Challenges in co-extraction of marine algal active ingredients:An exemple of co-extraction of alginates and fucoidans

Franck Hennequart,Director Research & Innovation,ALGAIA St Lô

Algaia™ portfolio

Red Seaweed ExtractsCarrageenansSold under Carra™ trade nameProduced in Chile at our strategic partner Gelymar

Brown Seaweed ExtractsAlginatesSold under Satialgine™, AlgoGel™ and Cecalgum™ Produced at Algaia’s French factory in Lannilis

Tailor made solutionsCustomized SolutionsSold under Algogel™ tradename Produced in France

R&D services with partnersAlgaia ServicesBiomass, analytical, bioactivity etc…Provided from our R&D center in Saint Lô (France)

Specialty Seaweed extractsUnder Developmentwith specific partners or through JDATo be produced at Algaia’s French facility in Lannilis

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Production facility at LannilisNext to Brest

▪ 80,000 m2 land space – 15,000 m2 occupied▪ Over 40 years experience in Alginate production▪ ISO 9001 since 1994▪ FSSC 22000 certification

Algaia™ at a glance

R&D center at Saint LôNext to Caen

▪ 1000 m2 state-of-the-art R&D facility ✓ Extraction laboratory✓ Analytical laboratory✓ Application laboratory✓ Pilot room (in development)

Headquarter in Paris

Strategic Alliance with GelymarLocated in Chile

▪ 4th largest Carrageenan producer globally▪ ISO 9001 ▪ FSSC 22000 certification▪ Pharma cGMP production line

Saint Lô

Lannilis

Paris

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Laminaria digitata

Laminaria hyperborea

➢ France, 2nd processor country of seaweedin volumes in Europe (about 80,000 Ton/year)

➢ Algaia: 40,000 Tons of fresh seaweed/ year

Algaia™ at a glance

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Functional Systems

Carrageenansin Europe

InnovativeNatural

Ingredients

Red Seaweed& other natural

extracts

Algaia’s Services

Brown Seaweed

AlginatesgloballySpecialties

GreenBiorefineryAlgogel Customized™ Brand Name

Carra™ Brand Name

AnalyticalBiomass Potential

Bioactivity

R&D services

Process development

. Satialgine™ range

. Algogel™ range

. Cecalgum™ range

Upon Cargill’s acquisition :

Enhance functionalities

Algaia™ at a glance

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Industries we serve…

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ALGAIA Services – Analyses & BioactivitiesAnalyses

Saccharides• Total sugars• Identification and quantification of polysaccharides• Characterisation in the composition• Study of the size range

Antioxidant compounds• Phenolic compounds• Vitamins• Pigments

Ionic Profile• Anions• Cations• Heavy metals• Isotopic ICPMS

Proteins• Total Proteins• Amino Acids

Pigment profile• Chlorophylls• Fucoxanthin• Astaxanthin• Phycocyanin• ….

Genomic identification• Microalgae strains• Seaweeds• ….

Fatty acid profile• Omega 6• Omega 3• Phospholipids• …

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ALGAIA Services – Analyses & BioactivitiesBioactivities

Prebiotic• Resistance to gastric enzymes • Selective fermentation of intestinal bacteria strains

Anti-microbial• Antiviral• Antibacterial• Antifungal

Anti-inflammatory• Enzymatic assays• Cytokines, interleukines• ….

Plant biostimulation• Germination• Defense elicitors• Plant nutrition / Stress• Plant defense gene expression

Anti-cholesterol• Influence on activity of selected genes involved in cholesterol absorption • Influence on activity of selected genes involved in cholesterol refining• Intestinal Absorption of cholestérol in vitro

Anti-cancer• Influence on selected genes involved in apoptosis• Proliferation• Adhesion• Migration and invasion• High flow screening

Cosmetic• Anti-ageing• Anti-UV• Antioxidant• Depigmentation• Cicatrisation

Cardiovascular• Atherosclerosis• Heart failure• hypertension• Obesity• Diabetes

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ALGAIA Services – boost R&D and markets

Culture/Wild stock of Macro & Micro algae

Producers of algal extractsUsers of algal extracts

Active formulae

• Analyses of batch composition in production

• Study on batch variability• Statistic studies on influence of

key cultivation parameters• Study on the valorisation of the

production• Support on potential markets of

interest• Support on wild biomass

assessment and management• Support in cultivation system

setup

• Studies on compatibility of extracts in formulated products

• Studies on products stability based on algal extracts

• Test on bioactivities of formulated products

• Identification of active algal extracts

• Development of novel algal extracts

• Analyses of batch composition in extract production

• Study on batch variability• Statistic studies on influence of

key production parameters• Tests on extract bioactivities• Valorisation of by-products

Get into sustainable commercial developmentas soon as possible !

