Biomedical Engineering Reference
In-Depth Information
Achouri and co-workers (2010) reported that initial extraction of soy proteins using
isoelectric precipitation at pH 6.8 followed by cryo-precipitation yielded 4.2% product
recovery for the 11 S soybean protein fraction with 98% protein purity for a control extracted
with sodium hydroxide, and average yields of 4.4% and 5.17%, respectively, when sodium
sulfate (Na
2
SO
4
) and ammonium sulfate [(NH
4
)
2
SO
4
] were used. Addition of calcium
chloride (CaCl
2
) doubled the extraction yield to 9%.
As some salts may be toxic, an appropriate salt must be selected for the target end-product
application (industrial, food, pharmaceutical). The type of proteins precipitated in a given
solution will also vary as a function of the salt concentration used.
In a modified version of the salting out process, salt proteins may be preferentially
separated out of solution by dilution of the protein extract. In this case, after extraction
of protein using an appropriate salt solution at desired ionic strength, the solution is
extensively diluted to decrease the solubility of the salt soluble proteins, inducing protein
precipitation.
Paredes-López and co-workers (1991) extracted protein from a 10% (w/v) solution of
defatted chickpea with sodium chloride (0.5 M, pH 7.0) and obtained a chickpea protein
isolate containing 87.8% protein using this method. After concentration of the extract by
ultrafiltration, protein was flocculated by the addition of water (4 °C, pH 7, 1:4 v/v ratio of
protein extract: water). Márquez and co-workers (1996) reported protein contents ranging
from 74.7 to 84.2% for common bean protein extract using similar methods.
Microfiltation and ultrafiltration
Microfiltation (MF) and ultrafiltration (UF) are pressure-driven filtration processes that
utilize a porous membrane to selectively retain compounds larger than a nominal molar
mass (retentate) while allowing particles of lower molar mass to pass through the membrane
(permeate). It is a frequently used alternative to isoelectric precipitation.
During this process, solutions containing extracted or dissolved proteins are subjected to
MF and/or UF to concentrate the proteins. MF membranes retain larger molecular weight
compounds and are better suited for retaining particles with a molecular mass greater than
300 000 and in the 0.02-10 micron size range. They are, therefore, more useful for the sepa-
ration of cellular material, such as in biological, biochemical, pharmaceutical and nutraceu-
tical applications. A useful application of MF in food processing is the removal of yeasts
and bacteria without the requirement for thermal treatment. In protein purification work,
MF could be used to specifically retain larger molecular weight proteins of interest, or to
remove these interfering proteins. The permeate can be further processed to obtain a higher
purity protein extract.
UF membranes retain smaller molecular mass particles (300-300 000) with sizes ranging
from 0.001 to 0.02 microns and are frequently used in protein processing. UF membranes
are typically constructed from regenerated cellulose, cellulose acetate, ceramic composites,
polysulfone, polyethersulfone, polyamide, polyacrylonitrile or polyvinyl alcohol. The
molecular weight cut-off of ultrafiltration membranes is diffuse by about one order of mag-
nitude, which makes it difficult to achieve absolute retention. Thus, a UF membrane with a
molecular weight cut-off of 200000 could retain particles as small as 20000 molecular
mass. In addition to molecular mass the shape of particles will also influence their retention
or permeation. Membranes with specific molecular weight cut-offs must be carefully
selected in order to retain the desired protein of interest. Other factors affecting efficient
separation of proteins by microfiltration or UF include the type of membrane, the molecular
weight cut-off, the volume concentration ratio and diafiltration conditions. A major problem
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