sustainable extraction of bioactives from marine biomass at...
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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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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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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