25.01.2017
1
Grundlagen und Anwendung moderner Trennverfahren
Günther K. BonnInstitute of Analytical Chemistry and Radiochemistry, Leopold‐Franzens
University of Innsbruck, Innrain 80‐82, A‐6020 Innsbruck, Austria
1
Fällung + ADSI
Komplexität von biologischen Proben
1. enorme Komplexität der Proben
2. geringe Konzentration der Zielanalyten
3. beschränkter dynamischer Bereich der analytischen Messgeräte
4. Störkomponenten (Detergenzien, Puffersysteme etc.)
MS
biologische Probe
Herausforderungen:
2
EinleitungAnalytik der Biomoleküle
25.01.2017
2
3
22 Proteine machen 99% des gesamten Serumproteoms aus!
90% 10%
EinleitungAnalytik der Biomoleküle
1. Enorme Komplexität von biologischen Proben
z.B. Blutserum
4Anderson, N. L. (2002) Mol. Cell. Proteomics 1: 845‐867
Dynamischer Bereich von Blutplasma
EinleitungAnalytik der Biomoleküle
2. Geringe Konzentration vieler Zielanalyten
25.01.2017
3
Selektivität
Empfind‐lichkeit
Geschwin‐digkeit
Hoher Probendurchsatz
Verbesserte Detektion
Gezielte Analyse
Effizienz
Effiziente Probenvorbereitung
EinleitungAnalytik der Biomoleküle
Proteine können auf verschiedene Art in ihrer nativen Form gefällt werden:
• Fällung durch Aussalzen
• isoelektrische Fällung (Fällung am IEP)
• Fällung mit organischen Lösungsmitteln
• Co‐Präzipitation
Fällung von Proteinen
6
25.01.2017
4
Precipitation of Phosphoproteins by
Trivalent Lanthanide Ions
A Top‐Down Approach
7
Protein phosphorylation
Protein dephosphorylation
Berg JM et al. 2010
25.01.2017
5
Protein separation and isolation
2. Chromatographic techniques
IMACImmobilized Metal ion Affinity Chromatography
MOACMetal Oxide Affinity Chromatography
Berg JM et al. 2010 According to: Leitner A. 2010
Proposed Precipitation Mechanism
10
25.01.2017
6
Lanthanide Phoshates(Low Solubility Products)
solubility products [logKsp(M)]
ErPO4 -25.13 ± 0.11
EuPO4 -25.96 ± 0.03
TbPO4 -25.39 ± 0.04
Li, X. et al. Geochimica et cosmochimica acta,1997.61(8):p.1625-1633
11
Dissolvation of pellet
MALDI-MS
on-pellet digest
denaturation, tryptic digestion30% formic acid
washing
precipitation
10 min, 70W microwave‐assisted digest
MALDI-MS
PeptidesProteins
Scheme for Precipitation of Phosphoproteins by Trivalent Europium-, Terbium- and Erbium- Ions
PrecipitantKH2PO4
Yüksel et al. Anal Bioanal Chem (2012) 403:1323–133112
TOP‐DOWNBOTTOM‐UP
25.01.2017
7
Methods and instruments
MALDI‐TOF MS: linear mode
For TOP‐DOWN
Methods and instruments
MALDI‐TOF MS: reflector mode
Improved Resolution!
