Milk is considered the most complete food, providing most of the nutrients needed. Milk proteins are not only important for their function as a source of amino acids, but they are also a source of bioactive peptides. These are short amino acid sequences with different activities that have a positive impact on body functions or conditions and may ultimately influence health. Bioactivities such as ACE-inhibitory, antioxidant, opioid, antimicrobial and anti-inflammatory have been identified in milk derived peptides.
The purpose of this project is to identify novel bioactive peptides (ACE-inhibitory, antioxidant and antimicrobial peptides) released from milk proteins by mean of enzyme-catalysed hydrolysis.
Goat milk fractions (produced using microfiltration membranes) and bovine casein were used as substrates. The goat milk fractions (retentate, permeate and skimmed milk) were hydrolysed with two commercial enzymes. The bovine casein was hydrolysed using the supernatant of a Greenlandic bacterium (Arsukibacterium ikkense), produced in the NOVENIA project, which contains cold-active proteolytic enzymes. The hydrolysates were tested for the relevant bioactivities and active fractions were fractionated by semipreparative
chromatographic methods in order to identify the major peptides responsible for the activity.
A positive correlation between ACE-inhibitory activity and extent of hydrolysis and activity was seen with both substrates. The most active hydrolysates showed an IC50 similar to the most active milk protein hydrolysates made in other studies.
Regarding radical scavenging activity, the bovine casein hydrolysates also showed a positive correlation between extent of hydrolysis and activity, although the difference between the unhydrolysed sample and the hydrolysates was less marked. The goat milk hydrolysates, instead, showed a difference between the fractions more than the degree of hydrolysis, the retentate being the most active radical scavenger regardless the degree of hydrolysis and enzyme used. Both hydrolysates are among the most active radical scavenging milk protein hydrolysates known. The bovine casein did not show an increase in iron chelation capacity after hydrolysis, while the goat milk hydrolysates showed again a difference in the substrates and not in degree of hydrolysis, with the permeate as the most active substrate. Partial hydrolysis of the bovine casein increased the inhibition of lipid peroxidation, while for goat milk hydrolysates no effect of the degree of hydrolysis was observed and the permeate was the most active fraction.

Several novel peptides were identified in the most active hydrolysates. Regarding ACE-inhibitory activity the Cterminal part of the peptide sequence was confirmed to be of paramount importance, with aromatic and hydrophobic amino acids (Tyr, Phe, Pro, Leu) as favourite in the three C-terminal positions.
The identity of the antioxidant peptides showed that for both radical scavenging and lipid oxidation inhibition, Tyr was important, being present in the majority of the active peptides. For lipid oxidation inhibition, furthermore, also Phe was found to be of importance.
These results showed that hydrolysates with interesting bioactivity could be generated using novel enzymes or combination of commercial enzymes. These hydrolysates might be used as ingredients in functional foods.