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International Dairy Journal 32 (2013) 13e19

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International Dairy Journal

journal homepage: www.elsevier .com/locate/ idairyj

Casein hydrolysate and derived peptides stimulate mucin secretionand gene expression in human intestinal cells

Daniel Martínez-Maqueda, Beatriz Miralles, Elvia Cruz-Huerta, Isidra Recio*

Instituto de Investigación en Ciencias de la Alimentación, CIAL (CSIC-UAM, CEI UAMþCSIC), Nicolás Cabrera, 9, 28049 Madrid, Spain

a r t i c l e i n f o

Article history:Received 27 November 2012Received in revised form12 March 2013Accepted 25 March 2013

* Corresponding author. Tel.: þ34 9 10017940.E-mail address: i.recio@csic.es (I. Recio).

0958-6946/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.idairyj.2013.03.010

a b s t r a c t

The present study was undertaken to explore if a casein hydrolysate and four component peptides withprobable ability to interact with opioid receptors can exert a stimulatory effect on mucin production inhuman intestinal cells (HT29-MTX). aS1-Casein fragments 143e149 (AYFYPEL) and 144e149 (YFYPEL),and the casein hydrolysate, significantly increased expression of MUC5AC, the major secreted mucin genein this cell line, over 1.7-fold basal level after 4 h of exposure. The determination of mucin-like glyco-proteins showed a higher effect on mucin secretion by the casein hydrolysate (210% of controls) than thatof AYFYPEL and YFYPEL (around 160%). Therefore, peptides or other components may participate in theactivity of the hydrolysate in a synergistic way or through a non-opioid mechanism. In conclusion, acasein hydrolysate and two derived peptides, AYFYPEL and YFYPEL, promote the mucin production andmay support the development of functional foods to improve mucus barrier and its protective role ingastrointestinal diseases.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

The role of proteins, either directly or after hydrolysis, asphysiologically active components in the diet is being increasinglyacknowledged. Peptide sequences can be released from the pre-cursor food proteins by in vivo gastrointestinal digestion and byin vitro processes such as fermentation, controlled enzymatic hy-drolysis or reactions occurring during food storage (e.g., cheeseripening). The term “food-derived bioactive peptides” is related todietary peptide sequences that may exert an in vivo biologicalfunction. Among them, interest is growing in those peptides thatexhibit effects on the intestine, especially modulatory activities(Shimizu, 2010). There are several reports describing the role offood-derived bioactive peptides increasing gut secretory andabsorptive capacity (Moughan, Fuller, Han, Kies, & Miner-Williams,2007). For instance, the release of hormones such as cholecysto-kinin, gastrin, and somatostatin can be influenced by peptide hy-drolysates (Foltz et al., 2008). The intestinal epithelium constitutesa large surface between the body and the exterior milieu, beingcontinuously exposed to food toxins, pathogens and the changingluminal conditions such as pH changes or the action of proteolyticenzymes. These factors may have a negative impact on the func-tions of intestinal cells.

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The intestinal mucus layer plays a protective role as a barrierbetween the epithelium and the luminal content. Mucins, whichare high molecular weight glycoproteins, represent the maincomponent responsible for the intestinal mucus structure and itsprotective properties. Mucins are produced by goblet cells and anyquantitative or qualitative modification of their synthesis mayaffect the efficiency of the protection. Fortunately, certain dietarycomponents have been shown to positively influence the produc-ing of mucus. Examples of these dietary components are someshort-chain fatty acids and dietary fibres that increase mucin syn-thesis or the goblet cell number (Barcelo et al., 2000; Gaudier et al.,2004). Of note, a casein-derived peptide with reported opioid ac-tivity, b-casomorphin 7, exhibited an enhanced mucin secretionand mucin gene over-expression mediated by m-opioid receptors intwo models (human and rat) of intestinal goblet cells (Zoghbi et al.,2006). In rat jejunum, it has been also demonstrated ex-vivo thatluminal administration of b-casomorphin 7 and commercial hy-drolysates of casein and a-lactalbumin induced mucin releasethrough a nervous pathway and opioid receptor activation (Claustreet al., 2002; Trompette et al., 2003). In a screening with differentfood peptides, it was found that several peptides produced a sig-nificant increase in secretion of mucins. Furthermore, a-lactorphinshowed enhanced expression of the mucin gene 5AC (MUC5AC) inintestinal cells (Martínez-Maqueda et al., 2012). Recently, the totalpeptide pool from a fermented milk showed its effect on thestimulation of gel-forming MUC2 expression as well as mucinsecretion in HT29-MTX cells (Plaisancié et al., 2013).

