proceedings of the symposium "algae: the blue revolution for a sustainable chemistry"...

Olmix SymposiumSeptember, 10th 2012

FRANCE - Palais des Congrès of Pontivy

How Algae can bring solutions to GlobalNutrition & Health issues

«Algae: The Blue Revolution

for a Sustainable Chemistry»

This event is organized by Olmix with the support of its partners:

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Dear partner,

OLMIX, pioneer in algae use for animal nutrition since 1995 has the pleasure to invite you to attend the 1st olmix algae symposium dedicated to “global nutrition and health issues”.

Using algae extract for their polysaccharides and trace-elements materials became com-mon for Olmix in the beginning of the second millennium.

It all started when OLMIX‘s R&D team had the feeling that they could increase the binding potential of Clay by including inside algae polysaccharides. The idea was to increase the interlayer space of natural montmorillonite clay thanks to the ulvans, green algae poly-saccharides. A full program was then developed with CNRS (National Center of Scientific Research) and CEVA (Center for study and valorization of algae) and gave birth to Ama-deite®, the algae based revolutionary and worldwide patented hybrid material. The first great commercial success using Algae in animal feeding was born: MTX+.

These successes encouraged OLMIX to increase its use of Algae for animal and vege-tal health and nutrition. Today ULVANS, a new R&D program, is aimed to provide even more Algae based solutions to the field with more technology inside, using enzymatic hydrolysis and separation techniques from harvest to the final product.

How Can Olmix innovate so much in Algae use? Its location, based right in the middle of a region of the world where most of the Algae scientific knowledge, supply and diversity is concentrated: BRITTANY.

To share this Algae Blue revolution for a sustainable Chemistry with its partners OLMIX organize on September 10th the 1st OLMIX ALGAE SYMPOSIUM “How algae can bring solutions to global nutrition and health issues”. Speakers from the

most recognized specialized institutions will share with us the latest knowledge on Algae science and yet more…

On behalf of Hervé Balusson, OLMIX CEO, we are looking forward to welcoming you soon in Brittany.

Olmix team

1st Olmix Algae SymposiumPalais des Congrès of Pontivy (France)September, 10th 2012

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Morning sessions under the Chairmanship of Catherine Boyen Director of the “Marine plants and biomolecules” laboratory - Roscoff Biological Station- CNRS UMR 7139 - Steering committee of Biogenouest®

8.30 Welcome reception

9.00 Welcome speechHervé Balusson Olmix Group President and CEO

Session 1 Algae: A new world to discover

9.10

Introduction - Brittany, historic region in algae valorisation• History of algae situation in the region• Sea World professional organizations and institutes - Current actionsDr. Christine Bodeau-Bellion Science et Mer Laboratory - President of the Syndicate of Seaweed and Marine Plants - Le Relecq Kerhuon

9.30Brittany an area of excellence in algae knowledge• Research organizations in Brittany - Their expertise• Present and future research programsPr. Eric Deslandes University of Western Brittany - Brest

9.50Discovering the Blue Chemistry• General information on algae, their origin and biological characteristicsDr. Philippe Potin Research Director - Roscoff Biological Station - CNRS UMR 7139 - IDEALG project coordinator

Algae: A sugared treasureDr. Mirjaml Czjzek Research Director - Roscoff Biological Station - CNRS UMR 1931

11.00 Coffee break

Session 2 Algae in the service of Health

11.30Enzymatic hydrolysis in chemistry of seaweedsPr. Nathalie Bourgougnon LBCM (Biotechnology and Marine Chemistry Laboratory) - University of Southern Brittany - Vannes

PROGRAM

«Algae: The Blue Revolution for a Sustainable Chemistry»

10.20

How Algae can bring solutions to Global Nutrition & Health issues

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1st Olmix Algae SymposiumPalais des Congrès of Pontivy (France)

September, 10th 2012

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12.00Bioactivities of Marine Polysaccharides in human and animal health (Update)Dr. Henri Salmon Research Director - INRA Tours - Nouzilly (French National Institute for Agricultural Research)

12.30Marine lipids in amplifying cancers chemotherapyPr. Philippe Bougnoux Oncologist, Director of the Unit «Nutrition, growth and cancer» - INSERM(National Institute for Health and Medical Research) - Chief of cancerology service - CHU Tours(University Hospital Center)

13.00 Lunch (marine buffet)

Afternoon sessions under the Chairmanship of Dr. Christine Bodeau-BellionScience et Mer Laboratory - President of the French Syndicate of Seaweed and Marine Plants - Le Relecq Kerhuon

Session 3 Industrial applications originating from algae chemistry

14.15The algae industry in ChileEliana Henriquez Flores Agronomist - Head of the International Affairs Unit - CIREN(Centre for Renewable Natural Resources Information) - Santiago, CHILE

14.30 Algae, source of active principles in cosmeticsAlexis Rannou Deputy Managing Director in charge of Innovation - ARD Soliance - Pommacle

14.50Algae, source of nutriments for humansDr. Maria Hayes Scientific Project Manager - NutraMara - Teagasc Ashtown Food ResearchCentre - IRELANDChristine Le Tennier Algues de Bretagne - Globe Export SARL - Rosporden

15.25 Algae in the service of soils nutritionDr. Bruno Daridon Research and Development Director - PRP Technologies - Paris

16.00 Coffee break

Session 4 Industrial applications originating from algae chemistry (follow)

16.15 Algae in the service of terrestrial plants healthDr. Adeline Picot Plants Pathology Laboratory - VEGENOV BBV - St Pol de Léon

Algae, source of nutrients in animal nutritionPr. Simon Davies Professor of Aquaculture Nutrition at the University of Plymouth (UK).Member of World Aquaculture Society

17.10 Round table: From research to industrial application.

18.30 Cocktail: Marine algae in the castle20.00 Gala dinner and evening festivities - Pontivy castle - Palais des Congrès

16.40

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GUEST SPEAKERSIntroduction and presentation

Mr Hervé BalussonOlmix Group President and CEO

Hervé Balusson is the founder and Chairman of Olmix Group, specialized in the trace-elements feed additives and organic fertilizer. He has succee-ded in bringing Olmix from a regional stage into international coverage.Olmix products are now available in more than 50 countries.

Ms Catherine BoyenDirector of the “Marine plants and biomolecules” laboratory - Roscoff Biological Station- CNRS UMR 7139 - Steering committee of Biogenouest®.

Dr. Christine Bodeau-Bellion Science et Mer Laboratory - President of the Syndicate of Seaweed and Marine Plants - Le Relecq Kerhuon

Pr. Eric DeslandesUniversity of Western Brittany - Brest

Dr. Philippe PotinResearch Director - Roscoff Biological Station - CNRS UMR 7139 - IDEALG pro-ject coordinator

Philippe Potin (49), Docteur en biologie, HDR, Directeur de Recherche 2ème classe au CNRS depuis oct. 2006 (SBR, UMR 7139 CNRS-UPMC-Paris6)Dr. Philippe Potin, marine biologist and biochemist has obtained his Ph.D. from the University of Brest in 1992 and continued his post-doctoral research at the NRC Institute for Marine Biosciences in Halifax (NS Canada) and was hired by CNRS in Roscoff. P. Potin’s scientific interests (>70 primary publications) are in the bases of pathogen defense reactions and signaling in marine algae, with an emphasis of the specific traits of marine plants such as the halide metabolism. Research in his team investigates fundamental processes underlying interactions between seaweeds and pests. He was also interested in technology transfer with the Goëmar Laboratories, to develop the use of oligosaccharides for disease control in agricultural crops (4 patents, one product on the market) and during his mandate as a project manager for



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the marine biotechnologies at the Maritime Cluster “Pôle Mer Bretagne”. He is currently the scientific coordinator of IDEALG, a 10-year national integrative pro-ject, within the framework of the French Stimuli Program Investis- sements d’Avenir, to capitalize on the recent breakthroughs in algal genomics to develop seaweed genetics and biotechnology.Philippe POTIN, Station Biologique de Roscoff, BP 74 - 29680 Roscoff, Tel.33-2. 98.29.23.75, Fax.33-2. 98.29.23.85, Mail [email protected]

Dr. Mirjaml CzjzekResearch Director - Roscoff Biological Station - CNRS UMR 1931

Mirjam Czjzek has studied chemistry at the University of Frankfurt, then at the TH Darmstadt in Germany where she has obtained her PhD in crystallography. After one year of a post-doctoral position in the ‘Labo-ratory for crystallography of biological macromolecules’ (LCMB) of Mar-seille, she has been recruited at the CNRS in October 1992. She is crys-tallographer by education and has started to work on CAZymes solving the crystal structures of cellulases and beta-glucosidases during several years in the group of Bernard Henrissat in Marseille, France. In 2005 she moved to the Station Biologique de Roscoff, where she is now ‘directrice de recherche’ of CNRS in the laboratory for ‘Marine plants and biomolecules’. Her research program entitled ‘Marine Glycobiology’ currently focuses on the structures and functions of carbohydrate-active enzymes, including their CBMs, which are involved in marine algal cell wall polysaccharide depolymerization.

Pr. Nathalie BourgougnonLBCM (Biotechnology and Marine Chemistry Laboratory) - University of Sou-thern Brittany - Vannes

Nathalie Bourgougnon has been working in the Laboratoire deBio-technologie et Chimie Marines (LBCM) at Université de Bretagne-Sud since 2001. Previously, she was lecturer at the University de la

Rochelle during 8 years. The principal thematic of her research relates to the search for marine substances with biological activities mainly extrac-ted from algae. She has a good experience in the field of the extraction, purification, characterization and evaluation of biological (antiviral, antifouling, antiprolifera-tive…) activities of marine compounds. She has published ca. 55 papers in peer-reviewed journals and book chapters, in particular on antifouling or antiviral substances extracted

from seaweeds, extraction and purification of bioactive marine substances. She has deposit two patents about antiviral substances. She has been involved in


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several Euro- pean projects (e.g. FP4 Bioactive Marine Natural Products in the Field of Antitumo- ral, Antiviral and Immunomodulant Activity, MAST III; FP5 AVINSI- Anti Viral Infection Non Specific Immunity: Basis of non specific immunity against viral diseases in aquacultured species; FP6 Valbiomar Biotechnologique valorization of the marine resources; FP7 Biotecmar: integrated transregional project for communication, technical information and technology transfer in the domain of biotechnological exploitation of marine products and by-products) and recently in OSEO program ULVANS. She has coordinated the project ASEM-DUO from MAE between France and Malaysia (2007-2009). She is member of several networks, Two French networks: BioChiMar concerning marine substances with biological activity and SEAPro (Sustanaible Exploitation of Aquatic PRO-ducts) concerning biotechnological up-grading of fish, seaweeds or aquaculture by-pro-ducts; and an international network: RAQ Quebec Aquaculture Network. At the national level, Nathalie Bourgougnon has collaborated with Dr. JL Mouget (Université du Mans) for physiological approaches of antifouling substances extracted from seaweeds, V. Stiger (Université de Bretagne Occidentale) for marine substances extracted from red seaweeds, Dr T. Renault (IFREMER, La Tremblade) for defences mechanisms of oysters against bacteria and viruses , JP Bergé (IFREMER, Nantes) for upgrading of marine resources and at the international level, she gained experience in cooperation with Morocco (University Tétouan; Pr. H. Riadi), Institute of Marine Biotechnology of University Malaysia Terengganu (Pr. Effendy) for biological compounds extracted from seaweeds, ISMER (Québec, Dr. R. Tremblay) for biological compounds extracted from seaweeds, microalgae, invertebrates. She is Vice-president of International PhD School (Coordination of Doctoral program) of the Université européenne de Bretagne (UEB) www.ueb.eu. At University de Bretagne-Sud, she is in charge of research program and Coordinator of master «Biotechnology» (www-lbcm.univ-ubs.fr).

Dr. Henri SalmonResearch Director - INRA Tours - Nouzilly (French National Institute for Agricul-tural Research)

Dr. Henri Salmon is a Research Director in the Institute of National Agro-nomic Research (INRA) in France. He earned his DVM from the National Veterinary School-Alfort in Paris and his PhD in Immunology from the University of Paris.Prior to joining INRA, he served 6 years as a Research Assistant at Col-lege of Veterinary Medicine in Alfort. Since 1984, he has served as Director of Research, INRA, laboratory of Animal Infectiology and Public Health, Tours-Nouzilly. He served one year in Transplantation Research Biology Center, Harvard Medical School and Massasuchetts General Hospital (Boston, MA).




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The objective of his research is an understanding of the interrela- tions («immune links») between digestive, pulmonary and mammary mucosal immune responses to improve protection against pathogens. He has dissected the mechanisms under-lying the migration of IgA plasma cells from the mucosae to the mammary gland in the sow ; these mechanisms substantiate the production of IgA in colostrum and milk. and hence are responsible of passive mucosal protection of the suckling piglets. Now he is looking at the means to shorten the onset of IgA response in gut of weaned piglet. To replace the anti-biotics-growth factors in food, he designed «immunoprobiotic», as vectors to enhance the neonatal gut immunity which deliver enhacing factors of IgA immune response including pre- and probiotics.

Pr. Philippe BougnouxOncologist, Director of the Unit «Nutrition, growth and cancer» - INSERM (Natio-nal Institute for Health and Medical Research) - Chief of cancerology service - CHU Tours (University Hospital Center)

Philippe Bougnoux is a medical oncologist, specialized in breast and gynaecologic cancers. He performed his trainings in medicine in Tours and in immunology at the Pasteur Institute in Paris. After a 3 years post-doctoral staying as a Fogarty fellow at the National Cancer Institute in Bethesda, MD, he became professor of cancer biology at the university of Tours, and chief of the cancer outpatient unit at the university cancer centre Henry S. Kaplan. He belongs to the Inserm research Unit 1069 « Nutrition, growth and Cancer » and has been coordina-ting a consortium of research units in chemistry and biology on marine-derived anticancer agents within the canceropôle of the western part of France, which he heads now. His research interests are to understand how diet and lipid nutrients influence the molecu-lar alterations which result in malignant tumors and how they integrate to delay breast can-cer occurrence or individual response to anticancer agents. He does translational research

in the field of dietary lipids in relation to breast cancer prevention and treatment. He is currently carrying out randomized clinical trials of dietary intervention with omega-3 poly-unsaturated fatty acids to enhance the sensitivity of tumors to radiation or chemotherapy.

Address: INSERM U1069, Henry S. Kaplan Cancer Centre, University Hospital Breton-neau, 37044 Tours, FranceTelephone: +33 (0) 2 4747 8261Email: [email protected] site: www.n2c.univ-tours.fr


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Ms Eliana Henriquez FloresAgronomist - Head of the International Affairs Unit - CIREN (Centre for Re-newable Natural Resources Information) - Santiago, CHILE

Agronomist - Head of the International Affairs Unit - CIREN (Centre for Renewable Natural Resources Information) - Santiago, CHILEIn the period between the years 2004-2008, working as Chief of Labora-tory Sub-department and Quarantine Agricultural Station, from the Agricultural and Lives-tock Service, she has made an important management of inter-agency cooperation at the national and international level with different research institutions of great renown and reco-gnized academic prestige around the world. This has enabled that SAG, at present, has signed various «Memorandum of understanding» or Agreements of International Coope-ration with institutions of Spain, Scotland, Italy and United States. Thus, all the necessary efforts were made that will, in the near future, allow to establish agreements with England, Australia, France and New Zealand.The technological horizon for Laboratory Sub-Department and Agricultural Quarantine Station was expanded through the emphasis on inter-agency, both national and foreign cooperation. In this way, one can access to the techniques implemented and developed in important centers of research around the world

M Alexis RannouDeputy Managing Director in charge of Innovation - ARD Soliance - Pommacle

Ingénieur Agricole (ISAB) 1991Ingénieur d’études ARD (Agro industrie Recherche et développement) en charge de la sélection variétale de la betterave biotechnologie pour la fabrication d’acide galacturonique.1994 Responsable du pilote industriel ARD mise au point de tension-actifs verts (Uronate de sodium et Alkyl polypentosides)1997 Directeur technique SOLIANCE & développement industriel Amadéïte avec OLMIX2000 Formation IFG CGDPME (Gestion des entreprises) 2002 Directeur Général Adjoint en charge de la production et du compte l’Oréal2007 DGA en charge de l’innovation (8 Brevets) 20 bx produits et du marketing stratégique2012 DGA en charge de l’innovation Soliance et Wheat Oléo




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Dr. Maria HayesScientific Project Manager - NutraMara - Teagasc Ashtown Food Re-search Centre - IRELAND

NutraMara Scientific Programme Manager & Principle Investigator - Work Package 2 and 7.Main Research Interests: • Isolation, purification and characterisation of marine derived molecules, especially peptides and phlorotannins from marine seaweeds and by-products • Fermentation • Bioassay development with a particular focus on heart and mental health disorders – i.e., renin, ACE-I, PAF-AH, PEP and inhibition of other enzymes with heart and mental health effects • Generation of chitin and chitosan from marine shellfisheries waste streams • Isolation and characterisation of enzymes (in particular chitinolytic enzymes)Short Biography:Dr Hayes obtained her BSc (Hons) in Science, specialising in Industrial Microbiology and Chemistry from University College Dublin (UCD). She carried out her PhD at the Teagasc Food Research Centre, Moorepark and University College Cork in the area of bioactive peptide isolation and characterisation from milk proteins and waste streams (whey and casein). She then carried out Post-doctoral work at the Centre of Applied Marine Biotech-nology in Donegal where she worked on the isolation of chitinolytic enzymes from shell-fisheries crab and whelk waste streams. She is currently the NutraMara Scientific Pro-gramme Manager and supervises two NutraMara PhD researchers who are funded by the Teagasc Walsh Fellowship programme. These students are Mr Ciaran Fitzgerald and Ms Michelle Tierney.

Selected publications: Fitzgerald, C., Gallagher, E., Tasdemir, D., Hayes, M., (2011), Heart Health peptides from

macroalgae and their potential use in functional foods. Journal of Agriculture and Food Chemistry, DOI: 10.1021/jf201114d Di Bernardini, R., Harnedy, P., Bolton, D., Kerry, J., O’ Neill, E., Mullen, A. M., Hayes, M., (2011), Antioxidant and antimicrobial peptidic hydrolysates from muscle protein sources and by-products. Food Chemistry, 124, 1296-1307.

Tierney, M. S., Croft, A. K., Hayes, M., (2010) A review of antihypertensive and antioxidant activities in macroalgae, Botanica Marina, 53 (2010), 387-408.


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Hayes, M., Carney, B., Slater, J., Bruck, W., (2008), Mining marine shellfish wastes for bioactive molecules: Chitin and chitosan; Part B: Applications. Biotech-nology Journal, 3, 7, 871-877.Hayes, M., Barrett, E., O’Connor, P., Gardiner, G., Fitzgerald, G., Hill, C., Stanton C., Ross R.P. (2007), Salivaricin P: one of a family of two component anti-listerial bacteriocins pro-duced by intestinal isolates of Lactobacillus salivarius, Appl Environ Microbiol. 73, 11, 3719-3723.