for BOTTOM‐UP
25.01.2017
8
Bovine milk composition
87,1%
4,9%3,9%
3,4%0,7%
water
carbohydrates
fats
proteins
minerals
38%
9% 29%
9%
4%9%2%
α‐S1‐casein
α‐S2‐casein
β‐casein
κ‐casein
α‐lactalbumin
β‐lactoglobulin
others
according to Pavia DL. 1990 / Eigel WN et al. 1984
Phosphoproteine
Milk proteins
protein (variant)mol. weight /
Daamino acids
c / g∙L‐1P
per molSH | S‐S per mol
pI
αS1‐casein (B 8P) 23,614 199 12 – 15 8 0 | 0 4.4 – 4.8
αS2‐casein (A 11P) 25,230 207 3 – 4 11 2 | 0 −
β‐casein (A2 5P) 23,983 209 9 – 11 5 0 | 0 4.8 – 5.1
κ‐casein (B 1P) 19,023 169 2 – 4 1 2 | 0 5.3 – 5.8
α‐lactalbumin (B) 14,176 123 0.6 – 1.7 0 0 | 4 4.2 – 4.5
β‐lactoglobulin (B) 18,363 162 2 – 4 0 1 | 2 5.1
serum albumin 66,267 582 0.4 0 1 | 17 4.7 – 4.9
according to: Eigel WN et al. 1984 / Fox PF et al. 1998
25.01.2017
9
Precipitation of Phosphorylated Proteins – Top‐Down Workflow
17
Güzel, Y.; Rainer, M. (); Mirza, M.R.; Bonn, G.K. Highly efficient precipitation of phosphoproteins using trivalent Europium‐,Terbium‐ and Erbium Ions. Analytical and Bioanalytical Chemistry (2012) 403(5), 1323–1331.
Terbium
Wash 1
Wash 2
Pellet
Supernatent
milk
Erbium
Wash 1
Wash 2
Pellet
Supernatent
milk
Europium
PPC‐5 (m/z ~12–13 kDa)
α‐lactalbumin (m/z ~14.1 kDa)
β‐lactoglobulin (m/z ~18.3 kDa)
non‐phosphorylated milk‐proteins
αS1‐casein (m/z ~24.5 kDa)
β‐casein (m/z ~25.1 kDa)
phosphoproteins
αS2‐casein (m/z ~24.5 kDa)
κ‐casein (m/z ~20.0 kDa)
Wash 1
Wash 2
Pellet
Supernatent
milk
Precipitation of Phosphoproteins from Bovine Milk by Trivalent Europium-, Terbium- and Erbium- Ions
Yüksel et al. Anal Bioanal Chem (2012) 403:1323–133118
25.01.2017
10
Erbium
Wash 1
Wash 2
Pellet
Supernatent
eggwhite
Terbiumegg
white
Pellet
Wash 2
Supernatent
Wash 1
lysozyme (m/z ~14 kDa)
ovomucoid (m/z ~ 28 kDa)
ovoglobulins G2+G3 (m/z ~30‐45 kDa)
ovotransferrin (m/z ~80 kDa)
ovalbumin (m/z ~45 kDa)
non phosphorylated egg‐white proteins phosphoprotein
Europium
Wash 1
Wash 2
Pellet
Supernatent
eggwhite
Precipitation of Phosphoproteins from Egg-White by Trivalent Europium-, Terbium- and Erbium- Ions
Yüksel et al. Anal Bioanal Chem (2012) 403:1323–133119
2M Ln3+‐Chloride
UV/VIS
take supernatant
bicinchoninic acid (BCA)
chelation of two bicinchoninicacid molecules with Cu+1
reduction of Cu+2 to Cu+1 in the presence ofproteins (alkaline conditions)