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Dietary opioid peptides have not been shown to reach the bloodstream system in adult animals or humans (Read, Lord, Brantl, &Koch, 1990), although the physiological activities have been un-equivocally associated. Therefore, there is a growing line of evi-dence indicating that at least b-casomorphins (b-casein-derivedopioid peptides) may have a local regulatory role on the gastroin-testinal tract in adults (Teschemacher, 2003). Milk opioid peptidesare generated by hydrolysis of caseins and whey proteins(Teschemacher, Koch, & Brantl, 1997), that occurs both in enzymatichydrolysates and in vivo gastro-intestinal digestion of dairy prod-ucts (Svedberg, De Haas, & Leimenstoll, 1985). Furthermore, pro-cessing of commercial products can promote the release of opioidpeptides, for instance, in cheeses (De Noni & Cattaneo, 2010;Sienkiewicz-Sz1apka et al., 2009), and infant formulae(Jarmo1owska et al., 2007). b-Casomorphin 7 has also been identi-fied in simulated gastrointestinal digestion of an infant formula(Hernández-Ledesma, Amigo, Ramos, & Recio, 2004).

Interestingly, in a peptic casein hydrolysate with antihyperten-sive properties, the peptides with major activity were identified asaS1-casein fragments: 90e94 (RYLGY), which differ in one aminoacid at the C-terminal end from a previously described opioidpeptide (RYLGYL) (Loukas, Varoucha, & Zioudrou, 1983), and 143e149 (AYFYPEL), which has a favourable structure to bind opioidreceptors due to the presence of Tyr in the second position and Phetogether with Tyr in the third and fourth positions, respectively(Meisel & Fitzgerald, 2000). In a recent screening, both fragmentsshowed significant activity on mucin secretion in human intestinalcells (Martínez-Maqueda et al., 2012). Moreover, other peptidesidentified in this hydrolysate, although not previously reported asopioid peptides, exhibit amino acid sequences that could be pre-dicted to interact with opioid receptors, such as aS1-casein f(144e149) (YFYPEL) and aS2-casein f(89e95) (YQKFPQY). The presentstudy was undertaken to explore if this casein hydrolysate andderived peptides with probable ability to interact with opioid re-ceptors can exert a stimulatory effect on mucin secretion and geneexpression in human intestinal cells.

2. Materials and methods

2.1. Samples

The bovine aS1-casein fragments 90e94 (RYLGY), 143-149(AYFYPEL), and 144e149 (YFYPEL) were synthesised using con-ventional solid-phase FMOC synthesis with a 433A peptide syn-thesiser (Applied Biosystems, Warrington, UK). Their purity (>90%)was verified in our laboratory by reverse phase high performanceliquid chromatography and tandemmass spectrometry. Bovine aS2-casein fragment 89e95 (YQKFPQY) was synthesised by GenscriptCorporation (Piscataway, NY, USA). The casein hydrolysate (Low-pept�) was prepared by casein hydrolysis with food-grade pepsin(Biocatalysts, Cardiff, UK) as previously described (Contreras,Carrón, Montero, Ramos, & Recio, 2009). The resulting hydrolysedcasein was subsequently spray-dried to produce a dried powder.

2.2. Cell culture

HT29-MTX cell line, a human colon adenocarcinoma-derivedmucin-secreting goblet cell line (Lesuffleur et al., 1993), wasgrown as described previously (Martínez-Maqueda et al., 2012).Experiments were conducted between passages 17 and 23. Serum-free medium with or without peptide (0.05, 0.1, and 0.5 mM) orcasein hydrolysate (0.1 and 1%, w/v) was added to cells and incu-bated for 2e24 h at 37 �C. The supernatants were collected, frozenand stored at �70 �C. The total RNA was isolated with Nucleospin�

RNA II (Macherey-Nagel, Düren, Germany).

2.3. Enzyme-linked lectin assay

Mucin-like glycoprotein secretion was determined by anenzyme-linked lectin assay (ELLA), as previously reported(Martínez-Maqueda et al., 2012). All experiments were performedthree times with at least three biological replicates.

