Optimized extraction of microalgaes microalgaes metabolites: a - - PowerPoint PPT Presentation

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Optimized extraction of microalgaes microalgaes metabolites: a - - PowerPoint PPT Presentation

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Optimized extraction of microalgaes microalgaes metabolites: a crucial step in metabolites: a crucial step in Optimized extraction of High- High -Throughput Screening programs dedicated to Throughput Screening programs dedicated to

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Optimized extraction of Optimized extraction of microalgae’s microalgae’s metabolites: a crucial step in metabolites: a crucial step in High High-

  • Throughput Screening programs dedicated to

Throughput Screening programs dedicated to phytoplankton phytoplankton chemodiversity chemodiversity

Track 12-1 Marine natural products and biomolecules

Serive B., Kaas R., La Barre S., Bach S., Cadoret J-P.

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Bioresources exploration

Van de Peer et al, 2000

Microalgaes: a high potential of biodiversity

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Microalgae biodiversity

Dunaliella salina

Source : IFREMER

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Rhodomonas salina

Source : IFREMER

Microalgae biodiversity

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Tetraselmis sp.

Source : IFREMER

Microalgae biodiversity

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Closterium baillyanum

Source : IFREMER

Microalgae biodiversity

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Euglena proxima

Source : IFREMER

Microalgae biodiversity

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Porphyridium purpureum

Source : IFREMER

Microalgae biodiversity

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Unknown cyanobacteria

Source : IFREMER

Microalgae biodiversity

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Arthrospira platensis

Source : IFREMER

Microalgae biodiversity

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Staurastrum vestitum

Microalgae biodiversity

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Chaetoceros muelleri

Source : gt-online.pl

Microalgae biodiversity

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Skeletonema costatum

Source : IFREMER

Microalgae biodiversity

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Asterionella formosa

Source : planktonforum.eu

Microalgae biodiversity

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Alexandrium minutum

Source : Gert Hansen

Microalgae biodiversity

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Microalgae biodiversity

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= Pharmacodiversity Chemodiversity Biodiversity =

12 phyla eucaryota 1 phylum procaryota Polyketides, terpenes, alkaloides, isoprenoids,

  • xylipins, 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

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Lutein Zeaxanthin Landrum & Bone, 2001

Microalgae chemodiversity and pharmacodiversity

Anti Age-related Macular Degeneration

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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 Fucoxanthine

  • O. aurita
  • D. tertiolecta

Microalgae chemodiversity and pharmacodiversity

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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

Photoprotectant Photoprotectant Antiproliferative Antiproliferative activity activity

Usijirene Palythene Asterina-330 Mycosporine-glycine Palythine Palythenic acid Porphyra-334 Shinorine

Antioxidant Antioxidant

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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.

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Choice of recalcitrant biological models

For 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

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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

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Grinding optimisation on Retsch MM-400

Time (min) Weight (g) 5 10 15 20 25 30 35 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 Weight (g) Time (min) 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. purpureum P. tricornutum

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. Purpureum P. tricornutum

Control Optimized grinding

Isoresponses overlay Image analysis to evaluate acurately the particle size distribution Serive et al, 2012. Bioresource Technology 124

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→ 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 Score ROS

Isoplot absorbance

Time (min) Wavelenght (nm) Bioactivity for a same strain and different environmental conditions

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Methodology in the work flow

Metabolic snapshot Total extract Microalgae chemodiversity Efficient metabolite extraction OMICs studies ■ Genomic ■ Transcriptomic ■ Proteomic ■ Metabolomic ■ Pigmentomic Serive et al, 2012. Bioresource Technology 124

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Photomer project 1 2 3 4

Identify new photosensitizers from microalgae for PhotoDynamic Therapy

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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

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OCEAN CHemodiversity Against Cell cycle Targets

Research of new marine inhibitors targeted against desease-relevant proteins kinases

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Bruker SolariX 12.0 Tesla FTMS

C A B

J-B Charcot

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Thank you for your attention

Contact: Benoit.Serive@live.fr