In vitro-digested milk proteins: Evaluation of angiotensin-1-converting enzyme inhibitory and antioxidant activities, peptidomic profile, and mucin gene expression in HT29-MTX cells

摘要

Over the past decades, several studies investigatedthe health-promoting functions of milk peptides. However,to date many hurdles still exist regarding thewidespread use of milk-derived bioactive peptides, asthey may be degraded during gastrointestinal digestion.Thus, the aim of our study was to in vitro digestintact whey protein isolate (WPI) and casein proteins(CNP), mimicking in vivo digestion, to investigate theirbioactive effects and to identify the potential peptidesinvolved. Whey protein isolate and CNP were digestedusing a pepsin-pancreatin protocol and ultra-filtered(3-kDa cutoff membrane). A permeate (＜3 kDa) anda retentate (＞3 kDa) were obtained. Soy protein wasincluded as a control (CTR). Angiotensin-1-convertingenzyme inhibitory (ACE1-I) and antioxidant activity(AOX) were assessed and compared with those observedin undigested proteins and CTR. Furthermore, the permeatewas characterized by nano-liquid chromatographyelectrospray ionization tandem mass spectrometry(LC-nano ESI MS/MS) using a shotgun peptidomic approach,and retentate was further digested with trypsinand analyzed by MS using a shotgun proteomic approachto identify potentially bioactive peptides. Further,the effects of WPI, CNP, and CTR retentate on cellmetabolic activity and on mucus production (MUC5ACand MUC2 gene expression) were assessed in intestinalgoblet HT29-MTX-E12 cells. Results showed that WPIpermeate induced a significant ACE1-I inhibitory effect[49.2 ± 0.64% (SEM)] compared with undigested WPI,CNP permeate, and retentate or CTR permeate (10.40± 1.07%). A significant increase in AOX (1.58 ± 0.04and 1.61 ± 0.02 μmol of trolox AOX equivalents permg of protein, respectively) upon digestion was foundin WPI. Potentially bioactive peptides associated withACE1-I and antihypertensive effects were identified inWPI permeate and CNP retentate. At specific concentrations,WPI, CNP, and CTR retentate were able tostimulate metabolic activity in HT29-MTX-E12 cells.Expression of MUC5AC was increased by CNP retentateand unaltered by WPI retentate; MUC2 expressionwas significantly increased by 0.33 mg/g of CNP andreduced by 1.33 mg/g of CNP. Our results confirm thatmilk proteins may be rich sources of bioactive compounds,with the greatest beneficial potential of CNPat the intestinal goblet cell level.