Ms Christine Le TennierAlgues de Bretagne - Globe Export SARL - Rosporden

«“I was born an entrepreneur”Christine Le Tennier is a dynamic person. An impulsive one. She is com-plete. Political cant, she does not know it. “I was born an entrepreneur.” A witticism? Not at all. Before she was 20 years old, Christine Le Tennier did not have any idea about what the wage system was. “My grandpa-rents were corporate managers.” Farmer on her paternal side, tinsmith in Alger for her maternal grandmother. When she was 20, she was hired by Hilton. As a barmaid. At age 22, she became a commercial executive. Still at Hilton. In Ontario – where she was born – and in New York State. When she was offered a big job in Africa, she turned it down. Went back to Brittany, met her future husband and went back to school to study international business.In 1986 Christine Le Tennier created Snc Glob’export with the aim of international consul-ting and trade. Globe export became Sarl Globe Export – Seaweed of Brittany in 1993, opening date of the first production factory of seaweed-based products.Today edible seaweed are lacking in Brittany, studies foresee a field for seaweed in 10 years. “But I do need seaweed here and now.” Meanwhile the development of this field, Christine Le Tennier imports a part of seaweed she transforms and makes a turnover of 2 million Euros with 13 to 15 employees. Customers of Seaweed of Brittany: industry, catering, retail, export (20%), mail order selling. The strategy developed in 2012 can be summarized in 2 major divisions: innovation and interna-tional.




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Dr. Bruno DaridonResearch and Development Director - PRP Technologies - Paris

Bruno Daridon, 51 years old, integrated PRP Technologies in March 2007 as R&D manager, and then joined the executive committee. He is an agronomist (ENSAIA Nancy-1984) and Doctor in Biotechnology and Food Process Engineering (INPL Nancy- 1988). From 1993 to 1997, he created as a R&D engineer, Prabil S.A., a society of research on hire about extraction and functionalization of plant molecules and valuations of non-food agricultural products, then became its general manager in 1997. From 2004 to 2007, he was in charge of the site Novasep Brabois where he developed processes of fractionation and purification of biomolecules for the pharmaceutical industry.

Dr. Adeline PicotPlants Pathology Laboratory - VEGENOV BBV - St Pol de Léon

After graduating with a PhD in plant pathology from the University of Paris-Sud 11 in 2010, Adeline Picot has been working at Vegenov as a Plant pathology assistant for one year. Her field of research focuses on the evaluation of plant defense elicitors and the optimization of their use in several pathosystems including grey molds and powdery and downy mildews in tomato, strawberry… She is involved in the French network Elicitra which aims at understanding, developing and promoting the strategy of plant de-fense elicitors.

Pr. Simon DaviesProfessor of Aquaculture Nutrition at the University of Plymouth (UK). Member of World Aquacul-

ture Society.


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La Bretagne : Terre historique de la valorisation des algues

Christine BODEAU

Présentation dédiée à : • Jean DUGOUJON, créateur de la Chambre Syndicale des Algues et Végétaux Marins • Pierre ARZEL, Chercheur à IFREMER • Jean-Yves FLOCH, Professeur à l’Université de Bretagne Occidentale

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Olmix Algae Symposium - Sept. 10th, 2012

3 entreprises dans années 1960’

21 entreprises aujourd’hui : diversité des applications

La Chambre Syndicale des Algues et Végétaux Marins

Bret’algue

Olmix Algae Symposium 2


Source : Google Earth

Source : Google Earth

La Bretagne : Une situation géographique unique

Olmix Algae Symposium

Source : Google

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Olmix Algae Symposium - Sept. 10th, 2012 Olmix Algae Symposium

Jusqu’à 13 m de marnage dans la Manche !

Marnages dans le Monde. - Source : SHOM Plus petits = verts / Plus grands = rouges

Les algues se répartissent sur tout l’estran :

La Bretagne : Une région au marnage exceptionnel

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Les débuts de la récolte en bateau



La récolte à pied

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M.Philippe

La récolte à pied

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L’algoculture

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Le séchage

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Le brulage



Extraction d’iode



Les utilisations industrielles et domestiques

XVIIème fabrication du verre

Engrais

Début XIXème, fabrication de l’iode (jusqu’en 1955)

Nourriture du bétail (depuis le XIXème)

Literie

Épaississant et gélifiant : alginates début du XXème / carraghénanes 1960

Alimentaires, légumes de la mer, 1980 (essor)

Cosmétique, pharmaceutique

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Molécules à rôle physiologique

de survie : antibiotiques,

antioxydants…

Oligoéléments Sels minéraux

Polysaccharides Sucres

osmolytes

Algues et biochimie

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Oligoéléments Sels minéraux

• Engrais • Cosmétiques • Thalassothérapie • Alimentation • Diététique (calcium) • Médecine (iode) • Nutrition animale

Algues et biochimie

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Polysaccharides Sucres

Alginates (Algues brunes)

Carraghénanes (Algues rouges)

Laminarine (accélérateur de

croissance)

Produits épaississants et

gélifiants

Engrais

Divers Pharmacie

Alimentation Diététique

Algues et biochimie

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Olmix Algae Symposium - Sept. 10th, 2012 Olmix Algae Symposium Algues et Mer ©

Molécules à rôle

physiologique de survie :

antibiotiques, antioxydants…

Molécules à haute valeur

ajoutée

Algues et biochimie

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Bord à Bord ©

Algues de Bretagne©

Alimentaire

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La récolte aujourd’hui en bateau

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Discovering the Blue Chemistry of algae:

Their origin, biology and metabolism

Philippe POTIN - Team Algal Defenses CNRS-UPMC UMR 7139 - Marine Plants & Biomolecules

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Seaweeds from

Roscoff: an exceptional

place for

studying biodiversity

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Seaweed belts on the shore

3 Floc'h, J.-Y. (1964). Distribution verticale et écologie des algues marines sur les côtes Bretonnes. Penn ar Bed 4(37)

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Reds and greens

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Phaeocystis globosa

Brown’s

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Modern stromatolites in Shark Bay, Western Australia http://en.wikipedia.org/wiki/Stromatolite

2.724 billion years ago as far back as 3.450 billion years ago

Blue green algae

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Algae are photosynthetic organisms. Based on the pigment and food reserve, algae are classified into different types, namely, blue green algae (BGA), green algae, red algae and brown algae.

Algae origins are not only a matter of pigments

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The tree of life (Haeckel, 1866, c/o Simonetta Gribaldo).

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Olmix Algae Symposium - Sept. 10th, 2012 Olmix Algae Symposium Lane et al, TREE, 2008

Algae in the eukaryotic tree

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Endosymbiotic gene transfer: organelle genomes forge eukaryotic chromosomes Timmis et al. (2004) Nature Reviews Genetics 5, 123-135

The endosymbiotic origins of eukaryotes

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between 2 and 1.5 billion years

Endosymbiotic gene transfer: organelle genomes forge eukaryotic chromosomes Timmis et al. (2004) Nature Reviews Genetics 5, 123-135

The endosymbiotic origins of eukaryotes

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? about 1,2 billion years

Secondary endosymbiotic origin of brown’s

12 The life of diatoms in the world's oceans E. Virginia Armbrust Nature 459, 185-192

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Secondary endosymbiotic origin of other algae

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Seaweeds belong to independant lineages

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Olmix Algae Symposium - Sept. 10th, 2012 Olmix Algae Symposium Dr Kathleen Drew-Baker

Porphyra life cycles (1949)

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Porphyra aquaculture in Asia (nori)

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Ulva life cycles

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Olmix Algae Symposium - Sept. 10th, 2012 Sauvageau 1917

Kelp forests and life cycles

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Kelp aquaculture

Source: Ifremer 19


www.seaweedenergysolutions.com/ -

Saccharina latissima farming

Kelp aquaculture

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Algal metabolisms

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Kelp biorefinery

22 Find an alternative to the storage of fresh biomass in formaldehyde

Acid Calcium

Na2CO3

LAMINARIN MANNITOL

FUCANS Na- ALGINATE

Co-extraction of laminarin, mannitol , fucans with alginates


Iodine first discovered in 1811 by Courtois in kelp ashes

Production of « soda » bricks by seaweed harvesters by burning dried kelps in stone ovens

Kelps : a major source of iodine

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Iodine may serve as an inorganic antioxydant in kelps Küpper,. et al. (2008) Proc. Natl. Acad. Sci. USA 105, 6954-8

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P. Rouzé / Y. van der Peer

+ Es Genome Consortium

Brown algal genomics

GENOME SEQUENCING: 3,000,000 reads (10X, shotgun)

cDNA SEQUENCING: 100,000 reads (full-length cDNAs)

J. M. Cock, Roscoff

- STRAIN SELECTION - BIOLOGICAL MATERIAL - LIBRARIES

- LIBRARIES - SEQUENCING - ASSEMBLY

- AUTOMATIC ANNOTATION - EXPERT ANNOTATION

The Ectocarpus genome (200 Mb) project J.-M. Cock

Cock JM et al. (74 authors) Nature. 2010 Jun 3; 465:617-21 and more than 20 papers during

the two last years.


The origin of alginate synthesis route in brown algae is likely the result of an horizontal gene transfer (HGT) from Actinobacteria

Michel et al. New Phytol. 2010 PMID: 20618907


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Hypothetical biosynthetic pathway for the formation of phloroglucinol derivatives in marine brown algae

(based on Ectocarpus genome data)



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Fungi (9) 81

97

88

Bacteroidetes (3) 78

99 Amoebozoa (2)

95

98

Plants (28)

81

100

9898Bacteria (10)

Actinobacteria (7) 100

89 E. siliculosus PKS2

E. siliculosus PKS3 E. siliculosus PKS1

F. vesiculosus PKS F. spiralis PKS S. binderiPKS

76 Brown algae

Chalcone synthases Stilbene synthases

Resveratrol synthases Pyrone synthases Acridone synthase

Bisphenyl synthases …

100

Beta-ketoacyl synthases (outgroup)

Type III Polyketide synthases


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The origin of phlorotannin in brown algae is likely the result of an horizontal gene transfer (HGT) from Actinobacteria

Phylogenetic relationships of brown algal Type III Polyketide synthases

Olmix Algae Symposium - Sept. 10th, 2012 Olmix Algae Symposium 29

Phloroglucinol synthesis in Pseudomonas fluorescens

Type III Polyketide synthase (PKS)

Boiled control

1 hour

3 hours

5 hours

Abundance

Time →

Extracted ion m/z 342 → Phloroglucinol

CNRS patent sept.2011 n° 11 58728 Meslet et al submitted soon.

Phloroglucinol synthesis in Ectocarpus siliculosus


Other brown seaweeds

A kelp with major ecological importance

A maricultured kelp

A proposal for a project between Chile, Germany, USA & France submitted to JGI

30

Macrocystis pyrifera (L.) C.Agardh

28


Red algal genomics

31

The Chondrus genome project

The Porphyra genome projects

Jonas Collén Catherine Boyen


Hana-Tsunomata™ when translated from Japanese means "Flower Chondrus". Other common references to Hana-Tsunomata™ sea vegetables include Hana-nori™ ("Flower seaweed"), Cultivated Chondrus, Kaede-nori ("Maple seaweed") and Hana-sakura-sou ("Cherry blossom sea plant").

Chondrus crispus flowering (NS, Canada, ASP)

An industrial reality



The Ulva genome project 150 Mbp

FSU Jena

John Bothwell

Ulva genomics

33

29


Ulva mariculture

34


Trends in seaweed blue chemistry

35


IDEALG

36

18 partners: UEB coordination, SBR: UMR 7139-6 teams, UMR 7144-3 teams+ FR2424, CEVA Pleubian, AMURE-UBO, IFREMER Brest, UBS-LBCM Lorient, Agro-Campus, ENSCR, IRISA-Symbiose (Rennes), CNRS U-Nantes, INRA-LBE, 1 large company : Danisco & 4 SMEs, C-Weed, Aleor, France Haliotis, Bezhin Rosko

IDEALG 2011-2020 Seaweed biotechnology and bioresources

A national 10-year large integrative project to merge

algal genomics with mariculture, biotechnology and chemistry.

30


Knowledge transfer:

37

Apply knowledge of metabolic pathways and networks, enzymes, molecules, biocatalyzers, bioconversion, bioengineering and aquaculture of domesticated seaweeds, Pre-pilot scale projects and technological research

Develop basic research on brown, red and green seaweeds toward domestication of local crops and improvement of seaweeds uses in biotechnology and blue-green chemistry

Domestication

Biotechnology Sustainable Chemistry

Seaweedomics toward

Systems biology

seaweed.ie

Thanks for your attention!

38 G. Michel B. Kloareg

J.M. Cock

M. Czjzek

C. Leblanc

J. Collén

C. Boyen

31

Marine macroalgae: a sweet treasure

Dr. Mirjam Czjzek 1


Marine seaweeds: a large diversity of original polysaccharides

2 Baldauf (2003) Science

1 cm

Ectocarpus siliculosus

Chondrus crispus


Marine seaweeds: a large diversity of original polysaccharides

3 Baldauf (2003) Science

fucans

Me

-3OSO

OOSO3-

O

O

OH

Me OOSO3-

Me

-3OSO

OOSO3-

O

O

OH

Me OOSO3-

O

O S O 3 -

O

O H

O

O S O 3 -

O H

O H O

O S O 3 -

agar/carrageenans

O H O

O H

O

O O

O H H O

O

H O H O

O O

H O O H

O H O H

O

xyloglucan

OOO

OH OH

O CH3

OOH

OHO2C

n

X+ -O3SX+ -O3S

ulvan

O

O

OH

OH

O H

O S O 3 -

OH O

O

O

NH

O

dermatan

32


The diversity of sugars and their linkages in polymers….

1

4

3 2

5 6

4

1

1 2

….goes hand in hand with the diversity of enzymes

GH GT glycosyl transferases or GT

glycosyl hydrolases or GH

Biosynthesis Catabolism, remodeling

glucomannan

lichenan

cellulose glucose

mannose

galactose

4


Chlorophyta (Green algae)

Rhodophyta (Red algae)

Heterokonts (Brown algae & diatoms)

Neutral polysaccharides

(mostly shared with land

plants)

Cellulose

Xylan Mannan Chitin

Cellulose

Mannan

Cellulose

Chitin (Diatoms)

Starch Floridean starch Laminarin

Polyanionic polysaccharides

(unique to marine algae)

Macroalgae

Ulvans Agars

Carrageenans Alginates Fucanes

Unicellular algae Capsulans

(Prasinococcus)

Sulfated glucogalactoxylans

(Porphyridium)

Carboxysulfated polysaccharides (Phaeodactylum)

Sulfated carboxylated Carboxy-sulfated

Algal polysaccharides: the complexity of organic matter in ocean

5


Red algal galactans

agarose

k-carrageenan

i-carrageenan

l-carrageenan

b(1-4) a(1-3) O

O O

O

OH

O

OH OH

OH

O

O OO

OSO3-

O

OH

OH

OH

O

O OO

OSO3-

O

OH

OH

OSO3-

O

OSO3-

O

OH

O

OSO3-

OH

OHO

OSO3-

porphyran

Increase in density of negative charge

Decrease in gel strength

Gels of 1 %

6

33


Conformational state of k-carrageenan (iono- and thermo-reversible gels)

NaCl NaI KCl

Helices

Variables Temperature [carrageenan]

[salt] Type of salt

Random coils or Flexible rods

Aggregation of helices Gel formation

7


b-agarase

Extended loops

k-carrageenase

Structural determinants of the k-carrageenase

8


Extended loops

k-carrageenase

Structural determinants of the k-carrageenase

9

Trp95 Arg151

34


soluble substrate

endo

15 20 25 30 Time (min)

DP4 DP6 DP8

Anion exchange chromatography

µS 0

36

47 49

60

51

10

% hydrolysis

citrate

µS

processive! jellified

substrate

Time (min)

DP4

15 20 25 30

0

18

40

42

56 62

% hydrolysis

10

citrate

0 40 10 20 30 0

0,1

0,2

0,3

Hydrolysis (%)

DP

4/C

itrat

e

Mode of action of the Pseudoalteromonas k-carrageenase

10


Mode of action of the Pseudoalteromonas k-carrageenase

DP4 DP4 DP4 11


Agarose sol/gel transition

Electronic Scanner microphotograph of a 2 % agarose gel

Attack of colloidal microgel by Zobellia galactanivorans

12

35


Agarose sol/gel transition

Handbook of hydrocolloids (Medin, A. S. 1995)

a(1-3)

agarose

b(1-4) O

O

C H 2 O H O H

O O

O

O

O H O H ) ( n

13


Zobellia galactanivorans, (Bacteroidetes) a specialized marine polysaccharide degrader

Barbeyron et al. 2001

114 glycoside hydrolases (GH), 12 Polysaccharide lyases (PL), 17 carbohydrate esterases (CE) and 72 sulfatases !

several polyspecific familes such as 16 GH16

Genome project : counting the carbohydrate active enzymes (CAZymes)

Genome Project – MPI Bremen F. O. Glöckner, M. Bauer, R. Amann

CeBiTech

14


Marine bacteria secrete specific enzymes for the degradation of algal polysaccharides

15

b-Agarases AgaB

k-Carrageenases

i-Carrageenases

GH96 CBM6 TPS3 CBM6 TPS3 TPS3 TPS3 TPS3 CBM6 a-Agarase

l-Carrageenase

Pseudomonas agaralyticus Flament et al. (2007) Appl Environ Microbiol

Zobellia galactanivorans Jam et al. (2005) Biochem J

AgaA

P. carrageenovora, Z. galactanivorans Barbeyron et al. (1998) Mol Biol Evol

Alteromonas fortis, Z. galactanivorans Barbeyron et al. (2000) J Biol Chem

GH-16 X-70 UNK GH-16

GH-16 Big2 GH-16 CBM16

GH-82 GH-82

WD repeat domain New GH

Pseudoalteromonas carrageenovora Guibet et al (2007) Biochem J

Fucanase

Alginate lyase

GH107 Cad Cad Cad UNK

New PL

Mariniflexile fucanivorans Colin et al. (2006) Glycobiology

Pseudomonas alginovora Chavagnat et al. (1996) Biochem J

AgaB

36


Phylogenetic analysis of Zobellia enzymes from family GH16

16


The variable compositions of agaroids

17

Neutral, ideal

Various modifications can be observed like, pyruvate groups, methylation, sulfatation, or even branching .... These modified agar components are often called agaropectins

O

O

CH2OMeOSO3-

O O

O

O

OHOH

O

O

CH2

O O

O

O

OH

OO

CH3HO2C

OH

O

O

CH2OHOH

O O

O

O

OHOH

O

O

CH2OMeOH

O O

O

O

OHOH

O

O

OH

O O

O

O

OHOH

O

HOOH

O

HO

Me

OOMe

CH3HO2COSO3-

a(1-3) b(1-4) b(1-4) a(1-3)

O

HOOH

Agarose

b(1-4) a(1-3)

M. Lahaye and C. Rochas 1991


Correlation with increasing L-galactose-6-sulfate units Maximum activity on red algae Porphyra sp.

L6S = L-galactose-6-sulfate LA = 3,6-anhydro-L-

galactose

Activity screening on algal cell wall extracts

18

37


Structural analysis by crystallography


W131

L6S L6S

G

G

-4

-3

-2 -1

E144

E139S

W56

H53

R133

R59

catalytic residues

Basic residues are conserved at -2 critical for porphyran recognition


Z. galactanivorans contains a complex agarolytic system



21

38

Enzymatic hydrolysis in chemistry of seaweeds

Nathalie Bourgougnon, Kevin Hardouin, Loannes Le Bars, Gilles Bedoux, Christel Marty, Justine Dumay*, JP Bergé **

Laboratoire de Biotechnologie et Chimie Marines, UBS, PRES UEB, IUEM * Mer Molécules Santé, Université Nantes ** IFREMER, Nantes 1


“Marine biofilm: biological and chemical approaches” program Study of the interactions between organisms and abiotic surfaces Study of physical and chemical parameters involved in bacterial adhesion by

conceiving model substrates Development of antifouling systems combining efficiency and environmental

respect Cell-cell interfaces Complex bacterial biofilms Procaryote-eucaryote interactions and communications

Biotechnology: valorization of marine molecules

Purification and characterization of compounds of interest from invasive marine organisms (sponges, algae, bacteria, echinoderms...)

