Saccharomyces cerevisiae
oxidative response by cyclic
voltammetry and GC-MS
*Corresponding author!
E-‐mail: [email protected]!
Carla Maria Oliveira
1,2Cris/ana C. Castro
3, Caitriona Gunning
1, Jos
é
A. Couto
1, Jos
é
A.
Teixeira
3, Rui C. Mar/ns
4and Ant
ó
nio C. Silva Ferreira
1,5*!
1. Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Rua Dr. António Bernardino de Almeida, 4200-072 Porto, Portugal! 2. Departamento de Química & QOPNA, Universidade de Aveiro, 3810-193 Aveiro, Portugal!
3. IBB - Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal!
4. BioInformatics - Molecular and Environmental Biology Centre, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal! 5. Stellenbosch University, Private Bag X1, Matieland, 7602, Stellenbosch, South Africa!
Objectives:
!
The interpretation of the overall metabolic process by a non-target approach!
The study of the resistance to oxidation during an alcoholic fermentation using a synthetic grape juice!
The identification and quantification of target compounds related with this metabolic process!
Why use
SGJ?!
To reduce
complexity!
Clearer
precursor-product relationship!
Reproducibili
ty!
"Solution A !
•
D-glucose 110g
•
D-fructose 110g
•
Ergosterol 10mg
•
Tween 80 1 ml
Solution B !
•
L (+) tartaric acid 6g
•
L-(-)malic acid 3g
•
Citric acid 0.5g
Solution C!
!• YNB (Difco) 1.7g
• CAA (Difco) 2g
• CaCl
20.2g
• Arginine-HCl 0.8g
• L-(-)proline 1g
• L-(-)tryptophan 0.1g
• Tyrosine 0.1g
• Phenylalanine 0.1g
CAA (casamino acids) is the group of amino acids obtained when casein (from milk) is broken down by enzymes
(Ciani and Ferraro 1996)!
1. Synthetic Grape Juice (SGJ)!
2. Phenolic acids addition
!
3. Hydroxycinnamic
acids!
3. Hydroxybenzoic
acids!
Aprox.15 mg/L each! Gallic acid ! O H OH OH O HO Vanillic acid ! OH OH O HO Protocatechuic acid ! O H OH O Coumaric acid ! O H OH OH O Caffeic acid ! O H OCH3 OH O Ferulic acid ! OH OCH3 O HO
Study of the resistance to
oxidation during the
3. Yeast addition
!
Portuguese Yeast Culture Collection
Saccharomyces cerevisiae
Experimental protocol
Control
V = 900 ml
!
SGJ + ! Yeast additionSupplemented 1
V = 900 ml
!
SGJ + Phenolic acids + Yeast additionSupplemented 2
V = 900 ml
!
SGJ + Phenolic acids + Yeast addition!
!
!
!
n
The antioxidant profile
was monitored:!
n
Target approach: using HPLC/DAD detection!
Current!
Intensity !
I/ A
!
Applied Potential Difference!
E (vs SCE) / V
!
3 mm diameter glass carbon electrode!Scan Range: 100 mV / sec (diffusion)!
-5.0E-06 0.0E+00 5.0E-06 1.0E-05 1.5E-05 2.0E-05 2.5E-05 3.0E-05 3.5E-05 0 0.4 0.8 1.2 E (mV) 815 Coumaric acid 800 Vanillic acid 680 Syringic acid 670 Ferulic acid 500 Ru>n 470 Gallic acid 460 Caffeic acid 425 Querce>n 420 Catechin
O. Makhotkina, P.A. Kilmar9n / Analy9ca Chimica Acta 668 (2010) 155–165
Cyclic voltammetry !
Scanning of electroactive
n
T
h
e
a r o m a t i c
profile was
e x p l o r e d
using
HS-SPME /
GC-MS analysis
conducted
by:!
!
n
Target
approach!
n
Non-target
approach!
!Retention Time
m/z
Target
versus
Non-Target Approach ...!
Varian Saturn 2000! ion-trap GC-MS!
Retention Time
m/z
Target
versus
Non-Target Approach ...!
Varian Saturn 2000! ion-trap GC-MS!
Voltammetric results !
Changes in the overall antioxidant profile!
!
•
A decrease in the amplitude of the broad band at 1 V;
-‐5.0E-‐06 0.0E+00 5.0E-‐06 1.0E-‐05 1.5E-‐05 2.0E-‐05 2.5E-‐05 3.0E-‐05 0 0.4 0.8 1.2 Cu rr en t I nt en si ty (A) PotenFal (V) Control_T0 Control_T11 -‐5.0E-‐06 0.0E+00 5.0E-‐06 1.0E-‐05 1.5E-‐05 2.0E-‐05 2.5E-‐05 3.0E-‐05 0 0.4 0.8 1.2 Cu rr en t I nt en si ty (A) PotenFal (V) Supplemented_T0 Supplemented_T11
•
The end of alcoholic fermentation shows a pattern like that of normal
wines.
