sustainable extraction of bioactives from marine biomass at...

Sustainable extraction of bioactives from marine biomass at iBET

Naiara Fernández, Maha Abdallah, Liliana Rodríguez, Ana Nunes, Miguel Batista, Ana A. Matias

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www.ibet.pt

iBETInstituto de Biologia Experimental e Tecnológica

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→ THE LARGESTBIOTECHNOLOGY RESEARCH ORGANIZATION IN PORTUGAL

→ BRINGS TOGETHER AS PARTNERS AND COLLABORATORS, PUBLIC INSTITUTIONS AND PRIVATE COMPANIES

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iBETInstituto de Biologia Experimental e Tecnológica

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CircularEconomy

• ZERO WASTE

• OPTIMIZATION OF NATURAL SOURCES

• WASTE VALORIZATION

• DESIGN OF GREENER TECHNOLOGIES

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iBETInstituto de Biologia Experimental e Tecnológica

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Bioactivity /Health benefits / Safety / Quality / Sensory

Raw materials

Extract/Compound

FinalProduct

Formulation

ExtractionPurification

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• hydrogen bond formation between the different

components of the mixture

Deep Eutectic Solvents (DES)

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• Two or more compounds that upon mixing at a certainmolar ratio suffer a high melting temperature depression

A

B

+

• Natural DES (NADES) are DES constituted by primary or secondary metabolites

• Low-cost • Use of new green solvents• Flexible design

• Expensive • Use of organic solvents• Time-consuming

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Deep Eutectic Solvents (DES)

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Mussels

Dissolved HA/CS

Marine by-products

Codfish bones

Natural DES

50°C

24h

Hyaluronic acid and chondroitin sulfate extraction from codfish and mussels

Percentage of total HA and CS disaccharides isolated in comparison to the conventional method

% Menthol: Borneol Menthol: Camphor Thymol: Borneol Thymol: Camphor

Codfish

bones

HA 16.1 17.3 27.7 18.0

CS 25.3 25.1 24.6 28.0

MusselsHA 63.5 48.1 44.6 43.1

CS 66.3 67.5 63.8 76.8

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Deep Eutectic Solvents (DES)

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Astaxanthin extraction from crab shell

NADES extraction

PA:CA (1:1)ME:PA(1:1)ME:CA(1:1)

ME:EU (1:1)ME:MA (8:1)

S/L ratio

0.25 gresidue/gNADES

Temperature

30oC 45oC 60oC

Extraction time (h)

2h 6h 24h

g

AX

T/

gd

ry r

esi

du

e

S o xh let

(ac e to

n e )

P A:C

A (1

:1)

ME :P

A (1

:1)

ME :C

A (1

:1)

ME :E

U (1

:1)

ME :M

A (8

:1)

0

2

4

6

8

1 0

1 2 S o x h le t ( a c e to n e ) , 6 h

3 0 C , 2 h 3 0 C , 6 h 3 0 C , 2 4 h

4 5 C , 2 h 4 5 C , 6 h 4 5 C , 2 4 h

6 0 C , 2 h 6 0 C , 6 h 6 0 C , 2 4 h

Soxhlet extraction

Acetone

Extraction time (h)

6h

PA: Perillyl alcoholm; CA: Camphor; ME: Menthol; EU: Eucalyptol; MA: Myristic Acid

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Deep Eutectic Solvents (DES)Astaxanthin extraction from crab shell

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Cytotoxicity of DES was evaluated in Caco-2 cells, as amodel of the human intestinal epithelium

0 1 2 3 4 5

0

2 0

4 0

6 0

8 0

1 0 0

1 2 0

C o n c e n t r a t io n ( m g / m L )

Ce

ll v

iab

ilit

y (

% o

f c

on

tro

l) P A :C A ( 1 :1 )

M E :P A ( 1 :1 )

M E :C A ( 1 :1 )

M E :E U (1 :1 )

M E :M A ( 8 :1 )

ME:MA (8:1) showed to be theleast toxic NADES in Caco-2 cells

MIC

* (

gA

XT

/m

L)

P A:C

A (1

:1)

ME :P

A (1

:1)

ME :C

A (1

:1)

ME :E

U (1

:1)

ME :M

A (8

:1)

P A:C

A (1

:1)

ME :P

A (1

:1)

ME :C

A (1

:1)

ME :E

U (1

:1)

ME :M

A (8

:1)

0 .0 0

0 .0 5

0 .1 0

0 .5

1 .0

1 .5

2 .0 A X T S t a n d a r dS . a u r e u s

E . c o li

C r a b s h e ll e x t r a c t s

When comparing the extracts obtained with an astaxanthinstandard solubilized in each one of the systems, extractsshowed a much better (10 – 98 fold) antimicrobial activity

Aantimicrobial activity using bacterial strains relevant to foodand cosmetic applications

MIC: Minimum inhibitory concentration

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Supercritical CO2

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Gas-like viscosity

Liquid-like density

Enchanced solubility

High miscibility

Low viscosity

High diffusivity

SUPERCRITICAL CO2

Absence of oxygen Extract Stability

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Supercritical CO2 with cosolvent

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Astaxanthin extraction from crab shell

Equilibration time Pressure Temperature Flow rate

0 - 30 min 200 - 500 bar 40 - 60 °C 30 - 50 g/min

Full Factorial Design 4 factors, 2 levels and 3 center points

Response surface methodology

Astaxanthin Extract Concentration

0.2 – 0.7 % 0.03 – 6.02 µg/g dried residue

Total ExtractionYield

Astaxanthin ExtractionYield

8 – 1025 µg/g dried extract

Carbon Dioxide95%, wt.

Ethanol5%, wt.

Brown crab residues25g

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Microencapsulation with Spray Drying

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STABILIZATION PROTECTION CONTROLLED RELEASE

External stimulus

solubility

reactivityvolatility

hygroscopicity

pHlight

oxygen

Temperature

Encapsulation matrixActive ingredient

• Sugars• Gums • Proteins • Polysaccharides • Lipids• Synthetic polymers

Natural-based pigments

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Outlet T

Intlet T

Feed emulsion

Powder

Hydrophilic particles present an intenseorange colour and good water dispersibility

Microencapsulation of carotenoid pigments from Dunaliella salina into natural polysaccharides

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Microencapsulation with Spray Drying

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Supercritical CO2 Drying

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Alginate-Chitosan aerogel fibres

Emulsion-Gelation

Alginate

Chitosan

Hydrogel/Alcogel

scCO2

CO2Ethanol

Supercritical drying*

Aerogel Fibres

alg:chit (99:1) aerogel fibres

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Scratch assay: incubation of 1.7 mg/mL in NCTC clone 929 fibroblasts, during 8hat 37ºC and 5% CO2 humidified atmosphere (mean ± SD, n=4). Statisticallysignificant differences when compared to control conditions are indicated by**** (p < 0.0001).

t=0h

t=8h

Supercritical CO2 Drying

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STIMULATION OF CELL MIGRATION (in vitro scratch assay) and ANTIBACTERIAL ACTIVITY

Percent reduction of S. aureus and K. pneumoniae resultingfrom contact with samples, at a concentration of 0.8 mg/mL,during 2.5 h at 37ºC (mean ± SD, n=3; except controls n=6).Cotton disks were used as untreated control.

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Nutraceuticals and Bioactives Process Technology

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info.ibet@ibet.pt

Av. República, Qta. do Marquês

Estação Agronómica Nacional

Edifício iBET/ITQB

2780-157 Oeiras - Portugal

(+351) 214 427 787

(+351) 214 421 173

Fax: (+351) 214 421 161