Laboratoire de Biotechnologie et Chimie Marines

15 teachers/researchers - 10 PhD students - 2 technical assistants

2


Laboratoire de Biotechnologie et Chimie Marines

Professional Licence Biotechnologie Ingénierie des produits cosmétiques et de santé

Master Biotechnologie Biomolécules, micro-organismes and bio-procédés

3

39


Which biomasses to screen?

Marine Drug design ?

Extracts with biological activities

Identification of the producer? extraction and purification, Synthesis very difficult, limited resource

Resources halieutics, by-products…?

Few active molecules

Interesting molecules

Many ways of upgrading

Biotechnologic biomass ?

Lot of biological molecules

Simple extraction

Inexhaustible resource

Many ways of upgrading

Selection of phyla know to synthesize cytotoxic molecules

4


French Brittany Seaweed A good biomass

Long tradition of the use of seaweeds

Harvest of marine macroalgae in Brittany (2009)

Brown Red Green Others

94.7%

3.9% 1.1%

0.3%

Important biodiversity

Harvested wild seaweed 70 000T/year CSAVM 2010

Sangiardi 2010

Good economic context with a lot of active companies using macroalgae

Invasive seaweeds

5


Animal and human foods Health market

Cosmetics

Chemical synthesis of natural substances

Extraction from marine organisms

Technologies

6

40


Extraction from marine organisms

Maceration in a mixture of solvants Liquid/liquid partition Fractionation by chromatography Purification by HPLC Identification RMN, Malditof….

Extraction technologies

Olmix Algae Symposium - Sept. 10th, 2012 Olmix Algae 7


Obtain compounds which can be the object of later upgrading from seaweeds

Low energy consummation

Low cost

Processes environment-friendly (no toxic, biodegradable)

Enzymatic hydrolysis

8


Enzyme molecules

Protein nature Product was not consumed during the reaction Active in small proportion Do not modify the balance thermodynamics reaction Accelerate only the speed of the reaction Specific of a reaction Large-scale production

9

41


Numbering EC

Enzyme classification

10


pH stat method

pH Regulation Temperature Control Calculation of Hydrolysis degree

Temperature Quantity enzyme Time of hydrolysis pH H2O Volume

Parameters studied

Enzymatic hydrolysis technology

11


Enzymatic hydrolysis technology

12

Raw material (seaweed) (1v)

Enzyme (large spectra) 1.0% Water (1v)

Temperature 60°C

Time 3 hours

Reaction

42


Example of enzymatic hydrolysis

13


Leu-Trp-Lys-Arg-Glu-Ile-Tyr-Phe-Arg-Gln-Ser-Val-Asp-Thr-Ala-Pro-Asn


14

Alcalase EC 3.4.21.62




15

Alcalase EC 3.4.21.62

Protamex EC 3.4.21.62 &

EC 3.4.24.28

43



Alcalase EC 3.4.21.62 Flavourzyme

EC 3.4.11.1

Protamex EC 3.4.21.62 &

EC 3.4.24.28

Enzymes tested only or in combination


16


Enzymatic hydrolysis Process

17

Identification of seaweed biomass Good knowledge of biomass composition

SEAWEEDS

Analysis

Protein Lipid Polymer Mineral

Quality

Food grade Non Food Grade

Quantity

Seasonality Geography …



18

Define the objectives of upgrading

SEAWEEDS

Analysis


Quality


44



19

Define the objectives of upgrading

SEAWEEDS

Analysis


Quality




General Strategy 20

Estimate the availability of the biomass

SEAWEEDS

Analysis


Quality


Quantity




21

Analysis

Protein 10-24% Lipid Polymer

38-60% Mineral 14-29%

Quality


Quantity


g / 100 g MS

45



22

Total protein by Kjeldhal

Soluble protein by Lowry and Bradford method

0 1 2 3 4 5 6

Temps (min)

Ala

GlyVal

IS

Leu

IleThr

SerPro

Asp

Met

Glu

Phe

Lys Tyr

Liquid chromatography

Gaz chromatography FID



23

Total sugars by Dubois

Anion exchange chromatography HPAEC

Characterization monosaccharides

Steric exclusion Low pression chromatography

Polysaccharides profil



24

AQUEOUS

SLUDGE

OILY 15% Proteins

0% Lipids

5% Minerals 24% Proteins

2% Lipids

20% Minerals

23% Sugars

46



25

Analysis


38-60% Mineral 14-29%

Quality


Quantity


Animal nutrition and animal Health Vegetal health



26

Studies of seasonality and geographic parameters

Analysis


38-60% Mineral 14-29%

Quality


Quantity




27

Enzyme 2

Enzyme 1

Composés minoritaires

Amadéite®

Protéines, Glucides, Lipides, Minéraux

Oligosaccharides -

Polysaccharides

Protéines

Enzyme 1

Hydrolyse 1

Algues

Culot

Enzyme 2

Hydrolyse 2

Culot

Composés

Minéraux, lipides,…

Nutrition

Autres (non hydrolysable)

Oligosaccharides

Peptides

Protéines

Polysaccharides

47


Proposed value chains (France) Seaweeds - Recovery of molecules with industrial interest

28 J. Fleurence & J. Dumay (MMS), E. Deslandes &V. Stiger (LEMAR), N. Bourgougnon (LBCM), R. Baron & JP Bergé (Ifremer)

Seaweed biomass

Clarified extract

Solid residues

Seaweed harvesting

Algoculture

SEPARATION CONCENTRATIONPURIFICATION

(membranes or chromatography

processes)

EXTRACTION Enzymatic

digestion, reactive extrusion, or

classical extraction with aqueous solvent in mild

conditions AGRI-FOOD

AQUACULTURE COSMETICS PHARMACY

HEALTH

Enzy

mat

ic tr

eatm

ent

Proteins Bioactive peptides

Pigments Oligosaccharides polysaccahrides

Phenolic compounds


Proposed value chains (France) Seaweeds - Recovery of molecules with industrial interest

29

Enzymatic extraction of protein from Palmaria palmata Different enzymatic treatment of seaweed

R-Phycoerythrin

Recovery Yield : 4 % expressed in regard of the dry weight instead to 0.4 % with a classical extraction


30

Young researchers: Kevin Hardouin & Loannes Le Bars Technical assistant: Christel Marty

Collaboration:

48

Bioactivities of Marine Polysaccharides (MSP)

in human and animal health - Uptade

Dr Henri SALMON - Director of research - INRA 37380 Nouzilly Dr Hervé DEMAIS - Scientific advisor - OLMIX 56580 Bréhan

1


Polysaccharides: Introduction

Polysaccharides = biologically active substances in biochemistry and medicine (aside from their rheological properties).

Today, the most promising biopharmacological activities of polysaccharides are their immunomodulatory and antitumor effects [1].

The most actives being extracted from primitives plants like mushrooms or algae.

2


Polysaccharides = macroamolecules

Repetitive structural features which are polymers of monosaccharide units joined to each other by glycosidic linkages

3

49


Classification of the bioactive sulfated polysaccharides

Seaweed

Red Green Brown

Ulvan

Kappa, Iota, Lambda types

Galactan and Carrageenan Fucoidan

Fucose, Xylose,Uronic acid, Galactose, Sulfate

Sulfated agalctose and 3,6 Anhydrogalactose

Sulfated rhamnose, sulfated aldobiuronic

acid

Marine Sulphated polysaccharides (MSP)

4


Polysaccharides = primary structural variability

Great potential for structural variability due to their interconnections (glycosidic bonds) at several points to form a wide variety of linear or branched structures [2].

Glycogen

α-1,4 ->LINEAR α-1,6 ->BRANCHED

1

2 3

4 5

6

1

1

2 3

4

Amylose

α1,4 glucose-> LINEAR

1 2

3

4

5

6

5


Marine Sulphated polysaccharides (MSP): ULVANS

6

Sulphated Polysaccharides -> Poly-anionic structure -> various bioactivities

. [→4)-α-l-Idop-(1→4)-α-l-Rham3S-(1→]n

[→4)-β-d-Glcp-(1→4)-α-l-Rham3S-(1→]n

Rare sugar such as Rhamnose (also in plants)

Glucuronic and iduronic acids (sugars from the mammalian glycosaminoglycans

family)

High sulfate content (Sulphated rhamnose)

A

50


Marine Sulphated polysaccharides (MSP): FUCANS

7

Sulphated Polysaccharides -> Poly-anionic structure -> various bioactivities

1→2, 4-O-sulphated fucopyranose)


Bioactivities of MSP

Antiviral Activities

Anti-Inflammatory Activities

Immuno Activities Antioxydant Activities

Anticoagulant and Antithrombotic Activities

Antilipidemic Activities

8


Antiviral Activities Generalities

Inhibition of the replication of enveloped viruses: blocks the viral entry into the cell by binding to glycoprotein C (gC) and glycoprotein D (gD) of HSV-1(37)

-> Antiviral activity of sulfated polysaccharides increases with sulfation and their molecular weight [34]

Chemical structure of disaccharides (VI and VII) relevant for binding

9

51


Antiviral Activities Antiviral Activities

10

In vitro inhibition of influenza A virus infection by marine microalga-derived sulfated polysaccharide p-KG03 Meehyein Kim a, Joung Han Yim , So-Yeon Kim a, Hae Soo Kim ,Woo Ghil Lee a, Sung Jin Kim ,Pil-Sung Kang b, Chong-Kyo Lee


Immuno-Inflammatory Activities of MSP: Generalities

MSP may affect multiple targets in the immune and inflammatory systems that can have impact on disease progression and outcome including tumor progression and metastasis [41]. Sulfated polysaccharides play two-edged roles, inhibitor and promoter, in immune response.

General Effects : • Stimulation of the immune response / control of immune cell activity to

mitigate associated negative effects such as inflammation [40].

• Anti-inflammatory Growing body of evidence illustrating their ability to interfere with the migration of leukocytes to sites of inflammation. [42,43].

• Inhibit tissue degradative enzymes such as heparanase and elastases that are involved in the breakdown of basement membrane integrity during inflammation [45,46].

11


Intravital microscopic images of a rabbit mesenteric venule. (A)Basal leucocytes rolling along the venular endothelium (B) demonstration that the rolling was abolished 3 min after systemic administration of fucoidin (10 mg/kg).

Changes in basal rolling leucocytes flux without change in arterial blood pressure over time in the rabbit after systemic treatment with fucoidin

By binding of fucoidan to L- and P selectins, cell adhesion molecules essential in the recruitment process., Fucans also inhibit leukocyte recruitment to the abdominal cavity during acute peritonitis in rats [44].

Anti-Inflammatory Activities of MSP: Anti-migration of leukocytes to sites of inflammation

12

interfering with the migration of leukocytes to sites of inflammation. [42,43].

52


Anti-Inflammatory Activities of MSP By Anti-Complement activity

Fucoidan fractions (low MW) inhibit both the classical and alternative pathways in human serum [47] by binding to the C1q and C4 of the complex

-> interaction with the complement ->reduce the pro-inflammatory state

X D, B, F :Area of

interaction with C4

13


λ-carrageenan stimulate mouse T cell cultures in a toll-like receptor-4 (TLR4) dependent manner generating a T helper 1 (Th1) patterned cytokine response. However, splenocytes prepared from TLR4-deficient mice still retain some ability to produce interferon-γ in response to λ-carrageenan suggesting that PRRs other than TLR4 are also elicited. [52]

[40].

Immuno-Activities of MSP: Binding to PRR

Pattern Recognition Receptors (PRRs)

14

mannose Toll-Like Receptors


These and other reports of algal sulfated polysaccharides directly stimulating the innate immune system [53,56,57] suggests that they may find therapeutic use in opposing T helper 2 (Th2)-based pathologies such as autoimmune disorders and allergy.

(56) (56)

Glial cells + TNFα +IFN=>iNOS (inducible nitric oxide synthase )

Inbition iNos with doses of Fucoidan

TNFα +IFN

TNFα +IFN TNFα +IFN+IFNTNFα +IFN+IFNFucoidan (µg/ml)

Fucoidan(µg/ml)

Anti-Inflammatory properties of MSP: Inhibition of iNOS production

15

53


Immuno- Activities of MSP: Via Macrophage activation

16

Botanical polysaccharides: Macrophage immunomodulation and therapeutic potential Igor A. Schepetkin, Mark T. Quinn *

Fucoidan+DC A pro-inflammatory cytokine/chemokine profile [53, 54, 55,56].

Chemokines

MCP-1

MSP

ROS=reactive oxygen production


57. Kim, M.H.; Joo, H.G. Immunostimulatory effects of fucoidan on bone marrow-derived dendritic cells. Immunol Lett 2008, 115, 138-143.

58. Choi, E.M.; Kim, A.J.; Kim, Y.O.; Hwang, J.K. Immunomodulating activity of arabinogalactan and focoidan in vitro. J Med Food 2005, 8, 446-453.

Fucoidans, carrageenan

tumoricidal activity

Anti-tumoral activities of MSP

17


Immuno-Inflammatory Activities of MSP: Summary of immune cells Activation by MSP

18

54


MSP: Anticoagulant Activity

MSP (Fucoidans, and ulvan)

and Red seaweeds >Heparin-like anticoagulant activity

[8,9,10 ,11,12]. [13,14,15,16,17].

Some high rhamnose-containing MSP are

more potent than standard heparin [16]. Control: Clotting time: 40s

Anti-thrombic 19


MSP: Antioxidant Activities

Ferric reducing/antioxidant power [23]

Superoxide radical scavenging ability [24,25, 26, 22].

Fucans’ Superoxide radical scavenging activity correlated positively high sulfate content of the polysaccharide fractions [24,26]. Antioxidant properties of Carrageenans [24] and Ulvans [27 ] also appeared related to sulfate content.

Inhibition of lipid peroxydation of rat liver microsome

20


MSP: Antilipidemic Activities

21

Lipid-lowering (serum triglyceride and total cholesterol) in hyperlipidemic animal models (28,29,30).

In rats fed a high cholesterol diet for 21 days, supplementation of the diet with ulvans from U. pertusa led to reductions in serum total cholesterol and LDL-cholesterol with no significant alteration in serum triglycerides . The effects of ulvans were modified when it was degraded into lower molecular weight fractions (29).

Illustration of the function of hepatic lipase as a lipolytic enzyme. Santamarina-Fojo S et al. Arterioscler Thromb Vasc Biol 2004;24:1750-1754

M.S.P

Certainly fucoidan and other algal sulfated polysaccharides may influence LPL (Lipoprotein Lipase) and HL (Hepatic Lipase) through interaction with well-characterized heparin-binding sites on these enzymes (31).

Conclusion:Algal sulfated polysaccharides are showing promising effects in addressing the hyperlipidemia associated with certain drug toxicities (32, 33).

55


Conclusions 1

The study of algal sulfated polysaccharides structures is challenging, because of their diversity and heterogeneity.

This may also have hindered their development as therapeutic agents to date in spite of various biological activities already demonstrated. (Immunity, inflammation, coagulation, oxidation, lipidemia….)

Due to the difficulties in identifying the precise chemical structure of algal sulfated polysaccharides, the relation between their structures and biological activities is far from fully understood.

The production of a standardized commercial product based on algal sulfated polysaccharide constituents can also be a challenge since their structural and pharmacological features may vary depending on species and on location and time of harvest [66].

22


Conclusions 2

23

Important points can already be mentioned regarding the conditions of MSP bioactivities:

Importance of understanding the structural requirements for biological activity

Do low molecular weight derivatives, which are potentially more bioavailable, remain active?

Therapeutic use by oral route can be limited due to low bioavailability given often high molecular weights of MSP(64).

At the opposite :

Advantage for the hypolipidemic effects due to bile acid sequestration in the intestinal lumen.

With regards to some immunomodulatory activities, site of activation of the immune system may also be within the intestinal lumen (e.g., at Peyer’s patches).

Hypothesized for immunomodulatory effects of polysaccharide constituents from Chlorella pyrenoidosa [65].


Conclusions 3

= increased activity = reduced activity = increased activity = reduced activity= increased activity = reduced activity= increased activity = reduced activity

Algal sulfated polysaccharides are a new source of numerous biological activities that may find in human and animal health many prophylactic and

therapeutic benefits in the near future.

24

56


25


6. Lahaye, M.; Robic, A. Structure and functional properties of ulvan, a polysaccharide from green seaweeds. Biomacromolecules 2007, 8, 1765-1774.

5. Percival, E.; McDowell, R.H. Chemistry and Enzymology of Marine Algal Polysaccharides; Academic Press: New York, NY, USA, 1967; p. 219.

7. Lahaye, M.; Brunel, M.; Bonnin, E. Fine chemical structure analysis of oligosaccharides produced by an ulvan-lyase degradation of the water-soluble cell-wall polysaccharides from Ulva sp. (Ulvales, Chlorophyta). Carbohydr Res 1997, 304, 325-333.

2. Sharon, N., Lis, H. Scientific American, 1993, pp. 74-81.

3. Hodgson, J. Biotechnology, 1991, 9, 609-613.

1. Ooi, V.E.C; Liu, F. Immunomodulation and Anti-Cancer Activity of Polysaccharide-Protein Complexes. Current Medicinal Chemistry, 2000, 7, 715-729.

4. Lahaye, M.; Ray, B. Cell-wall polysaccharides from the marine green alga Ulva rigida (Ulvales, Chlorophyta)-NMR analysis of ulvan oligosaccharides. Carbohydr Res 1996, 283,161-173.

8. Bernardi, G.; Springer, G.F. Properties of highly purified fucan. J Biol Chem 1962, 237, 75-80.

9. Springer, G.F.; Wurzel, H.A.; McNeal, G.M.; Ansell, N.J.; Doughty, M.F. Isolation of anticoagulant fractions from crude fucoidin. Proc Soc Exp Biol Med 1957, 94, 404-409.

10. Kusaykin, M.; Bakunina, I.; Sova, V.; Ermakova, S.; Kuznetsova, T.; Besednova, N.; Zaporozhets, T.; Zvyagintseva, T. Structure, biological activity, and enzymatic transformation of fucoidans from the brown seaweeds. Biotechnol J 2008, 3, 904-915.

11. Li, B.; Lu, F.; Wei, X.; Zhao, R. Fucoidan: structure and bioactivity. Molecules 2008, 13,1671-1695.

12. Pomin, V.H.; Mourao, P.A.S. Structure, biology, evolution, and medical importance of sulfated fucans and galactans. Glycobiology 2008, 18, 1016-1027.

13. Matsubara, K.; Matsuura, Y.; Bacic, A.; Liao, M.L.; Hori, K.; Miyazawa, K. Anticoagulant properties of a sulfated galactan preparation from a marine green alga, Codium cylindricum. Int J Biol Macromol 2001, 28, 395-399.

14. Farias, E.H.C.; Pomin, V.H.; Valente, A.P.; Nader, H.B.; Rocha, H.A.O.; Mourao, P.A.S. A preponderantly 4-sulfated, 3-linked galactan from the green alga Codium isthmocladum.Glycobiology 2008, 18, 250-259.

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16. Hayakawa, Y.; Hayashi, T.; Lee, J.B.; Srisomporn, P.; Maeda, M.; Ozawa, T.; Sakuragawa, N. Inhibition of thrombin by sulfated polysaccharides isolated from green algae. Biochim Biophys Acta 2000, 1543, 86-94.

18. Farias, W.R.L.; Valente, A.P.; Pereira, M.S.; Mourao, P.A.S. Structure and anticoagulant activity of sulfated galactans. Isolation of a unique sulfated galactan from the red algaeBotryocladia occidentalis and comparison of its anticoagulant action with that of sulfated galactans from invertebrates. J Biol Chem 2000, 275, 29299-29307.

