THE USE OF MALDI-TOF MS FOR IDENTIFICATION AND CHARACTERIZATION OF NOVEL STRAINS WITH PROBIOTIC PROPERTIES

Transcription

1 FOOD AND FUNCTION THE USE OF MALDI-TOF MS WHOLE CELL PROTEIN/PEPTIDE PROFILES FOR IDENTIFICATION AND CHARACTERIZATION OF NOVEL STRAINS WITH PROBIOTIC PROPERTIES 1 Andrea Teshim a,b, O. Šedo c I. Sedláček a, P.Svec a, Z. Zdráhal c, V. Dráb d a Czech Collection of Microorganisms, Institute of Experimental Biology, MU, Czech Republic b Department of Microbiology, Institute of Experimental Biology, MU, Czech Republic c Department of Functional Genomics and Proteomics, Institute of Experimental Biology, MU, Czech Republic d MILCOM, Dairy Research Institute, Tábor, CZ

2 m /z Matrix-Assisted Laser Desorption/Ionization - Time of Flight MALDI-MS Mass Spectrometry in MICROBIOLOGY simple, rapid, reliable chemotaxonomical technique high sensitivity (10-16 mol) and reproducibility unknown pre-identified Bacterial cells organic solvent + + strong organic acid modified RELEASE biochemical components and unique biomarkers (peptides and proteins) gas-phase charged ions a.i CHARACTERISTIC FINGERPRINT m/z measurements identification 2 0 classification

3 Intens. [a.u.] MALDI - MS profiles MALDI-MS protein/peptide profile is a characteristic strain-specific fingerprint Proteins 4776 Lactobacillus delbrueckii ssp. lactis 847 T 8000 = highly characteristic markers with great discriminatory 6000 power accessible by the analysis 4408 of intact bacterial cells % of bacterial cell dry weight molecular species possibility to distinguish closely related species, that are unidentifiable genotypically and by biotyping 3 m/z

4 Identification and diffenertiation of some problematic LAB within L. acidophillus group L. casei vs L. paracasei L. buchneri vs L. brevis SPECIES AND SUBSPECIES IDENTIFICATION. DIFFERENTIATION OF STRAINS MALDI-MS CHALLENGE: Differentiation of probiotic strains at the subspecies and strains level monitoring: PROTEOLYTIC and LIPOLITIC patterns strain-dependent properties MUTAGENESIS, DESINFECTION: - monitoring of culture changes Controls of microorganisms CONTAMINATION in dairy food processing identification of new enzymes ability to produce specific flavours, texture metabolic products 4

5 Overall aims of our study Optimization of culture conditions, sample preparation and MALDI-MS analysis To develop a reliable standardised protocol for Lactic Acid Bacteria MALDI-MS profiling Database developement 150 type and reference cultures MALDI-MS analysis of novel strains with probiotics properties (MILCOM, Tábor, CZ) and classification / identification of samples to evaluate the ability of the technique to - delineate species within genus - differentiate strains within individual species 5

6 Reference database developement Sample preparation 150 type and reference CCM and CCDM strains - Bifidobacterium, Lactobacillus, Lactococcus, Pediococcus overnight 30 / 37 C individual bacterial strain liquid MRS / MRS+cys or solid medium for anaerobes washed cells + water : abs.ethanol centrifugation pelet + 70% formic acid + pure acetonitrile analyzed or stored for months at -20 C Supernatant on MALDI target air dried centrifugation 6

7 MALDI-TOF MS analysis Each strain - 3 spots on MALDI target - air dried - α-cyano-4-hydroxy-cinnamic acid (CHCA) in 50%ACN and 2,5%TFA 384 potential samples MALDI-TOF MS analysis Reflex IV (Bruker DALTONICS) SOURCE N 2 laser, 337 nm MALDI target plate DETECTOR sample + matrix ionization accelerated, protonated ions Voltage 25 kv Time-of-Flight ANALYZER Lighter ions = faster 7

8 Intens. [a.u.] Spectra analysis 1 spectrum - sum of laser shots x Lactobacillus delbrueckii 1 spot 5 spectra ssp. lactis CCM CLUSTER ANALYSIS SOFTWARE 0.75 Biotyper Software (Bruker Daltonics) 0.50 m/z signals Spectra preprocessing (generating peak lists) Construction of mainspectra libraries Identification, analysis and classification of spectra Creation of a dendrogram m/z

