Original Article

High hydrostatic pressure pre-treatment of whey proteins enhances whey protein hydrolysate inhibition of oxidative stress and IL-8 secretion in intestinal epithelial cells

André F. Piccolomini1, Michèle M. Iskandar1, Larry C. Lands2 and Stan Kubow1*

1School of Dietetics and Human Nutrition, McGill University, Ste. Anne de Bellevue, QC, Canada; 2Division of Pediatric Respiratory Medicine, Montreal Children's Hospital McGill University Health Centre, Montreal, QC, Canada


Background: High hyperbaric pressure treatment of whey protein isolate (WPI) causes changes in the protein structure that enhances the anti-oxidant and anti-inflammatory effects of WPI.

Objective: The aim of this study was to compare the anti-oxidant and anti-inflammatory effects of pressurized whey protein isolate (pWPI) vs. native WPI (nWPI) hydrolysates in Caco-2 cells exposed to hydrogen peroxide (H2O2).

Design: Cells were cultured with different concentrations of pWPI or nWPI hydrolysates either 1 h before or 1 h after H2O2. Cell viability, IL-8 secretion, intracellular reactive oxygen species (ROS), and the medium anti-oxidant capacity (FRAP assay) were measured.

Results: Prior to and after H2O2 exposure, pWPI and nWPI hydrolysates inhibited IL-8 secretion and ROS generation, and increased FRAP activity in a dose-dependent manner. The maximal inhibition of H2O2-induced IL-8 secretion was greater with 2000 µg mL−1 of pWPI (50%) vs. nWPI (30%) hydrolysates. At the latter concentration, inhibition of H2O2-induced ROS formation reached 76% for pWPI, which was greater than for nWPI hydrolysates (32.5%).

Conclusion: These results suggest that WPI hydrolysates can alleviate inflammation and oxidative stress in intestinal cells exposed to oxidative injury, which is further enhanced by hyperbaric pressure pre-treatment of WPI.

Keywords: anti-oxidant; anti-inflammatory; reactive oxygen species; caco-2 cells; pressurized whey protein isolate hydrolysates

Received: 23 February 2012; Revised: 15 May 2012; Accepted: 15 May 2012; Published: 19 June 2012

Food & Nutrition Research 2012. © 2012 André F. Piccolomini et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution-Noncommercial 3.0 Unported License (http://creativecommons.org/licenses/by-nc/3.0/), permitting all non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Citation: Food & Nutrition Research 2012. 56 17549 - http://dx.doi.org/10.3402/fnr.v56i0.17549


Oxidative stress has been implicated as a potential etiological factor for inflammatory bowel disease (IBD) due to the well established detrimental effects of reactive oxygen species (ROS) in the gut inflammatory process (1). Oxidative stress can be caused by ROS such as hydrogen peroxide (H2O2), superoxide anion, singlet oxygen, or hydroxyl radicals (2) generated both in the lumen and in the intestinal mucosa (3). H2O2 is one of several ROS which causes oxidative stress that contributes towards colonic damage (2). H2O2 is generated via a variety of metabolic pathways and its intracellular accumulation can cause dysregulation of the normal metabolic processes partly by modifying proteins, lipids, and DNA (4). Also, H2O2 can activate transduction pathways such as mitogen-activated protein kinases that contribute to the production of pro-inflammatory cytokines such as (IL)-8 (5). The secretion of IL-8 is part of the immune response, particularly in inflammatory processes as IL-8 regulates the recruitment and activation of neutrophils in inflamed tissue (6). The dysregulation of IL-8 balance can lead to the type of serious tissue damage that has been reported in IBD (7) and in patients with active colitis (8). Oxidative stress has been shown to promote IL-8 production in several cell types, including the human colon adenocarcinoma Caco-2 cell line (9), which has been widely used as an in vitro model of the intestinal epithelium (10). Exposure of Caco-2 cells to H2O2 can represent the pro-inflammatory response of intestinal cells following their exposure to oxidants generated in the lumen during digestion. Caco-2 cell cultures have been effectively used as an in vitro model for the investigation of toxicants, food mutagens, and their toxicological mechanisms affecting gut health (11, 12) and testing the protective action of anti-oxidants and nutraceutical agents (13, 14).

Bioactive proteins and peptides obtained from bovine milk proteins are being used as nutraceuticals to improve human health (14). Whey proteins, which comprise approximately 20% of the total proteins in cow's milk, have been found in vitro and in vivo studies to exert potent anti-oxidant and anti-inflammatory effects (15). Extensive disulfide linkages in whey proteins, however, increase their resistance to proteolysis and subsequent digestibility (16). The high resistance of the most prevalent whey protein, β-lactoglobulin (β-lg), to pepsin-mediated hydrolysis is considered to be a key factor that impedes whey protein digestibility (17, 18). Various studies have shown a low accessibility to peptide bonds localized to the interior of the globular structure of β-lg to the hydrolytic action of pepsin (19).

