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Emerging Trends in

Molecular Techniques forIdentification,

Characterization andTyping of Novel Probiotics

V.K.Batish

Molecular Biology Unit, Dairy

Microbiology Division,National Dairy ResearchInstitute, Karnal-132001

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Pre-eminence of probiotics inhuman health and nutrition

Food-grade organisms with multiple health promotingfunctions Role in human health well documented Restoration of disturbed gut flora Suppression of enteric pathogens

Immense potential-functional food ingredient/therapeutics Heterogeneous group Lactic Acid bacteria (human gut origin) as probiotics Lactobacilli and Bifidobacteria - the two most

important members

Health promoting functions highly strain specific Proper identification - a desirable prerequisite for

application Commercial value

Functional /Health foods / Formulations

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Applications of LAB

Starter cultures

Health promoting products (probiotics)

Flavour, texture and food preservation

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FAO-WHO Guidelines for theevaluation

of probioticsStrain identification

(genotypic and phenotypic methods)

Functional Safety

characterization assessment

Human trials Effectiveness trials

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Limitations of Phenotypicidentification

Slow

Laborious

Costly

Need of pure cultures

Not clear, Unreliable, Inacurate results

Huge data analysis

Unsatisfactory at a strain level

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techniques for probiotic

identification?

Reliable/accurate identification atstrain level extremely crucial to lendsupport to purported health claims

Powerful tools - better discriminatorypower

Safety and QA

False labeling/identity/disputes

Boosting consumer confidence

Licensing of novelstrains/ atentin /IPR issues

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Known

UnKnown

DGGE/TGGE

FISH

Genotypic identification ofprobiotics

16S rRNA

sequencin

PCR

(genus &

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Technique Principle Work load

Dp Rd

Morphological

analysis

Microscopic analysis L Genus level or less M

Physiologicalanalysis

Growth characteristics M Genus level or less L

Biochemicalcharacterizatio

n

Assimilation andfermentation

patterns (API,BIOLOG,. . .)

L Genus/ species level M

Proteinprofiling

SDS/PAGE of Cellularproteins

H Species level H

Specificprimers

PCR with group-specificprimers

L Depends on primer H

Sequencing Determination of genesequences (16S rDNA. . .)

H Genus to specieslevel

H

RFLP Restriction EnzymeAnalysis (REA)

M Species to strainlevel

H

RAPD-PCR Randomly primed PCR L Species to strain level L

-Dp: Discriminatory power, Rd: Reproducibility, L: Low, M: Moderate, H: High

comparis

ion

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16S rRNA Sequencing

The rRNA is the most conserved (least variable) gene inall cells.

Portions of the rDNA sequence from distantly-relatedorganisms are remarkably similar.

This means that sequences from distantly related

organisms can be precisely aligned, making the truedifferences easy to measure. For this reason, genes that encode the rRNA (rDNA)

have been used extensively to determine taxonomy,phylogeny (evolutionary relationships), and to estimaterates of species divergence among bacteria.

Thus, the comparison of 16s rDNA sequence can showevolutionary relatedness among microorganisms. Thiswork was pioneered by Carl Woese in 1987 whoproposed the three Domain system of classification -Archaea, Bacteria, and Eucarya - based on suchsequence information.

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rRNA genes

rec A gene

ldhD gene(lactate dehydrogenase in case

of LAB)

hsp gene (heat shock protein genes)

rpoA gene (RNA polymerase gene)

pheS gene

Most powerful region: rRNA genes

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Bacterial 16S, 23S, and 5S rRNA genes are typically organized

as a co-transcribed operon.

One or more copies of the operon dispersed in the genome

E coli has 7

Archaea contains either a single rDNA operon or multiple

copies of the operon.

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P1P2 T1T

2

1542

2904

120

16SrRNA

23SrRNA

15S

76

tRNA

Spacer region

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well characterized in as many species

has hyper variable regions

flanked by strongly-conserved regions

Primers are designed to bind to conserved

regions and amplify variable regions.

Whole gene analysis for identification for

identification of unknown isolates

National Center for Biotechnology

Information (www.ncbi.nlm.nih.gov) and

the Ribosomal Database Project

(www.cme.msu.edu/RDP/html/index.html).

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AJ418906Lactobacillus gallinarumAJ418905Lactobacillus crispatusAJ418904Lactobacillus amylovorusAJ418902Lactobacillus acidophilusAM490847Lactobacillus kefiranofaciens

lb69lb71

N4AJ418919Lactobacillus delbrueckii

AM295065Lactobacillus orisAJ459482Lactobacillus paraplantarumAJ418920Lactobacillus pentosusAJ418936Lactobacillus vaccinostercusAJ418934Lactobacillus suebicus

AJ459391Lactobacillus paracaseiAJ418912 Lactobacillus caseiAJ459386Lactobacillus caseiAJ418939Lactobacillus rhamnosusAJ459829Lactobacillus rhamnosus

AY372038Lactobacillus caseiAJ418933Lactobacillus zeaeLb101Lb104

CP000419 Streptococcus thermophilusN3

CP000023 Streptococcus thermophilusLb84Lb56

D12Lb21

LGGAP007281Lactobacillus reuteri

AL935258 Lactobacillus plantarumLb66

CP000416 Lactobacillus brevisCP000517Lactobacillus helveticusCP000033 Lactobacillus acidophilusLb68N2N1

67

100

98

89

92

95

61

33

54

55

23

27

57

64

68

99

88

100

89

43

49

92

99

88

99

69

48

46

100

97

100

54

85

99

0.2

Phylogenetic tree showing the

relationship between the isolates tuf

sequences and those of NCBI

database sequences.