9 Optimization process Type of matrix (CHCA) Influence of cultivating medium Influence of frozen sample storage Influence of prolonged cultivation Reproducibility and repeatability testing L.casei ssp.casei 7088T 20h b L.casei ssp.casei 7088T 20h c L.casei ssp.casei 7088T 20h a L.casei ssp.casei 7088T 46h c L.casei ssp.casei 7088T 46h a L.casei ssp.casei 7088T 46h c L.casei ssp.casei 7088T 46h b 48 hours Distance Level hours 9

10 Intens. [a.u.] Intens. [a.u.] Intens. [a.u.] Intens. [a.u.] Intens. [a.u.] Intens. [a.u.] x x x x x x Lactobacillus casei ssp. casei CCM 7088T Influence of cultivating medium Lactobacillus casei subsp casei 7088T 20h I MRS Lactobacillus casei subsp casei 7088T 20h II MRS Lactobacillus casei subsp casei 7088T 20h III MRS Lactobacillus casei subsp casei 7088T cys I MRS+cys Lactobacillus casei subsp casei 7088T cys II MRS+cys Lactobacillus casei subsp casei 7088T cys III MRS+cys m/z 10

11 Lactobacillus casei ssp. casei CCM 7088T Influence of cultivating medium Lactobacillus casei subsp casei 7088T III MRS+cys Lactobacillus casei subsp casei 7088T II MRS+cys Lactobacillus casei subsp casei 7088T 20h III MRS Lactobacillus casei subsp casei 7088T 20h II MRS MRS MRS+cys Lactobacillus casei subsp casei 7088T I MRS+cys Lactobacillus casei subsp casei 7088T 20h I MRS Distance Level Small variations in spectra do not influence the certain sume of protein/peptide markers that remain constant (Valentine et al. 2005)

12 Lactobacillus casei ssp. casei CCM 7088T Influence of prolonged cultivation Lactobacillus casei subsp casei 7088T 20h II Lactobacillus casei subsp casei 7088T 20h III Lactobacillus casei subsp casei 7088T 20h I Lactobacillus casei subsp casei 7088T 46h III A h cultivation 46h cultivation Lactobacillus casei subsp casei 7088T 46h I Lactobacillus casei subsp casei 7088T 46h III B Lactobacillus casei subsp casei 7088T 46h II Distance Level

13 In a total dendrogram different samples of the same strain cultivated different period of time in different media form common branche Lactobacillus casei ssp. casei CCM 7088T MRS, and MRS+cys 20h and 46h cultivation 13

14 Reproducibility testing Lactobacillus Repeated cultivation after period of time Pediococcus Lactobacillus Reproducibility = sample A and B Bifidobacterium 14

15 Intens. [a.u.] Intens. [a.u.] Intens. [a.u.] Intens. [a.u.] 4 x Comparison of spectra of 4 genera Lactobacillus delbrueckii subsp delbrueckii 7191T Bifidobacterium bifidum 559T x Lactococcus raffinolactis 4543T Pediococcus acidilactici m/z 15

16 Differentiation of genera Lactococcus Lactobacillus Bifidobacterium Bifidobacterium Lactobacillus Pediococcus 16

17 L.casei ssp. casei L. brevis 17

18 18

19 19

20 MALDI-MS fingerprints use in species discrimination Which bacterial species are present in different parts of technology process? Reuteri group: - at least 6 species isolated from sourdough (L. frumenti, L. pontis, L. panis, L. reuteri, L. rossiae, L. siliginis, L. secaliphilus) = species adapted to the particular niche interesting challenge for a proteomic study to link ecological specification to functional characteristics embedded in the proteome of the strains. 20

21 MALDI-MS profiles of pure culturable cultures through technology proces Species and strains identity confirmation control of microorganisms contamination With the other methods undistinguishable mutants or related species Analysis without cultivation? Limits: mixtures of strains 21

22 MALDI-MS fingerprints use in: Identification and diffenertiation of some problematic LAB L. acidophilus group 16S RNA - not the method of choice API 50CH, FAME profiling: cultivation conditions, dissimilarity in data sets L. bulgaricus, L. delbrueckii, L. lactis, L. indicus sharing 78%DNA similarity only a few ways to discriminate L. delbrueckii / L. jensenii L. casei group L.casei / L. paracasei L. reuteri group DNA-DNA homology L. fermentum / L. reuteri type strain L. celobiosus shown to be identical to L. fermentum L. thermotolerans and L. ingluviei - 1 or 2 taxons? 22

