ENVIROPAK WP2 LARGE SCALE
EXTRACTIONS
Corinda Erasmus
Chaven Yenketswamy
Daniel Menu
Michael Barkhuyzen
Sonya Buchner
November 2004
CSIR Bio/Chemtek
CSIR Responsibilities
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Supply of extracted kafirin
Re-use of ethanol
Testing of lab-scale method on a large scale
Modification of large scale extraction to improve
protein quality and yield
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Simplified
Solvent
preparation
extraction process, large scale
Milling
Washing
Reactor
Extract
Extract
Extracted
separation
cereal
Washing
with solvent
Extracted
cereal
precipitation
Evaporation
Dissolved
Ethanol
protein extract
Acidification
Precipitate
Water
separation
Full
fat protein
Air
drying
fraction
Defatting
Pure
Hexane
protein, milled in ball mill
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Batch Record
Batch 1
16 litres
Batch 2a
16 litres
Batch 2b
16 litres
Batch 3
400 litres
Batch 4
400 litres
Batch 5
300 litres
Raw material weight
(kg)
2.5 (as is)
2.5 (as is)
2.5 (as is)
85.0 (as is)
69.0 (dry base)
65.4 (dry base)
Sorghum type
Decorticated
white
Decorticated
white
Decorticated
white
Decorticated
red
Decorticated
red
Decorticated
red
Hammer mill sieve
size (mm)
2
2
2
2
3
3
Pre-wash
No
No
No
No
Yes
Yes
100% ethanol mass
(kg)
8.75
8.75
8.75
238.0
224.0
161.0
Water mass (kg)
3.75
3.75
3.75
99
96
69
Cereal:solvent ratio
1:5
1:5
1:5
1:4
1:4.6
1:3.4
Maximum extraction
temperature (°C)
70.0
70.0
70.0
74.4
70.0
70.0
Heating medium
Oil mantel at
70°C
Oil mantel at
70°C
Oil mantel at
70°C
Steam at
96°C
Water mantel at
70°C
Water mantel at
70°C
Filtration method
Basket
centrifuge,
5µm nylon
cloth
Basket
centrifuge, 5µm
nylon cloth
Basket
centrifuge, 5µm
nylon cloth
Basket
centrifuge,
5µm nylon
cloth
Basket
centrifuge, 5µm
nylon cloth
Basket
centrifuge, 5µm
nylon cloth
Maximum evaporation
temperature (°C)
61°C
61°C
61°C
64°C, 5
hours
39°C, 6 hours
48°C, 6.5 hours
Final product protein
(%)
79.2 (full fat)
88.6 (fat free)
88.6 (fat free)
76.0 (fat free)
83.6 (fat free)
82.4 (fat free)
Drying method
Spray dried
Freeze dried
Freeze dried
Centrifuged
and air dried
(ambient)
Centrifuged
and air dried
(ambient)
Centrifuged and
air dried
(ambient)
Milling method (final)
Ball mill
Ball mill
Ball mill
Ball mill
Ball mill
With metabisulphite
Yes
Yes
No
Yes
Yes
Ball mill
CSIR Bio/Chemtek
Yes
Reactor
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Glass-lined double jacketed reactor with stirrer
Pre-heating of solvent was done from second
batch onwards
Reacted for 45 minutes
Reflux system to prevent excessive ethanol
evaporation during extraction
CSIR Bio/Chemtek
Re-use of ethanol
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Ratio of ethanol/water to cereal
Calculated on a cereal dry-base
Reduced ratio further to below 1:4 cereal:solvent
Discovered pH effect – due to recovery,
suspected dissociation of sulphuric compounds
from metabisulphite
During extraction – add NaOH to specific endpH (minimum of 11.0) in batch 5
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Filtration and centrifugation
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To separate solvent with solubilised protein from fine
solids
Niesch filter with filter paper was not feasible;
extraction mixture cooling caused premature protein
precipitation
Alfa-Laval flame-proof basket centrifuge (basket
diameter 1.5 m, height 0.8m) with a 5µm nylon liner
was highly effective, 500 rpm initially, increased to
1200 rpm to recover more solution
Centrifugation done immediately after drainage
Centrifuging was done until cake was dry (31%
moisture content, Infra-red moisture analyser)
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Acidification
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After evaporation, the product was cooled rapidly
inside the same reactor using cold water and
glycol to 2 degrees Celsius
1N HCl was used for acidification and it was
done slowly to prevent heat formation during the
neutralisation of the NaOH
Final product pH was 5.0 in the wet medium, but
the dry protein pH was between 4.4 and 4.6 in
both cases
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Defatting
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Defatting was done with hexane – product was
shaken in excess hexane for two hours followed
by washing and filtration
Defatted product was left at room temperature in
a fume cabinet to dry
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Results
Protein yield comparisons
Yield (mass %)
100
90
Batch 2a
80
Batch 2b
70
Lab-scale
extraction
Batch 3
60
50
40
Batch 4
30
Batch 5
20
10
0
Product protein
content, full fat
Product yield (%
of total protein)
Pure protein
recovery
Kafirin recovery
CSIR Bio/Chemtek
Discussion
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Evaporation of ethanol during process – will change ratio
during extraction – sealed reactor off as tightly as possible
Moisture content of starting material (dry base vs. as is
base) – not a factor on laboratory scale
Clarity of solution – variable after extraction, not sure if a
turbidity value for quality control can be implemented
Washing step – successful in improving clarity and final
product protein content, but what else did we loose?
