Download Conclusions

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
•
•
•
•
Introduction and context
Aim
Materials and methods
Results
•a.Introduction
and ofcontexting
Influence of the concentration
yeast extract
b. Influence of pH
•c.Aim
Influence of stirring or shaking
Degradation ofand
steviolbioside
•d.Materials
methods
e. Isolation of interesting bacterial consortia
Results
• •Conclusions
Conclusions
• •Results
f.
Gramstaining and growth on solid medium
• -Glucosidases: a molecular approach
• Conclusions
2
Introduction and contexting
3
Introduction and contexting
1.Steviol glycosides (>Stevioside) = from Stevia rebaudiana bertoni
leaves
Low dosis (max. 200 mg/day)
Application:
-Natural sweetener
-Alternative for aspartame
- Up to 300 times sweeter than sugar
High dosis (max. 3 ×500 mg/day)
Possible pharmacological effects:
- Lowering blood pressure
- Reducing glucose in blood
- Increasing insuline sensitivity
4
Introduction and contexting
2.Ingestion steviol glycosides  steviol  steviol glucuronide
(intestines)
(liver)
3.Possible active component = steviol glucuronide
4.Further fundamental research = necessary
 enough glucuronide required
through the production of steviol
upscaling production of steviol
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)
5
 : Flavobacterium johnsonae (Okamoto et al., 2000)
·······: Clavibacter michiganense (Nakano et al., 1998) and Flavobacterium johnsonae (Okamoto et al., 2000)
6
•
•
•
•
Introduction and context
Aim
Materials and methods
Results
•a.Introduction
and contexting
Influence of the concentration
of yeast extract
b. Influence of pH
•c.Aim
Influence of stirring or shaking
Degradation ofand
steviolbioside
•d.Materials
methods
e. Isolation of interesting bacterial consortia
Results
• •Conclusions
Conclusions
• •Results
f.
Gramstaining and growth on solid medium
• -Glucosidases: a molecular approach
• Conclusions
7
Aim
1.Isolation of bacterial consortia who produce glucosidases able to hydrolyze stevioside to steviol
2.Better insight in influence of incubation parameters
on hydrolysis pathway and hydrolysis rate
Incubation parameters:
• pH
• Concentration yeast extract
• Stirring
3.Production of steviol
8
•
•
•
•
Introduction and context
Aim
Materials and methods
Results
•a.Introduction
and contexting
Influence of the concentration
of yeast extract
b. Influence of pH
•c.Aim
Influence of stirring or shaking
Degradation of steviolbioside
•d.Materials
and methods
e. Isolation of interesting bacterial consortia
Results
• •Conclusions
Conclusions
• •Results
f.
Gramstaining and growth on solid medium
• -Glucosidases: a molecular approach
• Conclusions
9
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)
Construction of calibration curves
2. Stevioside preparation (95% with traces of reb A, rub and steB)
3. Solvents for HPLC: acetonitrile and 25 mM H3PO4
10
Materials and methods
• Bacteria and soil samples: Stevia plantation in Paraguay
• Culture media (‘stevioside minimum medium’):
− pH7:
o
o
o
o
o
o
0,2% NH3NO3
0,1% stevioside
0,1% KH2PO4
0,1% K2HPO4
0,05% NaCl
0,05% MgSO4
− pH8:
o
o
o
o
o
o
0,2% NH3NO3
0,1% stevioside
1,2% Trisbuffer
0,1% K2HPO4
0,05% NaCl
0,05% MgSO4
• % yeast extract ([YE]) depending on the experiment (between 0 and 0,2%)
• 1,5 % agar (solid medium)
− Incubation temperature: 37°C
11
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
12
•
•
•
•
Introduction and context
Aim
Materials and methods
Results
a.
b.
c.
d.
e.
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
f.
Gramstaining and growth on solid medium
• -Glucosidases: a molecular approach
• Conclusions
13
Results
a) 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
14
Results
b) 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
15
Results
d) 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
16
Results
e) 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
% Steviolglycosides or steviol:
Hydrolysis through formation or Rub
100
100
90
90
80
80
70
Stevioside
Rubusoside
Steviolbioside
SteE
SteM
Steviol
Totaal
60
50
40
30
20
10
(mmol/l) / Total (mmol/l)
(mmol/l) / Total (mmol/l)
% Steviolglycosides or steviol:
Hydrolysis through formation or SteB
70
60
50
40
30
20
10
0
0
0
100
200
Time (h)
300
400
0
50
100
*Efficient = fast and complete; without accumulation of any intermediary product
150
200
250
300
Time (h)
17
350
•
•
•
•
Introduction and context
Aim
Materials and methods
Results
•a.Introduction
and contexting
Influence of the concentration
of yeast extract
b. Influence of pH
•c.Aim
Influence of stirring or shaking
Degradation ofand
steviolbioside
•d.Materials
methods
e. Isolation of interesting bacterial consortia
Results
• •Conclusions
Conclusions
• •Results
f.