23 Main L. acidophilus groups Lactobacillus acidophilus group Lactobacillus casei group Lactobacillus reuteri group Lactobacillus animalis 663T Lactobacillus jensenii 243T Lactobacillus delbrueckii ssp delbrueckii 7191T Lactobacillus delbrueckii ssp lactis 847T Lactobacillus delbrueckii ssp indicus 845T Lactobacillus delbrueckii ssp bulgaricus 7190T Lactobacillus kefiranofaciens ssp kefiranofaciens 244T Lactobacillus kefiranofaciens ssp kefirangum 245T Lactobacillus helveticus 3806 Lactobacillus kitasatonis 630T Lactobacillus acidophilus 4833T Lactobacillus reuteri 777T Lactobacillus fermentum 7192T Lactobacillus coryniformis subsp torquens 646T Lactobacillus sakei subsp sakei 7203T Lactobacillus sakei ssp carnosus 776T Lactobacillus salivarius 7274 Lactobacillus pontis 4540T Lactobacillus amylophilus 7001 Lactobacillus paracasei ssp paracasei 1837 Lactobacillus casei ssp casei 7088T Lactobacillus paracasei ssp paracasei 792T Lactobacillus paracasei ssp paracasei P1630 Lactobacillus casei ssp casei 4798 Lactobacillus casei ssp casei Distance Level

24 The use of MALDI-MS fingerprints in comparing of strains with specific patterns = characterization of the strain Strain-dependent properties: probiotics: up (host mucin production) and down regulation (virulence factor expression in pathogens), production of specific antibiotics and antibiotic-like substances (reuterin ) technology: proteolytic and lipolytic patterns: flavour, ripening, gaps forming outputs of comparable fingerprints of strains, that are unidentified genotypically/phenotypically By novel or uncharacterized strain: prediction of strain-specific properties 24

25 The use of genomics in comparing of strains with specific patterns characterization and typing of the strain Complications To understand the properties of a probiotic strain: we do not know which pathway are required for a particular probiotic characteristics. Encoded sequence may not be expressed: Variability in time dependend on the environment Posttranslational modifications 25

26 Intens. [a.u.] Intens. [a.u.] Intens. [a.u.] m/z x MALDI-MS protein profiles x Proteins = very informative markers of present state of the strain x m/z Determination of protein turnover m/z screening of changes of proteome induced by various environmental situations or stresses Systematic mapping of proteins - useful for taxonomy, for function assignment of proteins High resolution reference map of cytoplasmic and membrane-associated protein. Available proteome data integrated into a public database. The level of expression of some protein; isoforms of proteins; the structure of proteins, stress responses 26

27 The use of MALDI-MS fingerprints in comparing of strains with specific patterns = characterization of the strain Protein turnover Through technology process Stress conditions - during production of the culture - incorporation into milk products or dietary supplements - during transport and storage - consumtion of the product with probio organisms - location of exerting the effect in GIT 27

28 The use of MALDI-MS fingerprints in the evaluation of industrial strains improvement MALDI-MS characteristic profile of the novel strain obtained Mutagenesis Recombinant DNA technology (F.e. Sǿrensen (2000) changed a serine codon to an amber codon in the pyrf gene of several industrial Lac.lactis ssp. lactis strains..) Complex monitoring of possible changes in mutant or recombinant strain Phenotypic or genotypic screening less sensitive 28

29 The use of MALDI-MS fingerprints in differentiation of strains (characterization and typing of strains) Score Oriented Dendrogram for Bifidobacterium_catenulatum_4989T_A_I Bifidobacterium_catenulatum_4989T_B Bifidobacterium_catenulatum_4989T_A Bifidobacterium_bifidum_3762_M Bifidobacterium_bifidum_3762 Bifidobacterium_adolescentis_4987T_B_M Bifidobacterium_adolescentis_4987T_B Bifidobacterium_adolescentis_4987T_A_M Bifidobacterium_adolescentis_4987T_A Lactobacillus_casei_subsp_casei_7088T_B Lactobacillus_casei_subsp_casei_7088T_A Pediococcus_pentosaceus_1578 Pediococcus_acidilactici_2425 Lactobacillus_brevis_3805T Lactococcus_lactis_subsp_lactis_1877T Lactobacillus_acidophilus_4833T Lactococcus_raffinolactis_4543T_I Lactococcus_raffinolactis_4543T Distance Level

