| In the past, many studies have been carried out to show that casein was a good source of ACE-inhibitory peptides. In order to enhance ACE-inhibitory activity of a prepared casein hydrolysate, plastein reaction was applied on the casein hydrolysate in the presence of proline in the present work. Solvent fractionation was employed to treat the modified casein hydrolysate. Enzymatic resistance of the obtained products was analyzed to investigate the impacts of the conducted treatment on ACE-inhibitory activity of the modified or fractionated products.A casein hydrolysate was prepared by hydrolyzing casein with Alcalase and then modified by an Alcalase-catalyzed plastein reaction. The prepared casein hydrolysate of a reaction time of6h had a degree of hydrolysis of10.9%and IC50value of52.6μg/mL. With the decreased amount of free amino groups of the modified hydrolysate as the response and the reaction time fixed at6h, and response surface methodology was used to optimize the reaction conditions. The optimized conditions were an enzyme addition of3.1kU/g proteins, reaction temperature of25℃, and casein hydrolysates concentration of50%(w/v), respectively. The maximum decreased amount of free amino groups of the modified hydrolysate prepared under these optimized conditions was112.1μmol/g proteins.Casein hydrolysate was modified at different extents by the plastein reaction in proline addition leve of0.6mol/mol free amino groups. The ACE-inhibitory activities and IC50values of the modified hydrolysates were assayed. The ACE-inhibitory activities of all the modified hydrolysates increased as the reaction extent increased and were higher than the original hydrolysates. A modified hydrolysate without proline had free amino groups about1668.8μmol/g proteins and a decreased IC50value of14.9μg/mL. A modified hydrolysate in the presence of proline had free amino groups of1593.3μmol/g proteins and a decreased IC50value of13.0μg/mL.The original hydrolysate and the modified hydrolysates of the highest activity were fractionated into supernatant and precipitation by ethanol-or methanol-water solvents in volume ratios of3:7,5:5and7:3respectively. The modified hydrolysate was also fractionated by pure water and served as a control. The peptide recovery, content of free amino groups, ACE-inhibitory activity and IC50value of the fractionated hydrolysates were analyzed. Fractionation of the original hydrolysate by the two solvents (7:3ethanol-or methanol-water, v/v) led to the soluble (or precipitate) fraction higher (or lower) activity. Fractionation of the modified hydrolysate shared the similar profile in the variation in the activity. The supernatant fraction of the fractionated hydrolysate without praline addition by ethanol-water (7:3, v/v) had free amino groups of1684.4μmol/g proteins and a decreased IC50value of11.8μg/mL, while the precipitation fraction by water had free amino groups of1577.8μmol/g proteins and a decreased IC50value of12.6μg/mL. The supernatant fraction of the hydrolysate modified in the presence of proline had free amino groups of1582.7μmol/g proteins and an IC50value of10.0μg/mL, while the precipitate fraction by water had free amino groups of1544.7μmol/g proteins and an IC50value of11.3μg/mL. Therefore, a mixed solvent containing lower polarity solvent is applicable to separate part of the hydrolysate with enhanced ACE-inhibitory activity.Enzymatic hydrolysis of the modified or fractionated hydrolysates by Alcalase, papain, pepsin or trypsin for10～30min led to a decrease in their ACE-inhibitory activities. The resultant products treated by trypsin, pepsin or papain (but not for alcalase) had a residual ACE-inhibitory activity of about31～50%, higher than that of the original hydrolysate (27.8%). This fact shows that further hydrolysis of the modified or fractionated hydrolysate by four proteases would damage their activities. Alcalase has a specificity mainly for hydrophobic amino acids, which might result in significant release of hydrophobic amino acids from the peptides, and result in the modified or fractionated hydrolysate with lowest activity upon digestion. The hydrolyzed products of the modified or fractionated hydrolysate produced by digestive enzymes showed impaired but higher activities than the original hydrolysate, implying that the plastein reaction gave casein hydrolysate an improved ACE-inhibition and enzymatic resistance. Tricine-SDS-PAGE electrophoresis showed that the peptide profiles between the original casein hydrolysate, modified casein hydrolysate and fractionated hydrolysates were very different, indicating that some new peptides were generated during Plastein reaction and played the most important role in increasing ACE-inhibitory activity of the modified casein hydrolysates. |
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