Annelies Smedts *, Stijn Ceunen** , Ruis Amery*, Jan M.C. Geuns** and Boudewijn Meesschaert*/***

1. Isolation of different bacterial consortia from Paraguayan soil samples containing steviol related -glucosidase activity to degrade stevioside to steviol Annelies Smedts*, Stijn Ceunen**, Ruis Amery*, Jan M.C. Geuns** and Boudewijn Meesschaert*/*** *: Department of Industrial Sciences and Technology, Katholieke Hogeschool Brugge-Oostende Zeedijk 101, B 8400 Oostende, Belgium **: Laboratory of Functional Biology, Katholieke Universiteit Leuven, Kasteelpark Arenberg 31,B 3001 Heverlee-Leuven, Belgium ***: Centre of Surface Chemistry and Catalysis and Leuven Food Science and Nutrition Research Centre (LFoRCe) Department of Molecular and Microbial Sciences, Katholieke Universiteit Leuven Kasteelpark Arenberg 20,B 3001 Heverlee-Leuven, Belgium

2. Introduction and context • Aim • Materials and methods • Results • Influence of the concentration of yeast extract • Influence of pH • Influence of stirring or shaking • Degradation of steviolbioside • Isolation of interesting bacterial consortia • Conclusions • Results • Gramstaining and growth on solid medium • -Glucosidases: a molecular approach • Conclusions • Introduction and contexting • Aim • Materials and methods • Results • Conclusions

3. Introduction and contexting

4. Introduction and contexting Steviol glycosides (>Stevioside) = from Stevia rebaudiana bertoni leaves Low dosis (max. 200 mg/day) High dosis (max. 3 ×500 mg/day) • Application: • Natural sweetener • Alternative for aspartame • - Up to 300 times sweeter than sugar Possible pharmacological effects: - Lowering blood pressure - Reducing glucose in blood - Increasing insuline sensitivity

5. Introduction and contexting Ingestion steviol glycosides  steviol  steviol glucuronide (intestines) (liver) Possibleactive component = steviol glucuronide Furtherfundamental research = necessary  enough glucuronide required through the production of steviol upscaling production of steviol Chemical synthesis Low yield (10%) Stevioside + NaIO4 (partiallybreakingoff the boundedsugars)  mixture refluxedwith KOH Bio-organicsynthesis Severalpossibilities (anaerobic and aerobic)

6.  : Flavobacterium johnsonae (Okamoto et al., 2000) ·······: Clavibacter michiganense (Nakano et al., 1998) and Flavobacterium johnsonae (Okamoto et al., 2000)

7. Introduction and context • Aim • Materials and methods • Results • Influence of the concentration of yeast extract • Influence of pH • Influence of stirring or shaking • Degradation of steviolbioside • Isolation of interesting bacterial consortia • Conclusions • Results • Gramstaining and growth on solid medium • -Glucosidases: a molecular approach • Conclusions • Introduction and contexting • Aim • Materials and methods • Results • Conclusions

8. Aim • Isolation of bacterial consortia who produce -glucosidases able to hydrolyze stevioside to steviol • Better insight in influence of incubation parameters on hydrolysis pathway and hydrolysis rate • Incubation parameters: • pH • Concentration yeast extract • Stirring • Production of steviol

9. Introduction and context • Aim • Materials and methods • Results • Influence of the concentration of yeast extract • Influence of pH • Influence of stirring or shaking • Degradation of steviolbioside • Isolation of interesting bacterial consortia • Conclusions • Results • Gramstaining and growth on solid medium • -Glucosidases: a molecular approach • Conclusions • Introduction and contexting • Aim • Materials and methods • Results • Conclusions

10. Materials and methods • Chemicals: • Pure standards of the steviol glycosides • Rebaudioside A (Reb A) • Stevioside (Ste) • Rubusoside (Rub) • Steviolbioside (SteB) • Steviolmonoside ether (SteM) • Steviolmonoside ester (SteE) • Stevioside preparation (95% with traces of reb A, rub and steB) • Solvents for HPLC: acetonitrile and 25 mM H3PO4 Construction of calibration curves

11. Materials and methods • Bacteria and soil samples: Stevia plantation in Paraguay • Culture media (‘stevioside minimum medium’): • pH7: • 0,2% NH3NO3 • 0,1% stevioside • 0,1% KH2PO4 • 0,1% K2HPO4 • 0,05% NaCl • 0,05% MgSO4 • pH8: • 0,2% NH3NO3 • 0,1% stevioside • 1,2% Trisbuffer • 0,1% K2HPO4 • 0,05% NaCl • 0,05% MgSO4 • % yeast extract ([YE]) dependingon the experiment (between 0 and 0,2%) • 1,5 % agar (solid medium) • Incubationtemperature: 37°C