30 The use of MALDI-MS fingerprints in differentiation of strains (characterization and typing of strains) 30

31 The use of MALDI-MS fingerprints in differentiation of strains (characterization and typing of strains) Lactobacillus paracasei ssp paracasei P1630 Lactobacillus paracasei ssp paracasei 792T Lactobacillus casei ssp casei 7088T Lactobacillus fermentum 7192T Lactobacillus parabrevis 778T Lactobacillus brevis 3805T Lactobacillus brevis 1815 Lactobacillus delbrueckii ssp lactis 847T Lactobacillus delbrueckii ssp indicus 845T Distance Level

32 Intens. [a.u.] m/z The use of MALDI-MS fingerprints in differentiation of strains (characterization and typing of strains) MALDI-MS analysis of novel strains Identification and describing new microorganisms with probiotic properties with aim to extend specific genetic sources for civilizing diseases prevention (GASTROPROBIO). Optimized sample preparation MALDI-TOF MS analysis x Spectra analysis Classification by comparing with database 32

33 Intens. [a.u.] Lactobacillus A CCDM strains pre-identified at the genus level Lactobacillus strains Bifidobacterium strains m/z MALDI-MS: - Almost all Lactobacillus CCDM strains clustered together with Lb. salivarius - 6 strains did not confirm definite classification at the species level 33

34 26 Lactobacillus CCDM strains were all clustered together to the sub-branche with Lactobacillus salivarius CCM 7274 very similar spectra of CCDM strains to each other 34

35 Lactobacillus A Green signals identical to reference signals Yellow signals identical to reference signals with abberation m/z Red signals distant signals 35

36 21 Bifidobacterium CCDM strains were all clustered together to the sub-branche with Bifidobacterium thermoacidophillum ssp. thermoacidophillum CCDM 609T 36

37 37

38 Are changes in taxonomy important in novel food production? Delineation of species, strains typing: polyphasic approach frequent name switches (f.e. L. casei/l. paracasei replacing a type strain; L. bulgaricus, Str. thermophilus..) In food production handling with pure, stabile and safe culture is more important than taxonomy The active application of living microorganisms in foods requires proper identification and labelling, nomenclature is essential for producers to communicate in a formal way. MALDI-MS classification = confirmation of pure and stabil culture and its classifiing to individual species in an appropriate database Pot B., Tsakalidou E. (2008): Taxonomy and metabolism of Lactobacillus. In: Ljungh A., Wadström T. (Eds.): Lactobacillus Molecular Biology. Williams a Wilkins Co., Baltimore. MD s 38

39 MALDI-TOF MS Modern approach in microbiology high-throughput analysis without on-line separation, pretreatement methods, filtering or cleavage of cells great discriminatory power, high reproducibility capability to differentiate and classify strains at the species and sub-species level rapid analysis or selective monitoring of bacteria, their metabolic products and unique proteins low consumption of chemicals; storage of samples combination with off-line separation methods 39

40 MALDI-TOF MS Modern approach in microbiology FUTURE? the development of specific protein expression databases. Envision the development of biofilm- or probiotic-specific proteome databases magnetic microbeads enrichment of proteins; detecting of bacterial strains in mixtures of cultures 40

41 Thanks to Mgr. Ondrej Šedo, Ph.D. analysis of samples, spectra analysis, generation of experimental data Assoc. Prof. RNDr. Ivo Sedláček, CSc. specialist, supervisor, consultations This study was supported by the Ministry of Education, Youth and Sports of the Czech Republic projects No. 2B08068 (NPVII), MSM , MSM and LC06034.

42 Thank You for Your attention Contact adress:

43 possible discussion after lecture

44 Lactobacillus_casei_subsp_casei_7088T_B Lactobacillus_casei_subsp_casei_7088T_A Lactobacillus paracasei subsp paracasei 792T Pediococcus_acidilactici_2425 Pediococcus_pentosacens_1578 Pediococcus pentosacens 1578_oct Lactobacillus ruminis 783T Lactococcus_raffinolactis_4543T Lactococcus raffinolactis 4543T_oct Lactobacillus rennini 651T Lactobacillus vaginalis 782T Lactobacillus coleohominis 789T Lactobacillus coryniformis subsp torquens 646T Lactobacillus mucosae 690T Lactobacillus acidipiscis 784T Lactobacillus_brevis_3805T Lactobacillus parabuchneri 779T Lactobacillus ultunensis 838T Lactobacillus parabrevis 778T Lactobacillus sakei ssp carnosus 776T Lactobacillus curvatus 834T Lactobacillus plantarum ssp argentoratensis 806T Lactobacillus plantarum ssp argentoratensis 1p 806T Lactobacillus_lactis_subsp_lactis_1877T Lactobacillus lactis subsp lactis 1877T_oct Lactobacillus oris 781T Bifidobacterium_bifidum_3762 Bifidobacterium_adolescentis_4987T_B Bifidobacterium_adolescentis_4987T_A Lactobacillus jensenii 243T Lactobacillus paralimentarius 848T Lactobacillus kalixensis 837T Lactobacillus intestinalis 773T Lactobacillus delbrueckii subsp. lactis 847T Lactobacillus delbrueckii subsp. indicus 845T Lactobacillus kitasatonis 630T Lactobacillus galinarum 793 Lactobacillus_acidophilus_4833T Lactobacillus acidophilus 4833T_oct Bifidobacterium catenulatum 4989T_oct Bifidobacterium_catenulatum_4989T_B Bifidobacterium_catenulatum_4989T_A Distance Level

