Abstract
Milk is an excellent source of nutrients, especially protein. Milk protein hydrolysates and
peptides are known for their health benefits, particularly antioxidative effects. The aim of
this study is to determine the physical properties and antioxidant activities of cow’s milk
subjected to enzymatic hydrolysis. Pasteurized skim milk (3.5% protein) was hydrolyzed
by adding Alcalase 2.4L, Protamex 1.5MG, Flavourzyme 1000L or porcine trypsin at the
enzyme-to-substrate ratios of 0.1-1% (w/w). Hydrolysis was conducted for ≤8 h at
refrigerated (4ºC), and enzyme’s optimal temperatures (50ºC, except 37ºC for trypsin) prior
to heating at 72ºC for 3 min and immediate cooling to inactivate the enzyme. Progress of
hydrolysis was monitored by determining the degree of hydrolysis (DH). Color values (L*,
a*, b*), total color difference against non-hydrolyzed milk (ΔE*), pH, and physical stability
of the hydrolyzed samples were measured. Antioxidant activities were determined as ferric
reducing antioxidant power (FRAP) and oxygen radical absorbance capacity (ORAC). DH of
hydrolyzed milk increased with enzyme concentration, hydrolysis temperature, and time.
The highest DH of 25% was observed in samples hydrolyzed with Alcalase, followed by
Flavourzyme, Protamex and trypsin. L*, a* and pH values of milk were unaffected by
hydrolysis. Protein hydrolysis of milk led to the increase in b* and the samples prepared
with trypsin showed the highest ΔE*. Hydrolysis with Alcalase or trypsin resulted in phase
separation of the milk, especially at high DH. Only Protamex- and Flavourzyme-hydrolyzed
milk with 10-20% DH, in which separation was absent, were analyzed for antioxidant
activities. FRAP and ORAC of non-hydrolyzed milk were 17.71 and 177.02 µmol TE/ml,
respectively and the values increased with DH. At any similar DH, Protamex-hydrolyzed
milk showed higher FRAP than those hydrolyzed with Flavourzyme, which had higher ORAC
value. The highest FRAP (25.23 µmol TE/ml) and ORAC (262.65 µmol TE/ml) were obtained
by hydrolysis with 0.5% Protamex at 50ºC for 30 min and 1% Flavourzyme at 50ºC for 90
min, respectively. Our findings indicated that enzymatic hydrolysis of protein under suitable condition could slightly improve the antioxidant activities of cow’s milk, while causing
minimal changes in physico-chemical characteristics as compared with non-hydrolyzed
milk.
