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

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

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

CSIR Bio/Chemtek Batch RecordBatch 1 16 litres Batch 2a 16 litres Batch 2b 16 litres Batch litres Batch litres Batch 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) Pre-washNo Yes 100% ethanol mass (kg) Water mass (kg) Cereal:solvent ratio1:5 1:41:4.61:3.4 Maximum extraction temperature (°C) 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

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

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

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)

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

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

CSIR Bio/Chemtek Results

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

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

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

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

CSIR Bio/Chemtek

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 D powder E powder F powder G powder