High hyperbaric pressure treatment of whey proteins can cause changes in their secondary and tertiary structure leading to denaturation (20). The most sensitive whey protein to denaturation induced by high hyperbaric pressure is β-lg, which contains two disulfide bonds and one free sulfhydryl group (21). Pressurization at 550 MPa has been demonstrated to increase the rate of β-lg hydrolysis due to partial unfolding of β-lg as the unfolded molecules are susceptible to proteolytic degradation (22). One study has shown that high hydrostatic pressure at 550 MPa causes a substantial increase in the rate of pepsin-mediated hydrolysis of whey protein isolates (WPI) (17). Cell culture, animal and human studies have indicated that hyperbaric pressure treatment of WPI increases their anti-oxidant and anti-inflammatory effects, which are linked to increased whey protein digestibility and the greater release of bioactive peptides (17, 23, 24).

A recent human trial has demonstrated that 1 month supplementation of pressure-treated WPI resulted in lowered inflammatory markers in patients with cystic fibrosis (25). Such supplementation has also been effective in countering gut associated inflammation as piglets with dextran sulfate-induced colitis fed pressure-processed WPI showed lower pro-inflammatory cytokine concentrations, decreased myeloperoxidase activity and improved antioxidant status in colonic tissue relative to both native WPI and skim milk protein-fed piglets (26). Whey proteins could diminish gut tissue inflammation via effects exerted on macrophages as a recent study showed that hydrolysates of a formula containing 41% whey protein concentrate, along with a combination of vitamins and minerals, vegetable oils and inulin, down-regulated macrophage-mediated secretion of the pro-inflammatory cytokine IL-8 and tumor necrosis factor (TNF)-α in a co-culture with Caco-2 intestinal cells (27). The majority of peptides generated during the hydrolysis of whey proteins are not generally considered to be readily absorbed intact systemically. It is possible, however, that whey peptides could exert direct anti-oxidant and anti-inflammatory effects within intestinal cells since they have been shown to survive simulated human gastric, duodenal and jejuneal digestive processes (18). The capability of hydrolysates of native or pressure-treated WPI, however, to directly decrease oxidative stress and pro-inflammatory cytokine release within Caco-2 cells has not been previously examined. The present work therefore investigated the effects of hydrolysates of native and pressure-treated WPI on oxidative damage and inflammation induced by H2O2 in intestinal Caco-2 epithelial cells.


Whey protein isolate high hyperbaric pressure treatment and sample preparation

Whey protein isolate produced by membrane ultrafiltration was purchased (Vitalus Nutrition Inc., Abbotsford, BC, Canada). Whey protein isolate was pressurized in 12.5% (w/v) in double-distilled water and pressurized with an Avure High Pressure Processing System model QFP 215L-600 (Avure Technologies, Columbus, OH) by applying a single cycle of hyperbaric pressure of 550 MPa. This acts to unfold the protein conformation of the whey proteins enabling them to be more susceptible to digestive enzymes (17). The native whey protein isolate (nWPI) 12.5% (w/v) and the pressurized WPI (pWPI) 12.5% (w/v) WPI solutions were lyophilized using the Flexi-Dry MP Lyophilizer (FTS Systems, Stone Ridge, NY, USA) and freeze-dried under −80°C and 90 mTorr (mT) vacuum in preparation for future cell culture studies.

In vitro enzymatic digestion

The enzymatic digestion method was adapted from Vilela et al. (17) with some modifications (28). This technique mimicked the in vivo gastrointestinal digestion of milk proteins. Briefly, lyophilized native and pressurized WPI were diluted in 390 mL of distilled water to reach a final concentration of 3 mg protein mL−1 and the pH was adjusted to 1.9 with 12N HCl. The WPI digestion was started by adding freshly prepared pepsin (Sigma-Aldrich, Oakville, ON, Canada) solution to reach an enzyme:substrate (E:S) ratio of 1:200. Subsequently the pH was raised to 7.4 using 10N NaOH, which inactivated pepsin irreversibly. After pepsin inactivation, the pancreatic enzymes trypsin (Sigma-Aldrich, Oakville, ON, Canada) and chymotrypsin Sigma-Aldrich, Oakville, ON, Canada) were dissolved in 10 mM NaH2PO4 to reach an E:S ratio of 1:200 and 1:87, respectively and immediately added to the solution. At the end of the digestion pH was increased to 10.5 to inactivate the pancreatic enzymes. The bottle was immediately chilled on ice under continuous stirring to decrease the temperature to 4°C.

Peptide isolation

After the digestion described above, the hydrolysates from native and pressurized WPI were subjected to an ultrafiltration system as described by Vilela et al. (17) and Iskandar (28). Briefly, to remove high molecular weight peptides a membrane filter of molecular weight cut-off (MWCO) of 10 kilodalton (kDa) (Millipore, Nepean, ON, Canada) was used in a stirred ultrafiltration membrane reactor (Model 8050, Millipore, Nepean, ON, Canada) under N2 pressure of 40 psi. The major advantage to use this filter is to retrieve amino acids rich in leucine, tyrosine, phenylalanine, lysine, arginine, proline and aspartate (29). The filtrate was freeze-dried (Flexi-Dry MP Lyophilizer, FTS Systems, Stone Ridge, NY, USA) at −80°C and 90 mT vacuum for further cell culture experiments. The lyophilized filtrates were flushed under N2 and kept at −20°C.

