1. Prebiotic effect of fructans with different structure and polymerization degree M. Mueller, H. Viernstein, R. Loeppert, W. Praznik Department of Pharmaceutical Technology and Biopharmaceutics, University of Vienna, Austria

2. Outline  Background: Prebiotics, Fructans  Materials and methods  Prebiotic effect of the tested fructans  Influence of polymerization degree and structure  Degradation pattern of fructans  Summary

3. Outline  Background: Prebiotics, Fructans  Materials and methods  Prebiotic effect of the tested fructans  Influence of polymerization degree and structure  Degradation pattern of fructans  Summary

4. Background: Pro-/Prebiotics  Probiotics: live microorganism with beneficial effect to the health of the host

5. Probiotics Stimulation of immune response Increase of the lactose tolerance Improvement of the intestinal microflora Cholesterol reduction, increase of insulin resistance Restoration of microflora after antibiotic treatment Treatment / prevention of diarrhea Improvement of allergic reactions Improvement of chronic fatigue symptom

6. Background: Pro-/Prebiotics  Probiotics: live microorganism with beneficial effect to the health of the host  Prebiotics : “selectively fermented food ingredient that stimulates specific changes in the composition of and/or activity in the gastrointestinal microflora, which benefits on the host well-being and health”.  Requirements: stable in the acidic gastric conditions, upper gastrointestinal tract  only a few prebiotics fully meet this definition: fructooligosaccharides (FOS), inulin, galactooligosaccharides, and glucooligosaccharides  other nondigestible food ingredients studied: branched fructans, galactans, xylo-oligosaccharides, ß-glucans or arabinoxylans Gibson and Roberfroid, 1995, J Nutr., 125, 1401-1412. M. Roberfroid, 2010, Br J Nutr. 104, S1-63.

7. Background: Fructans  well known prebiotics  antioxidants and immunomodulators  structure of fructans: fructose-derived oligo- and polysaccharids inulin type (ß2-1 linkage) mixed type (ß2-1 and ß2-6 linkages)  accumulated in  dicots such as composites (e.g. chichory, Jerusalem artichoke)  monocots such as liliales (e.g. garlic, onion) asparagales (asparagous) or agavacaee (agave species).

8. Outline  Background: Prebiotics, Fructans  Materials and methods  Prebiotic effect of the tested fructans  Influence of polymerization degree and structure  Degradation pattern of fructans  Summary

9. Fructans tested fructans from different plant sources: chicory jerusalem artichoke agave diverse structures: inulin-type (only ß2-1 linkages) mixed-type (ß2-1 & ß2-6 linkages with branching)

10. Fructans tested fructans from different plant sources: chicory (Raftilose, Raftiline) jerusalem artichoke agave (Metlos, Metlin) diverse structures: inulin-type (only ß2-1 linkages) mixed-type (ß2-1 & ß2-6 linkages with branching)

11. Polymerization degree (dp) - distributions harvest March dpn = 7, dpw =10 harvest Oct. dpn = 10, dpw =22 Raftilose® dpn = 4, dpw =5 Raftilin® dpn = 9, dpw =15 Metlos® dpn = 9, dpw =19 Metlin® dpn = 15, dpw =25 Inulin from J. artichoke Inulin from chicory Fructan from agave

12. Probiotics used  Bifidobacterium animalis  Lactobacillus paracasei sp. paracasei 1.) CRL 431 (Hansen) 2.) DN114001 (Danone) 3.) Shirota (Yakult)  Lactobacillus rhamnosus GG  Lactobacillus reuteri  Lactobacillus acidophilus

13. Methods Bioscreen turbidity increase due to growth of bacteria  measurement of OD 600 - turbidity caused by low solubility of sugars + low amount of sugar needed Agar plates conventional method  for confirmation of Bioscreen results - high amount of sugar needed + high reliability

14. Methods: Bioscreen Preincubation of probiotics Incubation: 48 h, 37°C Measurement: per 1 h, OD 600 + + + + Growth curve Inoculation Start OD 600 : 0.1 Sugar conc: 1% & Preparation of stocks from isolated fructans or inulins

15. Methods: Agar plates Preincubation of probiotics Incubation + + + + Dilution to OD 600 10 -5 - 10 -7 Preparation of stocks from isolated fructans or inulins Preparation of agar plates with 1% sugar 48 h, 37°C &

16. Outline  Background: Prebiotics, Fructans  Materials and methods  Prebiotic effect of the tested fructans  Influence of polymerization degree and structure  Degradation pattern of fructans  Summary

17. Growth curve of B. animalis

18. Growth curves L. rhamnosus GG L. acidophilus L. reuteri

19. Growth curves L. paracasei (1) L. paracasei (2) L. paracasei (3)

20. Summary growth curves B. animalisL. acidophilus L. paracasei ssp. paracasei L. reuteriL. rhamnosus GG DN 114001ShirotaCRL431 Glucose9979679 Inulin (J. artichoke - March)151318109 29 Inulin (J. artichoke - Oct)15133111 1633 FOS (agave)13121397820 Fructan (agave)14 2313121630 FOS (chicory)141217128927 Inulin (chicory)14132011 930 Time after which the half maximum OD 600 is reached = medium effective time ET 50 [h]

