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PRODUCT OVERVIEWNatural products

CREDITS

Cover Figure: Dunaliella cultivation in open raceways at Monzón Biotech (Spain)

This project has been funded by the European Commission under the Seventh Framework Programme for Research and Development Collaborative project - GA 613870

Dunaliella salina is the richest source of natural orange-, yellow- or red-pigmented carotenoids. Their antioxidant and pro-retinoic acid activity may protect humans from compromised immune response, premature ageing, cancers, cardiovascular disease and arthritis.

D-FACTORY products are derived from strains of Dunaliella salina and include powders, carotenoid-rich extracts and fresh or frozen algae.

Food and feed supplements and additives • Colorants, • Sources of nutritional antioxidants and pro-

Vitamin A,• Fibre, protein and starch-rich extracts.•

Pharmaceuticals and Cosmeceuticals• Actives ,• Excipients,• Emulsifiers.

D-FACTORY - THE PROJECTThe D-Factory has set a world benchmark for a sustainable CO2 algae biorefinery. The D-Factory is based on biomass from strains of the halotolerant microalga Dunaliella salina. Dunaliella cultivation is one of the largest amongst microalgae. The biorefinery elements are integrated and optimized using sustainability assessments. Key biomass processing technologies are being applied to the biomass:

• Innovative spiral plate technology for dynamic settling;

• Ultra membrane filtration;• Supercritical CO2 extraction techniques;• High performance countercurrent chromato-

graphy.

The D-Factory demonstration serves as a robust manifestation of the business case for global investment in algae biorefineries and in large-scale production of microalgae using raceways, lagoons, lakes and to a limited extent, photobioreactors.

INTRODUCTION

D-FACTORY PRODUCTSFRESHFresh Dunaliella salinaFrozen Dunaliella salina

POWDERS Dunaliella salina - spray dried Dunaliella salina - freeze driedDefatted Dunaliella powder10% carotenoid-rich extract + Carnauba wax4% carotenoid-rich extract + Cocoa butter

SEMI-PURIFIED CAROTENES AND XANTHOPHYLLS* Supercritical CO2 extracts of β-carotene stereoisomersEnriched extracts of lutein and zeaxanthin isomers9-cis β-carotene All-trans β-carotene α-carotene

OTHER SEMI-PURIFIED EXTRACTS*LuteinZeaxanthinAll-trans β-carotene9-cis β-carotene in development

* These samples are not commercially available yet. Please contact Pat Harvey (P.J.Harvey@greenwich.ac.uk) or Lena Brive (lena.brive@ri.se) for more information.

Legal StatusDunaliella bardawil (NBT Ltd,.Israel and Nikken Sohonsha Co, Japan) is classified GRAS (Generally Regarded as Safe) food sources by the U.S. Food and Drug Administration; Dunaliella bardawil powder a product of NBT Israel was granted GRAS status in 2008. Consequently, many preparations can be administered as a powder of spray dried or freeze-dried biomass with no extraction undertaken in the US. Dunaliella bardawil is listed as having no toxins under GRAS and the Ministry of Health, Japan.

CAROTENOIDS

Carotenoids are a class of more than 600 naturally occurring pigments biosynthesized by plants, algae, and photosynthetic bacteria, and are broadly classified into two classes, carotenes and xanthophylls.

Carotenoids comprise a wide range of hydrocarbon carotenes including α-carotene, β-carotenes and lycopene and many hydroxy-carotenoids such as lutein, violoxanthin and zeaxanthin. Carotenoid production from natural sources is in its infancy compared to the available mass of synthetic carotenes. Both the synthetic and the natural carotene show antioxidant and pro-vitamin A properties. Pro-vitamin A is converted by the body to vitamin A (retinol). Vitamin A is essential for normal growth and development, immune system function, and vision. In Dunaliella salina, β-carotene may reach as high as 12% of the ash-free dry weight, around 200-fold more than the amount found in orange fruits and vegetables.

β-carotene is currently used extensively as a natural colouring agent in many food products such as margarine, baked foods, soft drinks and animal feeds. It is also the main ingredient in demand for antioxidant supplements with health benefits that have been linked specifically to preventing night blindness and cataract, preventing toxin build-up in the liver and improving the immune system.