High-performance liquid chromatography (HPLC) profile

Reagents were HPLC grade and purchased from Sigma Chemical Co. (St Louis, MO, USA). Samples were analyzed using a Varian HPLC system with a tertiary gradient pump, a variable wavelength UV/VIS detector, and an autosampler with refrigerated sample compartment (Varian Canada Inc, Mississauga, ON, Canada). Samples were eluted using a Gemini-NX reverse-phase (RP)-HPLC column (100×4.5 mm) (Phenomenex, CA, USA), using a solvent flow rate of 1 mL min−1 and detection was at 215 nm. Gradient elution was carried out with a mixture of two solvents. Solvent A: 0.05% trifluoroacetic acid (TFA) in 10% aqueous acetonitrile (ACN) and solvent B: 0.05% TFA in 60% aqueous ACN, (v/v) starting with 100% solvent A and reaching 40% solvent A and 60% solvent B in 30 min.

Cell culture

The human colonic adenocarcinoma cell line Caco-2 was obtained from American Type Culture Collection (Manassas, VA, USA). All cell culture medium was obtained from Invitrogen (Carlsbad, CA, USA). Caco-2 cells passage numbers 21–29 were seeded at a density of 2.5×105 cells/well cultured in Dulbecco's modified Eagle's medium (DMEM, pH 7.4) with 25 mM glucose, supplemented with 20% heat inactivated fetal bovine serum (HFBS), 1% penicillin-streptomycin and 1% non-essential amino acid solution in a humidified atmosphere containing 5% CO2 at 37°C. All experiments were carried out in Minimal Essential Medium (MEM) with 2% HFBS and devoid of antibiotics.

Mitochondrial succinate dehydrogenase (MTS) assay

The effects of H2O2 on cell survival were measured using the MTS assay. Caco-2 cells were seeded in 24-well plates at a concentration of 2.5×105 cells/well. After 24 h of culturing, the cells were treated with different concentrations of H2O2 (0–2 mM) at 37°C for 1 h. Cells were then washed twice with MEM and incubated for 23 h. Subsequently, 10 µL of MTS reagent (Cayman Chemical, Ann Arbor, MI, USA) was added to each well and incubated for 3 h at 37°C. Finally, 100 µL per well of isopropanol containing 40 mM HCl was added to dissolve the formazan crystals. The absorbance was read at a wavelength of 560 nm using the Wallac Victor2 1420 Series microplate reader (Harlow Scientific, Arlington, MA, USA). Treatments were compared with untreated control cells. Results are expressed as percentage of untreated control cells. Each point represents the mean of three experiments with each individual treatment being run in triplicate.

Enzyme-linked immunosorbent assay

Confluent Caco-2 cells on the 24-well plate were exposed for 1 h to a culture medium containing H2O2 (Sigma-Aldrich, Oakville, ON, Canada) ranging from 0 to 2 mM. After H2O2 was removed, cells were washed twice with MEM, and allowed to recover for 23 h. The supernatants were collected after 24 h of the treatment to determine IL-8 release using a commercially available ELISA kit (BD Biosciences, Mississaga, ON, Canada) according to the manufacturer's instructions.

Exposure of Caco-2 cells to WPI and H2O2

To investigate the protective effect of WPI, 0.25 mM H2O2-stimulated Caco-2 cells were exposed to different concentrations of nWPI or pWPI hydrolysates (0, 500, 1000 and 2000 µg mL−1) in different 24-well plates. In this first set of experiments, Caco-2 cells were exposed to different concentrations of nWPI and pWPI hydrolysates for 1 h. WPI was removed, cells were washed twice with MEM, nWPI or pWPI hydrolysates were dissolved in MEM with 0.25 mM H2O2 and incubated for 1 h. Subsequently, Caco-2 cells were washed twice and nWPI or pWPI hydrolysates alone were added to the cells for 22 h. On the next day, supernatants were collected for IL-8 determination. A second set of experiments was performed to determine the recovery effect of nWPI or pWPI hydrolysates on pre-stimulated Caco-2 cells. Cells were exposed to 0.25 mM H2O2 for 1 h. H2O2 was removed, cells were washed twice with MEM, and nWPI or pWPI hydrolysates were added and cells were allowed to recover for 23 h. The supernatants were collected for IL-8 determination 24 h after the treatment.

Ferric reducing anti-oxidant power (FRAP) assay

FRAP was used to determine the total anti-oxidant potential in the supernatant of Caco-2 cells exposed to 0.25 mM and different concentrations of nWPI or pWPI hydrolysates (0–2000 µg mL−1). Briefly, in this assay the electron-donating capacity of the anti-oxidant was measured by the change in absorbance at 593 nm when a blue-colored Fe2+-tripyridyltriazine (Fe2+TPTZ) compound is formed from a colorless oxidized Fe3+ form. A standard curve was prepared from aqueous solutions of 1 mM FeSO4·7H2O at different concentrations ranging from 0.1 to 1.0 mM. The working reagent was prepared with 300 mM acetate buffer pH 3.6, 10 mM TPTZ (2, 4, 6-tripyridyl-s-triazine) solution dissolved in 40 mM HCl at 50°C, and 20 mM FeCl3·6H2O solution, at a 10:1:1 ratio, wrapped in foil. Once prepared it was immediately incubated for 10 min at 37°C. Thirty µL H2O, 10 µL standards or samples, and 200 µL FRAP working solution were added in a 96 well plate. The reaction occurred at room temperature for 30 min and absorbance was taken at 593 nm in a microplate reader (Infinite PRO 200 series, Tecan Group, San Jose, CA, USA).

