PRODUCT OVERVIEW Natural products

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1 PRODUCT OVERVIEW Natural products

2 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

3 INTRODUCTION 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, carotenoidrich 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 PROJECT The 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 chromatography. 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. D-FACTORY PRODUCTS FRESH Fresh Dunaliella salina Frozen Dunaliella salina POWDERS Dunaliella salina - spray dried Dunaliella salina - freeze dried Defatted Dunaliella powder 10% carotenoid-rich extract + Carnauba wax 4% carotenoid-rich extract + Cocoa butter SEMI-PURIFIED CAROTENES AND XANTHOPHYLLS* Supercritical CO2 extracts of β-carotene stereoisomers Enriched extracts of lutein and zeaxanthin isomers 9-cis β-carotene All-trans β-carotene α-carotene OTHER SEMI-PURIFIED EXTRACTS* Lutein Zeaxanthin All-trans β-carotene 9-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 Status Dunaliella 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 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.

4 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. Provitamin 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 agerelated macular degeneration and cataracts Dunaliella carotenoids Certain 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 difficult 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 effects in a number of diseases as well as possessing a good adverse effect profile. Dunaliella salina is the richest source of natural β-carotene and produces 9-cis-βcarotene 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

5 APPLICATION OVERVIEW MATERIAL MARKET END PRODUCTS ROLE β -carotene rich Dunaliella salina powder Food supplement Tablets, capsules, drinks and more Active Carotenoid-rich extract. High in stereoisomers of β-carotene, superior grade Nutraceuticals Food fortification Tablets, drinks, soft gel capsules Functional drinks Active Active Defatted powder Feed Cosmetics Food Pellets/granulates for animal feed Scrubs used for skin exfoliation Gluten-free bread Tablets Protein & carbohydrate source Abrasive powder Protein source Starch (powder) Nutraceuticals Tablets Excipient: tablet filler Food Sauces, drinks etc Functional ingredient: Thickener, emulsifier, dispersion stabilizer 9-cis β-carotene Possible pharmaceutical Possible active pharmaceutical ingredient (API) Active with anti-inflammatory effect. Nutritional and medical interest as a 9-cis retinoic acid precursor and pro-vitamin A α-carotene Pharmaceutical Anti-metastatic action All trans β-carotene Food Food colorant and provitamin A Lutein Nutraceutical/ pharmaceutical. Active - Clinically proven to prevent cataract and macular degeneration and may function as an anti-oxidant to decrease around 60 chronic disease risks. Zeaxanthin + lutein Cosmeceuticals, Nutraceuticals Tablets, topical formulations Active

6 APPLICATION OVERVIEW MATERIAL MARKET END PRODUCTS ROLE Polyunsaturated Fatty Acids (PUFAS) Nutraceuticals Food Soft gel capsules Margarines, functional drinks (low-fat, dairy-type) Active Ingredient Galactolipids Cosmetics Gel, emulsions, cream formulations Excipient: emulsion stabilizer, encapsulating agent Nutraceuticals Functional drinks Excipient: emulsion stabilizer, encapsulating agent Pharmaceutical Gel, emulsions, creambased formulations Excipient: emulsion stabilizer, encapsulating agent Pharmaceutical Possible Active Pharmaceutical Ingredient Possible immuno-stimulatory properties Feed Possible active ingredient Possible immuno-stimulatory properties Fatty acids Chemicals Surfactants/emulsifiers Raw material for surfactant synthesis Chlorophyll Food Drinks, desserts, breakfast cereals, confectionary Food colorant Peptides Food Nutraceuticals Pharmaceutical Active with therapeutics properties Active with therapeutics properties Glycerol Multiple Multiple Natural compound with uses in pharmaceutical, personal care, food industry, chemical intermediate

7 DUNALIELLA SALINA POWDERS FREEZE DRIED D. SALINA (MONZON BIOTECH) SPRAY DRIED D. BARDAWIL (NBT LTD.) Raw material: Drying method: Colour: Odour: Flavour: Consistency: D. salina and D. bardawil freeze drying or spray drying orange powder Average Algal Composition - Organic matter (ash free matter) analysis normalised to 100% W-powder: ash content 4-5% of dry weight E-powder: ash content up to 50% of dry weight Algal Composition W-powder E-powder glycerol lipid 9 18 protein equivalents 3 18 carbohydrate total carotene cis β-carotene all trans β-carotene α-carotene Lutein Zeaxanthin Chlorophyll Typical lipid composition of powder (% of those higher than 1%) % Lipid W-powder E-powder C11:0 Undecylic Acid C14:0 Myristic Acid C16:0 Palmitic Acid C17:1 Heptadecenoic Acid C18:0 Stearic Acid C18:1 Oleic Acid C18:2 Linoleic Acid C18:3 Linolenic Acid C20:1 Gadoleic Acid % of total Fatty Acids FA Total Saturated FA Total Monounsaturated FA 11 9 Polyunsaturated FA % Ash Free Dry Weight Total Lipid 9 14 Typical amino acid composition (mg/g protein) Aa WHO & FAO* W-powder E-powder Lys Met Met + Cys Thr Phe + Tyr Val Ile Leu Trp His * WHO and FAO (2007) Protein and amino acid requirements in human nutrition, World Health Organization technicalreport series, (935), pp

