4.3 Chromatographic Techniques
Chromatographic techniques are primarily used in the separation of peptide mainly
in the laboratory and at a small scale. The high purity of the target peptide for the
pharmaceutic industry is required, but its high added value offsets the high associated costs in its purification when using chromatography (90% of the total cost).
Although chromatographic processes allow achieving high separation and selectivity of target peptide, these processes are characterized by low productivity and
high operating and investment costs, which make their extensive use at the
industrial-scale prohibitively expensive [31]. Ion exchange chromatography
(IEC) and size exclusion chromatography (SEC) are some of the chromatographic
techniques tested for target peptides purification. In contrast, high-performance
liquid chromatography (HPLC) is crucial for peptide identification and production
in the laboratory and on a small scale. However, the most efficient process for
peptides purification continues undoubtedly to be reverse-phase HPLC chromatography [78].
Salvado [79] carried out the hydrolysis of sweet whey with chymotrypsin for
10 min, at temperature 37 °C and initial pH of 6.7, reducing the protein concentration in about 43% (as assessed by the Bicinchoninic Acid (BCA) assay method).
Tricine SDS-PAGE showed the efficiency and selectivity of this hydrolytic process.
The soluble k-casein (k-CN) and b-LG were not hydrolyzed under these experimental conditions, as shown by the bands in lanes 3 and 4 in Fig. 4. On the other
hand, a-LA was hydrolyzed as the respective band in lane 4 disappear in the gel
(Fig. 4). The resulting sweet whey hydrolysate presents several small fragments
with a molecular weight in the range 2.8–16.7 kDa and additional lower molecular
weight peptides, which are not visible by Coomassie staining after electrophoresis
in Tricine SDS polyacrylamide gel (line 4 Fig. 4).
Almost all proteins, fragments, and peptides present in the sweet whey hydrolysate are positively charged at pH 4.5, according to the sample loaded into the
isoelectric focusing gel (lane 4 Fig. 5).
After centrifugation, the soluble fraction of the sweet whey hydrolysate at pH
4.5 was loaded into a cationic exchanger column (SP-Sepharose XL
™ from GE
Health Care). All small fragments and peptides of the sweet whey hydrolysate
adsorbed on the anionic support that exchange cations in opposition to what happened with soluble k-casein (k-CN) and b-LG that did not adsorb as the respective
stained bands are present in the gel (lane 5 Fig. 4). The elution of the fragments and
peptides occurred only with citrate buffer 25 mM pH 4.5 containing 250 or
350 mM of NaCl (Fig. 6).
The different eluent fractions were mixed, and their antimicrobial activity was
assessed using the microdilution method. Penicillin–Streptomycin is used as control. The eluent fractions showed microbial inhibition, especially of the Staphylococcus aureus strain tested. However, the identification and quantification of
peptides with microbial activity require the use of the reverse-phase- HPLC.
488
A. F. A. Salvado et al.
Chromatographic techniques are primarily used in the separation of peptide mainly
in the laboratory and at a small scale. The high purity of the target peptide for the
pharmaceutic industry is required, but its high added value offsets the high associated costs in its purification when using chromatography (90% of the total cost).
Although chromatographic processes allow achieving high separation and selectivity of target peptide, these processes are characterized by low productivity and
high operating and investment costs, which make their extensive use at the
industrial-scale prohibitively expensive [31]. Ion exchange chromatography
(IEC) and size exclusion chromatography (SEC) are some of the chromatographic
techniques tested for target peptides purification. In contrast, high-performance
liquid chromatography (HPLC) is crucial for peptide identification and production
in the laboratory and on a small scale. However, the most efficient process for
peptides purification continues undoubtedly to be reverse-phase HPLC chromatography [78].
Salvado [79] carried out the hydrolysis of sweet whey with chymotrypsin for
10 min, at temperature 37 °C and initial pH of 6.7, reducing the protein concentration in about 43% (as assessed by the Bicinchoninic Acid (BCA) assay method).
Tricine SDS-PAGE showed the efficiency and selectivity of this hydrolytic process.
The soluble k-casein (k-CN) and b-LG were not hydrolyzed under these experimental conditions, as shown by the bands in lanes 3 and 4 in Fig. 4. On the other
hand, a-LA was hydrolyzed as the respective band in lane 4 disappear in the gel
(Fig. 4). The resulting sweet whey hydrolysate presents several small fragments
with a molecular weight in the range 2.8–16.7 kDa and additional lower molecular
weight peptides, which are not visible by Coomassie staining after electrophoresis
in Tricine SDS polyacrylamide gel (line 4 Fig. 4).
Almost all proteins, fragments, and peptides present in the sweet whey hydrolysate are positively charged at pH 4.5, according to the sample loaded into the
isoelectric focusing gel (lane 4 Fig. 5).
After centrifugation, the soluble fraction of the sweet whey hydrolysate at pH
4.5 was loaded into a cationic exchanger column (SP-Sepharose XL
™ from GE
Health Care). All small fragments and peptides of the sweet whey hydrolysate
adsorbed on the anionic support that exchange cations in opposition to what happened with soluble k-casein (k-CN) and b-LG that did not adsorb as the respective
stained bands are present in the gel (lane 5 Fig. 4). The elution of the fragments and
peptides occurred only with citrate buffer 25 mM pH 4.5 containing 250 or
350 mM of NaCl (Fig. 6).
The different eluent fractions were mixed, and their antimicrobial activity was
assessed using the microdilution method. Penicillin–Streptomycin is used as control. The eluent fractions showed microbial inhibition, especially of the Staphylococcus aureus strain tested. However, the identification and quantification of
peptides with microbial activity require the use of the reverse-phase- HPLC.
488
A. F. A. Salvado et al.