Enzymatic Modification Of Casein Hydrolysate In Ethanol-water Medium And Its Modified Properties

Casein was considered as one of the good sources to obtain bioactive peptides. In the presentstudy, plastein reaction was applied to modify casein hydrolysate in ethanol-water medium. Theeffects of plastein reaction on ACE-inhibitory activity, metal ions chelating activities andinhibition towards calcium carbonate precipitation were investigated. Solvent fractionation wasemployed to treat the modified casein hydrolysates with the highest ACE-inhibitory activity or thelargest reaction extent. Enzymatic hydrolysis was also applied to digest the modified orfractionated casein hydrolysates, to reveal their enzymatic resistance. The results obtained fromthe present study were listed as follows.(1) A casein hydrolysate with hydrolysis time of6h and a degree of hydrolysis of11.6%wasprepared. It exhibited the highest activity against ACE, with the calculated IC50value of42.8g/mL.(2) When the reaction time was fixed at6h, the optimal Alcalase addition, ethanol andsubstrate concentration or reaction temperature for plastein reaction in ethanol-water medium wereselected from response surface methodology as8.36kU/g proteins,56.8(v/v),56.8%(w/v) and37.5oC, respectively. When the reaction time and substrate concentration were fixed at6h and50%(w/v), respectively, the optimal ethanol concentration, papain addition and reactiontemperature of papain-catalyzed plastein reaction in ethanol-water medium were obtained fromsignal factor experiment as70%(v/v),3kU/g proteins and40oC, respectively.(3) Some modified casein hydrolysates were prepared at optimal conditions and named asTCH1-8(modified by Alcalase) or MCH0.5-8(modified by papain) according to different reactiontimes, respectively. The ACE-inhibitory activities of them were evaluated. TCH4and MCH1exhibited the highest ACE-inhibitory activity of62.5or64.5%, while TCH8and MCH8exhibitedthe greatest reaction extent but an activity of35.6%or41.7%, respectively. Kinetic evaluationindicated that casein hydrolysate and three modified casein hydrolysates were competitiveinhibitors to the ACE.(4) It was unexpectedly found that plastein reaction and reaction extent exhibited slightlyinfluence on metal ions chelating activities of the modified casein hydrolysate, as they showedlower zinc-or calcium-chelating activity but slightly higher iron (II)-chelating activity than that ofcasein hydrolysate. ACE-inhibitory activity of casein hydrolysate and TCH1-8were uncorrelatedto their zinc-chelation, while inhibition towards calcium carbonate precipitation was clearly correlated to the measured calcium-chelation.(5) Solvent fractionation of TCH4and TCH8by water and ethanol-water of3:7and7:3(v/v)showed impact on ACE-inhibitory activity. The increasing content of ethanol in the solventconferred the supernatant fractions higher ACE-inhibitory activity, while the precipitation showedthe opposite trend. The supernatant fractionated by ethanol-water in ratio of7:3(v/v) exhibitedhigher activity than the parent substrate, and the supernatant of TCH4exhibited the highestACE-inhibitory activity (69.5%). Therefore, a mixed solvent with lower polarity was applicable toseparate part of the modified casein hydrolysates with enhanced ACE-inhibitory activity.(6) The resulted62out of64digests in the present study exhibited higher ACE-inhibitoryactivities (45.1-74.8%) than casein hydrolysate (44.4%). This result indicated that the carried outplastein reaction in ethanol-water medium conferred the casein hydrolysate better proteaseresistance.