Maria Zakharova, Isabelle Gendron-Lemieux, Juliette Garcia, Éric Tamigneaux and Alain Tremblay. Nordic Seaweed Conference 2018 October 10,

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2 Extraction of value-added compounds from Saccharina longicruris and portrait of their biological properties Innovation and entrepreneurship in Quebec, Canada Maria Zakharova, Isabelle Gendron-Lemieux, Juliette Garcia, Éric Tamigneaux and Alain Tremblay Nordic Seaweed Conference 2018 October 10,

3 Canada s largest fisheries, aquaculture and processing innovation centre Applied research and development Technology transfer and et technical assistance Diagnostic and support services Processes and profitability optimization Analysis laboratories 3

4 9 expertise centres and 19 services dedicated to the innovation needs of the enterprises Located on the Gaspe Peninsula, the Magdalen Islands and Quebec s North Shore Gaspé Peninsula: Gaspé and Grande-Rivière North Shore Magdalen Islands 4

5 Context previous projects Seaweed research in Québec started 10 years ago with projects on kelp aquaculture 2012: NSERC Industrial Research Chair on macroalgae ( ) Wild harvest, aquaculture and processing/product development Work on sugar kelp culture, but also other sp: Sacchoriza dermatodea, Alaria esculenta, Laminaria digitata, Palmaria palmata, Chorda filum 6 industrial partners 2013: Kelp culture technology transfered to industrial partner Fermes marines du Québec : ALIMALGUE Food applications for seaweed cultivated in Québec. 5

6 Context - Research program in progress : OPTIMAL NSERC grant Support the development of an industrial integrated chain around cultured kelp Axis 1 Optimization of growing techniques in nursery and at sea Axis 2 Molecule extraction, drying techniques and food applications Development of products and processes Saccharina latissima and longicruris production Valuation of residues Axis 3 Test different applications for kelp co-products (food packaging, textile) 6

7 Context - Research program in progress : OPTIMAL NSERC grant Support the development of an integrated industry around cultured kelp Fermes Maximize marines and scale-up du Québec, a scallop hatchery hatchery production in the Gaspé peninsula, Identify best nowculture sells kelp sites seedlings Maximize culture yields Calendar, culture substrate and geometry, density control, broodstock origin Mechanize culture operations Two Develop companies conservation (Seabiosis and Salaweg) methods have (drying, developed blanching, food products freezing, (relish, smoking) tartare, pesto, meat Set and up a fish sensory spices) panel withof cultured seaweed kelpexperts in Canada Develop food products Develop mannitol and polyphenol extraction and follow monthly yields Characterize the extraction residues Develop a food packaging Develop a lactofermented product with stipes Develop a textile coating (flame retardant and UV protection) 7

8 Context Identifying best culture sites Shellfish aquaculture currently faces important environmental issues Need for diversification The existing culture lines and boats can be used for seaweed production in a co-culture scenario There is a lot of free space available in existing marine farms (red dots) About 2000 ha available MAPAQ (2018) Répertoire des sites maricoles du Québec. Données diffusées sur OGSL. Interest for the survey of sites with contrasting environmental conditions vs biomolecule study 8

9 Methodology - Identifying the best culture sites Culture trials over 3 years (2015 to 2017) at 5 existing shellfish sites and 1 experimental site Kelp plantlets produced in Fermes Marines du Québec in August-Sept. Outplanted on marine farms at the end of Sept. to Nov. (mean lenght of 2 mm) Mean weight (n=3) and length (n=50) recorded from May to July 2 to 10 kg of kelp blades harvested for drying at 40 C during each sampling Environnemental parameters recorded Hourly : Temperature and salinity In situ: Secchi disk, light intensity and water for N and P analysis. 9

10 Results - Identifying the best culture sites NORTH COAST (SEPT-ÎLES) Salinity stable, around Drifting ice TRACADIGACHE AND CASCAPÉDIA BAY Salinity below 20 in first 4-5 meters of water in Spring Drifting ice GASPÉ Freshwater in first 4-5 meters of water in Spring Permanent ice cover during 2-3 months MAGDALENE ISLANDS Salinity stable, around Drifting ice with permanent ice cover occasionnally Lowest NO 3-, below 0,3 µm PASPÉBIAC Salinity stable, around Drifting ice 10

11 Results Identifying the best culture sites Paspébiac Gaspé Traca Casca Magdalene Islands 11

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13 OPTIMAL PROGRAM: OBJECTIVES OF OLEOTEK Collaboration between 2 colleges (TTC) holding complementary expertise Extraction of active compounds present in Saccharina longicruris: Sugar (D-Mannitol) Polyphenols (brown algae: phlorotannins) Pigments (carotenoids) Fibers (alginates) Molecules with commercial interests and potential for rapid integration on the market Scale-up (pilot plant) and use of green chemistry process for the extraction of D-Mannitol 13

14 Global mannitol market was valued at USD 300 million in 2015 and is expected to reach USD 500 million by 2024 Growing demand for artificial sweeteners: fewer calories and similar sweetness as sugar Asia Pacific (India, China, Japan, Southeast Asian Countries, and Oceania) is the most dominant regional market in 2015 and held over 60% of global demand D-MANNITOL 14

