flowers (Cynara cardunculus) and Arctium minus [39] that are widely used in the
Iberian Peninsula in the manufacture of artisanal cheese [40]. These proteases,
named cyprosins or cynarases, break the peptide bonds within the polypeptide chain
of whey proteins next to hydrophobic amino acid residues, especially Phe, Leu,
Thr, and Tyr, and act mainly in a-LA [41]. These authors used aqueous extracts of
C. cardunculus in the hydrolysis of WPC, WPI, isolated a-LA protein, and casein
macropeptides with DH of 8–16% depending on the ratio enzyme/protein substrate,
and hydrolysis time between 1 to 7 h. In turn, Lafarga et al. [42] performed BSA
hydrolysis with food-grade enzyme papain (EC.3.4.22.2) extracted from Carica
papaya. Other proteins of plant origin, such as bromelain extracted from stems
(EC.3.4.22.32) and fruit (EC.3.4.22.33) of pineapples, were reported to have the
ability to hydrolyze the peptide bond in whey proteins [43].
3.3 Protein Hydrolysis Strategies
3.3.1 Using Free Enzymes in Solution
Soluble digestive enzymes used in an agitated reactor at large-scale is the simplest
method to perform protein hydrolysis, and once reached the desired DH, the process
is interrupted by disabling the enzyme activity by pasteurization. The next step is
the separation of peptides from the enzyme and non-hydrolyzed proteins by an
ultrafiltration (UF) process. The synthetic UF membrane is characterized by pore
size suitable for the free enzyme and non-hydrolyzed protein substrate to be
retained within the membrane module (concentrate) while the peptides generated
characterized by a much smaller size cross the membrane into the permeate. This
filtration step functions as the first step of isolation and purification in the production process of bioactive peptides. The combination of the agitated reactor with
a UF membrane module was the next step to implement a continuous process of
hydrolysis of whey proteins and the production of bioactive peptides [31]. This
strategy was also tested in the hydrolysis of concentrated protein solutions of
Alfalfa (Medicago sativa) [44] and wheat gluten [45]. This configuration allows
performing hydrolysis continuously, with better control of the reaction, minimizing
the loss of protein substrate and free enzyme, and simultaneously, separate the
fractions of protein hydrolysates that pass through the membrane into the permeate
[46].
The hydrolysis of the proteins results in the release of individual amino acids and
small peptides that gradually decrease the pH of the reaction medium as the degree
of hydrolysis (DH) increases. To work close to the optimal pH value in an enzymatic process and under more controlled hydrolytic conditions is added as a
chemical agent to maintain a constant pH value in the reactor. This pH correction
allows the monitoring of DH in real-time, known as the pH–STAT reaction
strategy.
Fernández and Kelly [47] compared the pH–STAT strategy with an uncontrolled
reaction with a free pH decrease in the hydrolysis of WPI with the commercial
proteolytic preparation Protamex
® . These authors concluded that under the non-pH
Enzymatic Production of Bioactive Peptides from Whey Proteins …
481
Iberian Peninsula in the manufacture of artisanal cheese [40]. These proteases,
named cyprosins or cynarases, break the peptide bonds within the polypeptide chain
of whey proteins next to hydrophobic amino acid residues, especially Phe, Leu,
Thr, and Tyr, and act mainly in a-LA [41]. These authors used aqueous extracts of
C. cardunculus in the hydrolysis of WPC, WPI, isolated a-LA protein, and casein
macropeptides with DH of 8–16% depending on the ratio enzyme/protein substrate,
and hydrolysis time between 1 to 7 h. In turn, Lafarga et al. [42] performed BSA
hydrolysis with food-grade enzyme papain (EC.3.4.22.2) extracted from Carica
papaya. Other proteins of plant origin, such as bromelain extracted from stems
(EC.3.4.22.32) and fruit (EC.3.4.22.33) of pineapples, were reported to have the
ability to hydrolyze the peptide bond in whey proteins [43].
3.3 Protein Hydrolysis Strategies
3.3.1 Using Free Enzymes in Solution
Soluble digestive enzymes used in an agitated reactor at large-scale is the simplest
method to perform protein hydrolysis, and once reached the desired DH, the process
is interrupted by disabling the enzyme activity by pasteurization. The next step is
the separation of peptides from the enzyme and non-hydrolyzed proteins by an
ultrafiltration (UF) process. The synthetic UF membrane is characterized by pore
size suitable for the free enzyme and non-hydrolyzed protein substrate to be
retained within the membrane module (concentrate) while the peptides generated
characterized by a much smaller size cross the membrane into the permeate. This
filtration step functions as the first step of isolation and purification in the production process of bioactive peptides. The combination of the agitated reactor with
a UF membrane module was the next step to implement a continuous process of
hydrolysis of whey proteins and the production of bioactive peptides [31]. This
strategy was also tested in the hydrolysis of concentrated protein solutions of
Alfalfa (Medicago sativa) [44] and wheat gluten [45]. This configuration allows
performing hydrolysis continuously, with better control of the reaction, minimizing
the loss of protein substrate and free enzyme, and simultaneously, separate the
fractions of protein hydrolysates that pass through the membrane into the permeate
[46].
The hydrolysis of the proteins results in the release of individual amino acids and
small peptides that gradually decrease the pH of the reaction medium as the degree
of hydrolysis (DH) increases. To work close to the optimal pH value in an enzymatic process and under more controlled hydrolytic conditions is added as a
chemical agent to maintain a constant pH value in the reactor. This pH correction
allows the monitoring of DH in real-time, known as the pH–STAT reaction
strategy.
Fernández and Kelly [47] compared the pH–STAT strategy with an uncontrolled
reaction with a free pH decrease in the hydrolysis of WPI with the commercial
proteolytic preparation Protamex
® . These authors concluded that under the non-pH
Enzymatic Production of Bioactive Peptides from Whey Proteins …
481