4.1 Precipitation
Selective precipitation of non-hydrolyzed proteins separate them from soluble
peptides, and the right conditions depend on protein solubility in a specific precipitating agent. The precipitation used in the first stage of peptide isolation from
protein hydrolysates involves the addition of a precipitating agent. These agents are
organic solvents such as ethanol or methanol, acids like trichloroacetic acid (TCA),
or salts such as ammonia sulfate. Centrifugation or filtration can then be used to
separate the insoluble compounds easily.
After this step, the isolation or fractionation of a target peptide is also possible
using the selective conditions such as adjusting pH to its isoelectric point where it is
insoluble and separates from other soluble peptides.
In some cases, the addition of chemical compounds can produce peptide
degradation and changes in its main physical and biological properties. However,
precipitation is a relatively fast, low-cost, and easy-to-scale process. Removal of
precipitation agents and other contaminants is essential because they can interfere
or hinder the purification of the target peptide and its subsequent identification.
4.2 Membrane Modules
UF membrane modules can also be used to separate peptides from non-hydrolyzed
proteins without the addition of precipitating agents. The proper selection of
membrane pore and physical properties allows some purification of the target
peptide [73]. Ultrafiltration (UF) and nanofiltration (NF) processes are commonly
used to fractionate mixtures of peptides and amino acids, in this way, improving
their biological or functional properties [74].
Arrutia et al. [75] observed a high flux of permeation through the membrane
when operating at a pH value of the isoelectric point of the target peptide. On the
other hand, the selection of the synthetic material constituting the UF membrane is a
relevant factor, as apolar peptides can adsorb on the membrane surface. Another
adverse effect occurs by the formation of the polarization layer on the surface of the
membrane, significantly decreasing the effective pore size. To prevent these phenomena, Doyen et al. [76] proposed the use of UF membranes combined with
electrodialysis processes. In this case, the electrical charge and size are responsible
for higher efficiency in the separation of the target peptide. For example, this greater
efficiency is due to a higher migration of anionic peptides from the concentrate
compartment when the opposite charge electrode (cathode) is localized in the
permeate [77].
Membrane technology has several advantages, and among them are (i) no
requirement of chemicals addition; (ii) relatively low energy consumption; (iii) low
processing costs, and (iv) easy scale-up. These advantages make membrane processes an important separation technology in comparison to chromatographic
techniques at industrial-scale applications.
Enzymatic Production of Bioactive Peptides from Whey Proteins …
487
Selective precipitation of non-hydrolyzed proteins separate them from soluble
peptides, and the right conditions depend on protein solubility in a specific precipitating agent. The precipitation used in the first stage of peptide isolation from
protein hydrolysates involves the addition of a precipitating agent. These agents are
organic solvents such as ethanol or methanol, acids like trichloroacetic acid (TCA),
or salts such as ammonia sulfate. Centrifugation or filtration can then be used to
separate the insoluble compounds easily.
After this step, the isolation or fractionation of a target peptide is also possible
using the selective conditions such as adjusting pH to its isoelectric point where it is
insoluble and separates from other soluble peptides.
In some cases, the addition of chemical compounds can produce peptide
degradation and changes in its main physical and biological properties. However,
precipitation is a relatively fast, low-cost, and easy-to-scale process. Removal of
precipitation agents and other contaminants is essential because they can interfere
or hinder the purification of the target peptide and its subsequent identification.
4.2 Membrane Modules
UF membrane modules can also be used to separate peptides from non-hydrolyzed
proteins without the addition of precipitating agents. The proper selection of
membrane pore and physical properties allows some purification of the target
peptide [73]. Ultrafiltration (UF) and nanofiltration (NF) processes are commonly
used to fractionate mixtures of peptides and amino acids, in this way, improving
their biological or functional properties [74].
Arrutia et al. [75] observed a high flux of permeation through the membrane
when operating at a pH value of the isoelectric point of the target peptide. On the
other hand, the selection of the synthetic material constituting the UF membrane is a
relevant factor, as apolar peptides can adsorb on the membrane surface. Another
adverse effect occurs by the formation of the polarization layer on the surface of the
membrane, significantly decreasing the effective pore size. To prevent these phenomena, Doyen et al. [76] proposed the use of UF membranes combined with
electrodialysis processes. In this case, the electrical charge and size are responsible
for higher efficiency in the separation of the target peptide. For example, this greater
efficiency is due to a higher migration of anionic peptides from the concentrate
compartment when the opposite charge electrode (cathode) is localized in the
permeate [77].
Membrane technology has several advantages, and among them are (i) no
requirement of chemicals addition; (ii) relatively low energy consumption; (iii) low
processing costs, and (iv) easy scale-up. These advantages make membrane processes an important separation technology in comparison to chromatographic
techniques at industrial-scale applications.
Enzymatic Production of Bioactive Peptides from Whey Proteins …
487