15 ACTIVE COMPOUNDS PRESENT IN SACCHARINA LONGICRURIS Alginates Pigments D-mannitol Polyphenols Added value increases 15

16 OLEOTEK Complete process: Alginates Pigments D-mannitol Polyphenols 6 1 Final residue valorization Algae pretreatment: -Drying -Grinding Added value increases 5 Extraction des alginates: -Aquatic treatment -ph swing method Extraction of polyphenols: - Soxhlet - UAE 2 4 Purification: -Hot filtration of sels -Recrystallization -Filtration of mannitol Extraction of mannitol: -Soxhlet -Ethanol 3 16

17 2015: BEGINNING OF A PROJECT Purification, identification and application of salts: Hot filtration TGA analysis: minerals Elemental analysis: ICP used to determine metals (mainly K + and Na + ) By ion chromatography (predominantly Cl - ) Composition: 57.5% KCl and 39.8% NaCl Possible valorization in food or chemicals markets Purification, identification and application of D-Mannitol (sugar): Extraction then recrystallization Analytical techniques used (NMR, melting point, TGA, HPLC, FTIR) Natural sweetener, food additive Use in medicine (reduce intracranial pressure) D-MANNITOL 17

18 2016: EXTRACTION OF D-MANNITOL Six sites (Paspébiac, Cascapédia, Tracadigache, North Coast, Gaspé, Magdalen Islands): harvest from April to July 2016 Each sample was extracted in ethanol in triplicate (Soxhlet 6 h) After recrystallization of D-mannitol: 0 to 13.6% (dry weigh) according to the sites and dates of harvest Purity: 95-99% Pilot plant test in progress 18

19 : EXTRACTION OF D-MANNITOL 3 sites (Paspébiac, North Coast, and Magdalen Islands): harvest from April to July 2017 Each sample was extracted in ethanol in duplicate or triplicate (Soxhlet 6 h) After recrystallization of D-mannitol: 0.2 to 18.6% (dry algae) according to the sites and dates of harvest (0 to 13.6% in 2016) Purity: 95-99% Scale up: extraction using 3 kg of dry algae, similar performance to the lab scale 19

20 2017: MANNITOL EXTRACTION SCALE-UP Good yield: 10%, small scale the same Purity: 73% After recrystallization in EtOH: 92% 2 nd recrystallization in EtOH: 96% Reactor 200 L 20

21 : EXTRACTION OF POLYPHENOLS Fragile molecules (photo- and thermosensitivity) Properties described in the literature: anti-oxidant 1, anti-inflammatory, allergy prevention, anti-bacterial 2, UV radiation protection 3 anti-cancer activity, anti-hiv, anti-wrinkle Extraction of polyphenols: Magdalen Islands: spring %; summer % (max-may) Paspébiac and North Coast: % (max-may) Previously published results: % (Scotland) Bio-activity tests in progress Alginate extractions started 2,00% 1,50% 1,00% 0,50% 0,00% ALGUES: IM Ultrasound, closed system Ultrasound, open system Soxhlet Hours 1. Wang T, Jónsdóttir R, Liu H, et al. (2012) Antioxidant Capacities of Phlorotannins Extracted from the Brown Algae Fucus vesiculosus. J Agric Food Chem. doi: /jf Lopes G, Sousa C, Silva LR, et al. (2012) Can phlorotannins purified extracts constitute a novel pharmacological alternative for microbial infections with associated inflammatoryconditions? PLoS One 7:e doi: /journal.pone Swanson AK, Druehl LD (2002) Induction, exudation and the UV protective role of kelp phlorotannins. Aquat Bot 73: doi: DOI: /S (02)

22 Light intensity (PAR) Date Paspébiac Magdalen Islands 29/05/ /06/ temperature 10/07/ Salinity Date Paspebiac Magdalen Islands 29/05/ salinity 26/06/ /07/ Temperature Date Paspebiac Magdalen Islands light intensity (PAR) 29/05/ /06/ /07/ Magdalen Islands The green horizontal bar indicates the depth of algae Paspébiac 22

23 : EXTRACTION OF ALGINATES 2HAlg + Na 2 CO 3 2NaAlg + H 2 O + CO 2 Extraction yield Color Texture FTIR Soxhlet residues Ultrasounds residues 26% Brownish Brittle 29% Bright Brittle Similar to commercial Very different from commercial Raw algae 23% Bright Brittle Similar to commercial Comparable yields but not the quality of alginates Ultrasound is destructive for alginates 23

24 CONCLUSIONS Possible transfer of the mannitol extraction process to industry: Technico-economic analysis in process Approach of potential partners in process Integration into an existing production schema in process Comparison of different drying methods for harvest of 2018 (mannitol) Improvement of scale-up purification (purity 99%) Exploration of new polyphenol extraction methods (microwave assisted extraction) Study of the effect of algae conservation methods on polyphenol pontent: lyophilization vs. hot air Activity study of polyphenol extracts (Transbiotech, beginning in November 2018) A dozen polyphenol extractions using edible vegetable oils will be carried out to produce vegetable oils enriched in polyphenols (directly marketable as functional foods or dietary supplements) 24

25 Special thanks to: 25

26 Optimal Program Partners 26

27 Soxhlet 27