optimized extraction of microalgae’s metabolites: a … r., lanneluc i., thiery v., lafferriere...
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Optimized extraction of Optimized extraction of microalgae’smicroalgae’s metabolites: a crucial step in metabolites: a crucial step in HighHigh--Throughput Screening programs dedicated to Throughput Screening programs dedicated to
phytoplankton phytoplankton chemodiversitychemodiversity
Track 12-1 Marine natural products and biomolecules
Serive B., Kaas R., La Barre S., Bach S., Cadoret J-P.
2- Laboratory of Physiology and Biotechnology of Algae
Bioresources exploration
Van de Peer et al, 2000
Microalgaes: a high potential of biodiversity
- Laboratory of Physiology and Biotechnology of Algae
Microalgae biodiversity
Dunaliella salinaSource : IFREMER
- Laboratory of Physiology and Biotechnology of Algae
Rhodomonas salinaSource : IFREMER
Microalgae biodiversity
- Laboratory of Physiology and Biotechnology of Algae
Tetraselmis sp.Source : IFREMER
Microalgae biodiversity
- Laboratory of Physiology and Biotechnology of Algae
Closterium baillyanumSource : IFREMER
Microalgae biodiversity
- Laboratory of Physiology and Biotechnology of Algae
Euglena proximaSource : IFREMER
Microalgae biodiversity
- Laboratory of Physiology and Biotechnology of Algae
Porphyridium purpureumSource : IFREMER
Microalgae biodiversity
- Laboratory of Physiology and Biotechnology of Algae
Unknown cyanobacteriaSource : IFREMER
Microalgae biodiversity
- Laboratory of Physiology and Biotechnology of Algae
Arthrospira platensisSource : IFREMER
Microalgae biodiversity
- Laboratory of Physiology and Biotechnology of Algae
Staurastrum vestitum
Microalgae biodiversity
- Laboratory of Physiology and Biotechnology of Algae
Chaetoceros muelleriSource : gt-online.pl
Microalgae biodiversity
- Laboratory of Physiology and Biotechnology of Algae
Skeletonema costatumSource : IFREMER
Microalgae biodiversity
- Laboratory of Physiology and Biotechnology of Algae
Asterionella formosaSource : planktonforum.eu
Microalgae biodiversity
- Laboratory of Physiology and Biotechnology of Algae
Alexandrium minutumSource : Gert Hansen
Microalgae biodiversity
- Laboratory of Physiology and Biotechnology of Algae
Microalgae biodiversity
17- Laboratory of Physiology and Biotechnology of Algae
O
CH3
O
N
O
O
CH3
O
CH3
CH3
CH3
CH3CH3 HO H
O
=
Pharmacodiversity
Chemodiversity
Biodiversity
=
12 phyla eucaryota 1 phylum procaryota
Polyketides, terpenes, alkaloides, isoprenoids, oxylipins, peptides, macrolides, microlides,
lectines, glycolipids, polysaccharides, PUFAs, mycosporine-like AA, pigments
Potential against cancers, Parkinson, Alzheimer, neurodegenerative diseases, bacterial and viral
diseases, as immunostimulant, as fluorescent probes
18- Laboratory of Physiology and Biotechnology of Algae
Lutein
Zeaxanthin
Landrum & Bone, 2001
Microalgae chemodiversity and pharmacodiversity
Anti Age-related Macular Degeneration
19- Laboratory of Physiology and Biotechnology of Algae
Violaxanthine
Pasquet V., Morisset P., Ihammouine S., Chepied A., Aumailley L., Berard J-B., Serive B., Kaas R., Lanneluc I., Thiery V., Lafferriere M., Piot J-M., Patrice T., Cadoret J-P., Picot L., 2011.
Antiproliferative activity of Violaxanthin isolated from bioguided fractionation of Dunaliella tertiolecta extracts. Marine Drugs, 9, 819-831.
Moreau et al, 2006 FucoxanthineO. aurita
D. tertiolecta
Microalgae chemodiversity and pharmacodiversity
20- Laboratory of Physiology and Biotechnology of Algae
Mimouni et al, 2012. The Potential of Microalgae for the Production of Bioactive Molecules of Pharmaceutical Interest. Current Pharmaceutical Biotechnology 13(15): 2733-2750.
Microalgae chemodiversity and pharmacodiversity
PhotoprotectantPhotoprotectant
AntiproliferativeAntiproliferativeactivityactivity
Usijirene Palythene Asterina-330 Mycosporine-glycine
Palythine Palythenic acid Porphyra-334 Shinorine
AntioxidantAntioxidant
21- Laboratory of Physiology and Biotechnology of Algae
For high-throughput screening of such metabolites, it is essential to reach the inner content of the cell.