colorimetric detection 562 nm
Recovery Study of Phosphoprotein
20
25.01.2017
11
Methods and instruments
Colorimetric assays: Bradford and BCA
Bradford reagent:Coomassie Brilliant Blue G‐250
BCA assay reaction
Lottspeich F. 2012
Bicinchoninsäure
Absorptionsmaximum von 562 nm
0
20
40
60
80
100
0,5 1 1,5 2 2,5 3
Rec
overy
[%
]
Volume Precipitant [µl]
Lanthanum
Europium
Terbium
Erbium
100%
Precipitation of Phosphoproteins by Trivalent Europium-, Terbium- and Erbium- Ions
Recovery Study
ccasein = 300µg/ml
cprecip. = 2M
22
25.01.2017
12
Precipitation of Phosphopeptides by
Trivalent Lanthanide Ions
A Bottum‐Up Approach
23
A Novel Strategy for Phosphopeptide Enrichment using Lanthanide Phosphate Precipitation
Workflow for the precipitation of phosphorylated peptides24
Bottom‐up
25.01.2017
13
A Novel Strategy for Phosphopeptide Enrichment using Lanthanide Phosphate Precipitation
MALDI mass spectra taken from digested milk peptides after precipitation with trivalent lanthanide ions. A,phosphopeptide enriched by precipitation with Er3+. B, phosphopeptide enriched by precipitation using Ho3+. C,phosphopeptide enriched by precipitation using Ce3+. α‐S1 and β‐S2 refers to first and second subunits of α‐caseinrespectively. β‐C refers to peptides form β‐casein
Erbium
Holmium
Cer
25
A Novel Strategy for Phosphopeptide Enrichment using Lanthanide Phosphate Precipitation
MALDI mass spectra taken from egg white peptides after precipitation with trivalent lanthanide ions. A,phosphopeptide enriched by precipitation with Er3+. B, phosphopeptide enriched by precipitation using Ho3+. C,phosphopeptide enriched by precipitation using Ce3+. Only phosphorylated peptides are labeled
Erbium
Holmium
Cer
26
25.01.2017
14
A Novel Strategy for Phosphopeptide Enrichment using Lanthanide Phosphate Precipitation
MALDI mass spectra of a sensitivity study using two synthetic phosphopeptides. A, representing 500 fmol/µL; B, 10 fold dilution (50 fmol/µL) and C, 100 fold dilution (5 fmol/µL)
500 fmol/µL
50 fmol/µL
5 fmol/µL
27
High Sensitivity!
[M+H]+ Da Phosphopeptide Sequencesa Phosho‐
groups
ErCl3 HoCl3 CeCl3 LaCl3 EuCl3 TmCl3 TbCl3 TiO2
1254.52
1331.53
1411.50
1466.61
1594.70
1660.79
1832.83
1847.69
1927.69
1951.95
2061.83
2088.89
2432.05
2511.13
2556.10
2619.04
2678.01
2703.50