12. Materials and methods • HPLC • Mobile phase: 30% acetonitrile (ACN)– 70% H2O • Elution with linear gradient: 25 mM H3PO4 and ACN: 0-10 min: 30-40% ACN; 10-20 min: 40-80% ACN; 20-30 min: 80% ACN • UV spectra recorded between 195 and 360 nm • Measuring growth • Optical density (OD) at 600 nm • Spectophotometrical, measuring turbidity • Characterization of metabolites • Identification using HPLC • Maintainance of interesting cultures • Solid medium (stevioside minimal medium with agar) • Incubated at 37°C until growth was observed • stored at 4°C

13. Introduction and context • Aim • Materials and methods • Results • Influence of the concentration of yeast extract • Influence of pH • Influence of stirring or shaking • Degradation of steviolbioside • Isolation of interesting bacterial consortia • Conclusions • Results • Gramstaining and growth on solid medium • -Glucosidases: a molecular approach • Conclusions

14. Resultsa) Influence of the concentration of yeast extract • Concentrations of YE from 0 to 0,2% added to stevioside minimal medium (slide 10) • [YE] = 0 : no growth observed  no -glucosidase production • [YE] < 0,02% : no garantee for hydrolysis or bacterial growth growth and hydrolysis: incomplete hydrolysis : initial moment of start = early (50h) • [YE]  0,02% : hydrolysis and bacterial growth •  [YE] : no faster hydrolysis or higher yield of steviol

15. Resultsb) Influence of pH • pH (7 or 8) = set before autoclaving • pH8 no effect on -glucosidase activity or on bacterial growth in the cultures c) Influence of stirring or shaking • Stirring (or shaking) the samples during incubation at 37°C • Stirring : + effect on the hydrolysis rate • Stirring : no additional effect on followed hydrolysis pathway

16. Resultsd) Degradation of steviolbioside • Addition SteB instead of Ste • Formation of small amount of SteM at same time as initial rise of steviol  Degradation of sophoryl residue = (partially) in 2 steps; degradation in 1 step = possible

17. Resultse) Isolation of interesting bacterial consortia • Interesting bacterial consortia = complete hydrolysis from stevioside to steviol in efficient way* Through formation of SteB Through formation of Rub *Efficient = fast and complete; without accumulation of any intermediary product

18. Introduction and context • Aim • Materials and methods • Results • Influence of the concentration of yeast extract • Influence of pH • Influence of stirring or shaking • Degradation of steviolbioside • Isolation of interesting bacterial consortia • Conclusions • Results • Gramstaining and growth on solid medium • -Glucosidases: a molecular approach • Conclusions • Introduction and contexting • Aim • Materials and methods • Results • Conclusions

19. Conclusions • Minimum of YE is required as N-source (0,02% = guarantee for complete hydrolysis) • Without YE: No growth, no -glucosidase production • In the future there will be worked with pH8 because precipitation of steviol is reduced to a minimum and growth is not hindered at this pH • Stirring: - accelerating hydrolysis - no influence on hydrolysis pathway

20. Conclusions • No correlation between bacterial growth and degradation stevioside • Split off sugar moieties: no additional advantage for bacteria • No evidence that bacteria ferment sugars • Degradation sophoryl residue (SteB): intermediate formation of SteM (at least partially) • Hydrolysis pathway ≠ influenced by incubation parameters = different selections of micro organisms?  16S rDNA

21. Introduction and context • Aim • Materials and methods • Results • Influence of the concentration of yeast extract • Influence of pH • Influence of stirring or shaking • Degradation of steviolbioside • Isolation of interesting bacterial consortia • Conclusions • Results • Gramstaining and growth on solid medium • -Glucosidases: a molecular approach • Conclusions • Introduction and contexting • Aim • Materials and methods • Results • Conclusions

22. Resultsf) Gram-staining and growth on solid media • Solid medium containing 0,01 - 5% Ste and 0,02% YE • After 7 hr incubation (37°C): small blue (circular) and white (circular, filamentous) colonies • Gram-staining: Consistent mixtures of Gram (+) and Gram (-) bacteria • Reinoculation in liquid medium containing 0.3% Ste • No hydrolysis was measured (t<300 hr)

23. Resultsc) Gram-staining and growth on solid media • Inoculation of the same solid media for 14 days: blue (circular) and thick white (irregular) colonies • Gram-staining: Blue colonies: Gram (-) bacilliWhite colonies: Gram (+) streptobacilli • RP-HPLC analysis on excised colonies (ACN-H2O gradient) 23

