Enzyme-assisted extraction and ultrafiltration of value ... · • Worlwide production: 1,199,139 tonnes (2017) • Harvested area: 188,888 ha harvested (2017) Source: Food and Agricultural

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


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


- 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.)


• 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


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


Effect of the solvent on the extraction


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


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

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1,5

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2,5

3

3,5

0

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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


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


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


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


Ultrafiltration in presence of enzymes

• The flux is relatively stable

• The use of enzymes results in higher permeate flux


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


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]

Documents

Enzyme-assisted extraction and ultrafiltration of value ... · • Worlwide production: 1,199,139 tonnes (2017) • Harvested area: 188,888 ha harvested (2017) Source: Food and Agricultural