2720.91
2747.10
2856.50
2901.32
2935.15
2966.16
3008.01
3042.27
3087.99
3122.27
3132.20
EVVGSpAEAGVDAA (Ov‐(340–352))
EQLSpTSpEENSK (α‐S2‐(141–151))
EQLSpTSpEENSK (α‐S2‐(141–151))
TVDMESpTEVFTK (α‐S2‐(153–164))
TVDMESpTEVFTKK (α‐S2‐(153–165))
VPQLEIVPNSpAEER α(‐S1‐(121–134))
YLGEYLIVPNSpAEER (α‐S1)
DIGSESpTEDQAMEDIK (α‐S1‐(58–73))
DIGSESpTEDQAMEDIK (α‐S1‐(58–73))
YKVPQLEIVPNSpAEER (α‐S1‐(119–134))
FQSpEEQQQTEDELQDK (β‐C‐(33–48))
EVVGSpAEAGVDAASVSEEFR (Ov‐(340–359))
IEKFQSpEEQQQTEDELQDK (β‐C‐(33–48))
LPGFGDSpIEAQCGTSVNVHSSLR (Ov‐(62–84))
FQSpEEQQQTEDELQDKIHPF (β‐C‐(48‐67))
NTMEHVSpSpSpEESpIISQETYK (α‐S2‐(17–36))
VNELSpKDIGSpESpTEDQAMEDIK (α‐S1‐(52–73))
LRLKKYKVPQLEIVPNSpAEERL(α‐S1‐(114–135))
QMEAESpISpSpSpEEIVPNSVEAQK (α‐S1‐(74–94))
NTMEHVSpSpSpEESpIISQETYKQ (α‐S2‐(17–37))
EKVNELSpKDIGSpESTEDQAMEDIK (α‐S1‐(50–73))
FDKLPGFGDSpIEAQCGTSVNVHSSLR (Ov‐(59–84))
EKVNELSpKDIGSpESpTEDQAMEDIK (α‐S1‐(50–73))
ELEELNVPGEIVESpLSpSpSpEESITR (β‐C‐(17–40))
NANEEEYSIGSpSpSpEESpAEVATEEVK (α‐S2‐(61–85))
RELEELNVPGEIVESLSpSpSpEESITR (β‐C‐(16–40))
NANEEEYSIGSpSpSpEESpAEVATEEVK (α‐S2‐(61–85))
RELEELNVPGEIVESpLSpSpSpEESITR (β‐C‐(16–40))
KNTMEHVSpSpSpEESpIISQETYKQEK (α‐S2‐(16–39))
Mono
Mono
Di
Mono
Mono
Mono
Mono
Mono
Di
Mono
Mono
Mono
Mono
Mono
Mono
Tetra
Tri
Mono
Penta
Tetra
Di
Mono
Tri
Tetra
Tetra
Tetra
Penta
Tetra
Tetra
‐
‐
‐
+
+
+
+
‐
+
+
+
+
+
‐
+
+
+
+
+
+
+
+
+
‐
+
+
+
+
+
‐
‐
‐
+
+
+
+
+
+
+
+
+
+
‐
+
+
‐
+
‐
‐
+
‐
+
‐
+
+
+
+
+
‐
‐
‐
+
+
+
+
+
+
+
+
+
+
‐
+
+
‐
+
‐
‐
+
+
‐
+
+
+
‐
+
+
‐
‐
‐
+
+
+
+
‐
+
+
+
+
+
‐
+
+
‐
+
+
‐
‐
+
+
‐
+
+
+
+
+
‐
‐
‐
+
+
+
+
+
+
+
+
+
+
‐
+
+
‐
+
+
‐
+
‐
+
‐
+
+
+
+
+
‐
‐
‐
+
+
+
+
+
+
+
+
+
+
‐
+
‐
‐
‐
‐
‐
‐
+
‐
‐
‐
‐
‐
‐
‐
‐
‐
‐
+
+
+
+
‐
+
+
+
+
‐
‐
‐
‐
‐
+
+
‐
‐
‐
‐
‐
+
+
‐
+
+
‐
‐
‐
+
+
+
+
‐
+
+
+
+
‐
‐
+
‐
+
+
+
‐
+
‐
+
‐
+
‐
+
+
+
Recovery of Phosphopeptides
23 20 19 20 21 12 14 18
25.01.2017
15
PP
P
PP
3.6% HCl
P P
P P
P
trypsin
Tryptic On‐Pellet Digest of Precipitated Phosphoproteins
Inte
nsity
m/z29
Workflow
Development of New Bioanalytical Tools and Methods for the Enrichment of Phosphorylated Peptides and Proteins
Güzel, Y.; Rainer, M. (); Mirza, M.R.; Messner, C.B.; Bonn, G.K. Highly Selective Recovery of Phosphopeptides using Trypsin‐Assisted Digestion of Precipitated Lanthanide‐Phosphoprotein Complexes. Analyst (2013) 138(10), 2897‐2905.
