTime for a paradigm shift

Supply viable beneficial bacteria or aSupply viable beneficial bacteria or asubstrate which enhances a specificb fi i l b t i i t d f t ibeneficial bacteria instead of tryingto eliminate the pathogen ?

“Bioecological control”Bioecological control

20.2.2009 32ILSI-India

Page 33: Efficacy of Indian Probiotic Cultures - ILSI Indiailsi-india.org/conference-on-feb20-2009/session1/Dr. Sunita Grover.pdf · Quantification of BSH activity by modified ninhydrin assay

ConclusionConclusion• “Thy Food Shall Be Thy Remedy”

HippocratesHippocrates

• Disease can be:– Prevented– Mitigated– Treated

• Appropriate Choice of :– Clinical proven probiotics

• We need to continue rigorous evaluation of assumptions and hypothesis to discover

l bi tinovel probiotics20.2.2009 33ILSI-India

Page 34: Efficacy of Indian Probiotic Cultures - ILSI Indiailsi-india.org/conference-on-feb20-2009/session1/Dr. Sunita Grover.pdf · Quantification of BSH activity by modified ninhydrin assay

AcknowledgementAcknowledgement• ILSI - India • Department of Biotechnology, Govt. of India, • Director, NDRI• Dr. V. K. Batish, Prof. and Head, DM Division • Raj Kumar, Ph.D student

R j h K Ph D t d t• Rajesh Kumar, Ph.D student• Ashok, SRF

Ashish SRF• Ashish, SRF

20.2.2009 34ILSI-India