-‐5.0E-‐06 0.0E+00 5.0E-‐06 1.0E-‐05 1.5E-‐05 2.0E-‐05 2.5E-‐05 0 0.4 0.8 1.2 Cu rr en t I nt en si ty (A) PotenFal (V) Control_T11 Supplemented_T11 Normal WineVoltammetric results !
HPLC-Data!
0 2 4 6 8 10 12 14 16 18 0 2 4 6 8 10 12 C o n ce n tr ati o n (m g /L ) Days ferulic acid p-coumaric acid O H OH O O H OCH3 OH O 8 10 12 14 16 18 20 22 24 0 2 4 6 8 10 12 C o n ce n tr ati o n (m g /L ) Days gallic acid vanillic acid O H OH OH O HO OH OCH3 O HO 2 4 6 8 10 12 14 16 18 0 2 4 6 8 10 12 C o n ce n tr ati o n (m g /L ) Days protocatechuic acid caffeic acid OH OH O HO O H OH OH O0 10000 20000 30000 40000 0 2 4 6 8 10 12 Area Days
One compound was found increasing in both control and supplemented media, after 5
days of alcoholic fermentation.
This result suggests that Saccharomyces cerevisiae has the ability to produce
antioxidants ‘de novo’ from simple precursors such sugars and amino acids.!
Chlorogenic acid confirmed by LC-MS
!
HPLC-Data!
O H OH O O OH OH COOH O HRetention Time
m/z
Target
versus
Non-Target Approach ...!
Varian Saturn 2000! ion-trap GC-MS!
Supplemented media! Control!
Target
Approach ...!
0! 0.2! 0.4! 0.6! 0.8! 1! 0! 1! 2! 3! 4! 5! 6! 7! 8! 9! 10! 11! Normalized area !Fermentation time / days!
(p-vinylguaiacol)!
CH2
H3CO
O
H
Product of ferulic acid
decarboxylation !
Target
Approach ...!
Supplemented media! Control! 0! 50! 100! 150! 200! 0! 1! 2! 3! 4! 5! 6! 7! 8! 9! 10! 11! mg/L !Fermentation time / days!
2-Phenylethanol! OH 0! 1! 2! 3! 4! 5! 0! 1! 2! 3! 4! 5! 6! 7! 8! 9! 10! 11! mg/L !
Fermentation time / days!
(Isoamyl acetate)! O CH3 O C H3 CH3 0! 10! 20! 30! 40! 50! 60! 0! 1! 2! 3! 4! 5! 6! 7! 8! 9! 10! 11! mg/L !
Fermentation time / days!
(Isoamyl alcohol)!
OH C
H3
Retention Time
m/z
PCA scores analysis: First versus second principal components!
Mass Spectroscopy Signal
Processing
PCA shows differences from day to day,
imposes a pattern on the fermentation!
Non Target : Mass Expression
C_1! C_11! S1_1! S1_11! S2_1! S2_11!
C: Control; S1:Supplemented1; S2:Supplemented2!
CH2 H3CO O H Control! Supp_1! Supp_2!
Non Target : Mass Expression
C_1! C_11! S1_1! S1_11! S2_1! S2_11!
C: Control; S1:Supplemented1; S2:Supplemented2!
Control! Supp_1! Supp_2!
High - Correlation!
low - Correlation!
“
Pathway Reconstruction”
!
!
Non-Target Approach : “Clustering” all MS Scans
MS Scans (4100)
MS
Sca
ns
(4
10
0)
Non-Target Approach : “Clustering” all MS Scans
Scan Corr 2250 0.8514 C10C2 1042 0.7088 C6C2 981 0.9432 976 0.9394 1712 0.7014 AceFc acid 975 0.9356 2220 0.9049 634 0.9043 2253 0.8993 2414 0.8280 2799 0.8279 C6 1048 0.7968 3319 0.7963 C8 1592 0.7551 3792 0.7549 C10 3351 0.7253 1657 0.7252 C8C2 83 128 O OH O H3C H3C SotolonMS Scans (4100)
MS
Sca
ns
(4
10
0)
981
0.9432 71 53 72 2-Phenylethanol! Scan 2983!2253
0.8993 101 73 69 171 116 High! low !“
Pathway Reconstruction”
Conclusion
A new biological interpretation can be achieve by the non-target approach that consists of a list of all compounds and their correlations!
Changes in the overall resistance to oxidation does indeed occur during alcoholic fermentation and shows a pattern like that of normal wines!
Chorogenic acid was found increasing in both control and supplemented media,
suggesting that Saccharomyces cerevisiae has the ability to produce antioxidants de
novo from simple precursors such as sugars and amino acids! !
The non-target approach could provide new knowledgement for the Pathway Reconstruction !