15. Mao, W.; Zang, X.; Li, Y.; Zhang, H. Sulfated polysaccharides from marine green algaeUlva conglobata and their anticoagulant activity. J Appl Phycol 2006, 18, 9-14.

17. Shanmugam, M.; Mody, K.H. Heparinoid-active sulphated polysaccharides from marine algae as potential blood anticoagulant agents. Curr Sci 2000, 79, 1672-1683.

20. Qi, H.; Zhang, Q.; Zhao, T.; Hu, R.; Zhang, K.; Li, Z. In vitro antioxidant activity of acetylated and benzoylated derivatives of polysaccharide extracted from Ulva pertusa(Chlorophyta). Bioorg Med Chem Lett 2006, 16, 2441-2445.

21. Ruparez, P.; Ahrazem, O.; Leal, J.A. Potential antioxidant capacity of sulfated polysaccharides from the edible marine brown seaweed Fucus vesiculosus. J Agric Food Chem 2002, 50, 840-845.

22. Wang, J.; Liu, L.; Zhang, Q.; Zhang, Z.; Qi, H.; Li, P. Synthesized oversulphated, acetylated and benzoylated derivatives of fucoidan extracted from Laminaria japonica and their potential antioxidant activity in vitro. Food Chem 2009, 114, 1285-1290.

23. Ruparez, P.; Ahrazem, O.; Leal, J.A. Potential antioxidant capacity of sulfated polysaccharides from the edible marine brown seaweed Fucus vesiculosus. J Agric Food Chem 2002, 50, 840-845.

24. Rocha de Souza, M.; Marques, C.; Guerra Dore, C.; Ferreira da Silva, F.; Oliveira Rocha, H.; Leite, E. Antioxidant activities of sulfated polysaccharides from brown and red seaweeds. J Appl Phycol 2007, 19, 153-160.

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35. Luescher-Mattli, M. Algae, A Possible Source for New Drugs in the Treatment of HIV and Other Viral Diseases. Curr Med Chem 2003, 2, 219-225.

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28. Vaquez-Freire, M.J.; Lamela, M.; Calleja, J.M. Hypolipidaemic activity of a polysaccharide extract from Fucus vesiculosus. Phytother Res 1996, 10, 647-650.

30. Huang, L.; Wen, K.; Gao, X.; Liu, Y. Hypolipidemic effect of fucoidan from Laminaria japonica in hyperlipidemic rats. Pharm Biol 2010, 48, 422-426.

29. Pengzhan, Y.; Ning, L.; Xiguang, L.; Gefei, Z.; Quanbin, Z.; Pengcheng, L. Antihyperlipidemic effects of different molecular weight sulfated polysaccharides from Ulva pertusa (Chlorophyta). Pharmacol Res 2003, 48, 543-549.

31. Yokota, T.; Nagashima, M.; Ghazizadeh, M.; Kawanami, O. Increased effect of fucoidan on lipoprotein lipase secretion in adipocytes. Life Sci 2009, 84, 523-529.

32. Raghavendran, H.R.; Sathivel, A.; Devaki, T. Effect of Sargassum polycystum(Phaeophyceae)-sulphated polysaccharide extract against acetaminophen-induced hyperlipidemia during toxic hepatitis in experimental rats. Mol Cell Biochem 2005, 276, 89-96.

33. Josephine, A.; Veena, C.K.; Amudha, G.; Preetha, S.P.; Varalakshmi, P. Protective role of sulphated polysaccharides in abating the hyperlipidemic nephropathy provoked by cyclosporine A. Arch Toxicol 2007, 81, 371-379..

25. Zhao, X.; Xue, C.; Cai, Y.; Wang, D.; Fang, Y. Study of antioxidant activities of fucoidan from Laminaria japonica. High Tech Lett 2005, 11, 91-94.

26. Wang, J.; Zhang, Q.; Zhang, Z.; Song, H.; Li, P. Potential antioxidant and anticoagulant capacity of low molecular weight fucoidan fractions extracted from Laminaria japonica. Int J Biol Macromol 2010, 46, 6-12.

27. Qi, H.; Zhang, Q.; Zhao, T.; Chen, R.; Zhang, H.; Niu, X.; Li, Z. Antioxidant activity of different sulfate content derivatives of polysaccharide extracted from Ulva pertusa(Chlorophyta) in vitro. Int J Biol Macromol 2005, 37, 195-199.

34. Witvrouw, M.; De Clercq, E. Sulfated polysaccharides extracted from sea algae as potential antiviral drugs. Gen Pharmacol 1997, 29, 497-511.

37. Harden, E.A.; Falshaw, R.; Carnachan, S.M.; Kern, E.R.; Prichard, M.N. Virucidal activity of polysaccharide extracts from four algal species against herpes simplex virus. Antiviral Res2009, 83, 282-289.

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47. Blondin, C.; Fischer, E.; Boisson-Vidal, C.; Kazatchkine, M.D.; Jozefonvicz, J. Inhibition of complement activation by natural sulfated polysaccharides (fucans) from brown seaweed.Mol Immunol 1994, 31, 247-253.

41. Groth, I.; Grunewald, N.; Alban, S. Pharmacological profiles of animal- and nonanimal-derived sulfated polysaccharides--comparison of unfractionated heparin, the semisynthetic glucan sulfate PS3, and the sulfated polysaccharide fraction isolated from Delesseria sanguinea. Glycobiology 2009, 19, 408-417.

42. Granert, C.; Raud, J.; Xie, X.; Lindquist, L.; Lindbom, L. Inhibition of leukocyte rolling with polysaccharide fucoidin prevents pleocytosis in experimental meningitis in the rabbit. J Clin Invest 1994, 93, 929-936.

39. Carlucci, M.J.; Scolaro, L.A.; Noseda, M.D.; Cerezo, A.S.; Damonte, E.B. Protective effect of a natural carrageenan on genital herpes simplex virus infection in mice. Antiviral Res2004, 64, 137-141.

43. Preobrazhenskaya, M.E.; Berman, A.E.; Mikhailov, V.I.; Ushakova, N.A.; Mazurov, A.V.; Semenov, A.V.; Usov, A.I.; Nifant’ev, N.E.; Bovin, N.V. Fucoidan inhibits leukocyte recruitment in a model peritoneal inflammation in rat and blocks interaction of P-selectin with its carbohydrate ligand. Biochem Mol Biol Int 1997, 43, 443-451.

45. Senni, K.; Gueniche, F.; Foucault-Bertaud, A.; Igondjo-Tchen, S.; Fioretti, F.; Colliec-Jouault, S.; Durand, P.; Guezennec, J.; Godeau, G.; Letourneur, D. Fucoidan a sulfated polysaccharide from brown algae is a potent modulator of connective tissue proteolysis.Arch Biochem Biophys 2006, 445, 56-64.

46. Parish, C.R.; Freeman, C.; Hulett, M.D. Heparanase: a key enzyme involved in cell invasion. Biochim Biophys Acta 2001, 1471, M99-M108.

44. Cumashi, A.; Ushakova, N.A.; Preobrazhenskaya, M.E.; D’Incecco, A.; Piccoli, A.; Totani, L.; Tinari, N.; Morozevich, G.E.; Berman, A.E.; Bilan, M.I.; Usov, A.I.; Ustyuzhanina, N.E.; Grachev, A.A.; Sanderson, C.J.; Kelly, M.; Rabinovich, G.A.; Iacobelli, S.; Nifantiev, N.E. A comparative study of the anti-inflammatory, anticoagulant, antiangiogenic, and antiadhesive activities of nine different fucoidans from brown seaweeds. Glycobiology 2007, 17, 541-552.

38. Damonte, E.B.; Matulewicz, M.C.; Cerezo, A.S. Sulfated seaweed polysaccharides as antiviral agents. Curr Med Chem 2004, 11, 2399-2419.

48. Clement, M.J.; Tissot, B.; Chevolot, L.; Adjadj, E.; Du, Y.; Curmi, P.A.; Daniel, R. NMR characterization and molecular modeling of fucoidan showing the importance of oligosaccharide branching in its anticomplementary activity. Glycobiology 2010, 20, 883-894.

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53. Leiro, J.M.; Castro, R.; Arranz, J.A.; Lamas, J. Immunomodulating activities of acidic sulphated polysaccharides obtained from the seaweed Ulva rigida C. Agardh. Int Immunopharmacol 2007, 7, 879-888.

49. Tissot, B.; Montdargent, B.; Chevolot, L.; Varenne, A.; Descroix, S.; Gareil, P.; Daniel, R. Interaction of fucoidan with the proteins of the complement classical pathway. Biochim Biophys Acta 2003, 1651, 5-16.

50. Tissot, B.; Daniel, R. Biological properties of sulfated fucans: The potent inhibiting activity of algal fucoidan against the human complement system. Glycobiology 2003, 13, 29G-31G.

51. Tissot, B.; Gonnet, F.; Iborra, A.; Berthou, C.; Thielens, N.; Arlaud, G.J.; Daniel, R. Mass spectrometry analysis of the oligomeric C1q protein reveals the B chain as the target of trypsin cleavage and interaction with fucoidan. Biochemistry 2005, 44, 2602-2609.

52. Tsuji, R.F.; Hoshino, K.; Noro, Y.; Tsuji, N.M.; Kurokawa, T.; Masuda, T.; Akira, S.; Nowak, B. Suppression of allergic reaction by lambda-carrageenan: toll-like receptor 4/MyD88-dependent and -independent modulation of immunity. Clin Exp Allergy 2003, 33, 249-258.

54. Nakamura, T.; Suzuki, H.; Wada, Y.; Kodama, T.; Doi, T. Fucoidan induces nitric oxide production via p38 mitogen-activated protein kinase and NF-kB-dependent signaling pathways through macrophage scavenger receptors. Biochem Biophys Res Commun2006, 343, 286-294.

55. Yang, J.W.; Yoon, S.Y.; Oh, S.J.; Kim, S.K.; Kang, K.W. Bifunctional effects of fucoidan on the expression of inducible nitric oxide synthase. Biochem Biophys Res Commun 2006,346, 345-350.

56. Do, H.; Pyo, S.; Sohn, E.H. Suppression of iNOS expression by fucoidan is mediated by regulation of p38 MAPK, JAK/STAT, AP-1 and IRF-1, and depends on up-regulation of scavenger receptor B1 expression in TNF-alpha- and IFN-gamma-stimulated C6 glioma cells. J Nutr Biochem 2010, 21, 671.

57. Kim, M.H.; Joo, H.G. Immunostimulatory effects of fucoidan on bone marrow-derived dendritic cells. Immunol Lett 2008, 115, 138-143.

58. Choi, E.M.; Kim, A.J.; Kim, Y.O.; Hwang, J.K. Immunomodulating activity of arabinogalactan and focoidan in vitro. J Med Food 2005, 8, 446-453.

59. Zhou, G.; Sun, Y.; Xin, H.; Zhang, Y.; Li, Z.; Xu, Z. In vivo antitumor and immunomodulation activities of different molecular weight lambda-carrageenans from Chondrus ocellatus.Pharmacol Res 2004, 50, 47-53.

60. Clement, M.J.; Tissot, B.; Chevolot, L.; Adjadj, E.; Du, Y.; Curmi, P.A.; Daniel, R. NMR characterization and molecular modeling of fucoidan showing the importance of oligosaccharide branching in its anticomplementary activity. Glycobiology 2010, 20, 883-894.

61. Nishino, T.; Yokoyama, G.; Dobashi, K.; Fujihara, M.; Nagumo, T. Isolation, purification, and characterization of fucose-containing sulfated polysaccharides from the brown seaweed Ecklonia kurome and their blood-anticoagulant activities. Carbohydr Res 1989,186, 119-129.

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63. Pomin, V.H.; Pereira, M.S.; Valente, A.P.; Tollefsen, D.M.; Pavao, M.S.G.; Mourao, P.A.S. Selective cleavage and anticoagulant activity of a sulfated fucan: Stereospecific removal of a 2-sulfate ester from the polysaccharide by mild acid hydrolysis, preparation of oligosaccharides, and heparin cofactor II-dependent anticoagulant activity. Glycobiology2005, 15, 369-381.

66. Bourgougnon, N.; Lahaye, M.; Quemener, B.; Chermann, J.C.; Rimbert, M.; Cormaci, M.; Furnari, G.; Kornprobst, J.M. Annual variation in composition and in vitro anti-HIV-1 activity of the sulfated glucuronogalactan from Schizymenia dubyi (Rhodophyta, Gigartinales). J Appl Phycol 1996, 8, 155-161.

64. Hiebert, L.M. Oral heparins. Clin Lab 2002, 48, 111-116

65. Ewart, H.S.; Bloch, O.; Girouard, G.S.; Kralovec, J.; Barrow, C.J.; Ben-Yehudah, G.; Suárez, E.R.; Rapoport, M.J. Stimulation of cytokine production in human peripheral blood mononuclear cells by an aqueous Chlorella extract. Planta Med 2007, 73, 762-768.

62. Nishino, T.; Nagumo, T. The sulfate-content dependence of the anticoagulant activity of a fucan sulfate from the brown seaweed Ecklonia kurome. Carbohydr Res 1991, 214, 193-197.

References

31

59

Marine lipids in amplifying cancers chemotherapy

Pr. Philippe Bougnoux - INSERM U 1069, Centre Henry S. Kaplan, University Hospital, Tours, France

[email protected] 1


Background

Tumor sensitivity to anticancer agents is highly variable

Associated with specific breast cancer pathological types (luminal type, basal-like type etc..)

Some of the variability is accounted for by the type of acquired somatic genetic alterations

or tumor-host interactions

or even the host-environment interactions

The prediction of tumor sensitivity to anticancer drugs remains uncertain

There must be dietary determinants of tumor chemosensitivity

How to identify potential dietary components associated ? 2


Overview

How was DHA identifed: an observational study in breast cancer DHA increases the sensitivity of breast cancer cell lines to anthracyclines Dietary DHA makes rat mammary tumors sensitive to anthracyclines DHA increases radiosensitivity of mammary tumors in rats

How to account for the tumor specificity of DHA action ? Loss of antioxidant defences during tumor progression Remodeling of tumor neovascular architecture

Translation: from the sea to the patient A phase I-II study of DHA supplementation in metastatic breast cancer during

chemotherapy In progress:... a randomized, phase III study

3

60


How was DHA identified as a lipid component of diet associated with tumor chemosensitivity ? An observational clinical study

Context: Breast cancer patients presenting with a tumor larger than 3 cm

receive neoadjuvant chemotherapy as their initial treatment

Some tumors will shrink, others will not

Hypothesis: If dietary lipids influence breast tumor sensitivity to chemotherapy, then patients with sensitive tumors should have past dietary intake of lipids different from that of patients with resistant tumors

4


The Adipose Tissue

Precise measure of dietary lipid intake difficult to establish

FA composition reflects past dietary intake of FA Low turnover : not influenced by the last meal

Use of adipose tissue fatty acid profile as a biomarker of past dietary intake of fatty acids

5


56 patients with locally advanced breast cancer (or T > 3 cm)

Adipose tissue fatty acid composition on biopsy

Neo-adjuvant chemotherapy (anthracyclins, cyclophosphamide, 5 FU)

Endpoint: Tumor response to neoadjuvant chemotherapy

DHA content of mammary adipose tissue is an independent predictive factor of tumor

chemosensitivity

0

1

2

3

4

5

median >

Odd

s R

atio

*

Adipose tissue DHA, (%)

15

20

Likelihood of response to chemotherapy *

*Adjusted for age, body mass index and tumour size; p=0.03

Bougnoux et al, Br J Cancer, 1999

Fatty acid profile

Study design

6

61


… Could DHA increase the sensitivity of breast cancer cell lines to anticancer

agents ?

DHA is associated with a greater efficacy of chemotherapy…

…causality ?

DHA, docosahexaenoic acid is a 22C, 6 double bonds PUFA In the diet, it is exclusively found in sea products

7


DHA enhances doxorubicin efficacy The effect is increased by prooxidant agents

and abolished by antioxidants Germain et al., Int J Cancer, 1998

Control Docosahexaenoic acid (DHA)

Prooxidants Antioxidants

Oleic acid

0 20 40 60 80 100

Cell Viability (%) Conditions

Doxo + DHA + Antioxidants Doxo+ oleic acid +Oxidants

Doxo + Oxidants *

* Doxo + DHA Doxo + DHA + Oxidants

Doxorubicin, 10-7 M (Doxo)

Culture medium enriched (5d) in DHA or oleic acid

Breast cancer cell line MDA-MB 231

Effect of DHA on chemosensitivity of MDA-MB 231

8


Fatty acids + oxidants

*

*

* *

*

Fatty acids + antioxidants

-10

10

30

50

70

90

% change in cell toxicity after doxorubicin

Fatty acids alone

*

*

*

Fatty acids , 10µg/ml

LA ALN n-3

AA n-6

EPA n-3

GLA n-6

DHA n-3 n-6

R 2 = 0,8293

0

20

40

60

80

100

0 10 20 30 40

hydroperoxides, pmol/µg proteins

Cyt

otox

icity

, %

… while producing lipoperoxides

PUFA enhance cytotoxic efficacy of doxorubicin…

Germain et al., Int J Cancer,1998 Doxorubicine is a quinone macromolecule

which generates an oxidative stress

What about other PUFA?

9

62


In vivo?

10


Dietary DHA is readily incorporated into adipose tissue

11 Olmix Algae Symposium - Sept. 10th, 2012 Olmix Algae Symposium Olmix AlgaeOlmix Algae Symposium

In-vivo dietary intervention with DHA

0

1

2

3

4 5

6

7 8

0.1

0.8

1.8

5.4

Palm DHA 0.1 g

DHA 0.3 g

DHA 0.8 g

Diet

Adipose tissue

Basal diet (7 % peanut/rapeseed) • Control (addition of 8 % palm oil) • DHA (addition of 8 % DHASCO)

•w or w/o antioxidants •w or w/o oxidants

Study design


Weeks

Cha

nge

in s

ize

of

Targ

et tu

mor

, %

Bougnoux et al, Lipids, 1999

Dietary DHA enhances the antitumor action of anthracyclins

DHA + oxidants

DHA

DHA + anti-oxidants

1 3 5

-40

-20

0

20

40

Palm oil

Epirubicin injections

The oxidative status of diet strongly influences the effect

Epirubicin efficacy according to dietary supplementation

12

63


Dietary DHA increases the sensitivity of mammary tumors to ionizing radiation

This effect is abolished by Vit. E

Tumor irradiation

Tum

or s

urfa

ce v

aria

tion,

(%)

-70 -60 -50 -40 -30 -20 -10

0 10

0 2 4 6 8 10 12 14 16 18

Days following irradiation

Palm oil

Diet :

Single dose, 18 Gy

DHA

Colas et al, Int J Cancer 2004

What about other anticancer treatments which generate ROS, such as radiation therapy ?

13

Study design • Dietary enrichment of rat tissues with DHA • Irradiation of mammary tumor with a single

dose of 18Gy • Measure of tumor regression according to diet

Context: Interaction of ionizing radiation with tissues H20 generates reactive oxigen species

Hypothesis: Dietary enrichment of rat tissues with DHA should make them more susceptible to lipid peroxidation induced by ROS


What about non tumor tissues ?


20

100

200mmHg

20

100

200mmHg

25mm/sec

60 50

40

30 20

10 0

LVDP (mmHg) Basal

+ pro-oxidant contrôle +anti-oxidant

60 50

40 30 20 10 0

LVDP (mmHg) After volume loading

+ pro-oxidant contrôle Diet with 15% fish oil

+anti-oxidant

Germain et al., Pharmacol. Res, 2003

Dietary omega-3 PUFA do not increase the cardiac toxicity of anthracyclins

Saline

ECG

Volume charge

Pressure transductor

Investigation of cardiac toxicity

15

64


Loss of antioxidant defences during tumor progression may account for the tumor specificity of DHA action

Maheo et al., Free Rad Biol & Med, 2004

Vibet et al., Free Rad Biol & Med, 2008

How to account for the tumor specificity of DHA action ?