45 Lactobacillus casei subsp casei 7088T_B Lactobacillus casei ssp casei 7088T_A Lactobacillus paracasei subsp paracasei 792T Lactobacillus paracasei ssp paracasei P1630 Lactobacillus casei ssp casei 7089 Lactobacillus casei ssp casei 4798 Lactobacillus paracesei ssp paracasei 7275 Lactobacillus casei ssp casei 4791 Lactobacillus ruminis 783T Pediococcus acidilactici 3449 Pediococcus acidilactici 2496 Pediococcus acidilactici 2482 Pediococcus acidilactici 2425 Lactobacillus vaginalis 782T Pediococcus pentosaceus 822 Pediococcus pentosaceus 835 Pediococcus pentosaceus 2483 Pediococcus pentosaceus 1578_oct Pediococcus pentosaceus 1578 Lactobacillus plantarum ssp argentoratensis 806T Lactobacillus plantarum ssp argentoratensis 1p 806T Lactobacillus parabuchneri 779T Lactobacillus parabrevis 778T Lactobacillus brevis CCM 1815 Lactobacillus brevis 3805T_oct Lactobacillus brevis 3805T Lactobacillus jensenii 243T Lactobacillus kalixensis 837T Lactobacillus intestinalis 773T Lactobacillus delbrueckii subsp. lactis 847T Lactobacillus delbrueckii subsp. indicus 845T Lactobacillus ultunensis 838T Lactobacillus kefiranofaciens ssp kefiranofaciens 244T Lactobacillus kefiranofaciens ssp kefirangum 245T Lactobacillus kitasatonis 630T Lactobacillus galinarum 793 Lactobacillus acidophilus 4833T_oct Lactobacillus acidophilus 4833T Bifidobacterium catenulatum 4989T_oct Bifidobacterium catenulatum 4989T_B Bifidobacterium catenulatum 4989T_A Lactococcus raffinolactis 4543T_oct Lactococcus raffinolactis 4543T Lactobacillus rennini 651T Lactobacillus hilgardii 828T Lactobacillus sakei ssp carnosus 776T Lactobacillus curvatus 834T Lactobacillus mucosae 690T Lactobacillus salivarius 7274 Lactobacillus coleohominis 789T Lactobacillus paralimentarius 848T Lactobacillus oris 781T Lactobacillus lactis subsp lactis 1877T_oct Lactobacillus lactis ssp lactis 1877T Lactobacillus coryniformis subsp torquens 646T Lactobacillus acidipiscis 784T Bifidobacterium adolescentis 4987T_B Bifidobacterium adolescentis 4987T_A Bifidobacterium bifidum 3762 Bifidobacterium psychraerophilum 582T Bifidobacterium animalis 4988T Bifidobacterium gallicum 565T Bifidobacterium breve 562T Bifidobacterium bifidum 3762_oct Bifidobacterium bifidum 559T Bifidobacterium 7209T Bifidobacterium thermacidophilum subsp porcinum 597T Bifidobacterium thermacidophilum subsp thermacidophilum 609T sed Bifidobacterium thermacidophilum subsp thermacidophilum 609T Distance Level