Intracellular reactive oxygen species (ROS)

The generation of intracellular ROS was evaluated by oxidation of 2′,7′-dichlorofluorescin-diacetate (DCFH-DA) (Invitrogen, Carlsbad, CA, USA). The DCFH-DA was cleaved in the cells to dichlorofluorescin (DCFH) and oxidized by ROS to fluorescent dichlorofluorescein (DCF). This experiment explored the stimulation of the Caco-2 cells with 0.25 mM H2O2 for 1 h and after being washed twice, cells were treated with different concentrations of nWPI or pWPI hydrolysates from 0 to 2000 µg mL−1 for 23 h. After treatment cells were incubated with 5 µM DCFH-DA in phosphate buffered saline containing 5% (w/v) dimethyl sulfoxide (DMSO) solution for 30 min at 37°C. The fluorescent DCF was monitored by spectrofluorimetry (fluorescence excitation and emission were 485 nm and 530 nm, respectively; Wallac Victor2 1420 Series spectrofluorimetry reader – Harlow Scientific, Arlington, MA, USA). The values were expressed as percentages of DCF fluorescence intensity to that of untreated control cells. Each point represents the mean of three experiments with each individual treatment being run in triplicate.


Statistical analysis was performed using SAS for Windows (version 9.2, 2008, SAS Institute Inc., Cary, NC). One-way and two-way ANOVA were performed with Tukey's post hoc test to determine significant statistical differences between groups after verification of normality using Shapiro-Wilk.


Effect of high hydrostatic pressure treatment on the peptide profiles of WPI hydrolysates

Figure 1 represents the peptide profiles of nWPI and pWPI with MWCO <10 kDa performed by HPLC analysis. Differences in the peak abundances as well as two new peaks were observed in the pWPI that were not present in the nWPI.

Fig 1
Fig. 1.  Chromatogram of peptides from reverse-phase HPLC obtained via UV/VIS detector wavelength of 215 nm. (A) Peptides with MWCO less than 10 kDa derived from pepsin, trypsin and chymotrypsin hydrolysis of nWPI. (B) Peptides with MWCO <10 kDa derived from pepsin, trypsin and chymotrypsin hydrolysis of WPI exposed to hydrostatic pressure at 1-cycle of 550 MPa. The * symbol identifies extra peaks found in pWPH hydrolysates and arrows indicate differences in the relative abundances of peptides as a result of pressure treatment (+, higher abundance, –, lower abundance in pWPI hydrolysates relative to nWPI hydrolysates).

Effects of H2O2 cell viability and IL-8 response in stimulated Caco-2 cells

Cell viability as measured by the MTS assay following exposure of the cells to different concentrations of H2O2 is described in Fig. 2. The higher concentrations of H2O2 decreased (P<0.05) Caco-2 cells viability; however, the H2O2 concentration of 0.25 mM was associated with a similar number of viable cells as the control treatment. Disturbed integrity of the Caco-2 cell layer and detaching cells were also seen at treatment with 2 mM H2O2. Figure 3 represents the IL-8 response in Caco-2 cells exposed to different concentrations of H2O2. Increased (P<0.05) IL-8 concentrations relative to control Caco-2 cells was observed in cells treated with H2O2 concentrations from 0.25 to 2.0 mM with an approximate 300% increase observed with the lowest effective H2O2 concentration of 0.25 mM. The 0.25 mM H2O2 dose was used in subsequent experiments as it was not associated with decreased cell viability as opposed to the higher H2O2 concentrations (Fig. 2).

Fig 2
Fig. 2.  Effects of different concentrations of H2O2 on Caco-2 cell viability. Values are means±SE of three independent experiments. Means within each treatment without a common letter differ P<0.05. H2O2=Hydrogen peroxide.

Fig 3
Fig. 3.  Effect of different concentrations of H2O2 on IL-8 secretion of Caco-2 cells. Values are means±SE of three independent experiments. Means within each treatment without a common letter differ P<0.05. H2O2=Hydrogen peroxide.

Effects of WPI hydrolysates on cell viability and inflammatory response

Figure 4 illustrates the effect of different concentrations of WPI hydrolysates on Caco-2 cell viability in the absence of H2O2. No cell toxicity was observed in cells treated with different concentrations of either nWPI or pWPI hydrolysates. The protective anti-inflammatory effects of nWPI and pWPI hydrolysates on H2O2-stimulated Caco-2 cells are shown in Fig. 5. Both types of WPI hydrolysates were associated with anti-inflammatory effects although pWPI hydrolysates showed lower (P<0.05) IL-8 release at each of the tested WPI hydrolysates doses. Stimulated Caco-2 cells treated with the highest dose of the hydrolysates of nWPI or pWPI of 2,000 µg mL−1 demonstrated a reduction (P<0.05) of IL-8 secretion of 26.3 and 37.7%, respectively, as compared with H2O2-stimulated Caco-2 cells alone. The Caco-2 cells stimulated with H2O2 had reduced (P<0.05) IL-8 concentrations when treated with either type of WPI hydrolysates for 1 h post-stimulation (Fig. 6). The inflammatory response in Caco-2 cells induced by 0.25 mM H2O2 after 1 h post-stimulation treatment with the highest dose of nWPI or pWPI hydrolysates showed decreased (P<0.05) IL-8 secretion of 30.4 and 50.1%, respectively.

