Protein Processing in Food and Bioproduct Manufacturing and Techniques for Analysis - Food and Industrial Bioproducts and Bioprocessing - page 95

will experience an increase in pH (Bazinet et al ., 1998 ). During processing, protons
generated by the bipolar membrane come into contact with proteins circulating in the
electrodialysis cells on the cationic side of the bipolar membrane. At the isoelectric point
when the net charge on the protein is zero, the proteins aggregate/precipitate allowing
for their selective separation. Proteins can be subsequently recovered by centrifugation or
filtration as is done in other conventional processes for protein separation.
Using this technique, Bazinet and co-workers (1998) lowered the pH of a soy protein
solution from 8.0 to 4.5 in a cell of 100 cm 2 effective electrode surface, at a constant current
of 25 mA/cm 2 and obtained a precipitate with 95% protein purity. Mondor and co-workers
(2004a, 2004b) also successfully applied bipolar membrane electrodialysis to separate soy
proteins.
Application of bipolar electrodialysis for the simultaneous removal of inhibitory acetate
and pH control during E. coli fermentation was investigated by Wong and co-workers
(2010). The final biomass and recombinant protein concentrations obtained increased by up
to 37 and 20%, respectively.
There has been limited application of bipolar membrane electrodialysis for protein
separation at the commercial scale due to cost, complexity of use and lack of availability of
units for large scale processing.

Protein precipitation using organic solvents
Organic solvents such as acetone and ethanol can be used to induce protein precipitation.
Similar to the salting-out phenomenon, addition of organic solvents removes water from the
hydration spheres of the protein allowing electrostatic forces to bring oppositely charged
regions of the protein together. Water is, thus, removed both by bulk replacement by the
organic solvent and by structuring of the water around the organic molecules (Singh, 1995).
Acetone, ethanol, acetone-methanol, chloroform-methanol, tricholoroacetic acid-ethanol
are examples of solvents and solvent mixtures frequently used for protein precipitation.
Modifications in protein functionality, safety of solvent handling and miscibility are some
of the challenges associated with organic solvent protein precipitation.

3.3.2.4 Protein Purification further downstream

Washing
After recovery, proteins can be further purified by repeated washing with an appropriate
solvent followed by centrifugation and/or filtration. This extra unit operation allows the
removal of non-protein soluble components trapped within the interstitial spaces of the
precipitate. The process can be further optimized to remove contaminating soluble proteins
prior to drying (e.g., by selecting an appropriate pH of the water used for washing).
By washing the acid precipitated soy protein with pH 4.5 wash water, then resolubilizing
and washing at pH 9.0 culminating with a final pH 4.5 precipitation, fat content was further
decreased from 7-9% to 3-6%, while increasing the protein content from ~81-85% to
~90-92% without the added washing step (Mattil et al ., 1979 ).
Lawhon and co-workers (1981b) also reported that solubilizing full fat soy flour in water
under controlled conditions (1:12 w/w solid:water ratio, pH 9 at 60 °C for 30 min), separating
the aqueous phase from the slurry by centrifugation and washing the residue with water
(1:5 solid:water ratio) at pH 9.0, precipitating the combined aqueous phase at pH 4.5.
Washing the curd with acid water (pH 4.5) then decreased the fat content to 3.2%, while
ensuring a protein content approaching 90%.