Aims
A part of our project was the research and development of a technique enabling a high extraction yield of any metabolite, taking into account the difficulty of extracting bound and or inaccessible molecules with a wide variety of polarities.
The challenge is therefore to identify molecules issued from this biodiversity which are often poorly known in terms of chemodiversity.
The final aim is to compare without extraction bias the potential of microalgae.
22- Laboratory of Physiology and Biotechnology of Algae
Choice of recalcitrant biological modelsFor the study of the chemodiversity extraction
Porphyridium purpureum Phaeodactylum tricornutum
Length : 5-8 µm
Phylum : Rhodophyta
Features :
- Exopolysaccharides
- Thick wall
Length : 3-4 x 15-20 µm
Phylum : Ochrophyta
Features :
- Silica wall
15 µm
23- Laboratory of Physiology and Biotechnology of Algae
Method Effectiveness Reproductibility Other considerations
Soaking - - Long processing
Cryogrinding - - Liquid nitrogen handling require
Potter homogeniser - - n.r.
Homogeniser + - Blade blunted by diatoms
Bead-beater + - Particles release
Planetary micro mill +++ +++ Particles release
Sonication ++ + ROS release, hot spots
Mixer mill – stainless steel jars + + n.r.
Mixer mill – plastic grinding tube +++ +++ n.r.
→
→→→
→→→
Chemodiversity extraction
→
Main studied parameters:
Effectiveness (1-(recalcitrant cells/total))
Reproducibility
Operating cost
Temperature stability
Easy to handling
Comparison of 9 disruption techniques
24- Laboratory of Physiology and Biotechnology of Algae
Grinding optimisation on Retsch MM-400
Time (min)
Wei
ght (
g)
0 5 10 15 20 25 30 350.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
Wei
ght (
g)
Time (min)0 5 10 15 20 25 30 35
0.2
0.3
0.4
0.5
0.6
0.7
0.8
A
B
+
+Size classes:( ) Fragments; ( ) Whole cells ; ( ) Agregates.
Optimisation of 3 studied variables by image analysis: creation of an index of desirability.
P. p
urpu
reum
P. t
ricor
nutu
m Optimum
Grinding time : 27 min
Beads weight : 0.92 g
Optimum
Grinding time : 30 min
Beads weight : 0.24 g
Control Optimized grinding
50 µm
50 µm
50 µm
50 µm
P. P
urpu
reum
P. t
ricor
nutu
m
Control Optimized grinding
Isoresponses overlay
Image analysis to evaluate acurately the particle size distribution
Serive et al, 2012. Bioresource Technology 124
25- Laboratory of Physiology and Biotechnology of Algae
→ In a total extract, we have different kind of molecules:
Known by the scientific community and known by us
Known somewhere in the world but unknown by our database
Unknown by everyone
→ Isoplot absorbance metabolic snapshot because of the microalgae cellular plasticity
Total extract, chemodiversity and dereplication
SK(1,2,3) SK-R(1,2,3) L(1,2,3,4)
6
7
8
9
10
11
12
13
Sco
re R
OS
Isoplot absorbance
Time (min)
Wav
elen
ght(
nm)
Bioactivity for a samestrain and different
environmental conditions
26- Laboratory of Physiology and Biotechnology of Algae
Methodology in the work flow
Met
abol
icsn
apsh
ot
Total extract
Microalgaechemodiversity
Efficient metabolite extraction OMICs studies
■ Genomic
■ Transcriptomic
■ Proteomic
■ Metabolomic
■ Pigmentomic
Serive et al, 2012. Bioresource Technology 124
27- Laboratory of Physiology and Biotechnology of Algae
Photomer project
1 2
3 4
Identify new photosensitizers frommicroalgae for PhotoDynamic Therapy
28- Laboratory of Physiology and Biotechnology of Algae
45 microalgae strains composition
→ Spectral screening of interest in pharmacognosy.
→ High interest for oceanography applications.
Pigment analysis and spectral screening
Examples of original spectra from the following strains:
A- Alexandrium minutum B- Alexandrium tamarense
C- Chlorarachnion reptans D- Phaeodactylum tricornutum
A B
C D
29- Laboratory of Physiology and Biotechnology of Algae
OCEAN CHemodiversity Against Cell cycle Targets
Research of new marine inhibitors targeted againstdesease-relevant proteins kinases
30- Laboratory of Physiology and Biotechnology of Algae
Bruker SolariX 12.0 Tesla FTMS
C
A
B
J-B Charcot
31- Laboratory of Physiology and Biotechnology of Algae
Thank you for your attention
Contact: [email protected]