control, the hydrolysis of whey proteins was faster, but in contrast, the pH–STAT
strategy was more suitable for trypsin. Additionally, they also reported that the
comparison of both hydrolytic strategies of WPI with papain achieved similar DH
at the end. However, the profile of peptides and their physical–chemical and
bioactive properties were very different.
The pH values during the hydrolytic process have a decisive influence on the
profile of the peptides generated by the different proteases [48]. The confirmation of
these results by other authors led to the conclusion that the production of bioactive
peptides should be controlled, not only temporarily or based on the degree of
hydrolysis (DH), but also by bioactivity tests [49].
3.3.2 Using Immobilized Enzymes
The immobilization of enzymes has been extensively studied and tested in the
synthesis of peptides. Protein hydrolysis performed with immobilized proteases
allows the overcome some difficulties identified on a large-scale and offers several
advantages over conventional hydrolysis with soluble digestive enzymes. Several of
these advantages can be highlighted (i) enzymatic hydrolysis in softer and controlled conditions, (ii) avoid autolysis activity of the protease itself minimizing the
contamination of bioactive peptides released from the protein parent molecules, and
(iii) easy separation and recycling of preparation with immobilized enzyme after the
hydrolysis step, making the process economically more viable.
The profile of peptides is also associated with the controlled and rapid stop of the
protein hydrolysis reaction. However, this is not always easy for the free enzyme
without using an aggressive method such as pasteurization, which can affect the
quality of the bioactive peptides generated. In contrast, preparation with immobilized protein can be removed easily and quickly from the hydrolytic reaction
medium by sedimentation, filtration, or centrifugation, and more recently, when the
biocatalysts have paramagnetic properties, a magnetic field was proposed. Inclusively, immobilized protease (e.g., trypsin) combining with ultrasonic probe-based
can enhance the assessment of the protein cleavage quickly [50].
Peptide profile differences observed in protein hydrolysates were also justified
by the alteration of protein substrate accessibility or affinity to the immobilized
enzyme. Therefore, the primary selection criterium for the immobilization of proteases is the choice of macroporous supports to ensure free access of the protein
substrate to immobilized proteases. However, the high costs associated with support
and the most common methods used in the immobilization of proteases often
decrease the economic advantage and has limited its application more intensely in
protein hydrolysis and bioactive peptide production.
Ittrat et al. [51] proposed that the use of agricultural waste materials function as
cheap alternative supports for protease immobilization. Bassan et al. [52] immobilized trypsin in particles resulting from the processing of the agro-industrial lignocellulosic waste material with great success in the production of bioactive peptides
from whey proteins. These authors also found that more than 80% of enzyme activity
remained after five cycles of reuse of this preparation with immobilized protease.
Other studies highlight the immobilization of trypsin by adsorption and covalent
482
A. F. A. Salvado et al.
strategy was more suitable for trypsin. Additionally, they also reported that the
comparison of both hydrolytic strategies of WPI with papain achieved similar DH
at the end. However, the profile of peptides and their physical–chemical and
bioactive properties were very different.
The pH values during the hydrolytic process have a decisive influence on the
profile of the peptides generated by the different proteases [48]. The confirmation of
these results by other authors led to the conclusion that the production of bioactive
peptides should be controlled, not only temporarily or based on the degree of
hydrolysis (DH), but also by bioactivity tests [49].
3.3.2 Using Immobilized Enzymes
The immobilization of enzymes has been extensively studied and tested in the
synthesis of peptides. Protein hydrolysis performed with immobilized proteases
allows the overcome some difficulties identified on a large-scale and offers several
advantages over conventional hydrolysis with soluble digestive enzymes. Several of
these advantages can be highlighted (i) enzymatic hydrolysis in softer and controlled conditions, (ii) avoid autolysis activity of the protease itself minimizing the
contamination of bioactive peptides released from the protein parent molecules, and
(iii) easy separation and recycling of preparation with immobilized enzyme after the
hydrolysis step, making the process economically more viable.
The profile of peptides is also associated with the controlled and rapid stop of the
protein hydrolysis reaction. However, this is not always easy for the free enzyme
without using an aggressive method such as pasteurization, which can affect the
quality of the bioactive peptides generated. In contrast, preparation with immobilized protein can be removed easily and quickly from the hydrolytic reaction
medium by sedimentation, filtration, or centrifugation, and more recently, when the
biocatalysts have paramagnetic properties, a magnetic field was proposed. Inclusively, immobilized protease (e.g., trypsin) combining with ultrasonic probe-based
can enhance the assessment of the protein cleavage quickly [50].
Peptide profile differences observed in protein hydrolysates were also justified
by the alteration of protein substrate accessibility or affinity to the immobilized
enzyme. Therefore, the primary selection criterium for the immobilization of proteases is the choice of macroporous supports to ensure free access of the protein
substrate to immobilized proteases. However, the high costs associated with support
and the most common methods used in the immobilization of proteases often
decrease the economic advantage and has limited its application more intensely in
protein hydrolysis and bioactive peptide production.
Ittrat et al. [51] proposed that the use of agricultural waste materials function as
cheap alternative supports for protease immobilization. Bassan et al. [52] immobilized trypsin in particles resulting from the processing of the agro-industrial lignocellulosic waste material with great success in the production of bioactive peptides
from whey proteins. These authors also found that more than 80% of enzyme activity
remained after five cycles of reuse of this preparation with immobilized protease.
Other studies highlight the immobilization of trypsin by adsorption and covalent
482
A. F. A. Salvado et al.