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Enzyme-assisted extraction and ultrafiltration of value-adedcompounds from sour cherry winepomaceMaria Cinta Roda-Serrat, Cecilie Lundsfryd, Simone Rasmussen, Rime B. El-Houri, Peter B. Lund and Knud V. Christensen
Dept. Of Chemical Engineering, Biotechnology and Environmental TechnologyUniversity of Southern Denmark
BIOCAS, circular BIOmass CAScade to 100%
M.C. Roda-Serrat 27/05/2019 ICheaP14, Bologna
• InterRegional project: The Netherlands, Belgium, Germany and Denmark
• Vision of the project: To reach the complete utilization of biomass through an integral approach
• Development of bio-cascades in which the waste from one process is the feed for the next
Fine chemicals
Nutrients
Bulk chemicals and bioplastics
Energy, soil enrichment
Valu
e
Source: WireTech World Online Magazine
M.C. Roda-Serrat ICheaP14, Bologna 27/05/2019
- Water soluble pigments (red-orange-purple hues)
- Common in fruits, berries, vegetables and some flowers
- Can substitute synthetic food colorants
- Reported health promoting effects
- Can be recovered from some industrial biowaste streams
Cyanidin-3-O-glucoside
Anthocyanins as food additives and nutraceuticals
Extraction- Conventional solvent extraction using methanol, ethanol or alcohol-water mixtures- Extraction in aqueous solutions aided by:
- Pressure (PLE)- Sonication (UAE)- Microwaves (MAE)- Enzymes (EAE)
- Supercritical solvent extraction
Purification- Chromatography- Membrane technology- Co-precipitation with polymers
Recovery of anthocyanins from plant material
M.C. Roda-Serrat ICheaP14, Bologna 27/05/2019
Extraction- Conventional solvent extraction using methanol, ethanol or alcohol-water mixtures- Extraction in aqueous solutions aided by:
- Pressure (PLE)- Sonication (UAE)- Microwaves (MAE)- Enzymes (EAE)
- Supercritical solvent extraction
Purification- Chromatography- Membrane technology- Co-precipitation with polymers
Recovery of anthocyanins from plant material
M.C. Roda-Serrat ICheaP14, Bologna 27/05/2019
- Degradation of the polysaccharides on the cell wall by a wide variety of enzymes:- Cellulases- Polygalacturonases- Beta-glucanases- Pectin lyases
- Effects reported- Increase in extraction efficiency- Decrease in the viscosity of the mash- Increase in filtration efficiency- Flavonoid glycosidase side activity: Anthocyanin degradation
Low selectivity, but not too
low!
Enzyme-assited extraction of anthocyanins
Sour Cherry (Prunus Cerasus L.)
M.C. Roda-Serrat ICheaP14, Bologna 27/05/2019
• It is mainly processed to juice, marmelade, liquour or wine due to its sour taste
• This results in the production of side streams that can
potentially be valorised.
• Worlwide production: 1,199,139 tonnes (2017)
• Harvested area: 188,888 ha harvested (2017)
Source: Food and Agricultural Organization of the United Nations
Top 10 producers of sour cherry (2017)
Case study: Sour cherry wine pomace
M.C. Roda-Serrat 27/05/2019 ICheaP14, Bologna
We refer to pomace as the residue after fermentation and pressing
Objective of the study:
- High extraction yield of anthocyanins and phenolic acids at a competitive cost
- Mild process to minimize degradation
- Compliance with current legislation, labelling, and market demands
- Water-based process (avoiding, when possible, the use or organic solvents)
- Purification strategy based in membrane filtration
Sour cherry wine pomace
Flesh; 31%
Stones; 65%
Stems; 2%
Sticks; 2%
Components % Fresh Weight
CoarseFiter Crusher Extraction
Unit
UltraFiltration
25 kDa
NanoFiltration
150 Da
Suspension Stones Crushed Solids
Fiber waste
Essential Oils
ParticlesPolysaccharides
Enzymes
AnthocyaninsPhenolic acidsFree sugarsSugar alcoholsBuffer salts
AnthocyaninsPhenolic acidsFree sugars
Free sugarsSugar alcoholsBuffer salts
ExtractionUnit
Bio-cascade for sour cherry wine biowaste
Production of biochars
Biogas upgrading
Concentrated anthocyanins
Essential oils
M.C. Roda-Serrat 27/05/2019 ICheaP14, Bologna
Effect of the solvent on the extraction
M.C. Roda-Serrat 27/05/2019 ICheaP14, Bologna
Extraction for 60 minutes at 50°C using:
• Water
• Citrate-phosphate buffer pH 3.0, 50 mM
• Citrate-phosphate buffer pH 3.0, 150 mM
• Citric acid solution 50 mM