1. Using two breast cancer cell lines at different degree of tumor progression, we found that the degree of tumor progression influenced tumor response amplification by DHA

2. DHA enhanced doxorubicine toxicity only in cell lines able to produce ROS in response to doxorubicine ….and to subsequently produce lipoperoxides

3. In these cell lines, there was a lack of GPx response to the oxidative stress

4. There was an inverse correlation between GPx1 activity and response of the cells to chemotherapy

16


Mechanisms other than ROS-induced lipid peroxidation?

… Is the antitumor action of docetaxel amplified by DHA ?

Docetaxel is a macrocyclic molecule without semi-quinone functions

17


MDA-MB-231

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.00

20

40

60

80

100 ControlDHAControl +Vit EDHA+VitE

Docetaxel nM

Cel

l via

bilit

y, %

… and the sensitizing effect of DHA to docetaxel

is not abolished by vit E

Breast cancer cell line MDA-MB 231

U 921 Tours – Unpublished data 18

Dietary DHA increases the antitumor action

of docetaxel

Chemotherapy with docetaxel Tumor Surface

- 4 - 3 - 2 - 1 0 1 2 3 4 5 6 7

- 70

- 60

- 50

- 40

- 30

- 20

- 10

0

10

20

Control

weeks

Docetaxel

varia

ti on

%

DHA

- 4 - 3 - 2 - 1 0 1 2 3 4 5 6 7

- 70

- 60

- 50

- 40

- 30

- 20

- 10

0

S. Vibet, PhD Theses Tours

Mammary tumors in rats

DHA sensitizes breast cancer cells to docetaxel …

65


DHA increases the activity of several drugs with distinct modes of action

19

The cancer cell Lipoperoxidation through amplified oxidative stress (Vibet et al, 2008)

Anthracyclins: doxorubicin, epirubicin (Germain et al, 1998, Hardman et al, 2001) Ionizing Radiations (Colas & Bougnoux, 2004)

Tubulin kinetics ? Vincristine (Ikushima et al, 1991), Vinorelbin (Menendez et al 2004),

NF-kappa B pathway ? Taxanes (Shaikh & Wahle, 2008) Restores apoptotic pathways ?

The host: Activation of reducing enzymes : Mitomycin C (Pardini et al, 1993) Alteration of drug metabolism through CYPs ..

Cyclophosphamide (Shao & Pardini , 1995)

The tumor microenvironment Generates secondary products with cytotoxic activities ? Changes the tumor microenvironment

- alters immune response against tumor cells (Calder et al, 2009) - alters angiogenesis, through lipid peroxidation ?


Investigating tumor neovascular architecture

20

Using Power-Doppler sonography of rat mammary tumors, sensitized with microbubles to quantify tumor vessels We found that dietary DHA led to decreased tumor vascularization, prior to any chemotherapy, and that effect of DHA was abolished by dietary Vit. E (Colas et al., Clin Cancer Res 2006)

Remodelling tumor neovessels may also account for the tumor specificity of DHA action

Denis et al., Clin Cancer Res 2003

Using polymer casts of tumor blood vessels, we found that fish oil diet induced remodeling of tumor vascularization with lower density and thiner blood vessels This led to decreased interstitial pressure within mammary tumors and increased diffusion

Goupille et al, Breast Cancer Res & Treat 2012


Translation : A pilot study of chemosensitization In metastatic breast cancer patients

Need for a proof of concept

21

66


Evaluation of the feasibility and safety of DHA administration during chemotherapy of metastatic breast cancer

Hypothesis: 1.8 g of daily DHA should lead to an enrichment of membrane lipids of breast cancer metastases, making them more sensitive to anthracyclin-based chemotherapy

Study design: Phase I-II study, with 25 patients – monocentric (Tours) – 2 years inclusion - Patients with metastatic breast carcinoma, OMS < 2 - DHASCO (Martek Inc) 9 cp/day, 10 days prior to chemotherapy, up to the end of CT - Chemotherapy with Epirubicine, Cyclophosphamide, 5-FU, 1 cycle every 3 weeks

End points: Safety & compliance, plasma + RBC levels of DHA Time to tumor progression

Secondary end points: Survival Bougnoux et al., Proc Am Ass Cancer Res, 2006

22


Interindividual variability in DHA incorporation after dietary intervention

Stratification into 2 groups

High DHA incorporators

Low DHA incorporators

DHA is rapidly incorporated after dietary intervention

Plasma level

Red Blood Cells

Results:



Elevated DHA incorporation during first line chemotherapy improves survival

Bougnoux et al, Br J Cancer 2009

Median follow-up = 18.6 months (range 3 to 53 months)

p<0.007 p<0.02

Survival according to the incorporation status of DHA

24

67


Vascularization ? The example of liver metastases



Discussion

Increasing plasma DHA level results in clinical benefit in patients undergoing chemotherapy for metastatic breast cancer

Supplementation with DHA is safe, and may reduce some side effects

The variability in DHA incorporation among patients may be circumvented by providing DHA within a food carrier and not as capsules of oil

26


No, this is premature PUFA should not be provided without defined clinical trials

A phase III randomized trial comparing the fatty acid to placebo is on-going in metastatic breast cancer patients to definitely assess the usefulness of fatty acid supplementation during chemotherapy

Finally, could dietary DHA or n-3 PUFA be provided safely during cancer treatments ?

Not with antioxidant molecules at pharmacological doses …may stimulate tumor growth

Effect may change according to the intake of other PUFA ?

EPA has been reported to alter immune response and promote melanoma growth in experimental systems

27

68


RAN

DO

MIZ

E

(Vegetal oil in food supplement)

Fish oil, 2 g / day

First line chemotherapy (anthracyclines, taxanes..)

Metastatic breast cancer

HER2 negative

RH positive

(Fish oil in food supplement) DHA 1.5 g / day

DHA 0 g / day

216 patients, 2 yrs inclusion

Dietary intervention, 4 to 6 months

Endpoints: Time to progression QoL Funding : PHRC 2011

15 cancer centers involved

The future… marine-derived lipids as adjuvant to breast cancer treatments Bougnoux et al, Prog Lipid Res 2010

ClinicalTrials.gov Identifier: NCT01548534

Aim of the study: To determine whether adjuvant nutrition improves the treatment of breast cancer (Dec 2011 - July 2015)

A randomized, double-blind, phase III multicentric clinical trial comparing fish oil supplementation versus vegetable oil during chemotherapy in metastatic breast cancer patients

28


Supports: Région Centre, French Research Ministry , DHOS, Inserm (ATC), INCa (Canceropôles)

Maastricht

• NICE

Participants

Inserm U 1069, Clinical oncology CHU Tours, France Charles Couet Caroline Goupille Marie-Lise Jourdan Emmanuelle Germain Séverine Colas Karine Mahéo Nawale Hajjaji

Oncology & Radiotherapy Olivier Le Floch Agnès Reynaud-Bougnoux

Surgical Gynecology Gilles Body Jacques Lansac

29

CIC Inserm 202, Tours Bruno Giraudeau

Tumor imaging Léandre Pourcelot François Tranquart Inserm U 930 Tours

Phase III randomized clinical trial Virginie Berger + CECO ICO-CPP, Angers France


30

69

CHILE Reliable Supplier of Quality Food

Eliana Henríquez Flores, Ing. Agrónomo Information Center of Natural Resources

Head of International Affairs Unit 1


Exceptional sanitary conditions: Phytosanitary Island

Southern hemisphere off-season agricultural production

Chile, long and narrow country, located in South America between latitudes 17 º 30 'and 56 º 30' south latitude.

Diversified Production: More than 5 000 km long, with 16 of the 24 climates present in the world.

5.1 million ha. arable land in an area of 75 million ha.

Population: 17,248,450 (13% rural)

Diversity of climates: Diversity production North: Desert West: Pacific Ocean East: Andes mountains South: Southern Ice

Chile

2


Chile

3

70


MINISTRY OF AGRICULTURE

Oficina de Estudios y Políticas Agrarias Servicio

Agrícola y Ganadero

Comisión Nacional de

Riego

Instituto de Desarrollo

Agropecuario

Corporación Nacional Forestal

Instituto de Investigaciones Agropecuarias

Fundación Comunicación Capacitación y Cultura del

Agro

Information

Center of Natural

Resources

Fundación para la

Innovación Agraria

Instituto Forestal

Agencia Chilena para la Calidad e Inocuidad

Alimentaria

Institutions of the Ministry of Agriculture

4


Information Center of Natural Resources

It is an institution that provides information on renewable natural resources, which has brought together the largest database of georeferenced soil, farms, water resources, agroclimate, fruit plantations and forest that exists in Chile, and other products are developed for different MINAGRI institutions.

5


General scenario of algae in Chile

In Chile, seaweeds are exported as raw material, used internally as alginates and agar and to a lesser degree, are also consumed as food.

The species that accumulate polysaccharides in their walls are a source of raw material for a variety of industrial products which the agar, carrageenan and alginates are the most used.

Seaweed synthesized polysaccharides in considerable amounts constituting an important renewable resource. A limited number of red and brown algae are exploited for the production of industrial application phycocolloids. It has been found that some seaweed sulfated polysaccharides have interesting biological properties such as anticoagulant activity, antitumor and antiviral.

The production of algae (gracilarias) borders the 90 000 t per year and there are only emerging projects that seek to increase diversity in the cultivation of these plants hydrobiological.

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SPECIE DESCRIPTION

Macrocrystis pyrifera The common name of this alga is "huiro" or "kelp". The contribution of this alga is in the production of alginates. In Chile extends from Cape Horn to Valparaiso. (SANTELICES, 1989).

Porphyra columbina Conocida como "luche rojo". Es explotado casi de toda la costa Chilena, comercializándose a nivel local para el consumo humano bajo la forma de precosido y seco. (RED ALGAS MARINAS CHILE, 1990).

Lessonia trabeculata Known as "red fight." Is exploited almost all the Chilean coast, locally marketed for human consumption in the form of pre-cooked and dry. (RED SEAWEED CHILE, 1990).

Ulva lactuca Its common name is "fight", "fight green" and "sea lettuce". One of the most common species off the coast of the Tenth Region of Chile. Consumption in the region is low, its main contribution of carbohydrates. (Ramirez, 1981.).

Gracilaria chilensis

Common name "nap". The main raw material used in production of Agar-Agar is unique to the genre Glacilaria. (SANTELICES, 1986). The main importing country of Chile is Japan, which absorbs about 80% of production. Coquimbo is the main area of export of this resource. (SANTELICES, 1990).

Durvillaea antartica

In our country known as cochayuyo. In Chile this plant is harvested for human consumption or for export as raw material for the production of alginates. Human consumption takes two forms. The conical stipe is consumed fresh, as part of salads or stews and is called "ulte".Fronds dry roasted and cooked as part of hot dishes, this is known as "cochayuyo". (SANTELICES, 1989). The main production areas are in regions VII and X. (RED, 1990).

FUENTE: http://www.angelfire.com/sd/Lasalgas/itcl001.html

Algae most important in Chile

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PRODUCT

VOL 2010 (Ton)

Value 2011 (USD Mill)

Algas (total) 65100 81,2

Lessonia (chascon) 52800

Macrocristis (Huiro) 3300

Gracilaria (pelillo) 2900

Carrageninas 5210 52,9

Agar Agar 2170 43,7

Alginatos 1756 20,6 Fuente: Intern. Trade Center – Trade Map

Description of exportable supply of Chile

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Chilean production of different algae and algal products for export FOB amount reached close to USD 200 million, with volumes close to the 75,000 tons.

Export supply of Chile, the most important product in volume are natural dried seaweed, especially the genus Lessonia. However, when analyzing the export earnings, are Derivatives from Algae who take the utmost importance and represent 59% of the returns.

Olmix Algae Symposium - Sept. 10th, 2012 Olmix Algae Symposium Tabla: Exportaciones de algas pardas 2010. FUENTE: Revista Aqua. Mayo 2011.

Description of exportable supply of Chile

The kelp industry has had a high growth worldwide and in this area, Chile contributes with 10% of the biomass. Of these, the ones with the greatest commercial importance are Lessonia nigrescens, Lessonia trabeculata, Macrocystis integrifolia and Macrocystis pyrifera Durvillacea Antarctic.

Some of these species are of great economic importance, since they are extracted aliginatos used in the pharmaceutical industry, food (animal and human), textile, biomedical and cultivation of land plants, among other things.

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China66%

Japan10%

France9%

Norway7%

Denmark3%

Others5%

Target Markets - ALGAE

Target markets: natural seaweed

10

The main destination markets for natural seaweed production in Chile in 2011 were China (with 67% of the tonnage shipped), Japan, France and Norway.

Different markets have virtually remained the volumes imported during the last 5 years, except China, which doubled its imports from Chile in this period.

Chile has had an average annual growth rate of 7% in these exports over the past 5 years.


Seaweed products

PHYCOCOLLOIDS

Definition: Colloids are complex polysaccharides capable of forming gels, viscous substances and stabilizers of suspensions, according to its concentration and type of colloid (agar, vegetable gums, starch, Peptinas, alginates, carrageenans, etc.). The FICO prefix means Algae (Latin).

Alginates or alginic acids: Alginic acid obtained from different types of algae (Macrocrystis, Lessonia, Fucus, etc.). Extracting with sodium carbonate and precipitating with acid treatment. The gels are formed in the presence of calcium, which must be added in a controlled manner to achieve the formation of ordered molecular associations are not reversible by heating, this property makes alginates unique among all gelling agents. It is used in canned vegetables and jams, confectionery, cakes and cookies and ice cream. Also used the development of cold meats, pates, dehydrated soups, to keep the pulp suspension in fruit nectars and soft drinks that contain as foam stabilizer in beer.

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Seaweed products

12

Carrageenan: Are obtained from various types of red algae (Gigartina, Gracilaria, Furcellaria). The carrageenans are acidic and form thermally reversible gels, and it is necessary to dissolve in hot. They are widely used in the preparation of desserts, and they interact very favorably with milk proteins. With concentrations of 0.025% of carrageenan, it can stabilized suspension and at around 0.15% already provide solid textures .

Agar-agar: In Chile It is obtained mainly from Gracilaria chilensis. Is a polysaccharide widely used in microbiology, as an excellent growth medium for microorganisms.

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Japan34%

USA20%

Russian Fed18%

Thailand7%

Others21%

Target markets - AGAR AGAR

Target markets: products of algae

13

Even when China is the largest importer of natural algae in Chile, that country is not important destination of the processed products derived from algae.

In the case of agar, and alginates, the main countries receiving exports from Chile are Japan and USA.

In the case of carrageenan, USA and Denmark are the main destinations of exports from Chile.

France is an importer of Natural Seaweed, alginates and carrageenan with a 9%, 6% and 8% respectively from the total export volumes from Chile.

Japan26%

USA20%

Brazil16%

Mexico12%

France6%

Others20%

Markets - ALGINATES

USA23%

Denmark22%

Mexico15%Spain

6%France

3%

Others31%

Markets - CARRAGEENAN


Producto

Precio prom (USD/Kg)

Rango según importador

Agar Agar (2010) 16,8 14,7 - 18,1

Carragenina (2011) 10,0 7,9 - 11

Alginatos (2010) 14,2 11,3 - 16,2

500

700

900

1 100

1 300

1 500

1 700

1 900

2007 2008 2009 2010 2011

Prices: ALGA NATURAL (USD/Ton)

China

Japan

France

Promedio

Export Price

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In 2011, the average export price of algae in the wild was 1.193/Ton USD (FOB).

This price has shown an average annual increase of about 11% over the past 5 years, however the trend over the past 2 years has been to stabilize.

Among processed products derived from seaweed, Agar Agar shows a higher average price per kg (USD 16.8), followed by Alginates and Carrageenan.


Conclusions from the point of view of Chile's export supply

1. There has been a sharp increase in world demand for Chilean supply of algae in their natural state and also for products derived from algae, due to its quality and export capacity in the country.

2. Chile has shown a very quick and efficient ability to increase their export volumes to meet rising world demand, yet there is great potential for further growth in the country.

3. The export supply in Chile is not only crude feedstock (algae naturally), but also products derived from algae, especially Agar Agar, Alginates and Carrageenan.

4. The export prices of Chilean Offer are convenient, especially for tariff benefits with importers, thanks to the many trade agreements Chile has signed with most countries.

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EMPRESA DIRECCION TELÉFONO WEB

Prodalmar Ltda. Calle General Velásquez 890 Of. 602, Antofagasta .

(55) 284148; (55) 224949

http://www.prodalmar.cl/

ALIMEX S.A. Isidora Goyenechea 3621 · Piso 17 · Las Condes, Santiago.

(56 2) 4301200 http://www.alimex.cl/espanol/

Guangjin Ltda. Doctor Johow 672, Santiago. (56-2) 2765935 http://www.bentalina.cl

Exportaciones M2 s.a. Melgarejo 750 Of. 74, Coquimbo. (56) - (51) 341758

Algas Cruz Alta S.A. Félix de Amesti 124, Of. 41, Santiago. (56-2) 2455757 www.algascruzalta.com

Costa Azul S.A. (32) 2972593 http://www.ipesca.co.cl/

Cultivos Acex S.A. Mardoqueo Fernández 156, Providencia, Santiago.

(2) 2331304

Algas Vallenar S.A. Vallenar: Panamericana Norte S/N Km. 665

MJ Chile Ltda. Vía 1 Sitio 2 Km.10 - Bajo Molle, Iquique. 412044

Pampamar S.A. Av. Las Condes 8060 Of. 203, Santiago. (56-2) 2297223 http://www.pampamar.cl/

Inversiones Kelp S.A. GENERAL VELASQUEZ 890, OF. 803, Santiago

(56-2) 3353309

CULTIVOS MARINOS CHILESUR S.A

Pacheco Altamirano, pasaje Canal Tenglo N° 2932, Puerto Montt

Terra Natur S.A. Hualqui (41) 2780987

Algae companies in Chile

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Chilean regulations

CHILEAN FOOD STANDARDS Chile has regularized within the aquaculture products, in the Chilean Norm 1857. Office 84, this standard applies to the algae commonly known in Chile for commercial purpose, with common and scientific nomenclature.

FOOD HEALTH REGULATIONS According to Health Regulations of food, derived from seaweed (phycocolloids) are considered as food additives as required by Title III, Paragraph I, Article 130, since they only meet a technological and nutritious. In Title III, Paragraph II The use of additives, Article 149 states that are allowed to use as thickeners and hydrocolloids with good manufacturing practices agar, alginate, ammonium, calcium and sodium, Carrageenan. In the provisions of Title II, Paragraph II of the labeling and advertising, Article 107, all food products must bear a label or tag on their packaging contain the following information: name of the product, net contents, name or business name and address of manufacturer, country of origin, number and date of the health decision, dates, and duration of the product, instructions for use and storage. (Food Health Regulations 1998).

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75

« Algae, source of active principles in cosmetics »

Alexis Rannou - Deputy Managing Director in charge of Innovation - ARD Soliance

1


La cosmétique Définition

[C’est une substance ou une préparation destinée à être mise

en contact avec les diverses parties superficielles du corps humain, notamment l'épiderme, les systèmes pileux et capillaire, les ongles, les lèvres et les organes génitaux externes, ou avec les dents et les muqueuses buccales, en vue, exclusivement ou

principalement, de les nettoyer, de les parfumer, d'en modifier l'aspect, de les protéger, de les maintenir en bon état ou de

corriger les odeurs corporelles.]