Fig 4
Fig. 4.  Effects of different concentrations of WPI on Caco-2 cell viability. Values are means±SE of three independent experiments. Survival is expressed as percentage of untreated control cells. WPI=Whey protein isolate. () Native WPI, () Pressurized WPI.

Fig 5
Fig. 5.  Protective effect of different concentrations of WPI hydrolysates on 0.25 mM H2O2 induced IL-8 secretion by Caco-2 cells. Means within each type of WPI treatment without a common letter differ P<0.05. The * symbol represents the comparison between nWPI and pWPI treatments and significantly differ (P<0.001). C=Control. WPI=Whey protein isolate. () Native WPI, () Pressurized WPI.

Fig 6
Fig. 6.  Recovery effects of different WPI hydrolysates treatment with pre-stimulation of 0.25 mM H2O2 on IL-8 secretion of Caco-2 cells. Values are means±SE of 3 independent experiments. Means within each type of WPI treatment without a common letter differ P<0.05. The * symbol represents the comparison between nWPI and pWPI treatments and significantly differ (P<0.001) C=Control. WPI=Whey protein isolate. () Native WPI, () Pressurized WPI.

Effects of WPI hydrolysates on the anti-oxidant capacity of cell culture medium

The ferric reducing anti-oxidant power (FRAP) measured in the cell culture medium of Caco-2 cells stimulated with 0.25 mM H2O2 after exposure to different concentrations of nWPI or pWPI hydrolysates is shown in Fig. 7. The medium of Caco-2 cells treated with either nWPI or pWPI hydrolysates showed increased (P<0.05) in FRAP activity at all hydrolysates treatment concentrations. In addition, pWPI hydrolysates-treated Caco-2 cell culture media showed increased (P<0.05) FRAP activity as compared to nWPI hydrolysates exposed Caco-2 cells at all tested concentrations.

Fig 7
Fig. 7.  The ferric reducing antioxidant power (FRAP) in the supernatant of Caco-2 cells exposed to 0.25 mM H2O2 and treated with either nWPI or pWPI hydrolysates. Values are means±SE of three independent experiments. Means within each type of WPI treatment without a common letter differ P<0.05. The * symbol represents the comparison between nWPI and pWPI treatments and significantly differ (P<0.001). C=Control. WPI=Whey protein isolate. () Native WPI, () Pressurized WPI.

Effects of WPI hydrolysates in the generation of reactive oxygen species (ROS)

The effects of different concentrations of WPI hydrolysates on Caco-2 cells exposed to 0.25 mM H2O2 are shown in Fig. 8. Caco-2 cells treated with either type of WPI hydrolysates showed a decrease (P<0.05) in intracellular ROS production. Caco-2 cells treated with the highest dose of hydrolysates of nWPI or pWPI, i.e. 2000 µg/mL demonstrated a reduction (P<0.05) in ROS formation of 32.5 and 76.1%, respectively, as compared with untreated Caco-2 cells. In addition, Caco-2 cells treated with pWPI hydrolysates had reduced (P<0.05) ROS generation as compared to nWPI hydrolysates treated cells.

Fig 8
Fig. 8.  Effects of different WPI hydrolysates treatment on 0.25 mM H2O2-induced intracellular ROS generation in Caco-2 cells. Values are means±SE of three independent experiments. Means within each type of WPI treatment without a common letter differ P<0.05. The * symbol represents the comparison between nWPI and pWPI treatments and significantly differ (P<0.01). WPI=Whey protein isolate. () Native WPI, () Pressurized WPI.


The results of the present study have demonstrated that WPI hydrolysates can exert anti-oxidant and anti-inflammatory effects in H2O2-stimulated Caco-2 epithelial cells. The anti-oxidant effects of WPI hydrolysates as shown by decreased intracellular ROS generation and increased anti-oxidant capacity in the cell culture medium likely involved improved intracellular glutathione (GSH) status and enhanced anti-oxidant enzyme activities. Whey proteins are rich in the cysteine precursor required for synthesis of GSH, which acts as a major intracellular anti-oxidant partly by donating sulfhydryl protons to unstable ROS molecules (30). The reduced form of GSH is a substrate for GSH peroxidase that catalyzes the reduction of different peroxides such as H2O2 to H2O (31). Previous work has shown that treatment with hydrolysates of pWPI was associated with improved intracellular status of the total and reduced form of GSH in cultured human lung epithelial cells (17). Caco-2 cells exposed to similar concentrations of H2O2 have shown attenuated intracellular oxidative stress when treated with the human whey protein lactoferrin (32); however, the present findings indicate that whey protein derived by-products of digestion such as peptides and amino acids can provide similar anti-oxidant protection.