LaPO4
PP
P
PP
washing
LaPO4
Overview of recovered phosphopeptides from a proteinmixture (lysozyme, cytochrome c, myoglobin, bovine serum albumin, a‐ and b‐casein) and bovine milk
proteinmixture milk 1:100 dilution
[M+H]+ Position Protein Phosphopeptide sequences Phospho groups LaCl3 CeCl3 TiO2 LaCl3 CeCl3 TiO2 LaCl3 CeCl31466.6 153‐164 α‐S2 TVDMESpTEVFTK mono + + ‐ + + + + +
1482.6 153‐164 α‐S2 TVDM*ESpTEVFTK mono + + ‐ ‐ + ‐ + +
1594.7 153‐165 α‐S2 TVDMESpTEVFTKK mono + + + + + + + +
1610.7 153‐165 α‐S2 TVDM*ESpTEVFTKK mono + + ‐ ‐ + ‐ + +
1660.7 121‐134 α‐S1 VPQLEIVPNSAEER mono + + + + + + + +
1832.8 104‐119 α‐S1 YLGEYLIVPNSpAEER mono + + + + + + + +
1847.6 58‐73 α‐S1 DIGSESpTEDQAMEDIK mono + + ‐ ‐ ‐ ‐ ‐ ‐
1927.6 58‐73 α‐S1 DIGSpESpTEDQAMEDIK di + + ‐ + + + + +
1943.6 58‐73 α‐S1 DIGSpESpTEDQAM*EDIK di + + + + + + + +
1951.9 119‐134 α‐S1 YKVPQLEIVPNSpAEER mono + + + + + + + +
1981.8 48‐63 β FQSpEEQQQTEDELQDK mono + + + + ‐ + + ‐
2000.0 118‐134 α‐S1 KYKVPQLEIVPNSpAEER mono + + ‐ + + ‐ ‐ +
2061.8 48‐63 β FQSpEEQQQTEDELQDK mono + + + + + + ‐ ‐
2080.0 118‐134 α‐S1 KYKVPQLEIVPNSpAEER mono + + + + + + + +
2432.0 45‐63 β IEKFQSpEEQQQTEDELQDK mono + + ‐ + + ‐ ‐ ‐
2548.2 119‐139 α‐S1 YKVPQLEIVPNSpAEERLHSMK mono + + ‐ + + ‐ + +
2560.1 44‐63 β KIEKFQSpEEQQQTEDELQDK mono + + ‐ + + ‐ ‐ ‐
2564.6 119‐139 α‐S1 YKVPQLEIVPNSpAEERLHSM*K mono + + ‐ + + ‐ + +
2598.0 52‐73 α‐S1 VNELSpKDIGSpESpTEDQAMEDIK di + + ‐ + + ‐ + +
2678.0 52‐73 α‐S1 VNELSpKDIGSpESpTEDQAMEDIK tri + + ‐ + ‐ + + +
2703.5 114‐135 α‐S1 LRLKKYKVPQLEIVPNSpAEERL mono + + ‐ + + + ‐ ‐
2716.2 130‐152 α‐S2 NAVPITPTLNREQLSpTSpEENSKK di + + ‐ + + ‐ + +
2720.9 74‐94 α‐S1 QMEAESpISpSpSpEEIVPNSpVEQK penta + + ‐ + + ‐ ‐ ‐
2747.1 17‐37 α‐S2 NTMEHVSpSpSpEESpIISQETYKQ tetra + + + + + + ‐ ‐
2856.5 50‐73 α‐S1 EKVNELSpKDIGSESTEDQAMEDIK di + + ‐ + + ‐ + +
2935.1 50‐73 α‐S1 EKVNELSpKDIGSpESTEDQAMEDIK tri + + ‐ + ‐ ‐ + +
2966.1 17‐40 β ELEELNVPGEIVESpLSpSpSpEESITR tetra + + + + + ‐ ‐ ‐
3008.0 61‐85 α‐S2 NANEEEYSIGSpSpSpEESpAEVATEEVK tetra + + + + + + + +
3024.2 16‐40 β RELEELNVPGEIVESLSpSpSpEESITR tri + + + + + ‐ ‐ ‐
3042.2 16‐40 β RELEELNVPGEIVESLSpSpSpEESITR tri + + + + + ‐ ‐ ‐
3087.9 61‐87 α‐S2 NANEEEYSpIGSpSpSpEESpAEVATEEVK penta + + ‐ + + ‐ ‐ ‐
3122.2 16‐40 β RELEELNVPGEIVESpLSpSpSpEESITR tetra + + + + + + + +
3132.2 16‐39 α‐S2 KNTMEHVSpSpSpEESpIISQETYKQEK tetra + + ‐ + + + + +
34 15 31 16 22
25.01.2017
16
Dephosphorylated HeLa cell lysate (1 mg/mL) with spiked α‐ and ß‐casein (5 μg/mL) after enzymatic on‐pellet digestion using trivalent cerium cations. α1, α2 and β correspond to the tryptic phosphopeptides deriving from αS1‐, αS2, and β‐casein, respectively
HeLa cell lysate ‐ digest
spiked cell lysate after precipitation
Development of New Bioanalytical Tools and Methods for the Enrichment of Phosphorylated Peptides and Proteins
On‐pellet digest of precipitated α‐/ß‐casein from spiked cell lysates
crude saliva digest
precipitated fractionCeCl3
Rainer, M. (), Güzel, Y., Messner, C., & Günther Bonn (2014). Co‐Precipitation of Phosphorylated Proteins Using Trivalent Cerium‐, Holmium‐, and Thulium Cations. Current Pharmaceutical Analysis, 10(3), 175‐184.