Final recovery of kafirin in centrifuge – losses and
overflow of cereal pieces from first centrifuging stage
No need for freeze-drying, but milling is very slow in ball
mill
Evaporation temperature during ethanol recovery – how
much can the protein endure?
Final product drying temperature – seems to have a
significant effect on protein structure
CSIR Bio/Chemtek
Unknown factors that may differ
between lab and large scale kafirin
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Final protein pH, may be influenced by washing steps with
tap water, pH of mixrure during extraction
Final levels of impurities (quantity and type for example
starch or fibre) – may need to design specific centrifuge
cloth (risk of blockages)
Final levels of sodiummetabisulphite – may be influenced
by long exposure to high vacuum
Thermal damage – so far the three large-scale systems
were similar – working at lowest practical temperature
Effect of final drying method – three batches were similar
Effect of protein recovery method – filtration (not practical
on large scale) vs. centrifuge (mechanical stress ?)
Particle size of milled protein – unknown, but all are ballmilled
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Samples taken for analysis (FT-IR)
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Samples taken at the following intervals during
batch 5:
 Solution of kafirin halfway during the
evaporation stage
 Wet protein paste after evaporation was
complete, before acidification step
 Wet protein paste after acidification
 Final product after drying
CSIR Bio/Chemtek
Samples prepared for FT-IR
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Sample A – wet kafirin solution during evaporation
Sample E – same as A, but dried at room temperature in
petri-dish (23°C)
Sample B – wet paste before acidification
Sample F – same as B, dried at room temperature
Sample C – kafirin after acidification (dried paste, 3°C)
Sample D – dried kafirin obtained from initial extract –
dried at room temperature
Sample G – final product
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FT-IR spectra of kafirin from big batch 5,
sample A and B
0.12
α-helix
random coil
Absorbance
0.1
0.08
0.06
0.04
0.02
0
-0.02
1700
1600
1500
1400
1300
Wavenumber (cm-1 )
Sample A (kafirin in solution)
Sample B (pastry)
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FT-IR spectra of Kafirin from big batch 5,
sample D, E and F
0.5
Absorbance
0.4
β-sheet
α-helix
0.3
0.2
0.1
0
-0.1
1700
1600
1500
1400
1300
-1
Wavenumber (cm )
Sample D
Sample E
Sample F
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FT-IT spectra of kafirin from big batch 5,
Sample C and G
Absorbance
0.5
0.4
0.3
α-helix
β-sheet
0.2
0.1
0
-0.1
1700
1600
1500
1400
1300
Wavenumber (cm-1 )
Sample C
Sample G
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Secondary structure analysis (peak intensity ratio) %
Samples
1650 cm-1
(α-helix peak)
1640 cm-1
(random coil
peak)
1620 cm-1
( β-sheet
peak)
C powder
35.2
31.9
32.9
D powder
38.4
33.1
28.5
E powder
38.3
33.6
28.2
F powder
36.2
32.6
31.3
G powder
33.5
36.2
30.3
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