2.4. Real-time quantitative RT-PCR assays (qRT-PCR)

Quantitative RT-PCR amplification was carried out using aLightcycler 480 (Roche, Mannheim, Germany) in 384-well micro-plates (Roche). RNA (375 ng) was reverse transcribed using a HighCapacity cDNA Reverse Transcription Kit (Applied Biosystems) ac-cording to the manufacturer’s instructions. For MUC5AC (accessionno. AJ001402), target gene primers 2870-2889/3109-3091 wereused. For reference genes cyclophillin (accession no. Y00052) andb-actin (accession no. NM_001101) primers 280-340/445-421 and879-896/1076-1053, respectively, were used (Tai et al., 2008;Zoghbi et al., 2006). The SYBR Green method was used and eachassay was performed with cDNA samples in triplicate. Each reac-tion tube contained 5 mL 2� SYBR Green real-time PCR Master Mix(Roche) 0.25 mL of a 10 mM of gene-specific forward and reverseprimers, 0.27 mL of cDNA and 4.23 mL of water. Amplification wasinitiated at 95 �C for 5 min, followed by 45 cycles of 95 �C for 10 s,60 �C for 10 s and 72 �C for 10 s. Control PCRs were included toconfirm the absence of primer dimer formation (no-templatecontrol), and to verify that there was no DNA contamination(without RT enzyme negative control). All real-time PCR assaysamplified a single product as determined by melting curveanalysis.

The relative expression levels of the target gene were calculatedusing the comparative critical threshold method (DDCt). Humancyclophilin and b-actin were tested as reference genes. Cyclophilingene was chosen to calculate the threshold cycles because it hadpreviously been shown to be constant under all conditions used. Allexperiments were performed at last three times in triplicate.

2.5. Analysis by reverse phase high-performance liquidchromatography-tandem mass spectrometry

Reverse phase high-performance liquid chromatography-tandem mass spectrometry (RP-HPLC-MS/MS) quantification ofpeptides AYFYPEL, YFYPEL, RYLGY, and YQKFPQY in the hydrolysatewas performed on an Agilent 1100 HPLC System (Agilent Technol-ogies, Waldbron, Germany) connected on-line to an Esquire 3000ion trap (Bruker Daltonik GmbH, Bremen, Germany) and equippedwith an electrospray ionisation source as previously described(Contreras et al., 2010). The column used was a reverse phaseXBridge PST C18 Column (150 � 2.1 mm i.d., 5 mm particle size)(Waters Corp, Milford, MA, USA). The signal threshold to performtandem mass spectrometry was 50,000. The hydrolysate was dis-solved at the concentration of 2.5 mg mL�1. The quantification wasperformed by representing the peak obtained byMS analysis versusthe peptide concentration. Plots were made with the peak area ofthe molecular ions with m/z value, corresponding to parental ion,and their sodium and potassium adducts. Five calibration pointswere obtained from 1 to 16 mg mL�1. Linear (y ¼ a þ bx) regressionfor the calibration curves was estimated. The following curves wereobtained (a) y¼ 1.40� 106 x� 1.92 � 106 (R2 ¼ 0.997) for AYFYPEL;(b) y ¼ 2.14 � 106 x � 1.27 � 106 (R2 ¼ 0.998) for YFYPEL; (c)y ¼ 6.59 � 106 x þ 2.49 � 106 (R2 ¼ 0.995) for RYLGY, and (d)y ¼ 2.21 � 106 x � 1.75 � 106 (R2 ¼ 0.999) for YQKFPQY.

To study peptide stability during experiments, cell supernatantswere also analysed by RP-HPLC-MS/MS using a reverse phaseMediterranea Sea C18 Column (150� 2.1mm i.d., 5 mmparticle size)

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(Teknokroma, Barcelona, Spain). The samples were eluted at0.2 mL min�1.

Data obtained were processed and transformed to spectra rep-resentingmass values using the Data Analysis program (version 4.0,Bruker Daltonik). To process the MS/MS spectra and to performpeptide sequencing BioTools (version 3.1, Bruker Daltonik) wasused.

2.6. Statistical analysis

Data were analysed by a two-way analysis of variance (ANOVA),followed by the Bonferroni test. For a better comparison of theconcentrations versus control data for each time, data were ana-lysed by a one-way ANOVA, followed by the NewmaneKeuls test.GraphPad Prism 4 software was used to find significant differencesbetween means and controls as P < 0.05 (*), P < 0.01 (**) orP < 0.001 (***).

3. Results

3.1. Effect of synthetic peptides on MUC5AC expression and mucinsecretion by HT29-MTX cells

HT29-MTX cells were exposed to three concentrations (0.5, 0.1,and 0.05mM) of casein-derived peptides which have structures thatfit the requirements to bind opioid receptors, i.e., RYLGY, AYFYPEL,YFYPEL, and YQKFPQY. These peptides were previously identified ina peptic casein hydrolysate with proven antihypertensive activity(Contreras et al., 2009). The level of MUC5AC mRNA was followedby qPCR up to 24 h (Fig. 1). Peptides AYFYPEL and YFYPEL producedan increase in the relative expression of the gene that reached thehighest level after 4 h of exposure. The maximal responsewas 1.79-fold basal level (P < 0.01) at the highest concentration (0.5 mM) ofYFYPEL. The homologous peptide, AYFYPEL, showed MUC5ACincreased expression at 0.1 mM at 4 h (1.74-fold basal level,P < 0.05). Peptide RYLGY showed expression values over 1.7-foldbasal level, although due to the high variability, it did not reachsignificance. The sequence YQKFPQY did not elicit a substantialchange in the level of expression of MUC5AC.