Xanthophylls are typified by lutein and zeaxanthin. These are polar, oxygenated compounds containing hydroxyl and epoxide substituents, which cannot be converted to retinol, so they have no vitamin A activity. However lutein and zeaxanthin are the only carotenoids found in the retina and lens of the eye and it is thought that diets rich in these two compounds may help slow the development of age-related macular degeneration and cataracts

Dunaliella carotenoidsCertain strains of the microalga Dunaliella salina have long been known as a source of β-carotene, comprising all-trans β-carotene, and the equivalent

stereoisomers, 9-cis, 13-cis and di-cis β-carotenes in equal amounts. These stereoisomers of β-carotene are diff icult to synthesize chemically. 9-cis β-carotene is of particular nutritional and medical interest as a retinoid precursor, the well known RXR and is associated with therapeutic eff ects in a number of diseases as well as possessing a good adverse eff ect profile. Dunaliella salina is the richest source of natural β-carotene and produces 9-cis-β-carotene and all-trans-β-carotene in equal amounts depending on culture conditions. It is currently cultivated commercially in Australia and in Israel (NBT).

MOLECULAR STRUCTURES OF SOME COMMON CAROTENOIDS

All-trans β-carotene

All-trans α-carotene

9-cis β-carotene

All-trans lutein

All-trans zeaxanthin

APPLICATION OVERVIEW

MATERIAL

β -carotene rich Dunaliella salina powder

Carotenoid-rich extract. High in stereoisomers of β-carotene, superior grade

Defatted powder

Starch (powder)

9-cis β-carotene

Lutein

Zeaxanthin + lutein

α-carotene

All trans β-carotene

MARKET

Food supplement

Nutraceuticals

Food fortification

Feed

Cosmetics

Food

Nutraceuticals

Food

Possible pharmaceutical

Nutraceutical/pharmaceutical.

Cosmeceuticals, Nutraceuticals

Pharmaceutical

Food

END PRODUCTS

Tablets, capsules, drinks and more

Tablets, drinks, soft gel capsules

Functional drinks

Pellets/granulates for ani-mal feed

Scrubs used for skin exfo-liation

Gluten-free breadTablets

Tablets

Sauces, drinks etc

Possible active pharmaceutical ingredient (API)

Tablets, topical formulations

ROLE

Active

Active

Active

Protein & carbohydrate source

Abrasive powder

Protein source

Excipient: tablet filler

Functional ingredient: Thickener, emulsifier, dispersion stabilizer

Active with anti-inflammatory effect. Nutritional and medical interest as a 9-cis retinoic acid precursor and pro-vitamin A

Active - Clinically proven to prevent cataract and macular degeneration and may function as an anti-oxidant to decrease around 60 chronic disease risks.

Active

Anti-metastatic action

Food colorant and pro-vitamin A

APPLICATION OVERVIEW

MATERIAL

Polyunsaturated Fatty Acids (PUFAS)

Galactolipids

Fatty acids

Glycerol

Chlorophyll

Peptides

MARKET

Nutraceuticals

Food

Cosmetics

Nutraceuticals

Pharmaceutical

Pharmaceutical

Feed

Chemicals

Multiple

Food

Food

END PRODUCTS

Soft gel capsules

Margarines, functional drinks (low-fat, dairy-type)

Gel, emulsions, cream formulations

Functional drinks

Gel, emulsions, cream-based formulations

Possible Active Pharmaceutical Ingredient Possible active ingredient

Surfactants/emulsifiers

Drinks, desserts, breakfast cereals, confectionary

Nutraceuticals

Pharmaceutical

ROLE

Active

Ingredient

Excipient: emulsion stabilizer, encapsulating agent

Excipient: emulsion stabilizer, encapsulating agent

Excipient: emulsion stabilizer, encapsulating agent

Possible immuno-stimulatory properties

Possible immuno-stimulatory properties

Raw material for surfactant synthesis

Natural compound with uses in pharmaceutical, personal care, food industry, chemical intermediate

Food colorant

Active with therapeutics properties

Active with therapeutics properties

Multiple

FREEZE DRIED D. SALINA (MONZON BIOTECH)

SPRAY DRIED D. BARDAWIL (NBT LTD.)