21. Overview growth curves B. animalisL. acidophilus L. paracasei ssp. paracasei L. reuteriL. rhamnosus GG DN 114001ShirotaCRL431 Glucose9979679 Inulin (J. artichoke - March)151318109 29 Inulin (J. artichoke - Oct)15133111 1633 FOS (agave - Metlos)13121397820 Fructan (agave - Metlin)14 2313121630 FOS (chicory - Raftilos)141217128927 Inulin (chicory - Raftiline)14132011 930 Time after which the half maximum OD 600 is reached = medium effective time ET 50 [h]

22. Overview growth curves B. animalisL. acidophilus L. paracasei ssp. paracasei L. reuteriL. rhamnosus GG DN 114001ShirotaCRL431 Glucose9979679 Inulin (J. artichoke - March)151318109 29 Inulin (J. artichoke - Oct)15133111 1633 FOS (agave - Metlos)13121397820 Fructan (agave - Metlin)14 2313121630 FOS (chicory - Raftilos)141217128927 Inulin (chicory - Raftiline)1413201211930 Time after which the half maximum OD 600 is reached = medium effective time ET 50 [h]

23. Confirmation with conventional cultivation negative… glucose….. agave…. agave

24. Outline  Background: Fructans  Materials and methods  Prebiotic effect of the tested fructans  Influence of polymerization degree and structure  Degradation pattern of fructans  Summary

25. Correlation dp – ET 50 All tested fructansFructans from agave

26. Separation of chicory inulin using SEC L. paracasei (1) B. animalis 0201010 403050 [min] 60 nC Raftilose Raftiline Fraction 1 Fraction 2 Fraction 3 Fraction 4 Fraction 5 dp=19 dp=12 dp=8 dp=5 dp=3

27. dpwdpn Metlos96 Metlin2210 Metlin F11411 Metlin F265 Metlin F343 Raftilose75 Raftiline167 Raftiline F13419 Raftiline F22012 Raftiline F3128 Raftiline F485 Raftiline F532

28. Separation of agave fructan using SEC L. paracasei (1) B. animalis dp=11 dp=3 dp=5

29. Correlation dp – ET 50 L. acidophilus L. rhamnoss GG Mueller et al. 2015. J Funct Food. submitted

30. Outline  Background: Fructans  Materials and methods  Prebiotic effect of the tested fructans  Influence of polymerization degree and structure  Degradation pattern of fructans  Summary

31. Degradation pattern  Incubation of bacteria with fructans for 24 hours  Measurement of the sugar composition in the medium every 2 hours  Continuous cleavage to mono- and disaccharids before uptake by the cells  No extracellular cleavage  fructans of a certain dp may be adsorped by the cells and cleaved intracellularely

32. Correlation degradation and growth curve 0h 2h 4h 6h ST 8h 10h 12h 14h 16h 18h 20h ST 22h 24h 40h L. paracasei (1): Glucose Growth curve  glucose was exhausted after 6 to 24 hours dependent on the strain. glucose

33. 0h 2h 4h 6h ST 8h 10h 12h 14h 16h 18h 20h ST 22h 24h 40h Correlation degradation and growth curve (2) L. paracasei (1): Inulin from chicory monosaccharide disaccharide

34. Correlation degradation and growth curve (3) L. paracasei (2) – Fructan from agave 0h 2h 4h 6h ST 8h 10h 12h 14h 16h ST 18h 20h 22h 24h 38h 48h 0h 2h 4h 6h ST 8h 10h 12h 14h 16h ST 18h 20h 22h 24h 36h 48h monosaccharide disaccharide  Degradation correlates with the growth dp<3 dp 4 dp 7 dp 6 dp 5

35. Degradation dependent on probiotic strain and fructan source  B. animalis cleaves fructose extracellularely; slow usage of disaccharides, 1-kestose, no usage of oligos dpn > 8.  correlates with delayed growth with fractions with high dp.  L. acidophilus and L. paracasei 1 cut monosaccharides extracellularly, also use disaccharides, 1-kestose and oligos with higher dp fast.  correlation with fast growth with all dps.  L. paracasei (2+3) and L. rhamnosus GG cut mono- and disaccharides extracellularly; 1-kestose usage slow.  delayed growth and low usage of fractions with high dp. Oligos from Metlos and Metlin may be taken up and used intracellularly.  L. reuteri cleaves monosaccharides extracellularly, delayed usage of oligosaccharides with a dp up to 6  corresponds to low, delayed growth with fractions with higher dp.

36. Outline  Background: Prebiotics, Fructans  Materials and methods  Prebiotic effect of the tested fructans  Influence of polymerization degree and structure  Degradation pattern of fructans  Summary

37. Summary  Fructans from agave, chicory and J. artichoke show a significant prebiotic effect on all tested strains.  Differences depending on the probiotic strain and the fructan type.  Higher growth promotion for fructans with lower dp and branched structure.  Degradation pattern and location dependent on structure and strain.

38. International Agave Symposium in Mexico: Thanks to Prof. Dr. Werner Praznik Dr. Renate Löppert Marie Stutzig Jacqueline Reiner Lisa Fleischhacker Prof. Dr. Helmut Viernstein

39. Thanks for your attention! Contact: monika.mueller@univie.ac.at Gracias por su atención!