The present study results showing an inhibited release of the pro-inflammatory cytokine IL-8 support the concept that hydrolysates of WPI can exert anti-inflammatory effects on intestinal Caco-2 cells exposed to oxidants (Fig. 5). IL-8 is the major cytokine measured in Caco-2 cells with respect to inflammation as compared to the inflammatory response assessed by the production of IL-6, prostaglandin E2, nitric oxide, IL-1β, TNF-α, and interferon-γ (33). IL-1β, an important mediator in intestinal inflammation, promotes IL-8 production (34). Oxidative stress induced via H2O2 has been shown to promote IL-8 production and increase IL-8 mRNA expression after H2O2-induced oxidative stress in Caco-2 epithelial cells (9). H2O2 up-regulates IL-8 gene expression via TNF receptors in the cell membrane, signaling the activation of the nuclear factor (NF)-κB pathway and IL-8 release (35). Our findings extend the results of a previous study showing that hydrolyzed whey protein formula exerted anti-inflammatory action on stimulated macrophages co-incubated with Caco-2 cells, which the authors attributed to synergistic effects of whey proteins and micronutrients present in the formula (27). The lowered IL-8 release in Caco-2 cell cultures shown in the present work could be related to the improved cellular redox status, which is closely regulated by the ratio of oxidized glutathione (GSSG) to the reduced form of GSH. Previous work has shown pretreatment of TNF-α stimulated human lung epithelial cells with hydrolysates of pWPI resulted in an improved cellular redox status that was related to decreased IL-8 secretion (17). When cells are exposed to increased levels of oxidative stress, GSSG accumulates and the ratio of GSH to GSSG decreases (36). A low GSH/GSSG ratio is linked with elevated levels of pro-inflammatory cytokines since redox status is a key regulator of the inflammatory response (37). Apart from direct stimulation of GSH synthesis induced by precursor thiol-containing peptides and amino acids, other endogenous antioxidant pathways may be stimulated by the pWPI hydrolysates. For example, whey protein treated C2C12 myoblasts showed activation of the transcription factor NF-E2-related factor 2 that upregulates synthesis of endogenous antioxidant proteins and enzymes (38).

The anti-oxidant and anti-inflammatory effects of the WPI hydrolysates were enhanced by hyperbaric pre-treatment of WPI. These findings are in agreement with recent observations that piglets with dextran sulfate sodium-induced colitis fed pressure-processed WPI showed significantly improved anti-oxidant and anti-inflammatory effects in the colonic tissue in comparison to native WPI and skim milk fed piglets, including decreased colonic content of IL-8, TNF-α and IL-18 (26). Similarly, hydrolysates of pressure-treated WPI exerted more effective anti-inflammatory effects than native WPI hydrolysates in stimulated cystic fibrosis transmembrane conductance regulator mutant respiratory cells (17, 39). Different peptide profiles were noted in hydrolysates resulting from the digestion of pressurized WPI including two new peaks (Fig. 1), which may have contributed to the greater anti-oxidant and anti-inflammatory effects of pWPI. Changes in the secondary and tertiary structures of pressure-processed whey proteins generates a unique profile of bioactive amino acids and peptides that enhances their anti-oxidant and anti-inflammatory effects (17, 23, 24, 26). In this manner, pressure processing of WPI might be comparable to the fermentation of whey proteins that has been shown to enhance the tissue anti-oxidant effects of whey proteins, which was related to an increased generation of the low molecular weight peptide γ-glutamylcysteine that can induce tissue GSH (40). Our previous studies have shown an increase in free sulfhydryl group content in WPI after high hydrostatic pressure treatment that we attributed to sulphide-disulphide interchange reactions (41). The antioxidant impact of an increase in free sulfhydryl groups in WPI is uncertain, however, as thiol-containing peptides may form aggregates via disulphide bond formation due to the high reactivity of thiol groups (42). Hydrolysates of pressurized WPI have also been shown to contain relatively higher free amino acid levels of alanine, histidine, glutamine, tryptophan, lysine and branched chain amino acids (BCAA) (17). It is conceivable that the combined effects of the above amino acids could have also contributed to the greater anti-inflammatory and anti-oxidant effects associated with pWPI since pre-treatment with those amino acids was shown to inhibit IL-8 secretion in H2O2-stimulated Caco-2 cells (39). Treatment with the BCAA valine, isoleucine, and leucine was also associated with elevated activities of both GSH S-transferase (GST) and catalase whereas histidine, tryptophan and lysine caused increase in GST activity (39). GST has ROS scavenging activity including the breakdown of lipid hydroperoxides (43) whereas catalase is involved in the decomposition of H2O2 (44). Glutamine has been reported to exert anti-oxidant effects in the Caco-2 and HCT-8 cell lines exposed to H2O2 by preserving cell membrane integrity and viability (45).


The findings from the present study have demonstrated anti-oxidant effects of hydrolysates of WPI in H2O2-treated Caco-2 cells that are improved with prior high hydrostatic pressure treatment of WPI. Furthermore, this study demonstrates an enhancement of the anti-inflammatory effects of WPI hydrolysates following by pressurization of WPI. More research is needed to measure a broader array of pro-inflammatory and anti-inflammatory cytokines to assess the extent of anti-inflammatory action associated with WPI hydrolysates. The above results, however, are consistent with the concept that whey hydrolysis products can act on intestinal cells directly to counteract oxidative stress and inflammation and so decrease tissue damage associated with inflammatory bowel conditions such as IBD. Our findings also indicate that the anti-inflammatory action associated with pWPI treatment in the piglet model of IBD (26) could be partly mediated via direct protective effects exerted on stimulated intestinal cells. Further studies are needed to investigate the specific native and pressurized WPI hydrolysate components involved in the protective effects as well as the mechanisms of action, which could involve down-regulation of the NF-κB pathway.

Conflict of interest and funding

This study was funded by a grant to Stan Kubow by the National Sciences and Engineering Council of Canada (NSERC).