Development of New Bioanalytical Tools and Methods for the Enrichment of Phosphorylated Peptides and Proteins
On‐pellet digest of precipitated proteins from Human Saliva
25.01.2017
17
33
Conclusion
simple and fast method
highly selective for phosphorylated peptides and proteins
enables top‐down and bottum‐up phosphoproteomics
no stationary phase or resin required (reduced unspecific binding)
trypsin was observed to be not affected by the lanthanide ions
the amount of precipitant can be adjusted to each application
MS and LC‐MS compatible
allows automation using liquid handling robotics
Highly efficient precipitation of phosphorylated peptides and proteins using trivalent lanthanide ions
ADSIAnalytische AbteilungVO Moderne Trennmethoden
Innsbruck, 25.01.2017
25.01.2017
18
• Medikamentenentwicklung
• Kosmetik
• Nutrition
Themenschwerpunkte
Phytokosmetik
(Phyto)-NutritionPflanzl.Lebensmittel-
zusatzstoffe
PhytoanalytikPhytopharmazie
Institut für Analytische Chemie
und Radiochemie
allows to see the Nature
Olive‐Net
Themenschwerpunkte „Phytovalley“ Tirol
25.01.2017
19
Medikamentenentwicklung
Ziel: Therapeutisch wirksames Pflanzenextrakt
1. Herstellung und Qualitätskontrolle von Pflanzenextrakten
2. In vitro Tests: ‐ Löslichkeitsexperimente
‐ Bioverfügbarkeit
‐ Toxizität
‐ Immunreaktionen
‐ Hepatische Metabolisierung
3. Charakterisierung der Inhaltsstoffe
4. Tiermodellversuche ausgewählter Kandidaten
Solubility Tests (WP 1)
• solubility in DMSO
• comparison of 100 mg/ml, 50 mg/ml, 20 mg/ml, 10 mg/ml 2 mg/ml
• determination by means of UHPLC-UV and visually
25.01.2017
20
MedikamentenentwicklungEndotoxinquantifizierung
• an integral part of gram-negative bacteria cell wall
• thermally stable (autoclaving is insufficient)*
• activate many transcription factors and septic shock mediators=> may lead to wrong results in cell culture experiments
• factors B and C in the LAL enzyme mix react with endotoxins, triggering a protease activity
• matching peptide releases p-nitroaniline upon cleavage
• direct quantification via UV/VIS absorbance measurement
Quantification via a LAL chromogenic assay
*Bang FB Biol Bull 1953 105 361**Amanda44 Wikipedia CC v3.0 license 2011 July 31st
Limulus Amoebocyte Lysate
**
MedikamentenentwicklungEndotoxinquantifizierung
• two-stage analysis (pre-testing and exact quantification)
• quantification limit of 0.5 EU/mg extract (10 EU/mg uncritical)
• false-positives due to β-1,3-glucans are eliminated by means of glucan blocker
• absorbance vs. concentration to eliminate quenching
Key facts of ADSI‘s workflow
25.01.2017
21
MedikamentenentwicklungAssoziierte Partner
MedikamentenentwicklungBioverfügbarkeit
In vitro Gastrointestinal‐Permeabilität
• PAMPAPermeabilitätsassay basierend auf einer Lipid Doppelschicht
Durchführung und LCMS Analytik am ADSI
• CaCo2Permeabilitätsassay auf Basis von colorektalen Darmepithelzellen
Durchführung extern, LCMS Analytik am ADSI
25.01.2017
22
MedikamentenentwicklungBioverfügbarkeit