Source: article L.5131-1 du code de la santé publique 2


La cosmétique Composition d’un produit cosmétique

EAU 65-85%

Huiles 5-25%

• Ingredients & Actives 0,5-5% • Conservateurs 0,1-0,5% • Parfums 0,1-0,5%

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La cosmétique Structure de la peau

La peau: un organe de 2,5 m² constituée de 70% eau

Collagène + GAG

HA + Chondroitine sulfate

Epiderme: 13% eau

Derme: 57% eau

4


Pourquoi les algues ? Une biodiversité exceptionnelle

1. Les algues, un potentiel d’innovation

Microalgues/phytoplanctons : Des milliers d’espèces dans plusieurs classes taxonomiques, très diversifiés du point de vue phylogénétique :

• Procaryotes = Cyanobactéries (autotrophes) • Eucaryotes = Algues vertes, rouges, dorées et brunes

Macroalgues : Pluricellulaires, attachés à des substrats :

• Vertes • Rouges • Brunes

5

NB : Les plantes supérieures comprennent plus de 250 000 espèces dont la plupart sont comprises dans une seule classe



2. Une biodiversité de morphologies

Microalgues : • Seules ou en colonies (sous forment de chaine ou de filament) • Certaines sont mobiles grâce à leurs flagelles

Macroalgues :

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3. Une diversité des habitats et des environnements

Habitat : • Eau de mer • Eau saumâtre ou douce • Ou même les sols, les rochers et les arbres

Environnements : • Sources hydrothermales • Arctique et Antarctique • Zone aride • Forte pression osmotique • UV


Solution clarifiée (Sucres et

polysaccharides…)

Extraits peptidiques

Autres extraits (sels, pigments, oligoélements)

Extraits lipidiques


8

4. Grande disponibilité

• Sécurité d’approvisionnement (production: 14,7 Mn Tonnes de macroalgues en 2009)

5. Potentiel d’activités et de fonctionnalités

• Production de métabolites + fonction des conditions du milieu



polysaccharides…)


Autres extraits (pigments, sel, oligoélements)

Extraits lipidiques

Polysaccharides sulfatés Rhéologie HPM Activité biologique

MPM et BPM

Actions nutritives Actions peptides Amincissants, etc.

Antioxydants Zinc, Manganèse, etc.

Potentiel important sur AGPUFAs


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polysaccharides…)


Autres extraits (sels, pigments, oligoélements)

Extraits lipidiques

Potentiel important AntioxydantsActions nutritivesPolysaccharides sulfatés

5. Potentiel d’activités et de fonctionnalités

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Pourquoi les algues ? Une source d’innovation à haute valeur ajoutée

10

BIODIVERSITE ALGUES = ENORME POTENTIEL

Nombre d’espèces

Nombre de molécules

Conditions de culture

HAUTE VALEUR AJOUTEE


Un besoin marché Les algues comme matières premières cosmétiques

Macroalgues

Ingrédient clé de la réussite de la cosmétique marine française, unique et reconnue dans le monde entier (instituts, thalasso, spas, masques, etc.)

Excellente dermo-compatibilité des algues

Rôle de texturant

Source de tensio-actifs naturels.

Source d’ingrédients actifs • Concernant les actifs cosmétiques issus de phycocolloïdes, on trouve les oligo-alginates

présentant des actions anti-pollution, anti-éruption cutanée, anti-acné, et antivieillissement.

• Les fucoïdanes agissent sur la réduction du vieillissement de la peau et favorisent la croissance capillaire.

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Un besoin marché Les algues comme matières premières cosmétiques

Microalgues

Cosmétique : un concept biomimétique

Anti-âge : puissants antioxydants (caroténoïdes, groupe d'enzyme SOD, les tocophérols ou l'ascorbate)

Protection UV : Synthèse des pigments anti-UV mycosporine et scytonemine (cyanobactéries «de l’extrême» du genre Nostoc*)

Hydratation : Synthèse d’exopolysaccharides en cas de sécheresse ou en présence d’un environnement agressif

Les microalgues sont riches en PUFAs : ils représentent une grande ressource pour des applications cosmétiques

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- Sélection après études bibliographiques

- Différentes provenances : algothèques privées, partenariats,

- Isolement sur site et identification de l’espèce (par PCR)

- Différents milieux : marines, eaux douces et eaux saumâtres

Centre de Biotechnologie Marine Des labos de R&D

Souches de microalgues

13


- Adaptation des souches au laboratoire

- Optimisation des paramètres de culture

- Cultures des microalgues jusqu’au stade du photobioréacteur de 300L

Centre de Biotechnologie Marine Un savoir-faire

14


Photobioréacteurs de 2100L

2 Bassins de 55 m3

Récolte par ultracentrifugation

+ Partenariats avec des sites de production industriels (capacités > 1 T MS)

Centre de Biotechnologie Marine Une unité de validation industrielle et de production

15

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Quelques exemples Les algues comme matières premières cosmétiques

16

Quelques exemples d’actifs d’origine marine


Soliance : Wakamine

17

Wakamine

Eclaircissez votre peau

Puissant actif blanchissant d’origine marine


La pigmentation de la peau résulte de la présence de mélanine dans l’épiderme.

Wakamine Contexte biologique

18

Deux types de mélanine : • l’eumélanine, brun-noir, • et la pheomélanine, jaune-orange.

La tyrosinase est l’enzyme indispensable pour les deux premières étapes de la mélanogénèse. Wakamine est un actif blanchissant performant Il inhibe la tyrosinase, l’enzyme clé impliquée dans la production de mélanine.

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Plusieurs noms lui sont donnés : Chine : Qun dai cai (Lembi & Waaland 1988)

Angleterre : Sea mustard (Kirby 1953), Precious sea grass (Rhoads & Zunic 1978)

Japon : Wakame (Madlener 1977)

Corée : Miyok (Madlener 1977), Miyeouk (Lembi & Waaland 1988)

Source : Algaebase.org

Alanine 7.6%

Arginine 5.7%

Acide aspartique 9.5%

Cystine 2.3%

Acide glutamique 12.4%

Histidine 5.9%

Isoleucine* 4.3%

Leucine* 8.0%

Lysine * 3.5%

Methionine* 3.4%

Proline 5.9%

Serine 4.7%

Tryptophane* 1.5%

Threonine* 5.0%

Tyrosine 3.7%

*Acides aminés essentiels

Wakamine Undaria pinnatifida

19

Wakamine est extrait d’une macroalgue brune, Undaria pinnatifida, de l’ordre des Laminariales.

Cette algue présente un profil unique en acides aminés, avec plus de 42% d’acides aminés essentiels.


Cette algue fut introduite en France en 1971 en Méditerranée (étang de Thau) et par IFREMER sur les côtes Bretonnes en 1983.

Wakamine Origine géographique

20

Undaria pinnatifida : Originaire des baies de la mer du Japon à l’ouest d’Hokkaido ainsi que des baies Coréennes et Chinoises, elle est traditionnellement cultivée et largement utilisée dans la cuisine asiatique.


Wakamine Culture de l’algue

21

Undaria pinnatifida est récoltée dans ses habitats naturels depuis des siècles, elle est également cultivée. Sa production est estimée entre 450 000 et 500 000 tonnes au Japon et en Corée.

Culture de l’algue sur l’île d’Ouessant Cette macroalgue est aujourd’hui cultivée sur l’île d’Ouessant en Bretagne, un site classé par l’UNESCO.


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En inhibant la tyrosinase, Wakamine limite la production de mélanine

Wakamine Mécanisme d’action

22


Wakamine inhibe la production de mélanine : -39%

Témoin: les mélanocytes sont visibles

Wakamine (100mg/ml) : Les mélanocytes sont légèrement

visibles

Wakamine Inhibition de la production de mélanine (test ex vivo)

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Protocole Les épidermes sont obtenus à partir d’explants de peau. Ils sont séparés après incubation dans du NaBr 2N pendant 100 minutes à 37°C. Les épidermes sont ensuite fixés avec une solution tampon de formaldehyde, rincés et traités avec un mélange de l’actif (100μg/ml) et d’une solution de DOPA (1mg/ ml) pendant 4 heures à 37°C. Après incubation, les épidermes sont rincés et visualisés au microscope. Une analyse quantitative est réalisée par traitement d’images.


Wakamine aussi efficace que l’Arbutine (test in vivo)

24

Protocole Une solution de Wakamine 3% est appliquée sur les explants de peau à l’aide de patchs de papier Whatmann une fois par jour pendant 6 jours. Les explants sont fixés dans le formol et montés dans la paraffine. La coloration due à la mélanine sur les coupes est traitée selon la technique de Masson. L’intensité de couleur est calculée par analyse d’images.

Wakamine est plus performant que l’Arbutine (référence quasi drug au Japon)

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Wakamine éclaircit la peau (test ex vivo)

25

-10

-8

-6

-4

-2

0J0 J28 J56

Varia

tion

de

l'Ind

ex m

élan

ique

(eff

et e

clairc

issan

t)2% Arbutine

1% Arbutine + 1% Wakamine

* p< 0.001 Chaque valeur de l’index mélanique est significativement différente par rapport à la valeur de J0.

* *

La diminution est effective pour 91% du panel

Protocole L’efficacité éclaircissante de Wakamine a été évaluée sur un panel de volontaires de type asiatique (22 volontaires, d’âge moyen 43 ans). Les panélistes appliquent 2 fois/jour pendant 56 jours.

En remplaçant 1% d’arbutine par 1% de Wakamine, des résultats in vivo similaires sont obtenus.

Wakamine permet de rendre vos formules plus innovantes


Soliance Microalgue - Phaeodactylum tricornutum

MEGASSANE Protection ciblée des protéines contre les rides

26

Extrait d’une microalgue appartenant à la famille des Diatomées.

Microalgue unicellulaire très répandue dans les eaux côtières et saumâtres des zones tempérées

Actif breveté (prix nobel) - Anti-photovieillissement

• Maintien de l’homéostasie de la peau • Purification des cellules • Nouvelle approche pour lutter contre le vieillissement.



Mécanisme d’action : Détoxifications des cellules Mégassane stimule les trois activités du

protéasome et les restaure après exposition aux UV pour limiter l’oxydation des protéines

Megassane est recommandé dans : Des produits anti-âge Protections solaires Gammes de soins purifiants/détoxifiants

27

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28

Stimulation des activités du protéasome (test in vitro) Protocole Les kératinocytes sont traités avec 2,5 μg/ml d’extrait de P.tricornutum pendant 24 h. Les activités peptidases du protéasome sont évaluées dans le lysat cellulaire en utilisant 25 μM de LLVY-amc, 150 μM de LLE-na, et 40 μM de LSTR-amc comme substrat peptidique fluorescent avec 20 μg de protéines totales.

En stimulant les activités du protéasome, Megassane diminue le taux de protéines oxydées

Diminution du taux de protéines oxydées (test in vitro) Protocole Un Oxyblot est réalisé avec 10 μg de protéines issues du lysat de kératinocytes, cultivés 7 h après traitement avec 2,5 μg/ml d’extrait de Phaeodactylum.



29

Restauration des activités du protéasome après exposition aux UV Protocole Une culture de kératinocytes est irradiée par des UVA UVB et/ou traitée par de P.tricornutum pendant 24 h. Les activités peptidases du protéasome sont évaluées dans le lysat cellulaire.

En restaurant les activités du protéasome Megassane répare les protéines endommagées par les UV

Réparation des dommages protéiques dus aux UV Protocole Une culture de kératinocytes, préalablement exposée aux UV, est traitée avec 2,5 μg/ml d’extrait de Phaeodactylum. Un Oxyblot est réalisé avec les protéines issues du lysat.


Complexes Argilo-Algales Brevet Mondial Amadéite®

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Revertime®

Antirides et anti-âge exceptionnel


Revertime® Antirides et anti-âge exceptionnel

Forte activité anti-oxydante protégeant efficacement la peau des attaques radicalaires occasionnées par le stress de la vie courante :

32

- 52% * de réduction de réaction anti radicalaire (* à 0,25%)



33

Activité anti-élastastique hors du commun : pouvoir inhibiteur de l’élastase (contribue à la dégradation des fibres élastiques):

- 100 %* de réduction de la dégradation des fibres élastiques (* à 0,1%)

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34

- 64% * synthèse collagène type 1

(* à 0,01%)

Forte production de pro collagène de type 1 l’un des constituants majeurs du derme :



35

La surface et le volume des rides : -12,3 % L’amplitude et la rugosité moyenne du tissu cutané : -5,5 et -2,6 %

In vivo, effet sur les rides de la pate d’oie après seulement 28 jours, réduction significative de :



Un actif antirides et anti-âge exceptionnel. Très facilement incorporable en formulation, Revertime® propose une solution efficace et pertinente dans la lutte contre le vieillissement cutané.

36

87


Emultime®

Auto Emulsifiant naturel, technologie à froid


Emultime® Auto Emulsifiant naturel, technologie à froid

Sous simple agitation à froid ou à chaud, Emultime® permet d’obtenir des mélanges « Eau/huile » homogènes et stables

Gain énergétique considérable par rapport à une émulsion classique à chaud

Mise en œuvre plus rapide et process simplifié : temps de production divisé par 4

100% naturel : approche écologique de la formulation cosmétique

Permet de réaliser des laits et des crèmes cosmétiques avec des textures originales

38


Conclusion

39

Les algues sont les MP cosmétique

du présent et du futur car elles ont

un véritable potentiel de découverte

en terme d’origine, d’activité et de

fonctionnalité.

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« Algae, source of nutriments for humans »

Dr Maria Hayes - Researcher and Principle Investigator of NutraMara Biodiscovery and characterisation and Scientific Programme Manager (2008-2012) -

Teagasc - The Irish Agricultural and Food Development Authority

1


Teagasc Food Research Centres

Teagasc Nutraceutical Research Facility

Introduction

2


State of the Art Teagasc represents in Ireland

3

“Provide a resource base of knowledge and skills to stimulate the innovation function”

Teagasc Ashtown Food Research Centre Teagasc Moorepark Food Research Centre

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Challenges facing stakeholders

4

Legal restrictions, high costs and environmental problems regarding the disposal of marine processing wastes.

European legislation (EU Council Directive 1999/31/EC, 1999) specific targets for disposal at landfills.

The Marine Functional Foods Research Initiative focuses on three main marine resources (i) fish processing waste streams, (ii) seaweeds and microalgae and (iii) aquaculture sources.

The Marine Functional Foods Research Initiative aims to exploit these marine resources for functional foods/ ingredients development.

Identification of bioactive compounds to the dietary intervention level in order to make a health claim.


Very diverse pool of potential food ingredients from aquatic sources

5


Significant interest in marine proteins

6

Fish protein Gelation

Water binding

Solubility

Emulsification Foaming

90


Market Trends The global market for functional foods $174 billion.

Functional foods and nutraceuticals offer opportunities for the agri-food sector… to become the ‘pharmacy for disease prevention’.

7


Key Areas based on Market and Scientific Opportunity



Traditional use of Seaweed in Ireland and worldwide

During Potato famine seaweed source of nourishment.

Sluichèan: a seagrass.

High nutrient content.

Dulse Bread: red seaweed.

Seaweed : in Asian countries for centuries in foods, such as sushi.

9

Dulse Bread

Sluichèan

91


Traditional use of Seaweed

“Nori” - Porphyra sp. (Sushi)

“Habi-Nori” - Petalonia binghamiae (Eaten dried and roasted).

“Hijiki” - Hizikia fusiforme (Soups, salads, vegatable dishes).

“Kombu” - Laminaria sp. (dashi soup)

“Wakame” - Undaria pinnatifida (Salads, soups).

10


Harvesting To the Dish

Animal products

Traditional use of Seaweed

11


(FAO, 2010)

Macroalgae production world-wide

12

Harvested - 1,045,000 T ww Cultivated - 15,781,159 T ww

CHINA, top seaweed producer • CHINA 62.8% • INDONESIA 13.7% • PHILIPPINES 10.6% • REPUBLIC OF KOREA 2.8% • JAPAN 2.9%

JAPAN is the second most important producer in terms of value = NORI production.

CHILE 21,700 T

Saccharina japonica, CHINA

92


Open-water system in Canada (IMTA)

Macroalgae production world-wide

13


New derivatives

Development of

extractions

Defining activities

Marketing and product development

Proteins, peptides and amino acids

Seaweed sources H H H H

Seafood sources H H H H

Carbohydrates

Chitin and chitosan L M H H

Agar L L L M

Carrageenan L L M M

Alginate L L L M

Fucoidan H H H H

Laminaran M M M M

Ulvan M M M M

Antioxidants M H H H

Omega-3 fatty acids M H M H

Innovation potential summary

14

H = high potential


Algal bioactive ingredients

15 HO

OH

HO

O

HO

OHOHO

O

HO

HO

HO

OHHO

n

Antioxidants

Blood pressure Cancer Inflammation Obesity Diabetes Mental health

Laminarin

Fucoidan

LIPIDS

93


Bioassays : Renin Inhibition

Aspartyl protease.

Sodium depletion, decreased blood volume and blood pressure and β-adrenergic stimulation.

Angiotensin II inhibits renin secretion and has a number of physiological effects.

Cleavage of angiotensinogen by renin is the rate determining step in the RAS.

Inhibition of renin attractive strategy for control of hypertension.

16

RAAS system


Platelet Activating Factor – Acetylhydrolase (PAF-AH) Inhibitors

Plasma PAF-AHs are located cytosolic and plasma.

Selective for phospholipids with very short acyl groups.

Linked to atherosclerosis and may be a positive risk factor for coronary heart disease in humans.

Screening for inhibitors of this from seaweed and marine extracts and bovine muscle extracts.

Process of atherosclerosis

Platelet-activating factor acetyl-hydrolase 17

Olmix Algae Symposium - Sept. 10th, 2012 18

Bioactive peptides sources

Marine by-products and algal sources…

Olmix Algae Symposium - Sept. 10th, 2012 Olmix Algae

94


Generation of bioactive peptides

Characterisation – UPLC & MS

Freeze-dry

Freeze-dry

Extraction of macroalgal, microalgal or marine by-product proteins

Hydrolysis with proteolytic enzymes

Purification RP-HPLC

Test hydrolysates for PAF-AH, PEP, ACE-I and renin inhibition

UF 10kDa MWCO/3kDa MWCO

19


Background

Hypertension is one of the major yet controllable risk factors in CVD but it can be controlled by inhibition of a number of enzymes in the Renin Angiotensin System (RAS).

20


Background

Platelet activating factor acetylhydrolase (PAF-AH) is a circulating enzyme produced and secreted by inflammatory cells centrally involved in atherosclerosis.

It generates two key pro-inflammatory mediators, lysophosphatidylcholine (LPC) and oxidized nonesterified fatty acids (oxNEFAs).

Evidence exists for a regulatory role of these lipids in promoting atherosclerotic plaque development that can ultimately lead to the formation of a necrotic core, a key determinant in atherosclerotic plaque vulnerability.

21

95


Algal proteins and peptides inhibit Renin

Isolation of protein from Palmaria palmata

Generation of peptides using food grade enzymes

Enrichment of peptide fractions and purification of peptides

Characterisation of bioactive peptides (bioassay and UPLC-MS)

22


Protein extraction

Crude protein was extracted from Palmaria palmata using the method previously described by Galland-Irmouli.

Following a cold water extraction the protein fraction of was precipitated with ammonium sulphate.

