Bioactive peptides in the pancreatin-hydrolysates of whey protein support cell proliferation and scavenge reactive oxygen species

Abstract

Whey protein (WP) in milk shows physiologically active functions such as cholesterol control and immune system strengthening. In this study, we performed hydrolysis and peptide polarity fractionation to enhance the efficacy and diversity of its physiological activities, using the digesting enzyme, pancreatin. Our results indicate that hydrolysis significantly increased the cell proliferation of the WP fractions, with the lower-polarity fractions showing greater efficacy in this regard. Our results indicate that hydrolysis significantly increases cell proliferation of the WP fractions. Additionally, we confirmed differences in the antioxidant activity of the WP fractions as a function of polarity was confirmed via scavenging 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assay in vitro. WP itself did not show anti-inflammatory efficacy. However, all the hydrolyzed fractions downregulated the mRNA expression levels of inflammatory cytokines in all treated cell lines and, based on a senescence-associated (SA)-β-galactosidase assay, the fraction with the lowest polarity (F6) inhibited cellular senescence to the greatest extent. Furthermore, we identified the peptide sequences with various physiological activities from whey protein hydrolysates through mass spectrometry. Taken together, our results indicate that the fractionation of WP via hydrolysis generates novel functions including promoting cellular cell proliferation, anti-inflammatory effects, and enhancing antioxidant and anti-cellular senescence.

Keywords: cellular senescence; hydrolysis; inflammation; milk; whey protein.

Figure 1.

HPLC chromatogram of hydrolyzed whey protein (WP). (A) WP hydrolyzed using pancreatin. Chromatogram of (B) WP and (C) hydrolyzed WP. Fractions were obtained according to acetonitrile concentration (1–31%). Elution time, 40 min; Flow rate, 1 mL/min; UV detection wavelength, 214 nm.

Figure 2.

Effect of hydrolyzed WP on cell proliferation. (A) A549, (B) HepG2, and (C) RAW 264.7 cells treated with the indicated concentrations of hydrolyzed WP for 36 h. Cell proliferation was measured via MTS assays. The results are presented as mean ± S.E.M relative cell proliferation compared with the control group. *p < 0.05, compared with non-treated control.

Figure 3.

Antioxidant activity of hydrolyzed WP. Radical scavenging activity of hydrolyzed WP measured at the indicated concentrations using an ABTS + radical scavenging assay. Resveratrol (Res.) was used as the positive control. ***p < 0.001.

Figure 4.

Anti-inflammatory activity of hydrolyzed WP. RAW 264.7 cells were treated with hydrolyzed WP for 36 h and thereafter, treated with LPS to induce an inflammatory response. The mRNA expression levels of (A) IL-1β, (B) IL-6, (C) iNOS, and (D) TNF-α measured via qRT-PCR. The results are presented as mean ± S.E.M. *p < 0.05, compared with the respective LPS-treated controls. Con., control.

Figure 5.

SA-β-galactosidase activity (SA-β-gal) of hydrolyzed WP. (A) HDFs treated with hydrolyzed WP for 24 h, followed by the induction of senescence using H₂O₂. The level of senescence in HDFs was measured using SA-β-gal assay. Senescent cells (SA-β-gal positive cells) were stained blue using X-gal. (B) Percentage of SA-β-gal positive cells. The results are presented as mean ± S.E.M. *p < 0.05, compared with the respective H₂O₂-treated control cells. # < 0.05, compared with the control cells (non-H₂O₂-treatment).

Figure 6.

Physiological activities of WP hydrolysates-derived peptides P1 and P2. (A) HepG2 and (B) RAW 264.7 cells treated with the indicated concentrations of VLVLDTDYKK (P1) and VGINYWLAHK (P2) for 36 h. Cell proliferation was measured by MTS assays. ***p < 0.001, compared with the non-treated control. (C) Radical scavenging activity of P1 and P2 measured at the indicated concentrations using an ABTS + radical scavenging assay. Resveratrol was used as the positive control. (D,E) RAW 264.7 cells were treated with P1 and P2 for 36 h and thereafter, treated with LPS to induce an inflammatory response. The mRNA expression levels of (D) IL-1β, (E) IL-6 were measured by qRT-PCR. The results are presented as mean ± S.E.M. *p < 0.05, ***p < 0.001, compared with the respective LPS-treated controls. (F) HDFs treated with P1 and P2 for 24 h, followed by the induction of senescence using H₂O₂. The level of senescence in HDFs was measured using a SA-β-gal assay. Senescent cells (SA-β-gal positive cells) were stained blue using X-gal. (G) Percentage of SA-β-gal positive cells. The results are presented as mean ± S.E.M. *p < 0.05, compared with the respective H₂O₂-treated control cells.