• sophisticated tri-layer of phospholipids and hexadecane*
• isothermal experiment (25 °C)
• two scenarios: - proton gradient (pH4.5 → pH7.4)- pH-eqilibrated (pH7.4 → pH7.4)
• three replicates per time step, three observations (2h, 4h, 6h)
• two internal standards*: - phenazone (MW 189 g/mol)- sulfasalazine (MW 398 g/mol)
Optimization for plant extracts
*Chen X et al. Pharm Res 2009 25 1511
MedikamentenentwicklungParallel Artificial Membrane Permebility Assay
25.01.2017
23
MedikamentenentwicklungParallel Artificial Membrane Permebility Assay
Vasicine Pe ≈ 2∙10‐6 cm/s (HI) (pH7.4)
Pe ≈ 2∙10‐7 cm/s (LO) (pH4.5)
MedikamentenentwicklungParallel Artificial Membrane Permebility Assay
sum formula, ID, m/z, r.t., Pe, SD, RSD, graphical rating
25.01.2017
24
Datenintegration und Bioinformatik
• Toxizität und Metabolismus der Wirkstoffe
• Identifizierung der bioaktiven Substanzen
• Information über Wirkungsmechanismus
Biologische DatenAnalytische Daten
+
Datenintegration‐ und Bioinformatik‐Plattform
• Quantifizierung der bioaktiven Substanzen
NutritionBeispiel Olivenöl
Ziel: Aussage über Qualität; EFSA Health‐Claim
1. Extraktion phenolischer Komponenten
2. Saure Hydrolyse
3. Quantifizierung von Tyrosol und Hydroxytyrosol
4. Bestimmung der Routine‐Qualitätsparameter
‐Säurezahl
‐Peroxidzahl
‐Iodzahl
ADSI, InnsbruckUniversity of Athens
Greece
Institute of ChemicalMethodologies
Rome
25.01.2017
25
NutritionBeispiel Olivenöl
Health claim: Commission Regulation No. 432/2012
Commission Regulation No. 432/2012 of 16th of May, 2012
Correlation between Olive‐Polyphenols and Protection of the LDL‐Cholesterols forOxidation (Oxidized LDL is strong atherogenic)
NutritionWorkflow – determination of phenolic compounds in EVOO
Native Olive Oil Extra
Direct Quantification ofHydroxytyrosol und Tyrosol
Acid Hydrolysis (1M H2SO4)
Hydroxytyrosol Tyrosol
Quantification of Hydroxytyrosol andTyrosol as Parameter of all Derivates
(EFSA: ≥5mg/20g)
Extraction with 60% MeOH/Water
EFSA Claim: Polyphenoles out from Olive Oil protect LDL‐Cholesterolfrom oxidative Stress (min. 5mg Polyphenoles per 20 g
Olive Oil)
25.01.2017
26
NutritionAcid hydrolysis
Acid hydrolysis of phenolic compounds in EVOO
Lecture Prof. Skaltsounis, Innsbruck 2014
Acid hydrolysis with 1 M H2SO4
HPLC‐UV analysis of a oleuropein standard (0.3 mg/ml) before and after acid hydrolysis
HPLC‐UV chromatograms of a oleuropein standard before and after acid hydrolysis at 280nm
0
5
10
15
20
0
5
10
15
20
0 2 4 6 8 10 12 Time [min]
Absorbance [mAu]
Absorbance [mAu]
Oleuropein
Hydroxytyrosol
Before hydrolysis
After hydrolysis
NutritionAcid hydrolysis of phenolic compounds in EVOO
25.01.2017
27
Horizon 2020Bioactive Compounds from Olea Europaea: Investigation and
Application in Food, Cosmetic and Pharmaceutical Industry
Olive‐Net
Call: H2020‐MSCA‐RISE‐2016(Marie Sklodowska‐Curie Research and Innovation Staff Exchange)
Topic: MSCA‐RISE‐2016
Proposal number: 734899
Proposal acronym: Olive‐Net