23


Generation and enrichment

24

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

Fr10 Fr11 Fr12 Fr13 Fr14 Fr15 Fr16 Fr17 Fr18 Fr19 Fr20 Fr21 Fr22 Fr23 Fr24 Fr25

RP-HPLC fractions

Perc

enta

ge In

hibi

tion

RP-HPLC

Renin bioassay 58.97 % inhibition (+/- 1.26) IC50 0.32mg/ml

F10-F25

96


Sequence Phylum Protein (assession number) Species sharing 100% homology

Calculated mass

Observed mass

Charged state

D.IRLIIVLMPILMA.A Rhodophyta Photosystem II protein Y (O19893)

Cyanidium caldarium,Galdieria sulphuraria 1494.93 499.29 (+3)

MNEIVALMI.I Rhodophyta Cytochrome b6-f complex (Q85FX8) Cyanidioschyzon merolae 1032.53 517.26 (+2)

P.ILMA.A Rhodophyta Photosystem II protein Y (O19893)


I.LMAASWAIY.N Rhodophyta Photosystem II protein Y (O19893)


Q.ILPSILVPLV.G Rhodophyta Photosystem I reaction center subunit VII (P58214) Chlorella vulgaris 1062.7 532.32 (+2)

L.PSIL.V Rhodophyta Photosystem I reaction center subunit VII (P58214) Chlorella vulgaris 428.26 429.09 (+1)

I.LVPLVGLV.F Rhodophyta Photosystem I reaction center subunit VII (P58214) Chlorella vulgaris 808.54 809.4 (+1)

V.PLVGLVFPAI.A Rhodophyta Photosystem I reaction center subunit VII (P58214) Chlorella vulgaris 1024.63 1025.48 (+1)

L.VFPAIAM.A Rhodophyta Photosystem I reaction center subunit VII (P58214) Chlorella vulgaris 747.39 748.4 (+1)

V.FPAI.A Rhodophyta Photosystem I reaction center subunit VII (P58214) Chlorella vulgaris 446.25 447.12 (+1)

F.PAIA.M Rhodophyta Photosystem I reaction center subunit VII (P58214) Chlorella vulgaris 370.22 371.11 (+1)

Peptide identification by tandem mass spectrometry

25


Renin inhibition assay of synthesised peptides

Sequence Renin inihibition +/-

D.IRLIIVLMPILMA.A 51.38 3.5

MNEIVALMI.I - -

P.ILMA.A 29.34 4.59

I.LMAASWAIY.N - -

Q.ILPSILVPLV.G 24.56 3.56

L.PSIL.V - -

I.LVPLVGLV.F 15.35 2.16

V.PLVGLVFPAI.A 17.5 3.21

L.VFPAIAM.A - -

V.FPAI.A - -

F.PAIA.M - -

26

IC50 (mM)

3.34

Fitzgerald & Hayes, 2012, J. Agriculture and Food Chemistry


Bioassays: PAF-AH inhibition

PAF-AH is an enzyme that converts PAF to the biologically inactive lyso-PAF.

PAF is a biologically active phospholipid that is involved in activation of platelets, monocytes, macrophages, polymorphonuclear leukocytes.

PAF-AH is linked to atherosclerosis and may be a positive risk factor for coronary heart disease in humans.

27

Figure 1: PAF-AH inhibitory activities presented as percentage inhibitory values for the trout myofibrillar protein thermolysin 10-kDa-UFH RP-HPLC fractions.

PAF-AH inhibition by RP-HPLC fractions generated from trout myofibrilar muscle hydrolysed with thermolysin and filtered

through a 10-kDa membrane filter

020406080

100120140

MAFP control F22 F10 F26 F25

Sample name

% P

AF-

AH

inhi

bitio

n

97


Bioassays: PEP inhibition

0102030405060708090

100

Berberine Trout fullhydrolysate

3kDa trout fitrate 10kDa troutfiltrate

Inhibitory sample name

% In

hibi

tion

of B

SA

28

Enzyme prolyl endopeptidase (PEP) plays a role in the degradation and metabolism of biologically active peptides containing proline such as oxytocin, vasopressin, substance P, bradykinin, neurotensis, and angiotensins .

Important biological functions in organs such as the brain and have been implicated to play a role in the development of neurodegenerative conditions such as AD.

Furthermore, specific inhibitors of PEP have anti-amnesic effects, and some of them have been synthesized as anti-amnesic drugs.

Figure 2: PEP inhibitory activities presented as percentage inhibition values for the trout myofibrillar protein thermolysin hydrolysates, 10-kDa and 3-kDa filtrates.


Mass spectrometry analysis and determination of peptide amino acid sequences

0

20

40

60

80

100

%Int.

800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000Mass/Charge

1[c].M22

45 mV[sum= 7655 mV] Profiles 1-171 Unsmoothed

Data: Actin 2 Hours0001.K19 16 Jan 2008 20:36 Cal: 160108_2 16 Jan 2008 20:23 Kratos PC Axima CFRplus V2.4.1: Mode reflectron, Pow er: 79, Blanked, P.Ext. @ 3000 (bin 132)

1827.83 {r4723}

2482.81 {r7464}

2394.94 {r8578}

720.08 {r6353}2406.82 {r4960}

1433.66 {r3009}1021.47 {r4537}2221.90 {r7462}1458.54 {r6203} 2504.81 {r5608}716.56 {r11121} 1093.47 {r3743} 1823.74 {r7250} 2866.00 {r9638}2218.80 {r10980}

29

Fingerprint Q-TOF MS and UPLC system


Peptide characterisation

Protein identified using Protein Lynx Global Server 2.4

and TurboSEQUEST

Peptide sequence Position Peptide mass (Da)

Charge state

Protein Lynx Global Server 2.4 & peptide

source

TurboSEQUEST and peptide source

Hemoglobin subunit beta

FGKEFTPVLQADFQK 117-131 1754.911 (3+) 10-kDa_UFH Full hydrolysate


FGDLSTADAVMNNPK 44-58 1579.742 (2+) 10-kDa-UFH RP-HPLC fraction 3 RP-HPLC fraction 4 RP-HPLC fraction 6


LHVDPENFKL 95-104 1211.641 (2+) 10-kDa-UFH RP-HPLC fraction 3

Hemoglobin subunit alpha

VLSAADKGNVKA 2-13 1172.663 (2+) 10-kDa-UFH Full hydrolysate

Catalase FSDVHPEYGSR 482-492 1293.585 (2+) 10-kDa-UFH Full hydrolysate

Catalase AQKPDVLTTGGGNPVGDKLNS 21-41 2068.066 (2+) 10-kDa-UFH Full hydrolysate, RP-HPLC fraction 3

Catalase AAQKPDVLTTGGGNPVGDKLNS 20-41 2139.104 (2+) 10-kDa-UFH Full hydrolysate

Catalase LVQDVVFTDEMAH 51-63 1503.714 (2+) 10-kDa-UFH Full hydrolysate

Fatty acid-binding protein

VGMPDDIIQKGKD 22-34 1415.719 (2+) 10-kDa-UFH Full hydrolysate

30

**Mora & Hayes, 2012, Identification of Peptide Sequences and Bioactive Characterization of Rainbow Trout Thermolysin Hydrolyzate Fractions with in vitro Inhibitory activities against Enzymes Important in Heart and Brain Health (Accepted in the Journal of Agriculture and Food Chem)

98


EPA (20:5 n-3)

Linolenic acid (18:3 n-3)

Palmitic acid (16:0) Palmitoleic acid (16:1 n-7)

Fatty acids Reduce risk of cardiovascular diseases

Possible health effects

Oleic acid (18:1 n-9) Antioxidant activity

Antioxidant activity

Antimicrobial activity Reduce risk of cardiovascular diseases

DHA (22:6 n-3) Improve childhood cognitive development

Reduce risk of old-age diseases such as Alzheimer Cancer prevention

Antiviral, antitumoral, antihyperlipidemia and anticoagulant

Sulfated polysaccharides

Insoluble fibers

Polysaccharides Possible health effects

Reduce total and LDL cholesterol

Potential functional products

31


Generate accelerated solvent extracts (ASE®) from algal species

32

(5 min.)(5 min.)

Flush with fresh solventFlush with fresh solvent(0.5 min.)(0.5 min.)

Extract readyExtract ready(total time, 12(total time, 12

Load cellLoad cell

Fill with Fill with solvent(ssolvent(s))(0.5(0.5--1 min.)1 min.)

Static extractionStatic extraction(5 min.)(5 min.)

Flush with fresh solventFlush with fresh solventFlush with fresh solventFlush with fresh solventFlush with fresh solvent(0.5 min.)(0.5 min.)(0.5 min.)(0.5 min.)

Flush with fresh solventFlush with fresh solvent(0.5 min.)(0.5 min.)

Extract readyExtract readyExtract readyExtract readyExtract ready(total time, 12(total time, 12(total time, 12(total time, 12

Extract readyExtract ready(total time, 12(total time, 12--14 min14 min.)

Heat and pressuriseHeat and pressurise(5 min.)(5 min.)

Purge with nitrogenPurge with nitrogen(1(1--2 min.)2 min.)


Hydrophilic ASE® extracts generated using ethanol: water 70:30 (v/v)

Code Name Date Location Sample weight

ISCG0365 Ascophyllum nodosum 08/02/2012 Finavara Co. Clare 100g DW

ISCG0238 Fucus vesiculosus 16/06/2011 Finavara Co. Clare 100g DW

ISCG0259 Fucus serratus 18/07/2011 Spiddal, Co. Galway 100g DW

ISCG0239 Fucus spiralis 16/06/2011 Finavara Co. Clare 100g DW

ISCG0257 Laminaria digitata 18/07/2011 Spiddal, Co. Galway 100g DW

ISCG0356 Ulva Intestinalis 24/01/2012 Spiddal, Co. Galway 750g WW

ISCG0355 Pelvetia caniculata 24/01/2012 Spiddal, Co. Galway 750g WW

ISCG0223 Fucus vesiculosus 08/06/2011 Golf Course, Galway 34 Kg WW

ISCG0072 Fucus vesiculosus 16/04/2012 Spiddal, Co. Galway 220g DW

ISCG0070 Cystoseira nodicaulis 30/03/2010 Finavara Co. Clare 42 g DW

ISCG0029 Codium fragile 02/02/2009 Spiddal, Co. Galway 15.75 g DW

ISCG0071 Palmaria palmata 04/11/2010 Spiddal, Co. Galway 70 g DW

33

99


Using NMR for metabolite screening

34 Test extracts for antioxidant activities


Using 2D-NMR for metabolite identification

35


Lipophilic ASE® extracts generated using chloroform: methanol 2:1 (v/v)

Code Name Date Location Sample weight

ISCG0356 Ulva intestinalis 08/02/2012 Finavara Co. Clare 100g DW

ISCG0269 Pelvetia canaliculata 16/06/2011 Finavara Co. Clare 100g DW

ISCG00223 Fucus vesiculosus 18/07/2011 Spiddal, Co. Galway 100g DW

ISCG0324 Fucus spiralis 16/06/2011 Finavara Co. Clare 100g DW

ISCG0353 Ascophyllum nodosum 18/07/2011 Spiddal, Co. Galway 100g DW

ISCG00283 Cytoseira tamariscofolia 24/01/2012 Spiddal, Co. Galway 750g WW

ISCG0029 Codium fragile 02/02/2009 Spiddal, Co. Galway 15.75 g DW

ISCG0071 Palmaria palmata 04/11/2010 Spiddal, Co. Galway 70 g DW

36

100


Lipophilic ASE® extracts generated using chloroform: methanol 2:1 (v/v)

Extraction conditions

5 min preheat time 1,500 psi 120ºC Heat time 5 minutes Flush volume, 50 % of cell volume Purge time 60 s Static cycle : 4

37


ASE® extracts have been enriched for phlorotannins

Fucus serratus ISCG0068 ASE® M16 (5C)

Ascophyllum nodosum ISCG0032 ASE® M15 (1B)

Fucus vesiculosus ISCG0023ASE® M17 (3B)

A nodosum ISCG0032 ASE® M17 (9B)

C. nodcaulis ASE® M17 (10B)

The figure in brackets refers to the phlorotannin enriched extract.

Phlorotannin enriched extracts were examined using Q-TOF-MS.

38


UPLC-Q-TOF-MS of ASE® enriched phlorotannin extracts

Heptamer of phloroglucinol identified but not pure (745.1).

Also contains Iodine (126.9).

Quinic acid derivative of Phloroglucinol (316).

Fingerprint of all samples obtained (10 in total).

Further purification required.

39

101


Impact of Research

40

Support small/medium sized

enterprises

They would provide a novel source of bioactives for inclusion into Irish diets.

Improve Irish publics health

May help lower the rate of high blood pressure.

Contribute new ingredients to the growing Irish and

International functional food industry

May encourage people to buy indigenous food products.

Ensure protection of the Irish coastline.

Health-promoting bioactives from

seaweed

Environmental benefits


41

102

« Algae, a “new” nutrient resource for human food »

Christine LE TENNIER - C.E.O - Globe Export - Rosporden

1


Founded on pioneering spirit and internationally oriented, Globe Export has specialised in the sale and transformation of nutritional marine plants for more than 25 years.

Used for ages, these plants are to expand their role in human nutrition over the next 15 years, thanks to their natural nutritional properties.

We develop healthy, nutritional, and tasty products which exceed expectations in terms of taste and nutrition.

High in protein, trace-elements and omega 3, combined with a low fat content, seaweed is a food source which can’t be beaten.

We cultivate and promote innovation & originality, thereby driving our development.

Algae: A “new” nutrient resource for human food

Globe Export, a vision for a future based on nutritional marine plants

2


Based in Brittany, in an unspoiled setting including one of the largest natural seaweed fields in the world, providing extremely high-quality raw materials, Globe Export covers all aspects of the food production process

Small-scale production / trade: Algues de Bretagne®

Research and development / innovation: Creativ'Concept®

Algoculture section department: Globe Sea Garden®

The key figures:

3 industrial sites (seaweed, pasta, molecular cuisine) 100 SKUs 20 employees 2 Millions € T.O. (2011)


3

Globe Export, a vision of a future based on nutritional marine plants

103


Dulse Palmaria palmata

Haricots de mer Himanthalia elongata

Laitue de mer Ulva lactuca

Nori Porphyra ombilicades

Wakamé Undaria pinnatifida

Kombu breton Laminaria digitata

With Algues de Bretagne, you can go beyond traditional nutrition.


4

Algues de Bretagne is the specialist in edible seaweed, with a wide range of products providing a plethora of tastes and ensuring high nutritional value.



With its three separate ranges, Algues de Bretagne brings its specialist’s expertise to the table.

1. Seaweed range : Raw material for niche products: Seaweed tartare, marine spices, glasswort and sea ”beans”, fish rillettes with seaweed, seaweed chutney, fresh salted seaweed, fish soup and seaweed toast

5



6

2. Pasta range:

Seaweed pasta using original shapes and tastes:

Dulse, Nori, Spirulina Flours.

Raspberry, cranberry, lemon-bergamot

Spicy colours, red beetroot, curry and Tomato.

Salmon ravioli.

104



7

3. Molecular range:

Flavour Pearls range (solid on the outside with a liquid centre which bursts in your mouth). Available in ambiant T° with a shelf life of one year or in frozen form(IQF) with a shelf life of 12 months at -18°C.

Flavour Clouds range (dried arometized oils melt in the mouth), and Emulsions range (vinaigrette, citrus bergamot, Tomato Coriander base).



Marine products are the future of the food industry

Seaweed provides an inexhaustible source of minerals and

high-quality nutrients.

Taste the difference delivered by Globe Export’s research into

the nutritional qualities of marine plants.

Our products and concepts represent a progressive, humanist

approach, a commitment to innovation and to the development of

marine products, which can meet the challenges of both taste

and health, so as to be fit for human consumption.

8


Globe Export Zi de Dioulan - BP 37 - 29140 Rosporden - France

Tel: +33 (0)2 98 66 90 84 - Fax: +33 (0)2 98 66 90 89 www.algues.fr

Algues de Bretagne: where the sea and gastronomy meet


9

105

« Seaweeds for soil and plant nutrition »

A fertilizer producing company case study

PRP Technologies - Dr. Bruno DARIDON - R&D manager 1


Position

PRP offer products for sustainable agriculture.

Design, produce and distribute products & services that improve soils vital functions and that stimulates plants, allowing an optimized use of natural resources.

2


The challenge : world agriculture has to face an increasing demand.

106


Agriculture is a main issue

4

1,8

2,8

6,3

9

0

1

2

3

4

5

6

7

8

9

10

1900 1965 2000 2050

Mill

iard

s in

habi

tant

s

Year

World population

Source: Population Division of the Department of Economic and Social Affairs of the United Nations Secretariat (2007)


Forcast

5

Increase neads for world production + 70% in 2050 + 1 milliard tons cereals and + 200 millions tons meat

Source: FAO


Forcast

10% « only » of the target can be acheived by surfaces increase.

90% of the target has to be achieved by Yield improvement & intensification.

Source: FAO 6

107


What does this chalenge means?

7

… Need of efficient soils with good fertility.

… Need of healthy plants, with high producing potential.


Constraints on the chalenge !

8

Produce with less :

Produce better :

Energy

Water

Fertilizers

Agrochemicals / pesticides

Preserve biodiversity Limit EGS Protect soils Preserve water ressource quality


Seeweads interests and their input towards the

agriculture challenges

108


Some R&D sustainable directions for seaweed uses in agriculture

10

Solid or liquid products for improving seed germination, root development together with a good rhizosphere microorganism-plant synergy.

Liquid products for improving photosynthesis and plant resistance to both biotic and abiotic stresses.


Seaweed in agriculture: an old story

11

Ancestral raw-material used since a long time in the fields near the coast areas.

All seaweed types (red, brown, green) where and are used. No known risk (toxic or ecotoxic) based on historical references and

long time use. Natural : -> Cosmetic, Food, Feed, Agriculture. Pérennial ressource if turnover & harvest are well managed. Composition:

– Nutritional interest for plants – Stimulation of natural plant defenses systems

-> How to developp in agriculture?


Several raw-materials for several market segments

12

Cost + Added value + biological activity Concentration

Seaweed compost – Partly stabilized but some lossess of C, N & S by lixiviation + gaz emissions (EGS) + losses

of biological activity.

Fermented seaweed « juce » – Difficult to manage + partial losses of biological activity

Whole seaweed, fresh or dried – Quality + preserved biological activity

Seaweed refined fractions obtained by downstream processing technologies

– Liquid extract -> polysaccharides, proteins, native soluble metabolites – Hydrolysates -> oligomers with controlled DP & DS (biological activity). – Purified fractions: UF, NF, OI, ED, adsorption, chromatography.

-> ULVANS project

109


Polysaccharides feedstocks = hydrocolloïdes from both marine and terrestrial sources

13

Feedstocks are of inegal importances Large established markets : Alginates (E400-405) : brown seaweeds and Laminaria Carraghenanes (E 407) : red seaweeds, Chondrius, Eucheuma Agar (E406) : red seaweeds, Agar-Agar, Gracillaires Pectin (E440) : apple, citrus, … Cellulose (E460) : wood, cereal straw,… Starch (E1400) : potatoe, wheat, maize, pea. No markets or lower size markets « yet » : ulvanes, xylanes, rhamnanes, mannanes, arabinogalactanes,

arabinoxylanes, fucanes, porphyran, …


Bioactive seaweed Oligosaccharides

Brown seaweeds •Laminarine (b1-3 glucane) •Oligoalginates (D-manuronate, L-guluronate) •Fucanes (sulfated polysaccharides)

Green seaweeds •Ulvanes (sulfated polysaccharides : glucuronic acid, iduronic acid, rhamnose, xylose, glucose)

Read seaweeds •Sulfated Galactanes

14


Origin of elicitors and stimulants? What is their impact

Traditionnals use « re-discovery » -> recent insite Technology transfert from food, healh or cosmetical domains to Agronomy Recent structured screening (physiology, proteomics) -> since the1980’s

Frequent common point with elicitors : they are inducing plant defense mechanism that are expressed at high level in the resistant varieties. A few secondary metabolites do have biocide, biostatic or direct inhibition properties on some pathogens organisms. Frequent point for stimulants : They can counteract some nutrion limitations from the soil or from the leaves.