46

47

48 Score Oriented Dendrogram Pediococcus_pentosacens_1578 Lactococcus_lactis_subsp_lactis_1877 Lactobacillus_brevis_3805_B Lactobacillus_brevis_3805_A Pediococcus_acidilactici_2425 Lactobacillus_acidophilus_4833T Bifidobacterium_catenulatum_4989 Lactobacillus_casei_subsp_casei_7088_II Lactobacillus_casei_subsp_casei_7088_I Bifidobacterium_adolescentis_4987_II_B Bifidobacterium_adolescentis_4987_II_C Bifidobacterium_adolescentis_4987_II_A Bifidobacterium_adolescentis_4987_III Bifidobacterium_bifidumum_3762_II_B Bifidobacterium_bifidumum_3762_II_A Bifidobacterium_bifidumum_3762_II_C Bifidobacterium_bifidumum_3762_II_D Distance Level - vsechny paralelni kultivace i analyzy jsou vzdy Lactobacillus_casei_subsp_casei_7088_I_B U nekterych vzorku jsme pro nasi vnitrni kontrolu provedli paralelni analyzy Bifidobacterium_bifidumum_3762_III znacene jako A-D. Dendrogram vytvoreny v BioTyperu Bifidobacterium_bifidumum_3762_I prokazal dobrou reprodukovatelnost mereni - seskupeny do jedne vetve. Na druhove urovni vsak vzorky rozdeleny nejsou spravne (ve spektrech napr. u pediokoku nelze pozorovat spolecne piky).

49 Score Oriented Dendrogram for _Ales Lactobacillus_casei_subsp_casei_7088T_B Lactobacillus_casei_subsp_casei_7088T_A Pediococcus_pentosaceus_1578 Pediococcus_acidilactici_2425 Lactobacillus_brevis_3805T Lactococcus_lactis_subsp_lactis_1877T Lactococcus_raffinolactis_4543T Lactobacillus_acidophilus_4833T Bifidobacterium_bifidum_3762 Bifidobacterium_adolescentis_4987T_B Bifidobacterium_adolescentis_4987T_A Bifidobacterium_catenulatum_4989T_B Bifidobacterium_catenulatum_4989T_A Distance Level

50 Intens. [a.u.] Intens. [a.u.] Intens. [a.u.] Intens. [a.u.] x x x x Lactobacillus casei ssp casei srovnání zástupců Lactobacillus casei ssp casei 7088 T (080911) Lactobacillus casei ssp casei 7089 Lactobacillus casei ssp casei 4798 Lactobacillus casei ssp casei m/z

51 Pediococcus acidilactici L. brevis

52 Intens. [a.u.] Intens. [a.u.] Intens. [a.u.] Intens. [a.u.] Intens. [a.u.] Intens. [a.u.] Intens. [a.u.] Intens. [a.u.] Intens. [a.u.] x10 4 * Comparison srovnáníof spectra within * * * * * Lactobacillus * * * * * Lactobacillus delbrueckii subsp. lactis 847T Lactobacillus delbrueckii subsp. indicus 845T Lactobacillus parabrevis 778T 0 Lactobacillus brevis 3805T Lactobacillus brevis 1815 Lactobacillus fermentum 7192T Lactobacillus paracasei subsp paracasei 792T 0 Lactobacillus paracasei ssp paracasei P Lactobacillus casei subsp casei 7088T m/z 17 /24

53 Rody a druhy od sebe

54

55 L. acidophilus group L. jensenii / L. delbrueckii L. reuteri group L. casei group Lactobacillus lactis ssp lactis 1877T Lactobacillus paracasei ssp paracasei P1630 Lactobacillus casei ssp casei 7088T Lactobacillus fermentum 7192T Lactobacillus coryniformis subsp torquens 646T Lactobacillus amylophilus 7001 Lactobacillus jensenii 243T Lactobacillus kefiranofaciens ssp kefiranofaciens 244T Lactobacillus kefiranofaciens ssp kefirangum 245T Lactobacillus helveticus 7193T Lactobacillus kitasatonis 630T Lactobacillus acidophilus 4833T Lactobacillus delbrueckii ssp bulgaricus 7190T Lactobacillus delbrueckii ssp lactis 847T Lactobacillus delbrueckii ssp delbrueckii 7191T Lactobacillus delbrueckii ssp lactis 2772 Lactobacillus delbrueckii ssp lactis Distance Level

56 L. casei group L.casei / L. paracasei Lactobacillus sakei subsp sakei 4393 Lactobacillus sakei ssp carnosus 776T Lactobacillus coryniformis subsp torquens 646T Lactobacillus paracasei ssp paracasei 1837 Lactobacillus casei ssp casei 7088T_B Lactobacillus paracasei ssp paracasei 792T Lactobacillus paracasei ssp tolerans 7092T Lactobacillus paracasei ssp paracasei P1630 Lactobacillus casei ssp casei 4791 Lactobacillus paracesei ssp paracasei 7275 Lactobacillus casei ssp casei Distance Level

57 Casei paracasei

58 The use of MALDI-MS fingerprints in differentiation of strains (characterization and typing of strains)