Raw material: D. salina and D. bardawilDrying method: freeze drying or spray drying Colour: orangeOdour: product typicalFlavour: product typicalConsistency: powder

Average Algal Composition - Organic matter (ash free matter) analysis normalised to 100%

W-powder: ash content 4-5% of dry weightE-powder: ash content up to 50% of dry weight

Algal Composition W-powder E-powderglycerol 0-3 6-30lipid 9 18protein equivalents 3 18carbohydrate 72 44total carotene 8 79-cis β-carotene 3.2 2.6all trans β-carotene 4 3.1α-carotene 0.7 0.7Lutein 0.2 0.2Zeaxanthin 0.1 0.1Chlorophyll 0.5 0.5

Typical lipid composition of powder (% of those higher than 1%)

% Lipid W-powder E-powderC11:0 Undecylic Acid 5.6 2.0C14:0 Myristic Acid 1.8 1.1C16:0 Palmitic Acid 33.7 25.4C17:1 Heptadecenoic Acid 3.2 1.8C18:0 Stearic Acid 1.8 1.5C18:1 Oleic Acid 5.8 5.6C18:2 Linoleic Acid 4.9 9.0C18:3 Linolenic Acid 17.6 28.2C20:1 Gadoleic Acid 1.2 0.00% of total Fatty Acids FATotal Saturated FA 45 32Total Monounsaturated FA 11 9Polyunsaturated FA 23 38% Ash Free Dry WeightTotal Lipid 9 14

Typical amino acid composition (mg/g protein)

Aa WHO & FAO* W-powder E-powderLys 45 21 57Met 16 21 21Met + Cys 22 42 35Thr 23 73 59Phe + Tyr 38 125 62Val 39 63 64Ile 30 57 50Leu 59 73 90Trp 6 26 11His 15 26 14

* WHO and FAO (2007) Protein and amino acid requiremen-ts in human nutrition, World Health Organization technical-report series, (935), pp. 1–265.

DUNALIELLA SALINA POWDERS

Contaminants: none detectedGMO: GMO-freeStorageHermetically-sealed container in the dark

Shelf life: Hermetically-sealed sealed light-protected container, 3 years

InformationDepending on growth conditions and species, the above values are subject to natural fluctuations. This specification does not release the purchaser of our products from his duty of care and responsibility. Produced from authentic raw material. Does not contain protein from animal source.

Contacts:

Monzón BiotechParc Cientific de Barcelonac/ Baldiri Reixac 4-8Torre I pl.408028 Barcelona (Spain)Phone 0034 932329455

NBTAmi Ben-Amotz, PhD, NBT Ltd.North Industrial ZonePOB 828Eilat, 8810602IsraelPhone: +972544384087Fax: +97235351378Email: ami@benamotz.com

DUNALIELLA SALINA POWDER

100% DUNALIELLA EXTRACT: PASTE

Raw material: Dunaliella salinaDrying method: freeze dryingExtraction method: supercritical CO2-extraction

Sensory property:Colour: dark redOdour: product typicalFlavour: product typicalDescription: oil soluble/dispersible pasteConsistency: viscous

Content100% Dunaliella extract. Essential N-3 fatty acids, α- and β-carotenes, lutein, zeaxanthin and isomers.

Carotenoid composition

Carotenoid % Lutein 0.55Zeaxanthin 0.14All-trans β-carotene 7.009-cis-β-carotene 14.0013-cis-β-carotene 2.60α-carotene 2.90 Total carotene % 27.00

Storage recommendation: Hermetically-sealed container in the dark, either cooled (8 °C) or frozen (-18 °C)

Shelf life: Min. 6 month when kept sealed and stored cooled and dark, min. 9 weeks aft er opening when kept cooled and dark, min. 24 month when kept frozen (-18°C) and dark.

CAROTENOID-ENRICHED POWDERS

Raw material: Dunaliella salina Drying method: freeze dryingExtraction method: supercritical CO2-extractionPulverisation: supercritical CO2 spraying

Sensory property:Colour: orangeOdour: product typicalFlavour: product typicalConsistency: free-flowing powder

10% extract + Carnauba waxRecommended for oily food stuff s & cosmetics. Shelf life 12 months, stored frozen (-18°C).

3% extract + Cocoa butterRecommended for oily food stuff s. Easy dosing & free flowing. Shelf life 12 months, stored frozen (-18°C).