  1. Spitz DR, Azzam EI, Li JJ, Gius D. Metabolic oxidation/reduction reactions and cellular responses to ionizing radiation: a unifying concept in stress response biology. Cancer Metastasis Rev 2004; 23: 311–22. [Crossref]
  2. Zhang K. Integration of ER stress, oxidative stress and the inflammatory response in health and disease. Int J Clin Exp Med 2010; 3: 33–40.
  3. Taha R, Seidman E, Mailhot G, Boudreau F, Gendron FP, Beaulieu JF, et al. Oxidative stress and mitochondrial functions in the intestinal Caco-2/15 cell lines. PLos ONE 2010; 5: 1–10. [Crossref]
  4. Chandra J, Samali A, Orrenius S. Triggering and modulation of apoptosis by oxidative stress. Free Radical Bio Med 2000; 29: 323–33. [Crossref]
  5. Grant JJ, Yun BW, Loake GJ. Oxidative burst and cognate redox signalling reported by luciferase imaging: identification of a signal network that functions independently of ethylene, SA and Me-JA but is dependent on MAPKK activity. Plant J 2000; 24: 569–82. [Crossref]
  6. Harada A, Sekido N, Akahoshi T, Wada T, Mukaida N, Matsushima K. Essential involvement of interleukin-8 (IL-8) in acute inflammation. J Leukoc Biol 1994; 56: 559–64.
  7. Atreya R, Neurath MF. Chemokines in inflammatory bowel diseases. Dig Dis 2010; 28: 386–94. [Crossref]
  8. Mahida YR, Ceska M, Effenberger F, Kurlak L, Lindley I, Hawkey CJ. Enhanced synthesis of neutrophil activating peptide-1/interleukin-8 in active ulcerative colitis. Clin Sci 1992; 82: 273–5.
  9. Yamamoto K, Kushima R, Kisaki O, Fujiyama Y, Okabe H. Combined effect of hydrogen peroxide induced oxidative stress and IL-1 alpha on IL-8 production in CaCo-2 cells (a human colon carcinoma cell line) and normal intestinal epithelial cells. Inflammation 2003; 27: 123–8. [Crossref]
  10. Quaroni A, Wands J, Trelstad RL, Isselbacher KJ. Epithelioid cell cultures from rat small intestine – characterization by morphologic and immunological criteria. J Cell Biol 1979; 80: 248–65. [Crossref]
  11. Okada T, Narai A, Matsunaga S, Fusetani N, Shimizu M. Assessment of the marine toxins by monitoring the integrity of human intestinal Caco-2 cell monolayers. Toxicol in Vitro 2000; 14: 219–26. [Crossref]
  12. Walle UK, Walle T. Transport of the cooked-food mutagen 2-amino-1-methyl-6-phenylimidazo-[4,5-b]pyridine (PhIP) across the human intestinal Caco-2 cell monolayer: role of efflux pumps. Carcinogenesis 1999; 20: 2153–7. [Crossref]
  13. Kameoka S, Leavitt P, Chang CS, Kuo SM. Expression of antioxidant proteins in human intestinal Caco-2 cells treated with dietary flavonoids. Cancer Lett 1999; 146: 161–7. [Crossref]
  14. Steijns JM. Milk ingredients as nutraceuticals. Int J Dairy Technol 2001; 54: 81–8. [Crossref]
  15. Krissansen GW. Emerging health properties of whey proteins and their clinical implications. J Am Coll Nutr 2007; 26: S713–S23.
  16. Alting AC, Hamer RJ, de Kruif CG, Visschers RW. Formation of disulfide bonds in acid-induced gels of preheated whey protein isolate. J Agr Food Chem 2000; 48: 5001–7. [Crossref]
  17. Vilela RM, Lands LC, Chan HM, Azadi B, Kubow S. High hydrostatic pressure enhances whey protein digestibility to generate whey peptides that improve glutathione status in CFTR-deficient lung epithelial cells. Mol Nutr Food Res 2006; 50: 1013–29. [Crossref]
  18. Picariello G, Ferranti P, Fierro O, Mamone G, Caira S, Di Luccia A, et al. Peptides surviving the simulated gastrointestinal digestion of milk proteins: Biological and toxicological implications. J Chromatogr B 2010; 878: 295–308. [Crossref]
  19. Kinekawa YI, Fuyuki T, Kitabatake N. Effects of salts on the properties of sols and gels prepared from whey protein isolate and process whey protein. J Dairy Sci 1998; 81: 1532–44. [Crossref]
  20. Hendrickx M, Ludikhuyze L, Van den Broeck I, Weemaes C. Effects of high pressure on enzymes related to food quality. Trends Food Sci Technol 1998; 9: 197–203. [Crossref]
  21. Patel HA, Singh H, Havea P, Considine T, Creamer LK. Pressure-induced unfolding and aggregation of the proteins in whey protein concentrate solutions. J Agr Food Chem 2005; 53: 9590–601. [Crossref]
  22. Alvarez P. Effect of applied hydrostatic pressure on the structure and rheological properties of whey proteins. Montreal: McGill University; 2004.
  23. Zavorsky GS, Kubow S, Grey V, Riverin V, Lands LC. An open-label dose – response study of lymphocyte glutathione levels in healthy men and women receiving pressurized whey protein isolate supplements. Int J Food Sci Nutr 2007; 58: 429–36. [Crossref]
