structure allow increasing access of endopeptidases to the bonds of the polypeptide
chain, therefore facilitating enzymatic hydrolysis. However, protein pre-treatment
can also produce protein aggregation and, consequently, hinder the release of
peptide bonds from the protein parent molecule(s), for example, due to
high-temperature processing [25] and high-pressure [26].
Additionally, processes involving strong acids have an adverse action on Gly
(Glycine for short according to the three-letter amino acids code) and Asn amino
acids. At the same time, alkaline conditions affect the Cis, Ser, and Thr amino acids,
significantly decreasing lysine bioavailability. Furthermore, Lys can be involved in
a Maillard reaction when it is present with 1% of lactose or other sugars that remain
after hydrolysis [27]. This last modification can significantly affect DH obtained
from isolated protein a-LA, either with trypsin or a-chymotrypsin, because these
enzymes are unable to perform the hydrolysis of peptide bonds with glycosylated
Lys [28]. However, the same authors found that hydrolysis of these peptide bonds
with Bacillus licheniformis protease (BLP) and subtilisin A was independent of the
degree of glycosylation.
In addition to the effect of pre-treatment of native protein solutions, the stability
of the peptides subjected to high variations of temperature, pressure, salts, pH, and
organic solvents used during hydrolysis or downstream processing should also be
evaluated. Stability is an essential factor in ensuring the maintenance of the biological and or physiological activity of bioactive peptides [20]. These stability
changes can alter or, at worst, destroy the beneficial bioactivity of the specific
peptide. Therefore, the discovery of new bioactive peptides at the level of laboratory research is essential to test conditions close to those one used at the
industrial-scale.
3.2 Protein Hydrolysis—The “Classical” Approach
Several physical, chemical, or biological hydrolytic processes release the bioactive
peptides from the protein parent molecules. The heat releases bioactive peptides in
protein-rich food processing, in the chemical methods acid or alkaline catalytic
agents are added, while in biocatalysis enzymes or fermentation processes are used
(Fig. 2). However, the classic hydrolytic processes of the protein parent molecules
presently in use to generate bioactive peptides are (i) hydrolysis using pure or in
mixture digestive enzymes; (ii) fermentation processes using microorganisms with
proteolytic activity, and (iii) the hydrolytic action of microbial or vegetable
proteases.
3.2.1 Digestive Enzymes
Currently, commercial preparations of food-grade proteases contain a diverse
number of endopeptidases divided mainly into six categories, according to amino
acid residues and co-factor involved in the catalytic process. Thus, there are serine-,
cysteine-, aspartic-, glutamic-, and threonine-proteases, as well as metallic-proteases
[29]. The mentioned amino acid residues act as nucleophilic agents attacking peptide
478
A. F. A. Salvado et al.
chain, therefore facilitating enzymatic hydrolysis. However, protein pre-treatment
can also produce protein aggregation and, consequently, hinder the release of
peptide bonds from the protein parent molecule(s), for example, due to
high-temperature processing [25] and high-pressure [26].
Additionally, processes involving strong acids have an adverse action on Gly
(Glycine for short according to the three-letter amino acids code) and Asn amino
acids. At the same time, alkaline conditions affect the Cis, Ser, and Thr amino acids,
significantly decreasing lysine bioavailability. Furthermore, Lys can be involved in
a Maillard reaction when it is present with 1% of lactose or other sugars that remain
after hydrolysis [27]. This last modification can significantly affect DH obtained
from isolated protein a-LA, either with trypsin or a-chymotrypsin, because these
enzymes are unable to perform the hydrolysis of peptide bonds with glycosylated
Lys [28]. However, the same authors found that hydrolysis of these peptide bonds
with Bacillus licheniformis protease (BLP) and subtilisin A was independent of the
degree of glycosylation.
In addition to the effect of pre-treatment of native protein solutions, the stability
of the peptides subjected to high variations of temperature, pressure, salts, pH, and
organic solvents used during hydrolysis or downstream processing should also be
evaluated. Stability is an essential factor in ensuring the maintenance of the biological and or physiological activity of bioactive peptides [20]. These stability
changes can alter or, at worst, destroy the beneficial bioactivity of the specific
peptide. Therefore, the discovery of new bioactive peptides at the level of laboratory research is essential to test conditions close to those one used at the
industrial-scale.
3.2 Protein Hydrolysis—The “Classical” Approach
Several physical, chemical, or biological hydrolytic processes release the bioactive
peptides from the protein parent molecules. The heat releases bioactive peptides in
protein-rich food processing, in the chemical methods acid or alkaline catalytic
agents are added, while in biocatalysis enzymes or fermentation processes are used
(Fig. 2). However, the classic hydrolytic processes of the protein parent molecules
presently in use to generate bioactive peptides are (i) hydrolysis using pure or in
mixture digestive enzymes; (ii) fermentation processes using microorganisms with
proteolytic activity, and (iii) the hydrolytic action of microbial or vegetable
proteases.
3.2.1 Digestive Enzymes
Currently, commercial preparations of food-grade proteases contain a diverse
number of endopeptidases divided mainly into six categories, according to amino
acid residues and co-factor involved in the catalytic process. Thus, there are serine-,
cysteine-, aspartic-, glutamic-, and threonine-proteases, as well as metallic-proteases
[29]. The mentioned amino acid residues act as nucleophilic agents attacking peptide
478
A. F. A. Salvado et al.