Factor Optimum Press 300.0 bar Temp 50.0 oC Optimize Desirability Optimum value = 0.662769 Optimum 3.98 % Yield 621.8 mg carotenoids/g extract 284.8 mg chlorophylls/g extract

OPTIMIZATION OF SUPERCRITICAL FLUID EXTRACTION

PROCESSES TO OBTAIN BIOACTIVES FROM

Isochrysis galbana MICROALGA

J.A. Mendiola1, G. Sullini1.2, A. Cifuentes1, E. Ibáñez1 1 Instituto de Investigación en Ciencias de la Alimentación CIAL (CSIC-UAM). C/Nicolás Cabrera 9, 28049 Madrid.

2 Università degli Studi di Messina, Messina, Italia.

MIRACLES WPs and Activities

WP1: Production and Harvesting

WP4: Product development and market assessment

Cell disruption Extraction

Hydrophobic compounds

(lipids)

Hydrophilic compounds (proteins,

carbohydrates)

Food, incl. functional foods, nutraceuticals

Aquaculture / aquafeed

Non-Food applications

WP3: Development of Integrated Biorefinery

Harvesting

WP2: Bio-prospecting, metabolic optimization, cultivation at extreme

locations

New production strains

Industrial strains

NATAC, URDV, DSM

EWOS, SPAROS

BIOPOL, IMENZ, CHIMAR, NATAC, CE

Marke

t assessm

en

t

VFT WU DLO CSIC ET

DNL DSM VFT WU, UT, UHU, VITO, FITO,

TMUC

WU, FCPCT, UIB, UA, UniRes, FITO

Sequential extractions

1st step: SC-CO2 extraction to recover omega-3 and highly lipophilic compounds, Experimental design

2nd step: SC-CO2+ ethanol extraction to obtain extracts rich in other bioactives (more polar).

Isochrysis galbana microalga

Isochrysis galbana is a marine flagellated microalga belonging to the phylum Haptophyta, class Coccolithophyceae, subclass Prymnesiophycidae, order Isochrysidales, family Isochrysidaceae. Its cells are easily assimilated by larval animals because of their small size (4-7 μm) and absence of a tough cell wall. I. galbana is represented by different strains, the most commonly cultured one is the Tahitian strain “T-iso”. Generally, this microalga is characterized by a fast growth rate, wide temperature and salinity tolerance and absence of toxins. As all microalgae, the growth and biochemical composition of I. galbana cultures is dependent on variations of culture conditions (e.g. nutrients, temperature, salinity, and photoperiod).

This Haptophyta is of substantial interest in aquaculture, principally to feed mollusk larvae, as well as fish and crustaceans in the early stages of growth.

Isochrysis galbana is a marine microalga well recognized as a natural rich source of EPA and DHA that are accumulated as oil droplets in prominent lipid bodies in the cell. This unicellular microalgae is investigated also for its high amount of the carotenoid Fucoxanthin.

Supercritical fluid extraction

Foodmics Laboratory activities

Sequential extractions: 1st step optimization

3-level factorial design (32). The studied factors were P (100-300 bar) and T (40-60 ºC) Other factors fixed: 10 g of algal material used with 30 g of sea sand as dispersive agent, 5 L/min CO2(gas) Extraction time optimization: Kinetic study. Extractions performed at different times in the center-point of experimental design (200 bar, 50 ºC). Extraction time fixed at 30 min (75 % extractable material recovered)

40 ºC 50 ºC 60 ºC

200 bar 40 ºC 50 ºC 60 ºC

100 bar 40 ºC 50 ºC 60 ºC

300 bar

Statistical optimization of experimental design for YIELD

Yie

ld %

Pressure (bar)

R2 of mathematical model 93.2% Significant factors: Pressure and P*P Goal: maximize yield Optimum value = 4.51 % Optimum for yield: P= 254.2 bar T= 55.4 ºC

Multiple response optimization: Yield, Carotenoids and chlorophyls Extraction conditions optimized for 1st extraction step to: maximize yield and carotenoids and minimize chlorophylls

• Extraction of polar compounds based on Gas Expanded Liquids

Liquid Gas Expanded Liquid (GXL)

• Extractions performed on the residue from 1st extraction step • Experimental design

• 15-70% Ethanol • Pressure high enough to expand EtOH, but not not to dissolve CO2 (ca. 60-80 bar) • Same temperature as 1st step optimum (50 ºC) to reduce cooling time

Composition of extracts: carotenoids up to 63%, chlorophylls up to 32 %. PolyUnsaturated Fatty Acids (linolenic, DHA…)

detected by GC-MS metabolomic method. Clear trend at 50 ºC: higher pressure, higher

carotenoid concentration.

1st

Ste

p

2n

d S

tep

Composition of extracts: Extracts very rich in chlorophylls Polyunsaturated Fatty Acids (linolenic,

DHA…) detected by GC-MS metabolomic method at lower concentration than 1st step

GC-MS metabolomic method also reported the presence of several carbohydrates

Acknowledgements This work has been financed by an FP7 project: 613588 MIRACLES http://miraclesproject.eu/

CONCLUSIONS

The recovery of bioactive compounds from the microalga Isochrysis galbana has been set up. In the 1st step pure supercritical CO2

was used and extracts rich in carotenoids were obtained. In the 2nd step the application of Gas Expanded Liquid Extraction was studied for the first time on this microalga, providing extracts with very high content in chlorophylls and carbohydrates.