8 DUNALIELLA SALINA POWDER Contaminants: GMO: Storage Hermetically-sealed container in the dark none detected GMO-free Shelf life: Hermetically-sealed sealed light-protected container, 3 years 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. Contacts: Monzón Biotech Parc Cientific de Barcelona c/ Baldiri Reixac 4-8 Torre I pl Barcelona (Spain) Phone NBT Ami Ben-Amotz, PhD, NBT Ltd. North Industrial Zone POB 828 Eilat, Israel Phone: Fax: ami@benamotz.com

9 CAROTENOID-RICH EXTRACT AND POWDER Raw material: Drying method: Extraction method: Sensory property: Colour: Odour: Flavour: Description: Consistency: 100% DUNALIELLA EXTRACT: PASTE Dunaliella salina freeze drying supercritical CO2-extraction dark red oil soluble/dispersible paste viscous Content 100% Dunaliella extract. Essential N-3 fatty acids, α- and β-carotenes, lutein, zeaxanthin and isomers. Carotenoid composition Carotenoid % Lutein 0.55 Zeaxanthin 0.14 All-trans β-carotene cis-β-carotene cis-β-carotene 2.60 α-carotene 2.90 Total carotene % CAROTENOID-ENRICHED POWDERS Raw material: Drying method: Extraction method: Pulverisation: Sensory property: Colour: Odour: Flavour: Consistency: Dunaliella salina freeze drying supercritical CO2-extraction supercritical CO2 spraying orange free-flowing powder 10% extract + Carnauba wax Recommended for oily food stuffs & cosmetics. Shelf life 12 months, stored frozen (-18 C). 3% extract + Cocoa butter Recommended for oily food stuffs. Easy dosing & free flowing. Shelf life 12 months, stored frozen (-18 C). DEFATTED POWDER Raw material: Drying method: De-Fatting method Dunaliella salina freeze drying supercritical CO2-extraction 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 after opening when kept cooled and dark, min. 24 month when kept frozen (-18 C) and dark. Sensory property: Odour: Flavour: Consistency: free-flowing powder

10 CAROTENOID-RICH EXTRACT AND POWDER Composition (%) W-powder E-powder Crude Fat Crude Protein Fiber 5 8 Starch incl sugars Ash 4 15 NaCl 0 2 Water 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: Nateco Hopfenveredlung St. Johann GmbH NATECO2 Auenstr Wolnzach / Germany Phone: Fax: contact@nateco2.de

11 MONZÓN & A4F BIOTECH PRODUCTS 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: A4F Estrada do Paço do Lumiar Campus do Lumiar, Ed. E R/C Lisboa Portugal Phone: info@a4f.pt For orange carotenoid-rich algae: Monzón Biotech Parc Cientific de Barcelona c/ Baldiri Reixac 4-8 Torre I pl Barcelona (Spain) Phone Closed flat-panel photobioreactors from A4F growing carotene-rich Dunaliella at Monzón Biotech production unit in Spain. Multilayer horizontal tubular photobioreactors growing carotene-rich Dunaliella cells at A4F pilot unit in Lisbon, Portugal Pond growing carotene-rich Dunaliella at Monzón Biotech production unit in Spain.

12 CUSTOMIZED CAROTENOIDS EXTRACTS 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. Packaging Hermetically sealed light protected containers according to customer requirements. Availability Minimum 100 g; maximum 1000 g containers. SEMI-PURIFIED COMPOUNDS FOR ORAL APPLICATION Lutein Zeaxanthin All-trans β-carotene 9-cis β-carotene in development Specification Purity >95%. Residual Solvents, metal and other impurities to ICHQ3A and ICHQ3B requirements. Packaging Hermetically sealed light protected containers Availability Minimum 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: Availability: hermetically sealed light protected vials 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 Extractions info@dynamicextractions.com Tel: +44(0)

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