ENVIROPAK WP2 LARGE SCALE EXTRACTIONS

1. ENVIROPAK WP2 LARGE SCALE EXTRACTIONS Corinda Erasmus Chaven Yenketswamy Daniel Menu Michael Barkhuyzen Sonya Buchner November 2004

2. CSIR Responsibilities • 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

3. Simplified extraction process, large scale Solvent preparation Milling Washing Reactor Extracted cereal Washing with solvent Extract separation Extracted cereal Extract precipitation Evaporation Ethanol Dissolved protein extract Acidification Full fat protein Air drying Precipitate separation Water fraction Hexane Defatting Pure protein, milled in ball mill

5. Reactor • 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

6. Re-use of ethanol • 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 end- pH (minimum of 11.0) in batch 5

7. Filtration and centrifugation • 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)

8. Acidification • 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

9. Defatting • 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

10. Results

11. Discussion • 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

12. Unknown factors that may differ between lab and large scale kafirin • 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 ball-milled

13. Samples taken for analysis (FT-IR) • 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

14. Samples prepared for FT-IR • 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

18. Samples Secondary structure analysis (peak intensity ratio) % 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