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ALGAIA – First Biorefinery studies

Macrocystis pyrifera

Lessonia nigrescens Lessonia trabeculata

Alginates

Polyphenols

Oligo-elements

Laminarans

Fucoidans

Iodine

Dietary Fibers

Pigments

Proteins

Lipids

Biorefinery / Cracking

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Brown Seaweeds Main Active Ingredients known

Proportionsin Dry Matter

Plant Biostimulant(liquids)

Feed Applications Food / dieataryApplications

Personal Care

Sugars(carbohydrates)

(total > 60%)

Alginates / Uronic ac.

> 30% Binding capacity, contact effects, root (?)

Binders / Stabilisers

Binders / Stabilisers

Binders / StabilisersCicatrisation

Laminarans 1 - 20% Elicitors, source of glucose bacteria

Elicitor, Prebiotics Elicitor, Prebiotics

unknown

Fucoidans 1 - 20% Elicitors, bindingcapacity, Antioxidants

Anti-microbial,Anti-inflammatory

Anti-microbial Skin Protection

Fucoxanthins 1 - 4% Unknown Dietary fibers Dietary fibers Slimming

Mannitol 1 - 20% Osmoregulant, antioxidant

Antioxidant Antioxidant,Cholesterol

Antioxidant

Ashes(Sea Minerals)

5 - 30% Oligo-element support, root physiology (?)

Oligo-elements+ Iodine

Oligo-elements+ Iodine

Natural mineral salts

Proteins / AA 2 - 8% Low amounts in liquids Marine Protein / Peptides

Marine Protein/ Peptides

Unknown

Phlorotannins 2 - 15% Antioxidant Antioxidants Antioxidants Antioxidants

Lipids(fatty acids)

2 - 5% Low amounts in liquids Marine lipids Marine lipids Oily solutions

Biorefinery / Cracking

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Feed Health (incl. aqua)Preventing shrimps diseasesPreventing chicken diseasesAntibiotic alternative

Could be the most active component of seaweed extracts used as Biostimulants

Anti-inflammatoryPersonal Care

Ex: anti eczema

Pharma applications (multiple)

Anti-microbial

COLLOIDSFunctional Systems

Biorefinery / Cracking

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Cracking

Extraction

Raw material Seaweed

Liquid

Fucoidan Others

Solid

Alginate

Objective:

Co-extraction proof of concept

2 to 3 products of interest

1st yield & feasability evaluation

Products quality characterisation

Others

Alginate

Fucoidan

Material & Methods

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Experimental design

Soft aqueous extraction

(T°C, H2O, Time h)

Raw material

Liquid fraction(Fucoidan)

Alginic acid protocol

Solid fraction(Alginate)

Ca neutralisation

Na Alginate

Raw material

Alginic acid protocol

Ca neutralisation

Na Alginate

Single extraction Co-extraction

X3 X3

SeaweedSpecies

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Sampling overview

Protocole development + Evaluation Macrocystis

pyrifera

Single extraction Co-extraction

Frozen Dried Frozen Dried

15 samples(alginate)

9 samples(alginate)

cracking

6 samples(alginate)

27 samples(fucoïdans)

12 samples(alginate)

45 samples(fucoïdans)

Lessonianigrescens

Protocole development + Evaluation

Single extraction Co-extraction

Frozen Dried Frozen Dried

6 samples(alginate)

6 samples(alginate)

cracking

6 samples(alginate)

27 samples(fucoïdans)

Total Samples studied >220 samples + FRESH SAMPLES

12 samples(alginate)

36 samples(fucoïdans)

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Macrocystis pyrifera – Classical extraction versusCo-extraction

Products yieldsMacrocystis pyrifera (frozen)

Trial 1 Trial 2 Trial 3 Average

Alginate yield, single extraction (% dry weight)

13 13.2 11 12.4

Alginate yield, co-extraction (% dry weight)

25.3 14 26.9 22.1

Fucoidan yield, co-extraction(% dry weight)

1.89 1.71 2.27 1.96

➢ Better extraction yields of Alginates with co-extraction ?