15

110


Ulva Sp : « Sea lettuce »

16

Allowed for food use : most consummed seaweed in Europe Tradionnaly harvested on the Britany costs Consummed fresh, finely chopped together with vegetables, cooked or dried High content in proteins (18 %) and polysaccharides High content in sulfur Vitamines A, B1, C Minerals (30 %) including :

– I, Ca, Cu, Si – Fe (12x lentils, = 2x wheat germ contents ), – Mg (up to 2 - 3 %)

-> ULVANS project


Advantages / weakness of seaweeds sources

17

Seaweeds flowed on the sand by the tide: -> legal status = waste – Decomposition is started, – High microbial load, – Hich sand load, – High variability, -> heavy and costly processes. Weak and variable quality.

Sea harvested seaweeds: -> legal status = natural ressource – Fresh and well preserved – Less microbial load – Low sand pollution -> better characterized raw-material with higher quality, simple

processes and more reliable starting material.

-> Prefered source for ULVANS project

-> Good source for composting


Seaweeds can help agriculture to lower some productivity

limitations

111


Produce more with less - Produce better

19

stresses on the phyllosphere or on the rhizosphere are inducing productivity losses and lower crop yield


Strategies to overcome crop plants stresses

Abiotic stresses •Genetic & plant selection • Stimulation & potentialising defense mechanisms -> seaweeds

Biotic stresses •Genetic & plant selection • Agrochemicals • Elicitors & potentialising defense mechanisms -> seaweeds • Biological pest control

Nutrition stresses • Agronomy • Fertilization •Germination stimulation -> seaweeds • Soil fertility and root development -> seaweeds

Chemical stresses • Pesticides Best-practices & more specific molecules • Formulation together with physiostimulants -> seaweeds

20


PRP EBV specific traits

- Mineral Inducer Process (coper, sodium, magnesium, sulfur, manganese, boron,…) - Major element: potassium

Improved response of plant to stress Rhizosphere stimulation

Improved plant growth, yield and yield components

Ulvans project will increase

theses effects

21

112


0

100 000

200 000

300 000

400 000

500 000

0 200 400 600 800 1000 1200

Tem ps (s)

Relative Luminescence Unit

H2O Tém oin

PRP Tém oin

H2O Chitine

H2O Chitine

PRP Chitine

PRP Chitine

H202 early signal after PRP EBV spraying

H2O2 production in response to oligo-chitosan (10 μg/ml) on young plants of Arabidopsis thaliana pre-treated with PRP EBV (2%) or with water (control)

22


Calcic response of plant after PRP EBV spraying

Choc mécanique lié à la

manipulation

Induced calcic response early signal in Arabidopsis thaliana after PRP EBV spraying at different doses

0

0,5

1

1,5

2

2,5

0 100 200 300 400 500 600 700

Time (s)

[Ca2

+] (m

icro

M) H 2 O

0,01% 0,10% 0,50% 1,00% 2,00%

23


Root growth stimulation after foliar spraying by PRP EBV under different stress conditions

0

0,5

1

1,5

2

2,5

3

3,5

4

Accroissement racinaire (m

m)

Tém oin sur m ilieu norm al

Tém oin sur m ilieu stressant

(NaCl)

Tém oin sur m ilieu stressant

(m annitol)

PRP EBV sur m ilieu norm al

PRP EBV sur m ilieu stressant

(NaCl)

PRP EBV sur m ilieu stressant

(m annitol)

Arabidopsis thaliana root elongation 24 hours after PRP EBV spraying on leaves in different conditions with or without stresses


113


Growth after severe hydric stress

Young plants of Arabidopsis thaliana after 10 days watering following 14 days of severe water stress.

Pre-treated with PRP EBV

Témoin

Control

25


Profitability of foliar spraying

26


Questions to be faced for a fertilizer producing compagny

regarding elicitors

114


Regulation is not harmonized and not favourable

28

Heterogenous regulations in the E.U. countries.

E.U. regulations (2012) • Fertilizers : Yes • Amendments : No (only national) -> E.C. in 2014 • Pesticides : Yes • Elicitors & phytostimulants : No, 3 exceptions (DE,IT,ES) + « PNP » in France

Paradoxal situation • Some farmers do have their own experimental basis + some good practices • Intentions (ex : ECOPHYTO-2018 in France) • Social opinion heavy tendency towards sustainability • Scientific litterature & patents on elicitors • Large and increasing number of R&D projects

Regulation is a limiting point for players together with the registration cost

This situation favors chemical groups (already structured + critical size)


Large offer diversity of « natural » molecules listed as elicitors or stimulant

29

Oligosaccharides -> seaweeds, vegetable, microbial, shellfish sources or sucrochemistry

Proteins -> ex : harpine, enzymes, … Aminoacides & related -> ex : L-proline, glycine bétaïne,… Lipids -> ex : brassinosteroides, phospholipides, … Metabolites -> ex : BABA, acetyl salicylique acid, ascorbique acid,

brassinostéroïdes, stilbènes, terpènes,… Plant extracts -> ex : Trigonella, Artemisia, dimers of ferulic acid, … Lignine-like -> ex : lignosulfonate, humic acids, fulviques acids, leonardite,… Microbial cell walls -> ex: yeast, bacteria, fungi. Living microorganisms -> large variety : producing elicitors, antagonists of

pathogènes, predators, nutrition promotors, mycorhizes, rhizobia,… Minerals -> phosphites, copper, silica, …

Effects are not fully understood. Interactions are not known. Which one to chose? Price/value ratio ? Which association and /or formulations are the best ?


What are industrials approaches for having a biological active solution?

30

Integrator : buying and formulate an already existing molecule -> Short-term (R&D is already done outside). -> Proportional cost. -> Exclusivity is diffcult to have or on short-term and limited area.

Screening available raw-material witch are not qualified for « agriculture » -> Midle term; -> Intermediate investment (R&D, formulation, agronomy), applicative IP possible; -> Risk on raw-material sourcing, variability (sometime).

Specific development of an active -> ex : ULVANS collaborative project -> Midle-long term -> significant investment -> Sourcing & quality are easier -> Exclusive, IP, homologation is possible.

115


Projects phases for a new seaweed product development

31

Regulation & Intellectual property surveys Screening & Characterisation of raw-materials Sourcing : choice, security, traçability, quality, cost

Added value and functions : biological & functional properties

Industrial goals : product & process -> process signature

Experiments & Agronomical trials (microcosm->field)

Formulations -> Applications

Development -> Scale-up, MKT, Commercial items

Homologation : Yes / No ?

Time to market : 3-5 years for fertilizers, 5 -10 years for elicitors


Seaweeds applications in agronomy

32

Seed coating Solid Fertilizers Ferti-irrigation Foliar spraying -> need for formulations design for each application -> need further segmentation for crop species


Seaweeds applications

33

Seed coating •Source of nutrients for seed

- -> Germination, growth, root developement -> direct •Source of nutrients for topical microflore microflora

- -> Root microbial ecosystem - > indirect nutrition + microbial consortia composition on rhizoplan

Solid fertilizers for soil •Nutrient source for microflora (base of soil food-web). •Elements & oligoelements of marine source, solubles & bio-availables. •Active on rhizosphere ecosystem composition -> nutrition & plant health.

Foliar spraying •Nutrients for leaves •Direct signals (if elicitors are recognized by leaves receptors). • Indirect signals (if hydrolysis on phyllosphere)

116


Interesting molecules from Ulva seaweed for agriculture

34

Polysaccharides & oligosaccharides – Source of energy, C, S & biological activity

Proteins & peptides – Source of N & biological activity

Minerals et Oligoelements – Bioavailable & soluble -> co-factors of enzymes – Naturaly complexed by ionic agropolymers (ex: carboxylic & amine functions).

Secondary metabolites and small molecules – Large number, diverse & bioactives – More documented in the health & cosmetic domains


Stabilization

35

Solid forms – Drying (energy cost for low AW)

Liquid forms – Concentration & osmotic pressure (! Solubility !) – pH – Conserving additives – Biocides (regulation + side effects issues))

-> aging testing for stability

!!! Good molecules for plants are also good for microorganisms !!! -> need for stabilized formulations.


Seaweeds & Industrial Quality

36

Raw-material freshness – Reproductibility (except seasonal and localizations factors) – Preserve lateral functions on polymers -> Activity – Supply chain

• Integrate : Harvest – Logistics - Process

-> cost = f(volume) + quality

Processes – Optimization of extraction-> selectivity & yield – Controlled DP et DS -> activity for ligands & receptors

Industrial limitations to be overcome !!! – sand -> abrasion ! – salt -> corrosion proof equipments

117


Ulva efficiency

37

Classical fertilizers positive list (N, P, K, Ca, Mg, K, Na, S, B, Co, Cu, Fe, Mn, Mo, Zn). Seaweed brings some more rare and “free” elements linked to natural molecules

– Directs nutrition effects Stimulants molecules (Improve efficiency of fertilizer

elements, improve photosynthesis and major element capture from soil by rhizospheric microflora,…).

– Both directs or indirects effects Elicitors of plant defense mechanisms

– Efficiency = f (Stress type and intensity) ? – Efficiency = f (patho-system) ? – Witch mechanisms & witch messagers ? – Robustness towards genetic variability of the crop

plants and of the pathogens too ? – Is it robust towards pedo-climatic conditions in the field?

Lot of question to solve!!!

LAB to

FIELD


PRP SOL impact on soil microflora

38

Project done with:

« Determine the impact of PRP SOL on some biological elements from the soil ecosystem : microorganisms, earthworms, plants ».


Impact of PRP SOL on soil microflora

39

3 indicators where measured in a randomized microcosm experiment followed by a 3 years field experiment:

Plant biomass

Enzymatic activities in soil

Bacterial population structure by

quantitative PCR analysis


118


Protocole

4 ecosystems are considered

soil* Soil +Earthworms**

Soil + plant***

Soil +Plant

+ Earthworms

* Soil : silt-clay type with no limitation (1 kg soil/ pot, no stress) ** Earthwoms: Nicodrilus giardi or Allolobophora terrestris or Aporrectodea terrestris, anecis specie (6 g/kg dry soil) *** Plant : Ray Grass (1 gramme / pot)

2 treatments / ecosystem: Control – PRP SOL

Microcosme where followed for 45 days at 20 – 23 °C, 3 pots / object

40


PCR Results

Écosystem Soil Soil

Earthwom Soil

Soil Plant

Soil Earthworm

Soil Earthworm

Plant

Soil Plant

Soil Earthworm

Plant

treatment Control control PRP SOL control PRP SOL Control PRP SOL PRP SOL 41


Similitude dendrogram from the PCR gels

S

S+M

S+P

S

S+M

S+P

Soil

Soil + PRP SOL

Soil + plant

Soil + earthworm + plant + PRP SOL

S+E

S+E+M

S+E+P

S+P+M

S+E+M

S+E+P

S+P+M

Soil + earthworm + PRP SOL

Soil + earthworm + plant

Soil + earthworm

Soil + plant + PRP SOL

42

119


Enzymatic Results in the ecosystem soil + plant + earthworm

0

20

40

60

80

100

Sol + plante + ver Sol + plante + ver + PRP SOL

Quantité de phénol libérée / g de sol / h

Activité de la phosphatase alcaline

0

50

100

150



Activité de l'α-glucosidase

0

100

200

300

400

500

600

700



Activité de la β-glucosidase

0

40

80

120

160

200



Activité de la β-xylosidase

PRP SOL: induce significant

increase for : Alacaline phosphatase (x 3),

β-xylosidase (x 2,5), α-glucosidase (x 4), β-glucosidase (x 1,5)

(P<0.05)

43


PCA Statistical analysis

Phosphatase acide

Phosphatase alcaline

-glucosidase

b-glucosidase

N-acetylglucosaminidase

b-xylosidase

FDA

-1

1-1 1

Axe 1: 35 %

Axe 2: 21%

solsol+plante

sol+vers

sol+PRP

sol+plante+PRP

sol+vers+PRP

sol+plante+vers

sol+plante+vers+PRP

A: Correlation circle B: Projection of objets on principal axes

sol sol+plante

sol+ver

sol+PRP SOL

sol+plante+PRP SOL

sol+ver+PRP SOL

sol+plante+ver

sol+plante+ver+PRP SOL

44


0,0

0,3

0,6

0,9

1,2

Tém oin PRP SOL

poids sec (mg)

Production de biom asse dans le dispositif sol + plante + vers de terre

x 2

0,0

0,4

0,8

1,2

1,6

2,0

Tém oin PRP SOL

poids sec (mg)

Production de biom asse dans le dispositif sol + plante

x 1,7

Plant biomass production

Under controlled conditions and without any stress, PRP SOL can induce a significant increase of plant biomass production.


120

« The use of seaweeds in plant protection »

Adeline Picot, Plant Pathology team - Vegenov-BBV

1


Outlines

1. Presentation of Vegenov

2. Elicitors: a promising strategy in plant protection

3. Seaweeds: a good source of plant defense elicitors

4. Seaweeds: additional uses in plant nutrition

2


1. Presentation of Vegenov

3

121


More than 20 years of experience on plant research and development

4


Main fields of investigation

5

Over more than 50 plant species

Main fields of investigation

Breeding (haplomethods, fingerprinting, marker-assisted selection and evaluation of plant disease resistance)

Plant product quality (sensory and nutritional analysis)

Crop protection (Epidemiology, Evaluation of phytosanitary products including plant defense elicitors)

- Pathogenicity assays - Biocide effects of plant products - Long-lasting and systemic effects - Study of the modes of action: biocide effect

and/or plant defense elicitor


2. Elicitors: a promising strategy in plant protection

6

122


Wind, Rain, hail …

UV ...

Pests

Micro-organisms

Hydric stress/osmotic stress…

Nematods

Parasitic Plants

Biotic and abiotic stresses encountered by plants during their life cycle

7


How to protect plants?

8

« Classical » methods

« Alternative » methods

Genetic resistance

Conventional Phytosanitary products

Use of beneficials

Prophylaxy

Induced resistance and the use of elicitors

Plan Ecophyto : to reduce the pesticide use by 50% before 2018


Definition of elicitors

Elicitors are molecules or non-pathogenic

microorganisms, able to induce physiological

modifications in plants, locally or systemically,

leading to the activation of plant defense

mechanisms.

9

123


Particular case: priming

10

priming Infection by a pathogen

Defense mechanisms are not, or slightly,

induced

Defense mechanisms are strongly induced,

compared to untreated plants


Wide diversity of elicitors

Wide range of molecules Ions, aa, proteins, glucids, lipids, secondary metabolites,…

Wide diversity of origins Microorganisms, plants, seaweeds, animal,… and chemical synthesis

11


Perception of the elicitor

Signal Transduction

Activation of plant defense pathways

Resistance

Induction of plant defense pathways after plant treatments with elicitors

12

124


Cell wall

Plasmic membrane

Ca2+

H+

Cl- K+

Ca2+

Oxidative burst

O2

H2O2

O2- Oxydases

1. Signal perception and transduction

13


Cell wall

Plasmic membrane

Ca2+

H+

Cl- K+

Ca2+

MAPK

Signal amplification

+ + +

SA-dependent pathway

Et/JA-dependent pathway

Other defense pathways….

Enhancement of physical and chemical barriers:

- Lignins - Phytoalexins

-PR Proteins: glucanase, chitinase, …

2. Activation of plant defense pathways

14


Cell wall

Plasmic membrane

Ca2+

H+

Cl- K+

Ca2+

MAPK

Signal amplification

+ + +

SA-dependent pathway

Et/JA-dependent pathway

Other defense pathways….

Infection Pathogen

Enhancement of physical and chemical barriers:

- Lignins - Phytoalexins

-PR Proteins: glucanase, chitinase, …

3. Resistance

15

125


Advantages and limits

Advantages • Supposed to be safer than conventional pesticides due to the

indirect mode of action

• Risk of developing resistant strains is limited

• Broader spectrum of action

Limits • Treatments are preventive

• Efficiency sometimes weak and unsteady, especially under conditions of production in the field (possibly linked to formulation problems, interferences with cultural conditions such as the variety, the abiotic and biotic stresses, plant nutrition , …)

16


3. Seaweeds: a good source

for plant defense elicitor

17


Seaweeds: a good source for plant defense elicitor

Treatments with seaweed extracts have been shown to enhance plant protection against a wide range of pests and diseases:

Nematodes

Fungal pathogens: Plasmopara viticola (in grapes), Sclerotinia sclerotiorum (Arabidopsis), Colletotrichum trifolii (alfalfa), …

Bacterial pathogens: Pseudomonas syringae (Arabidopsis), interference with quorum sensing…

Other pests: aphids and other sap-feeding insects (avoidance effect on plant treated with seaweed extracts)

18

126


Seaweeds: a good source for plant defense elicitor

A variety of molecules in algal extracts can be potent elicitors of plant defense against pests & diseases:

Oligo and polysaccharides: - Agars - Carrageenans - Alginates - Laminarans - Sulfated fucans - Other complex mucilages

Antioxidant polyphenols

…

19

Laminaran

Laminaria digitata

Λ-carrageen

an

Soliera robusta


Example of seaweed extracts evaluated at Vegenov on tomato gray mold (Botrytis cinerea)

20

~ 25 days

Sowing Preventive treatment

(4-leaf-stage)

5 days

Inoculation

5-7 days

Humidity at 100%

Rating of disease severity:

necrosis length


Protection efficiency, in % compared to water control10

0%

55%

53%52

%47

%50

%46

%

44%

0%

20%

40%

60%

80%

100%

SE1

SE2

SE3

BABA

Bion

50W

GSE

4SE

5

Rovr

al

Protection efficiency ≈ 50% with little to no impact on fungal growth and spore germination

Efficiency on tomato gray mold

21

127


Examples of mechanisms of action

22

Seaweed compounds

Pathosystem studied

Induction of plant defense

reactions References

Laminarin Tobacco/ Erwinia carotovora

PAL, LOX, SA caffeic acid, O-

methyl transferase

Klarzynski et al., 2000

Carrageenan Arabidopsis / S.

sclerotiorum Tobacco

JA-dependent response

Sangha et al., 2010;Mercier et

al. 2001

Extracts of Ascophyllum

nodosum

Arabidospis / P. syringae and S.

sclerotiorum

JA-dependent response

Subramanian et al., 2011

Extracts of A. nodosum Spinach / nematode

Phenolic components, flavonoids,…

Fan et al., 2010

Olmix Algae Symposium - Sept. 10th, 2012 Olmix Algae Symposium Olmix AlgaeOlmix Algae Symposium


4. Seaweeds : additional uses in plant nutrition

23


Fig 1. Schematic representation of physiological effects elicited by seaweed extracts and possible mechanism(s) of bioactivity, From Khan et al., 2009 (Journal of Applied Phycology) 24

128


Conclusions & Perspectives

Seaweeds: important source of nutrients and bioactive compounds, environmentally-friendly and safe, renewable bioresource

Promising results in plant protection against various plant and pest diseases

Other applications in plant nutrition as biostimulants of plant growth,…

More research is needed concerning: - the mode of actions - the identification of key gaps to their development to optimize the efficiency

in the field

25


26

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56580 Bréhan - FRANCE+33 (0)2 97 38 81 03

www.olmix.com

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