24. Resultsc) Gram-staining and growth on solid media -4,7%+3,2% +1,7% 24

25. Resultsc) Gram-staining and growth on solid media 25

26. Introduction and context • Aim • Materials and methods • Results • Influence of the concentration of yeast extract • Influence of pH • Influence of stirring or shaking • Degradation of steviolbioside • Isolation of interesting bacterial consortia • Conclusions • Results • Gramstaining and growth on solid medium • -Glucosidases: a molecular approach • Conclusions • Introduction and contexting • Aim • Materials and methods • Results • Conclusions

27. -Glucosidases: a molecular approach • -Glucosidases: overview • Carbohydrate-Active Enzyme (CAZy) database: 115 families based upon amino acid sequence homology • -Glucosidases: families GH-1, GH-3 and GH-9 • GH-1: (/)8 fold ; catalytic nucleophile: Glu ; +300 enzymes GH-3: different kind of folds; catalytic nucleophile: Asp ; +100 enzymes GH-9: (/)6 fold; catalytic nucleophile: Asp ; only few -glucosidases • Only few enzymes known with specific -1,2-glucosidase activity • -Glucosidase TMA7501 in cotyledons of germinated Tropaeolum majus seedlings • Tomatinase in tomato pathogen Septoria lycopersici • … 27

28. -Glucosidases: a molecular approach • Aim 28

29. -Glucosidases: a molecular approach • gDNA extractions 29

30. -Glucosidases: a molecular approach • gDNA extractions • High molecular weight gDNA is preferable for PCR ( agarose gel electrophoresis) • The greater the size, the less likely the formation of chimeras during PCR • Humic acids in fractions 1 and 2: 1 µl is enough for inhibition of DNA polymerases • Purity: (A260-A320)/(A280-A320) ratio > 1,7 (lower: protein contamination) A260/A230 ratio > 2,0 (lower: humic acid contamination) 0,01 < A320 < 0,1: background (phenol, humic acids) [gDNA] (µg/ml) : (50 µg/ml)*(A260 – A320)*dilution factor 30

31. -Glucosidases: a molecular approach • Degenerated primers • PCR-primers based upon reverse translation of conserved amino acid sequences show a degree of degeneracy (one or more of its positions can be occupied by one of several possible nucleotides) • Total degeneracy as low as possible: conserved domains with Trp, Tyr, Met, Asp, Glu, His,… • Little or no degeneracy at 3’ end  PCR assays: higher concentration of primer needed 31

32. -Glucosidases: a molecular approach Culture 1 Culture 2 • Results: gDNA extractions • Concentration • 35 – 130 µg/ml ; up to 300 µg/ml • Pre-treatment with lysozyme will extract more gDNA from Gram (+) bacteria • Purity • 1,2 < (A260-A320)/(A280-A320) < 1,8  CI extraction: less impurities • 0,9 < A260/A230 < 2,2  mainly due to humic acids in the first extractions • 0,0 < A320 < 0,3  mostly humic acids in first culture, phenol • Length • Minimal shearing ; > 10 kDa 32

33. -Glucosidases: a molecular approach • Results: degenerated primers • Alignment of 250 sequences of GH-1 and GH-3 -glucosidases identified several conserved domains 33

34. QIEGA ITENG YHWDLP C-terminal conserveddomain 34

35. -Glucosidases: a molecular approach • Results: degenerated primers • Alignment of 250 sequences of GH-1 and GH-3 -glucosidases identified several conserved domains • GH-1: 32 primers (FW: 21 ; RV: 11)  degeneracy between 16x and 1152x • GH-3: 10 primers (FW: 6 ; RV: 4)  degeneracy between 64x and 1152x 35

36. Introduction and context • Aim • Materials and methods • Results • Influence of the concentration of yeast extract • Influence of pH • Influence of stirring or shaking • Degradation of steviolbioside • Isolation of interesting bacterial consortia • Conclusions • Results • Gramstaining and growth on solid medium • -Glucosidases: a molecular approach • Conclusions • Introduction and contexting • Aim • Materials and methods • Results • Conclusions

37. Conclusions • Inoculation of solid medium: mixtures of Gram (+) and Gram (-) • Reinoculation in liquid medium: no measurable hydrolysis of stevioside after 300 hr • <10% of soil bacteria are culturable (“viable but not culturable” hypothesis) • After extended incubation of solid media: streptobacillus-shaped bacteria, showing minimal degradation of stevioside to steviol • Accumulation of rubusoside but no steviolbioside • Extended incubation in liquid medium: stevioside degradation? • gDNA extraction with PCI/CI gives mixed results • Relatively low yield • Pre-treatment with lysozyme might be necessary for higher yield • Moderate purity: contaminations with humic acids, phenol Some very good results, but further optimalization is needed! • Degenerated primers were developed, specifically for bacterial -glucosidases 37

38. Thank you for your attention ! 38