➢ Fucoidan extraction yields quite low

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Macrocystis pyrifera – Classical extraction versusCo-extraction

➢ Positive effect on final product[color and yield]

whitening = current challenge in alginate industry

➢ Significative quality difference= low viscosity

Extract viscosityMacrocystis pyrifera (frozen)

Trial 1 Trial 2 Trial 3 Average

Single alginate extraction (cPo at 60rpm)

512 1330 400 747

Alginate co-extraction (cPo at 60rpm)

28 20 25 24

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Experimental data Control

Alginate co-extraction yield

(% MS)15.8

Fucoïdans co-extraction yield

(% MS)-

Alginate viscosity (cPo)

1% at 25°C169

6h/X°C

25.6

2.53

127

3h/2X°C

26.6

2.78

145

1h/3X°C

26.9

2.69

114

1h/5X°C

32.1

3.48

78

Average values based on triplicats

➢ Improvement of final alginate viscosity (low viscosity)

➢ Pre-extraction step at 2X°C seems interesting

➢ Experimental conditions must be improved again regarding alginate polymerisation (MV)

Macrocystis pyrifera –Co-extraction at various conditions

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Importance of process condition on yields

Extraction

Controllable parameters(water, stirring intensity…)

Uncontrollable parameters(seaweed wash, age of seaweed…)

Studied parameters(time, temperature, ratio)

ResponsesExtraction yieldsExtract compositionPolysaccharide composition

EXPERIMENTAL PLAN SETUPS

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Importance of process condition on yields

PROPOSITION OF A MATHEMATICAL MODEL

Full factorial design: 2n experiments (3 factors: 23 = 8 extractions)

Effect calculation of extraction parameters:

Extractions done in triplicate: calculation of confidence interval (t.σ𝑠2

3

𝑘)

Definition of influential parameters: effect ± confidence interval ≠ 0

𝑥𝑎 =(−𝑌1 − 𝑌2 − 𝑌3 − 𝑌4 + 𝑌5 + 𝑌6 + 𝑌7 + 𝑌8)

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Importance of process condition on yields

Ash content Phenolic compounds content Mannitol content

Fucoidan content Laminaran content

0.26±0.64

0.08±0.56

-0.81±0.67

0.66±0.27

-0.58±0.61

EXPERIMENTAL PLAN SETUPS – Effect of extraction time

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Importance of process condition on yields

EXPERIMENTAL PLAN SETUPS – Effect of extraction temperature

Fucoidan content Laminaran content

Ash content Phenolic compounds content Mannitol content

-0.09±0.64-0.36±0.56

1.88±0.67

1.46±0.27

-2.68±0.61

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Importance of process condition on yields

Fucoidan content in extract expressed as a percentage of the total solid content (n=3)

-1 -1

+1

+1

Time Temperature Ratio

3.84%

5.16%

4.50%4.50%

5.97%

3.04%

4.50%

+0.66% + 1.46% + 0.00%

Tim

e

Temperature

Ratio

0.35%

2.30%

1.51% 1.13%

2.81%3.05%

Time/temperature = +0.49%Time/ratio = -0.09%Temperature/ratio = -0.06%

% Fucoidan = 4.50% + 0.66%A + 1.46%B + 0.00%C + 0.49%AB – 0.09AC – 0.06%BC

Confidence interval at 0.95% calculated = 0.27%

% Fucoidan = 4.50% + 0.66%A + 1.46%B + 0.49%AB

EXPERIMENTAL PLAN SETUPS – Effect of combined parameters

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IN SUMMARY

➢Co-extraction of functional alginates and fucoidan is viable

➢Extraction conditions affect yields and chemical functionality

➢Temperature, H2O Ratio & Extraction time must be assessed

➢All these elements need to be studied before industrial & marketpositionning to avoid business downturns

THANK YOU !

In Algae we trust…