In VitroAngiotensin I-Converting Enzyme Inhibition of Casein Hydrolysate Responsible for Plastein Reaction in Ethanol-Water Medium, Solvent Fractionation, and Protease Digestion

Abstract

A casein hydrolysate generated by Alcalase had in vitro ACE-inhibitory activity of 44.4%, and was treated by Alcalase-catalyzed plastein reaction in ethanol-water medium. Alcalase addition, ethanol, substrate concentration, and reaction temperature optimized from experimental design were 8.36 kU/g peptides, 56.8 (v/v), 56.8% (w/v), and 37.5 • C, respectively, when reaction time was fixed at 6 h. Two treated casein hydrolysates, namely TCH4 and TCH8, were obtained with reaction time of 4 and 8 h, and exhibited the highest ACEinhibitory activity of 62.5% or the greatest reaction extent but an activity of 35.6%, respectively. Fractionation of TCH4 and TCH8 by applying ethanol-water of 7:3 (v/v) conferred the obtained supernatant (precipitate) fractionates higher (lower) activity than the parent substrate, while applying ethanol-water of 3:7 (v/v) or water led to an opposite result in activity for the fractionates. In vitro digestion of TCH4, TCH8, and their fractionates revealed that they had resistance in activity towards the investigated four proteases, as the resulted 47 out of 48 digests had higher activities than casein hydrolysate. TCH8 exhibited better protease resistance than TCH4. It is concluded that the applied plastein reaction can enhance ACE inhibition and protease resistance of casein hydrolysate.

Biological activities of peptides obtained by pepsin hydrolysis of fishery products

“…In the studies presented here, it has been possible to obtain peptides with significant angiotensin inhibitory activity, similar to and even higher than that reported for peptides obtained from other raw materials and with other enzymes (for example, chickpea protein peptides with IC50 values ranging from 0.101 to 37.33 μg/mL prepared using papain, pancreatin or Alcalase [128], Alcalase casein hydrolysate with an angiotensin I-converting enzyme inhibitory activity of 62.5% [129], and Jatropha curcas peptide with an IC50 value of 4.78 g/mL obtained by Alcalase hydrolysis [130]. At this point, it is important to mention that the strong activity found for the peptides obtained with pepsin can be attributed to the pepsin specificity by the amino acid residues at position P1 and P1′ with a preferential cleavage in hydrophobic residues, since it has been reported that hydrophobic amino acids such Met, Val, Ala, and Tyr, increase the ACE inhibitory potential as they can bind to the catalytic site of ACE [115].…”

Use of Alcalase in the production of bioactive peptides: A review

“…The hydrolysates were later modified by Alcalase-catalyzed plastein reaction in an ethanol-water medium finding that most of the treated hydrolysates enhanced their angiotensin I-converting enzyme inhibition activities compared to the initial casein hydrolysate, mainly at 4 h of reaction time [502]. The same authors reported the optimization of the Alcalase-catalyzed plastein reaction in ethanol-water medium to improve the in vitro angiotensin I-converting enzyme inhibitory activity (44.4%) of the Alcalase casein hydrolysate [503]. The optimized conditions were Alcalase addition of 8.36 kU/g peptides, ethanol of 56.8% (v/v), substrate concentration of 56.8% (w/v), and 37.5°C, which led a casein hydrolysate with an angiotensin I-converting enzyme inhibitory activity of 62.5% [503].…”

“…The same authors reported the optimization of the Alcalase-catalyzed plastein reaction in ethanol-water medium to improve the in vitro angiotensin I-converting enzyme inhibitory activity (44.4%) of the Alcalase casein hydrolysate [503]. The optimized conditions were Alcalase addition of 8.36 kU/g peptides, ethanol of 56.8% (v/v), substrate concentration of 56.8% (w/v), and 37.5°C, which led a casein hydrolysate with an angiotensin I-converting enzyme inhibitory activity of 62.5% [503]. In another study, casein was digested with Alcalase, and the obtained hydrolysate presented an in vitro angiotensin I-converting enzyme inhibitory activity of…”