Gramstaining and growth on solid medium
• -Glucosidases: a molecular approach
• Conclusions
18
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
19
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
20
•
•
•
•
Introduction and context
Aim
Materials and methods
Results
•a.Introduction
and contexting
Influence of the concentration
of yeast extract
b. Influence of pH
•c.Aim
Influence of stirring or shaking
Degradation ofand
steviolbioside
•d.Materials
methods
e. Isolation of interesting bacterial consortia
Results
• •Conclusions
Conclusions
• •Results
f.
Gramstaining and growth on solid medium
• -Glucosidases: a molecular approach
• Conclusions
21
Results
f) 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)
22
Results
c) 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 (-) bacilli
White colonies: Gram (+)
streptobacilli
• RP-HPLC analysis on excised
colonies (ACN-H2O gradient)
23
Results
c) Gram-staining and growth on solid media
RebA
ST
RebC
DulcA
Rub
RebB
SB
SteE
SteM
SV
Total
t=0d
Standard
7,0%
90,9%
0,3%
0,4%
0,6%
0,1%
0,7%
0,0%
0,1%
0,0%
100%
t=14d
Control
6,7%
86,6%
0,3%
0,3%
0,9%
0,4%
4,5%
0,1%
0,1%
0,0%
100%
t=14d
Blue col.
6,6%
85,3%
0,3%
0,3%
1,8%
0,3%
4,6%
0,3%
0,1%
0,4%
100%
t=14d
White col.
6,5%
81,9%
0,3%
0,3%
4,1%
0,3%
4,4%
0,2%
0,1%
1,7%
100%
-4,7%
+3,2%
+1,7%
24
Results
c) Gram-staining and growth on solid media
[mAU]
Steviol
SVE
Steviolmonoside
Rubusoside
Rebaudioside B
Steviolbioside
Stevioside
Rebaudioside A
Absorbance
100
50
C:\ ClarityChromPrep\ Stijn2\ Data\ Plaat5ST _14d_witk ol_27_05_2009 16_23_05_0067 - S 2500: Channel 1
C:\ ClarityChromPrep\ Stijn2\ Data\ Plaat5ST_14d_blanco_27_05_2009 15_36_15_0045 - S 2500: Channel 1
A-B
0
5
10
15
20
[min.]
Time
25
•
•
•
•
Introduction and context
Aim
Materials and methods
Results
•a.Introduction
and contexting
Influence of the concentration
of yeast extract
b. Influence of pH
•c.Aim
Influence of stirring or shaking
Degradation ofand
steviolbioside
•d.Materials
methods
e. Isolation of interesting bacterial consortia
Results
• •Conclusions
Conclusions
• •Results
f.
Gramstaining and growth on solid medium
• -Glucosidases: a molecular approach
• Conclusions
26
-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
-Glucosidases: a molecular
approach
•
Aim
28
-Glucosidases: a molecular
approach
•
gDNA extractions
29
-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
-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
-Glucosidases: a molecular
approach
• Results: gDNA extractions
•
Concentration
– 35 – 130 µg/ml ; up to 300 µg/ml
– Pre-treatment with lysozyme will
Culture 1
Culture 2
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
-Glucosidases: a molecular
approach
• Results: degenerated primers
– Alignment of 250 sequences of GH-1 and GH-3 -glucosidases identified
several conserved domains
33
QIEGA
YHWDLP
ITENG
C-terminal
conserved domain
34
-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
•
•
•
•
Introduction and context
Aim
Materials and methods
Results
•a.Introduction
and contexting
Influence of the concentration
of yeast extract
b. Influence of pH
•c.Aim
Influence of stirring or shaking
Degradation ofand
steviolbioside
•d.Materials
methods
e. Isolation of interesting bacterial consortia
Results
• •Conclusions
Conclusions
• •Results
f.
Gramstaining and growth on solid medium
• -Glucosidases: a molecular approach
• Conclusions
36
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 !
38