Key words: olea europaea, olive mill waste, olive oil, extraction, analysis, profiling, cosmetics, inflammation, in vivo, ageing, supplements, nutraceuticals, oleacein, oleocanthal, obesity,
EU‐Evaluation Summary Report ‐ Results (0‐5)Excellence: 4.5, Impact: 4.6, Quality and Efficency of the Implementation: 4.7
Optimal capacity: yesFounded by the European Commision: September 2016
25.01.2017
28
List of Participants
UNIVERSITE D'AVIGNON ET DES PAYS DE VAUCLUSE France
UNIVERSIDAD COMPLUTENSE DE MADRID Spain
CONSIGLIO NAZIONALE DELLE RICERCHE Italy
ADSI‐Austrian Drug Screening Institute GmbH Austria
PHARMAGNOSE VIOTECHNOLOGIKI ANONYMI ETAIREIA Greece
UNI‐PHARMA KLEON TSETIS PHARMACEUTICAL LABORATORIES S.A. Greece
SERVIZO GALEGO DE SAUDE Spain
NATAC BIOTECH SL Spain
ALNuMed GmbH Germany
NEURO‐SYS SAS FranceECOLE NATIONALE D'AGRICULTURE DE MEKNES Morocco
INSTITUT NATIONAL DE LA RECHERCHE AGRONOMIQUE D ALGERIE Algeria
Rangsit University Thailand
ETHNIKO KAI KAPODISTRIAKO PANEPISTIMIO ATHINON Greece
CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE France
Natural ingredients and extracts discovery, technical extraction and purification technologies, analytical standards
Biomedical Research
Development of Drug Delivery Systems, API Synthesis, Clinical Studies, Drug Repositioning, Translational Research Novel routes of administration
Drug discovery, neurodegeneration
Development of functional foods and active ingredients from natural substances
Development of novel analytical methods, NMR and evaluation tools for rapid authenticity and quality testing
ICOA; Analytical strategies, molecular interactions and bioactivity tests
Laboratory for green extraction techniques of natural products
Physiology
Analysis, HPLC, CE‐technologysis
Extractions, Analytical methods, HPLC‐MS, MALDI, Screening technologies, Cell systems
Pharmacognosy and Natural Products Chemistry, Plant data base
Agrifood technology
ECOLE NATIONALE D'AGRICULTURE DE MEKNES
Oriental Medicine applications
Medicine and ApplicationsAgriculture
Extraction, Purification, Analysis
Expertise der Projektpartner im Olive‐Net EU‐Projekt
25.01.2017
29
Olive‐Net Roadmap
Olivenöl Abfälle
Olivenkultivierung Olivenölgewinnung
25.01.2017
30
Abfälle
Erzeugung von Wertstoffen aus Olivenöl‐Produktionsabfällen
Tocopherole Aldehyde
Polyphenole Flavonoide
Cyclische Mono‐ und Sesquiterpen‐kohlenwasserstoffe
Wertstoffe für Phytokosmetik‐, Pharma‐, Nahrungsmittel‐, Nahrungsergänzungsmittel
Abfälle
Fester Presskuchen, Waschwasser und einsehr dünnflüssiger Schlamm sind umwelt‐belastende Abfälle bei der Olivenölproduktion
Wertstoffe aus dem Abfall
Mehr als 2 Mio t/a Olivenöl werden in Europa produziert
Im Mittelmeerraum wird die Umwelt dadurchjedes Jahr mit über 30 Millionen TonnenPressrückstände, Waschwasser und Oliven‐blätter belastet.
Dort werden die flüssigen Reststoffe häufig inLagunen und Seen geleitet wodurch dasGrundwasser verunreinigt wird. Die Gewässerverfärben sich schwarz, sterben ab undstinken, da Olivenabfälle auch toxischeVerbindungen enthalten.
Top Related