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

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-OostendeZeedijk 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

• Introduction and contexting• Aim• Materials and methods• Results• Conclusions

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• Introduction and context• Aim• Materials and methods• Results

a. Influence of the concentration of yeast extractb. Influence of pHc. Influence of stirring or shakingd. Degradation of steviolbiosidee. Isolation of interesting bacterial consortia

• Conclusions• Results

f. Gramstaining and growth on solid medium

-Glucosidases: a molecular approach• Conclusions

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Introduction and contexting

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1.Steviol glycosides (>Stevioside) = from Stevia rebaudiana bertoni leaves

High dosis (max. 3 ×500 mg/day)Low dosis (max. 200 mg/day)

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3.Possible active component = steviol glucuronide

4.Further fundamental research = necessary enough glucuronide required

Chemical synthesis Low yield (10%)Stevioside + NaIO4 (partially breaking off the bounded sugars) mixture refluxed with KOH

Bio-organic synthesis Several possibilities (anaerobic and aerobic)

upscaling production of steviol

through the production of steviol

2.Ingestion steviol glycosides steviol steviol glucuronide (intestines) (liver)

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


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8

Aim


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Materials and methods• Chemicals:

1. Pure standards of the steviol glycosides– Rebaudioside A (Reb A)– Stevioside (Ste)– Rubusoside (Rub)– Steviolbioside (SteB)– Steviolmonoside ether (SteM)– Steviolmonoside ester (SteE)

2. Stevioside preparation (95% with traces of reb A, rub and steB)

3. Solvents for HPLC: acetonitrile and 25 mM H3PO4

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Construction of calibration curves

Materials and methods• Bacteria and soil samples: Stevia plantation in Paraguay

• Culture media (‘stevioside minimum medium’):

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− pH7:o 0,2% NH3NO3

o 0,1% steviosideo 0,1% KH2PO4

o 0,1% K2HPO4

o 0,05% NaClo 0,05% MgSO4

− pH8:o 0,2% NH3NO3

o 0,1% steviosideo 1,2% Trisbuffero 0,1% K2HPO4

o 0,05% NaClo 0,05% MgSO4

• % yeast extract ([YE]) depending on the experiment (between 0 and 0,2%)• 1,5 % agar (solid medium)

−Incubation temperature: 37°C

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

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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

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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

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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

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Resultse) Isolation of interesting bacterial consortia• Interesting bacterial consortia = complete hydrolysis from

stevioside to steviol in efficient way*

17*Efficient = fast and complete; without accumulation of any intermediary product

Through formation of SteB Through formation of Rub


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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

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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

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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)

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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)

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-4,7% +3,2% +1,7%

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RebA ST RebC DulcA Rub RebB SB SteE SteM SV Total

t=0dStandard 7,0% 90,9% 0,3% 0,4% 0,6% 0,1% 0,7% 0,0% 0,1% 0,0% 100%

t=14dControl 6,7% 86,6% 0,3% 0,3% 0,9% 0,4% 4,5% 0,1% 0,1% 0,0% 100%

t=14dBlue col. 6,6% 85,3% 0,3% 0,3% 1,8% 0,3% 4,6% 0,3% 0,1% 0,4% 100%

t=14dWhite col. 6,5% 81,9% 0,3% 0,3% 4,1% 0,3% 4,4% 0,2% 0,1% 1,7% 100%


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[min.]Time

5 10 15 20

[mAU]

Abso

rbance

0

50

100

Ste

vio

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Ste

vio

sid

e

Reb

aud

iosi

de

A

Ru

bu

sosi

de

Reb

aud

iosi

de

B

Ste

vio

lbio

sid

e

SV

E

Ste

vio

lmo

no

sid

e

C:\ ClarityChromPrep\ Stijn2\ Data\ Plaat5ST_14d_witkol_27_05_2009 16_23_05_0067 - S 2500: Channel 1C:\ClarityChromPrep\Stijn2\Data\Plaat5ST_14d_blanco_27_05_2009 15_36_15_0045 - S 2500: Channel 1A-B


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-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

• …

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-Glucosidases: a molecular approach• Aim

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-Glucosidases: a molecular approach• gDNA extractions

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-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

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-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

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-Glucosidases: a molecular approach• 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

Culture 1 Culture 2

-Glucosidases: a molecular approach• Results: degenerated primers

– Alignment of 250 sequences of GH-1 and GH-3 -glucosidases identified several conserved domains

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QIEGA

YHWDLP ITENG

C-terminal conserved domain

-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

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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

Thank you for your attention !

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

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