peptides used as food additives is preferably obtained by enzymatic hydrolysis from
milk and whey proteins with food-grade digestive or microbial proteolytic enzymes,
pure or a mixture of proteases, characterized by different specificities and low
production cost [20].
The most common method for evaluating and monitoring the release of peptides
in the laboratory and on the industrial scale is by quantifying the Degree of
Hydrolysis (DH), i.e., the percentage of peptide bonds broken during the hydrolytic process of proteins. According to Agyei et al. [17], DH influences the size of
the peptides generated, and peptides with biological functions require DH > 10%.
The hydrolysis of proteins produces multiple fragments from the primary sequence
of native proteins, and the number of smaller ones increases with DH. These
fragments (f) are assayed and then identified by a peptide chain according to the
single-letter amino acid code, for instance, containing the abbreviation of natural
amino acids f(GALMFWKQESPVICYHRNDT). The production of peptides on an
industrial scale continues with challenges in the face of the existence of several
bottlenecks due to the absence of technological environment-friendly, and economically efficient processes [20].
3.1 Effect of Industrial Pre-treatment of Proteins
Long hydrolytic processes are required to achieve high DH, more favorable for the
production of physiologically active peptides, which increases the probability of
microbial contamination. To avoid the addition of bactericides or pasteurization
processes, Tavares et al. [21] proposed and tested the pre-treatment of whey protein
concentrate (WPC) by microfiltration using a synthetic membrane with a weight
exclusion (MWCO) of 20 kDa to reduce microbial loading. Other strategies consist
of the research and use of new proteolytic enzymes that are resistant to high
temperatures or acid conditions and thus can help eliminate optimal microbial
growth conditions during the production of bioactive peptides. Other authors have
proposed enzymatic hydrolysis together with high-pressure homogenization
because enzyme activity is less sensitive to pressure compared to high temperature,
reducing in this way, the cost of industrial production of bioactive peptides [22].
New pre-treatment technologies of protein solutions that do not employ heat or
high pressure to minimize microbial contamination have currently been laboratorytested such as the use of UV, microwaves, ultrasonication, ozone, and application of
the pulsed electric field. For example, ultrasonication led to a more significant
release of antioxidant peptides from hydrolysates of whey proteins compared to
control (untreated whey proteins) in opposition to the use of microwave [23].
A comparison of the quality of industrial and research laboratory-level produced
b-LG hydrolysates showed very similar peptide profiles, although with very different biological activities. This unexpected behavior obtained after the release of
the peptides generated from the same protein molecule and with the same hydrolytic process is due, according to Li-Chan [24], to conformational changes in the
structure of proteins processed at the industrial level. These alterations in protein
Enzymatic Production of Bioactive Peptides from Whey Proteins …
477
milk and whey proteins with food-grade digestive or microbial proteolytic enzymes,
pure or a mixture of proteases, characterized by different specificities and low
production cost [20].
The most common method for evaluating and monitoring the release of peptides
in the laboratory and on the industrial scale is by quantifying the Degree of
Hydrolysis (DH), i.e., the percentage of peptide bonds broken during the hydrolytic process of proteins. According to Agyei et al. [17], DH influences the size of
the peptides generated, and peptides with biological functions require DH > 10%.
The hydrolysis of proteins produces multiple fragments from the primary sequence
of native proteins, and the number of smaller ones increases with DH. These
fragments (f) are assayed and then identified by a peptide chain according to the
single-letter amino acid code, for instance, containing the abbreviation of natural
amino acids f(GALMFWKQESPVICYHRNDT). The production of peptides on an
industrial scale continues with challenges in the face of the existence of several
bottlenecks due to the absence of technological environment-friendly, and economically efficient processes [20].
3.1 Effect of Industrial Pre-treatment of Proteins
Long hydrolytic processes are required to achieve high DH, more favorable for the
production of physiologically active peptides, which increases the probability of
microbial contamination. To avoid the addition of bactericides or pasteurization
processes, Tavares et al. [21] proposed and tested the pre-treatment of whey protein
concentrate (WPC) by microfiltration using a synthetic membrane with a weight
exclusion (MWCO) of 20 kDa to reduce microbial loading. Other strategies consist
of the research and use of new proteolytic enzymes that are resistant to high
temperatures or acid conditions and thus can help eliminate optimal microbial
growth conditions during the production of bioactive peptides. Other authors have
proposed enzymatic hydrolysis together with high-pressure homogenization
because enzyme activity is less sensitive to pressure compared to high temperature,
reducing in this way, the cost of industrial production of bioactive peptides [22].
New pre-treatment technologies of protein solutions that do not employ heat or
high pressure to minimize microbial contamination have currently been laboratorytested such as the use of UV, microwaves, ultrasonication, ozone, and application of
the pulsed electric field. For example, ultrasonication led to a more significant
release of antioxidant peptides from hydrolysates of whey proteins compared to
control (untreated whey proteins) in opposition to the use of microwave [23].
A comparison of the quality of industrial and research laboratory-level produced
b-LG hydrolysates showed very similar peptide profiles, although with very different biological activities. This unexpected behavior obtained after the release of
the peptides generated from the same protein molecule and with the same hydrolytic process is due, according to Li-Chan [24], to conformational changes in the
structure of proteins processed at the industrial level. These alterations in protein
Enzymatic Production of Bioactive Peptides from Whey Proteins …
477