Bootstrap values above 50% are shown. The scale bar represents the

number of changes per nucleotide position

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Genus specific primers (Dubernet et al., 2002)

LbLMA-1/R16-11

Product size : 250 bp

Species specific primers (Drake et al.,1996)

SS1/CA1 forLactobacillus casei

Product size:1200 bp

SS2/HE1 forLactobacillus helveticus / acidophilus

Product size:1200 bp, Differentiation based on EcoRI digestion

SS1/DB1 forLactobacillus delbreuckii

Product size:1200 / 1400 bp

niques based on 16SrRNA for identof probiotic Lactobacilli

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Lactobacilli

in MRS broth

spin Isolation of DNA

DNA

Taq

dNTPs

Buffer

PrimersAgarose gel

PCR protocol

Pospeich and Neumannsprotocol

PCR

PCR amplification

Id tifi ti f L t b illi t G l l b PCR

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Lanes: 1, J11; 5, 291; 6, 299; 7, 184; 11, J10; 12, 292; 13,Lr;

2, Vizylac; 3, Rinifol; 8, BSPOR; 9, SPORLAC;

4, A23; 10, A8; 14, Negative control; 15, 100 bp Marker

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

250 bp

Positive Isolates:

Human Milk Isolates : M1 / 12

Probiotic preparations: 9, MG2; 10, MG3; 11, MG4; 12,

MG5; / 5

1 2 3 4 5 6 7 8 9 10 11 12 1314 15 1 17 18 19 20

250 bp

Identification of Lactobacilli at Genus level by PCR

Fig. 1

Fig. 2

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PCR b d id tifi ti f L

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M 1 2 3 4 5 6 7 8 9 10 11

1400 bp2000 bp

Lanes: M, 500 bp marker; 1-7(La) 1, 11; 2, 13; 3, 14; 4,

15, 5, 16;6, 195; 7, 291; 8-11(Lh): 8, 292; 9,

11; 10, 288; 11, 5

M 1 2 3 4 5 6

Lanes: M, 500 bp Marker ;1,3,5 (uncut byEcoR1) 1, La 11; 3,

La 195; 5, Lh 288;2,4,6 (cut byEcoR1) 2, La 11; 4, La

195;6, Lh 288

2000 bp

PCR based identification ofL.acidophilus / L. helveticus

1400 bp

Lb.acidophilus GCT TGC CTA GAT TTC AGT GCT... ATA CAA CG

Lb.helveticus GCT TGC CTA GAA TTC GGT GCT... ATA CAA GCG

Fig. 3

Fig. 4

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M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

2000 bp

Lanes: M,500 bp Marker; 1-8 (Lc) 1, 3; 2, 9; 3, 13;4, 298;5, 299; 6, IS 3;7, IS 4;8, IS

5; 9-16 (100 ng/l) (Lc) 9, 3; 10, 9; 11, 13; 12, 298; 13, 299; 14, IS 3; 15, IS 4; 16 , IS 5

Lanes: M, 500 bp Marker ; 1-17 (Ldl/b): 1, 3; 2, 4; 3, 6;4, 10; 5, 27; 6, 26; 7, 277; 8,

281; 9, 293; 10, 307; 11, 184; 12, 253; 13, 2;14, 308; 15, 285; 16, 304; 17, 290

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

PCR based identification ofL. caseiand L.delbrueckiisubsp. bulgaricus

1200 bp

All the strainspositive

Total strains:18

Positive strains:121400/1200 bp

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Identification of isolates

Out of the 8 isolates from faecal samples, 5 belonged toLactobacillus delbrueckii, while the rest did not belong to

Lactobacillus casei, Lactobacillus helveticus / acidophilus or

Lactobacillus delbrueckii .

From human milk, one isolate was identified as

Lactobacillus.

Among the probiotic preparations, Lactobacillus was

identified 0nly in Microgenics and the four isolates were

identified asL. casei

The unidentified isolates from other probiotic formulations

were confirmed as Bacillus sp. by phenotypic examination

also (Grams staining and catalase test)

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PCR identification of isolates tospecies level1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

16

16 17 18 19 20 21 22 23 24 25 26 27 28 29 30Fig 6

Fig 7

Faecal

samples

Probiotics

Lanes :1..8

Lb. CaseiLanes :10..19

Lb. delbreuckii

Lanes :20..29

Lb. acidophilus

Lanes :1..6

Lb. Casei

Lanes :7..12

Lb. delbreuckii

Lanes :13..18

Lb. acidophilus

5 out of 8 faecal isolates are Lb. delbreuckii

4 out of 12 probiotic isolates are Lb. casei

1 2 3 4 5 6 7 8 910 112 1 3 14 15 16 17181920

assays or

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assays oridentification of BSH

positive lactobacilli LpBSHF / LpBSHR gave an amplifiedPCR product of 231 bp with Lb.plantarum but also with Shigella

dysentriae,L. monocytogenes andBifidobacterium bifidum

Multiplex PCR standardized usingLbLMA-1/R16-1 and LpF / LpR (975bp)

Twenty isolates gave amplification

product with LpBSHF/R

(975 bp)

(250bp)

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PCR assays for identificationof Mub

positive lactobacilli

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LTA based PCR

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Conclusion

Polyphasic approach based onadvanced molecular technologiesnecessary to achieve accurateinterpretation of the probioticidentity to elucidate the true healthimpact of these strains.

This approach will overcome theinherent limitations of each systemand will also throw some light on thecredibility of putative probiotic

strains to boost the consumer