Hydrophobically-modified casein derivatives were produced by using EDC-mediated
coupling reactions (Ha et al., 2013 (link); Ha et al., 2018 (link)). EDC can induce covalent
conjugations between the amino groups of sodium caseinate and carboxyl groups of
linoleic acid while NHS was used to enhance the stability of reaction
intermediate (O-acylisourea intermediate). Two hundred milligrams of sodium
caseinate were dissolved in 20 mL of deionized water and mixed with various
concentration of 10 mL of linoleic acid dispersed in ethanol. Linoleic acid
concentration levels in ethanol were 0 mg/10 mL, 17.9 mg/10 mL, 35.9 mg/10 mL,
and 53.8 mg/10 mL, which correspond to the molar ratio (charged amount of
linoleic acid) of 0%, 10%, 20%, or 30% to sodium caseinate solution,
respectively. Then, 5 mL of EDC and NHS dissolved in tetrahydrofuran was added
to sodium caseinate/linoleic acid mixture followed by stirring for 12 h at room
temperature (molar ratio of EDC:NHS:linoleic acid=1:1:1). To remove
unbound linoleic acid and residual reaction chemicals, caseinate/linoleic acid/
EDC/NHS mixtures were dialyzed against 50% ethanol and deionized water for 24
and 48 h using dialysis membrane (3.5 kDa molecular weight cut-off, Thermo
Scientific, Rockford, IL, USA) followed by freeze-drying.
coupling reactions (Ha et al., 2013 (link); Ha et al., 2018 (link)). EDC can induce covalent
conjugations between the amino groups of sodium caseinate and carboxyl groups of
linoleic acid while NHS was used to enhance the stability of reaction
intermediate (O-acylisourea intermediate). Two hundred milligrams of sodium
caseinate were dissolved in 20 mL of deionized water and mixed with various
concentration of 10 mL of linoleic acid dispersed in ethanol. Linoleic acid
concentration levels in ethanol were 0 mg/10 mL, 17.9 mg/10 mL, 35.9 mg/10 mL,
and 53.8 mg/10 mL, which correspond to the molar ratio (charged amount of
linoleic acid) of 0%, 10%, 20%, or 30% to sodium caseinate solution,
respectively. Then, 5 mL of EDC and NHS dissolved in tetrahydrofuran was added
to sodium caseinate/linoleic acid mixture followed by stirring for 12 h at room
temperature (molar ratio of EDC:NHS:linoleic acid=1:1:1). To remove
unbound linoleic acid and residual reaction chemicals, caseinate/linoleic acid/
EDC/NHS mixtures were dialyzed against 50% ethanol and deionized water for 24
and 48 h using dialysis membrane (3.5 kDa molecular weight cut-off, Thermo
Scientific, Rockford, IL, USA) followed by freeze-drying.