  24. Kishta O, Iskandar M, Lands L. Effects of pressurized whey protein in a murine model of Pseudomonas aeruginosa lung infection. Am Thorac Soc Int Conference 2009; 179: A2845.
  25. Lands LC, Iskandar M, Beaudoin N, Meehan B, Dauletbaev N, Berthiuame Y. Dietary supplementation with pressurized whey in patients with cystic fibrosis. J Med Food 2010; 13: 77–82. [Crossref]
  26. Piccolomini AF, Rodrigues A, Antunes J, Paquet M, Weiler H, Kubow S, et al. Pressurized whey protein isolate decreases inflammation in descending colon of piglets with dextran sulfate-induced colitis. Appl Physiol Nutr Metab 2011; 36: 477.
  27. Kanwar JR, Kanwar RK. Gut health immunomodulatory and anti-inflammatory functions of gut enzyme digested high protein micronutrient dietary supplement Enprocal. BMC Immunol 2009; 10: 1–19. [Crossref]
  28. Iskandar M. Effect of native and pressurized whey protein isolates on inflammation in respiratory epithelial cells expressing either wildtype or mutant cystic fibrosis transmembrane conductance regulator (CFTR). Montreal: McGill University; 2011.
  29. Agudelo RA, Gauthier SF, Pouliot Y, Marin J, Savoie L. Kinetics of peptide fraction release during in vitro digestion of casein. J Sci Food Agr 2004; 84: 325–32. [Crossref]
  30. Blokhina O, Virolainen E, Fagerstedt K. Antioxidants, oxidative damage and oxygen deprivation stress: a review. Ann Bot 2003; 91: 179–94. [Crossref]
  31. Aslund F, Zheng M, Beckwith J, Storz G. Regulation of the OxyR transcription factor by hydrogen peroxide and the cellular thiol – disulfide status. Proc Natl Acad Sci USA 1999; 96: 6161–5. [Crossref]
  32. Shoji H, Oguchi S, Shinohara K, Shimizu T, Yamashiro Y. Effects of iron unsaturated human lactoferrin on hydrogen peroxide induced oxidative damage in intestinal epithelial cells. Pediatr Res 2007; 61: 89–92. [Crossref]
  33. Van De Walle J, Hendrickx A, Romier B, Larondelle Y, Schneider Y-J. Inflammatory parameters in Caco-2 cells: Effect of stimuli nature, concentration, combination and cell differentiation. Toxicol in Vitro 2010; 24: 1441–9. [Crossref]
  34. Bai B, Yamamoto K, Sato H, Sugiura H, Tanaka T. Combined effect of 25-hydroxycholesterol and IL-1β on IL-8 production in human colon carcinoma cell line (Caco-2). Inflammation 2005; 29: 141–6. [Crossref]
  35. Li Q, Verma IM. NF-kB regulation in the immune system. Nat Rev Immunol 2002; 2: 725–34. [Crossref]
  36. Murphy MP, Hurd TR, Filipovska A, Costa NJ, Dahm CC. Disulphide formation on mitochondrial protein thiols. Biochem Soc Trans 2005; 33: 1390–3. [Crossref]
  37. Maccarrone M, Brune B. Redox regulation in acute and chronic inflammation. Cell Death Differ 2009; 16: 1184–6. [Crossref]
  38. Xu R, Liu N, Xu X, Kong B. Antioxidative effects of whey protein on peroxide-induced cytotoxicity. J Dairy Sci 2011; 94: 3739–46. [Crossref]
  39. Katayama S, Mine Y. Antioxidative activity of amino acids on tissue oxidative stress in human intestinal epithelial cell model. J Agr Food Chem 2007; 55: 8458–64. [Crossref]
  40. Zommara M, Tachibana N, Sakono M, Suzuki Y, Oda T, Hashiba H, et al. Whey from cultured skim milk decreases serum cholesterol and increases antioxidant enzymes in liver and red blood cells in rats. Nutr Res 1996; 16: 293–302. [Crossref]
  41. Hosseini Nia T. Structural and nutritional properties of whey proteins as affected by hyperbaric pressure. Montreal, QC, Canada: McGill University; 2000.
  42. Roblet C, Amiot J, Lavigne C, Marette A, Lessard M, Jean J, et al. Screening of in vitro bioactivities of a soy protein hydrolysate separated by hollow fiber and spiral-wound ultrafiltration membranes. Food Res Int 2012; 46: 237. [Crossref]
  43. Masella R, Di Benedetto R, Varì R, Filesi C, Giovannini C. Novel mechanisms of natural antioxidant compounds in biological systems: involvement of glutathione and glutathione-related enzymes. J Nutr Biochem 2005; 16: 577–86. [Crossref]
  44. Chelikani P, Fita I, Loewen PC. Diversity of structures and properties among catalases. Cell Mol Life Sci 2004; 61: 192–208. [Crossref]
  45. Ziegler F, Seddiki L, Marion-Letellier R, Lavoinne A, Déchelotte P. Effects of l-glutamine supplementation alone or with antioxidants on hydrogen peroxide-induced injury in human intestinal epithelial cells. Eur e-J Clin Nutr Metab 2011; 6: e211–e6. [Crossref]

*Stan Kubow
School of Dietetics and Human Nutrition
McGill University
21,111 Lakeshore Road, Ste-Anne-de-Bellevue
QC H9X 3V9
Tel: +1 514 398 7754
Fax: +1 514 398 7739
Email: stan.kubow@mcgill.ca