Quantification by HPLC
Calibration using external standards
Observations
• The extraction of AC is favoured at lower pH values
• The pH of the extraction mixture increases when adding the pomace, even when using buffered solutions
• The highest extraction yield was obtained using citric acid 50 mM (final pH 2.9)
0,00
0,50
1,00
1,50
2,00
2,50
3,00
3,50
4,00
0
10
20
30
40
50
60
70
80
Water Buffer 50 mM Buffer 150 mM Citric acid 50 mM
Amou
nt e
xtra
cted
(mg/
kg)
Phenolic acids Anthocyanins pH
finalpH
Effect of the pomace dosage on the extraction
M.C. Roda-Serrat 27/05/2019 ICheaP14, Bologna
Methodology:
Extraction for 60 minutes at 50°C using:
• Citric acid solution 50 mM
• Dosages of 200 and 500 g/L
Observations
• A lower dosage of pomace results in increased amount of products extracted
• The dosage was not lowered further due to the already low concentration
0
0,5
1
1,5
2
2,5
3
3,5
0
20
40
60
80
100
120
140
500 g/L 200 g/L
Amou
nt e
xtra
cted
(mg/
kg)
Phenolic acids AC pH
finalpH
Effect of enzyme addition on the extraction
M.C. Roda-Serrat 27/05/2019 ICheaP14, Bologna
Observation
• The use of enzymes seems to be counter productive. Flavonoid glucosidase activity is suspected
0
5
10
15
20
25
30
35
40
45
50
0 2 4 6 8 10 12
Ant
hocy
anin
sex
trac
ted
(mg
kg-1
)
Time (h)
No enzyme
Celluclast 1.5 L
Fructozym Flash-C0
10
20
3040
50
60
7080
90
100
0 2 4 6 8 10 12Ph
enol
icac
ids
extr
acte
d(m
g kg
-1)
Time (h)
No enzyme
Celluclast 1.5L
Fructozym Flash-C
Two commercial enzyme preparations were added to the reaction mixture at a dosage of 200 mL ton-1
• Fructozym® Flash-C (Erbslöh) : blend of pectinases
• Celluclast ® 1.5L (Novozymes) : single-enzyme product cellulase
Enzyme side-activity essay
M.C. Roda-Serrat 27/05/2019 ICheaP14, Bologna
Observations
• No degradation observed
Filtered samples containing anthocyanins and phenolic acids were incubated for 24 hours at 50 °C
• Control sample
• Sample spiked with Fructozym® Flash-C 200 mL ton-1
• Sample spiked with Celluclast® 1.5L 200 mL ton-1
0%
20%
40%
60%
80%
100%
120%
0 4 8 12 16 20 24
Ant
hocy
anin
con
tent
(%)
Time (h)
No enzyme
Celluclast 1.5L
Fructozym Flash-C
0%
20%
40%
60%
80%
100%
120%
0 4 8 12 16 20 24Ph
enol
ic a
cid
cont
ent (
%)
Time (h)
No enzymeCelluclast 1.5LFructozym Flash-C
M.C. Roda-Serrat 27/05/2019 ICheaP14, Bologna
Ultrafiltration set-up and parameters
The e-MBR: enzyme - Membrane BioReactor Membrane information and filtration parameters
Membrane cut-off 25 kDa
Membrane material ZrO2
Manufacturer Atech Innovations GmbH
Membrane area 0.011 m2
Trans-membrane pressure 0.5 bar
Cross-flow velocity 3.0 m s-1
Filtration performed on a Atech 25 kDa ZrO2 tubular ceramic membrane
0
20
40
60
80
100
0 20 40 60
Perm
eate
flux
(kg
m-2
h-1 )
Time (min)
No enzyme
0 20 40 60Time (min)
Fructozym® Flash-C
0 20 40 60Time (min)
Celluclast® 1.5L
M.C. Roda-Serrat 27/05/2019 ICheaP14, Bologna
Ultrafiltration in presence of enzymes
• The flux is relatively stable
• The use of enzymes results in higher permeate flux
M.C. Roda-Serrat 27/05/2019 ICheaP14, Bologna
Conclusions and perspectives
• Pomace from the sour cherry wine industry can be valorized to produce added-value extracts
• 82.1 ± 2.9 mg anthocyanins/kg and 48.6 ± 0.9 mg phenolic acids/kg were reported.
• The use of pectinases and cellulases seemed to result in lower extraction yields, even though enzymatic
degradation was not reported.
• The use of enzymes resulted in an increased permeate flux during ultrafiltration
• Enzymes can be used as a pre-treatment for the ultrafiltration step to increase the process throughput
• Other biowaste streams from the same process need to be investigated
• Different enzyme dosages need to be tested to find an optimum
• A technoeconomical evaluation is needed to assess the feasibility of this biocascade
M.C. Roda-Serrat 27/05/2019 ICheaP14, Bologna
Acknowledgements
• Frederiksdal Kirsebærvin (Lolland, Denmark) for supplying the raw material
• Ersblöh and Novozymes for providing the enzyme formulations tested
• European Regional Development Fund as part of the Interreg North Sea Region project 38-2-4-17 BIOCAS,
circular BIOmass CAScade to 100%
Thank you
Maria Cinta Roda-SerratPostdoctoral [email protected]