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

2. CSIR Bio/Chemtek CSIR Responsibilities n Supply of extracted kafirin n Re-use of ethanol n Testing of lab-scale method on a large scale n Modification of large scale extraction to improve protein quality and yield

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

4. CSIR Bio/Chemtek Batch RecordBatch 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) 85.0 (as is)69.0 (dry base)65.4 (dry base) Sorghum typeDecorticated white Decorticated red Hammer mill sieve size (mm) 222233 Pre-washNo Yes 100% ethanol mass (kg) 8.75 238.0224.0161.0 Water mass (kg)3.75 999669 Cereal:solvent ratio1:5 1:41:4.61:3.4 Maximum extraction temperature (°C) 70.0 74.470.0 Heating mediumOil mantel at 70°C Steam at 96°C Water mantel at 70°C Filtration methodBasket centrifuge, 5µm nylon cloth Maximum evaporation temperature (°C) 61°C 64°C, 5 hours 39°C, 6 hours48°C, 6.5 hours Final product protein (%) 79.2 (full fat)88.6 (fat free) 76.0 (fat free)83.6 (fat free)82.4 (fat free) Drying methodSpray driedFreeze dried Centrifuged and air dried (ambient) Milling method (final)Ball mill With metabisulphiteYes NoYes

5. CSIR Bio/Chemtek Reactor n Glass-lined double jacketed reactor with stirrer n Pre-heating of solvent was done from second batch onwards n Reacted for 45 minutes n Reflux system to prevent excessive ethanol evaporation during extraction

6. CSIR Bio/Chemtek Re-use of ethanol n Ratio of ethanol/water to cereal n Calculated on a cereal dry-base n Reduced ratio further to below 1:4 cereal:solvent n Discovered pH effect – due to recovery, suspected dissociation of sulphuric compounds from metabisulphite n During extraction – add NaOH to specific end- pH (minimum of 11.0) in batch 5

7. CSIR Bio/Chemtek Filtration and centrifugation n To separate solvent with solubilised protein from fine solids n Niesch filter with filter paper was not feasible; extraction mixture cooling caused premature protein precipitation n 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 n Centrifugation done immediately after drainage n Centrifuging was done until cake was dry (31% moisture content, Infra-red moisture analyser)

8. CSIR Bio/Chemtek Acidification n After evaporation, the product was cooled rapidly inside the same reactor using cold water and glycol to 2 degrees Celsius n 1N HCl was used for acidification and it was done slowly to prevent heat formation during the neutralisation of the NaOH n 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. CSIR Bio/Chemtek Defatting n Defatting was done with hexane – product was shaken in excess hexane for two hours followed by washing and filtration n Defatted product was left at room temperature in a fume cabinet to dry

10. CSIR Bio/Chemtek Results

11. CSIR Bio/Chemtek Discussion n Evaporation of ethanol during process – will change ratio during extraction – sealed reactor off as tightly as possible n Moisture content of starting material (dry base vs. as is base) – not a factor on laboratory scale n Clarity of solution – variable after extraction, not sure if a turbidity value for quality control can be implemented n Washing step – successful in improving clarity and final product protein content, but what else did we loose? n Final recovery of kafirin in centrifuge – losses and overflow of cereal pieces from first centrifuging stage n No need for freeze-drying, but milling is very slow in ball mill n Evaporation temperature during ethanol recovery – how much can the protein endure? n Final product drying temperature – seems to have a significant effect on protein structure

12. CSIR Bio/Chemtek Unknown factors that may differ between lab and large scale kafirin n Final protein pH, may be influenced by washing steps with tap water, pH of mixrure during extraction n Final levels of impurities (quantity and type for example starch or fibre) – may need to design specific centrifuge cloth (risk of blockages) n Final levels of sodiummetabisulphite – may be influenced by long exposure to high vacuum n Thermal damage – so far the three large-scale systems were similar – working at lowest practical temperature n Effect of final drying method – three batches were similar n Effect of protein recovery method – filtration (not practical on large scale) vs. centrifuge (mechanical stress ?) n Particle size of milled protein – unknown, but all are ball- milled

13. CSIR Bio/Chemtek Samples taken for analysis (FT-IR) n Samples taken at the following intervals during batch 5: n Solution of kafirin halfway during the evaporation stage n Wet protein paste after evaporation was complete, before acidification step n Wet protein paste after acidification n Final product after drying

14. CSIR Bio/Chemtek Samples prepared for FT-IR n Sample A – wet kafirin solution during evaporation n Sample E – same as A, but dried at room temperature in petri-dish (23°C) n Sample B – wet paste before acidification n Sample F – same as B, dried at room temperature Sample C – kafirin after acidification (dried paste, 3°C) n Sample D – dried kafirin obtained from initial extract – dried at room temperature n Sample G – final product

15. CSIR Bio/Chemtek

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 powder35.231.932.9 D powder38.433.128.5 E powder38.333.628.2 F powder36.232.631.3 G powder33.536.230.3