DEFATTED POWDER

Raw material: Dunaliella salina Drying method: freeze dryingDe-Fatting method supercritical CO2-extraction

Sensory property:Odour: product typicalFlavour: product typicalConsistency: free-flowing powder

CAROTENOID-RICH EXTRACT AND POWDER

Composition (%)

W-powder E-powderCrude Fat 4 2.5Crude Protein 18 26Fiber 5 8Starch incl sugars 60 40Ash 4 15NaCl 0 2Water 3 7

Storage recommendation: keep closed, cooled (8 °C) and dark.

Shelf life: min 24 month when kept closed, cooled and dark.

Information:Depending on growth conditions and species the above values are subject to natural fluctuations. This specification does not release the purchaser of our products from his duty of care and responsibility.Produced from authentic raw material. Does not contain protein from animal source.

Contact: NatecoHopfenveredlung St. Johann GmbH – NATECO2Auenstr. 18-2085283 Wolnzach / GermanyPhone: +49 8442 66-0 Fax: +49 8442 66-66e-mail: contact@nateco2.dehttp://www.nateco2.de

CAROTENOID-RICH EXTRACT AND POWDER

Dunaliella biomass cultivated in green, brown and orange growth phase conditions, in fresh, frozen, spray-dried and freeze-dried forms.

Availability: kilogram size samples

Contact: A4FEstrada do Paço do LumiarCampus do Lumiar, Ed. E – R/C1649-038 LisboaPortugalPhone: +351 218 072 499info@a4f.ptwww.a4f.pt

For orange carotenoid-rich algae: Monzón BiotechParc Cientific de Barcelonac/ Baldiri Reixac 4-8Torre I pl.408028 Barcelona (Spain)Phone 0034 932329455

Multilayer horizontal tubular photobioreactors growing carotene-rich Dunaliella cells at A4F pilot unit in Lisbon, Portugal

MONZÓN & A4F BIOTECH PRODUCTS

Closed flat-panel photobioreactors from A4F growing carotene-rich Dunaliella at Monzón Biotech production unit in Spain.

Pond growing carotene-rich Dunaliella at Monzón Biotech production unit in Spain.

Raw material: Dunaliella salina or defatted Dunaliella powder.

Process preparation method: Stage 1: a series of solvent extraction steps to prepare polar and non-polar concentrated fractions.

Stage 2: an automated liquid/liquid extraction process using proprietary hydrodynamic High-Performance Countercurrent Chromatography (HPCCC) to fractionate the concentrated extracts into the individual target products.

Stage 3: removal of process liquids to isolate the final products in either liquid concentrated form (depending upon customer requirements) or dried powder form. Additional crystallization steps are used to obtain analytical grade purity.

CUSTOMIZED LUTEIN/ZEAXANTHIN CONCENTRATED MIXTURES-TO CUSTOMER SPECIFICATION• Mixture standard ratio range: From 6:1

lutein:zeaxanthin to 2:1 lutein:zeaxanthin

• Other customized mixtures available on request

• Specifications according to customer requirements.

PackagingHermetically sealed light protected containers according to customer requirements.

AvailabilityMinimum 100 g; maximum 1000 g containers.

SEMI-PURIFIED COMPOUNDS FOR ORAL APPLICATION

• Lutein

• Zeaxanthin

CUSTOMIZED CAROTENOIDS EXTRACTS

• All-trans β-carotene

• 9-cis β-carotene in development

Specification Purity >95%. Residual Solvents, metal and other impurities to ICHQ3A and ICHQ3B requirements.

PackagingHermetically sealed light protected containers

AvailabilityMinimum 100 g; maximum 1000 g containers.

ANALYTICAL GRADE COMPOUNDS• Lutein

• Zeaxanthin

• All-trans β-carotene

• 9-cis β-carotene in development

Specification: Purity >99.5%

Packaging: hermetically sealed light protected vials

Availability: 2 mg to 2 g (further quantities in multiples of 2 g)

Note: Customised mixtures and purified compounds for oral application can be prepared to meet Good Manufacturing Practice (GMP) requirements in conjunction with a GMP licenced contract manufacturing organisation (CMO) as a separate specialised service

Contact: Dynamic Extractionsinfo@dynamicextractions.comTel: +44(0)1403 268404www.dynamicextractions.com