3.2.2 Microbial Proteases
Proteolytic enzymes of bacterial or fungal origin were also successfully tested to
release bioactive peptides from whey proteins, although their use is more limited
than digestive enzymes. There is a wide variety of these proteolytic enzymes
commercially available such as Proteinase K (alkaline endopeptidase of the
EC.3.4.21.64), thermolysin (metallic-endopeptidase EC.3.4.24.27) and subtilisin
(serine endopeptidase, EC.3.4.21.62), among many others. Furthermore, mixtures
(or cocktails) of proteases are also used for the production of bioactive peptides
derived from whey proteins. The advantages of using a cocktail of proteases in
contrast to enzymatic preparations with a pure enzyme are due to peptide diversity
that can generate increased bioactivity at a reduced cost. Ghosh et al. [35] tested the
hydrolysis of whey proteins with the preparation Flavourzyme, commercialized by
Novozymes, which contain endo- and exo-peptidases produced by Aspergillus
oryzae. These authors found that the number of small peptides increased with the
concentration of commercial enzyme preparation.
3.2.3 Fermentation Processes
Dairy starter cultures consist of Lactobacilli strains that contain various lactic acid
bacteria (LAB) with proteolytic activity in the production of bioactive peptides by
the hydrolysis of milk proteins. This group of bacteria includes Lactococcus lactis,
Lactobacillus helveticus, and Lactobacillus delbrueckii var. bulgaricus, characterized by containing proteases integrated into the cell wall and various intracellular
peptidases [36]. The interest in these Lactobacilli strains is associated with the fact
that they can be isolated from different ecological environments. Although they
belong to the same biological family, several studies reported that they hydrolyze
proteins differently and therefore produce a wide variety of bioactive peptides from
the same protein substrate. Different hydrolysis studies performed on the fraction of
milk and whey proteins using Lactobacilli strains at 30–37 °C for 24 h led to higher
DH for longer fermentation time. However, the bioactive peptides production yield
by the Lactobacilli strains is relatively small compared to what is possible to
achieve with the use of pure enzymes [37]. Therefore, some authors added
Prozyme E, a commercial cocktail of proteases, after 5 h of fermentation to increase
the efficiency of the fermentation process. However, these fermentation conditions
have not proven to be economically viable for the production of pure peptides in
large quantities. Nisin, an antibiotic, is a unique exception as it is a natural fermentation product of Lactococcus lactis [38].
3.2.4 Plant Proteolytic Enzymes
The current trend in the production of bioactive peptides is to search and test new
proteases able to hydrolyze peptide bonds in specific and less well-known sites
envisaging the production of almost tailor-made peptides with precise size, for uses
in medical therapies and specific diets. Plants have been investigated as alternative
sources of peptidases since their peptidases act with different specificity and in a
large variety of protein sources. In this research, particular attention focus on
aspartic proteases present in the flowers of the Asteraceae family, mainly thistle
480
A. F. A. Salvado et al.
Proteolytic enzymes of bacterial or fungal origin were also successfully tested to
release bioactive peptides from whey proteins, although their use is more limited
than digestive enzymes. There is a wide variety of these proteolytic enzymes
commercially available such as Proteinase K (alkaline endopeptidase of the
EC.3.4.21.64), thermolysin (metallic-endopeptidase EC.3.4.24.27) and subtilisin
(serine endopeptidase, EC.3.4.21.62), among many others. Furthermore, mixtures
(or cocktails) of proteases are also used for the production of bioactive peptides
derived from whey proteins. The advantages of using a cocktail of proteases in
contrast to enzymatic preparations with a pure enzyme are due to peptide diversity
that can generate increased bioactivity at a reduced cost. Ghosh et al. [35] tested the
hydrolysis of whey proteins with the preparation Flavourzyme, commercialized by
Novozymes, which contain endo- and exo-peptidases produced by Aspergillus
oryzae. These authors found that the number of small peptides increased with the
concentration of commercial enzyme preparation.
3.2.3 Fermentation Processes
Dairy starter cultures consist of Lactobacilli strains that contain various lactic acid
bacteria (LAB) with proteolytic activity in the production of bioactive peptides by
the hydrolysis of milk proteins. This group of bacteria includes Lactococcus lactis,
Lactobacillus helveticus, and Lactobacillus delbrueckii var. bulgaricus, characterized by containing proteases integrated into the cell wall and various intracellular
peptidases [36]. The interest in these Lactobacilli strains is associated with the fact
that they can be isolated from different ecological environments. Although they
belong to the same biological family, several studies reported that they hydrolyze
proteins differently and therefore produce a wide variety of bioactive peptides from
the same protein substrate. Different hydrolysis studies performed on the fraction of
milk and whey proteins using Lactobacilli strains at 30–37 °C for 24 h led to higher
DH for longer fermentation time. However, the bioactive peptides production yield
by the Lactobacilli strains is relatively small compared to what is possible to
achieve with the use of pure enzymes [37]. Therefore, some authors added
Prozyme E, a commercial cocktail of proteases, after 5 h of fermentation to increase
the efficiency of the fermentation process. However, these fermentation conditions
have not proven to be economically viable for the production of pure peptides in
large quantities. Nisin, an antibiotic, is a unique exception as it is a natural fermentation product of Lactococcus lactis [38].
3.2.4 Plant Proteolytic Enzymes
The current trend in the production of bioactive peptides is to search and test new
proteases able to hydrolyze peptide bonds in specific and less well-known sites
envisaging the production of almost tailor-made peptides with precise size, for uses
in medical therapies and specific diets. Plants have been investigated as alternative
sources of peptidases since their peptidases act with different specificity and in a
large variety of protein sources. In this research, particular attention focus on
aspartic proteases present in the flowers of the Asteraceae family, mainly thistle
480
A. F. A. Salvado et al.