Fig. 1. Time course effect at three different concentrations (,, 0.05 mM; , 0.1 mM;-, 0.5 mM

and aS2-casein fragment 89e95 YQKFPQY (D), on MUC5AC mRNA level in HT29-MTX cells dlevel of control (untreated cells), using cyclophilin as reference gene. Each point representsconcentration versus control were determined by two-way ANOVA applying the Bonferron

Mucin secretionwas determined in the supernatants of the cellssubmitted to the treatment with AYFYPEL and YFYPEL, where asignificant increase inMUC5AC expressionwas found. Themaximalpercentages of mucin-like glycoprotein were reached at 4 h with162% of control (P < 0.05) for AYFYPEL and 166% (P < 0.01) forYFYPEL (Fig. 2), which also showed a significant increase of mucinsecretion after 2 h of exposure (152%; P < 0.05).

3.2. Determination of the stability of peptides in the HT29-MTX cellculture

Peptide stability during treatment was studied by RP-HPLC-MS/MS analysis of the cell supernatants. This analysis aimed to esti-mate the degradation of the parent peptide by the action of thecellular peptidases and allowed the identification of the releasedpeptide fragments. Synthetic peptides were mostly stable up to 8 hof treatment and only small amounts of peptide fragments weredetected (Fig. 3). In general, an appreciable decrease of the syn-thetic peptide concentration in the mediumwas observed between8 and 24 h, although it was not always in accordance with therelease of the derived fragments. In addition to hydrolysis, thereduced peptide presence with incubation time could be alsoattributed to its localisation in the mucin layer or intracellularly.Peptide YFYPEL was hydrolysed to FYPEL, but 8 h after exposure,slight degradation was found (15% of the 2 h value), and a furtherYFYPEL decrease was only partially reflected in FYPEL occurrence.Interestingly, from peptide AYFYPEL, the active peptide YFYPEL wasformed besides FYPEL, but at too low rate to be consideredresponsible for the observed activity. Both RYLGY and YQKFPQYwere similarly stable for the first 8 h, giving a unique derivedfragment, YLGY and pyroglutamic acid derivative of parental pep-tide (m/z 793.4), respectively.

3.3. Effect of casein hydrolysate on MUC5AC expression and mucinsecretion

Given the effect on MUC5AC expression and mucin secretionshown by the two contained peptides AYFYPEL and YFYPEL,the casein hydrolysate was assayed at two different concentrations

) of aS1-casein fragments 90e94 RYLGY (A), 143e149 AYFYPEL (B), 144e149 YFYPEL (C),etermined by quantitative RT-PCR. Data are expressed as relative MUC5AC expressionthe mean � SE of three biological replicates in triplicate. Significant differences of eachi test: (*) P < 0.05, (**) P < 0.01.

Fig. 2. Time course effect at three different concentrations (,, 0.05 mM; , 0.1 mM; -, 0.5 mM) of aS1-casein fragments 143e149 AYFYPEL (A), and 144e149 YFYPEL (B), on mucinsecretion in HT29-MTX cells determined by enzyme-linked lectin assay. Data are expressed as mucin-like glycoprotein secretion as a percentage of control (untreated cells). Eachpoint represents the mean � SE of three biological replicates in triplicate. Significant differences of each concentration versus control were determined by one-way ANOVA applyingthe NewmaneKeuls test: (*) P < 0.05.

Fig. 3. Time course stability at 0.5 mM of: (A) aS1-casein fragment 90e94 RYLGY ( ) and derivative YLGY (-); (B) aS1-casein fragment 144e149 YFYPEL ( ) and derivative FYPEL(-); (C) aS1-casein fragment 143e149 AYFYPEL ( ) and derivatives YFYPEL ( ) and FYPEL (-); (D) aS2-casein fragment 89-95 YQKFPQY ( ) and the unidentified peptide derivativeof m/z 793.4 (-) in cell supernatants as determined by reverse phase high performance liquid chromatography and tandem mass spectrometry. Arbitrary units expressed as thepeak area of the molecular ions with m/z value, corresponding to parental ion and their sodium and potassium adducts.

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(0.1 and 1%) during 2, 4, 8, and 24 h (Fig. 4). The level of MUC5ACexpression was significantly increased at 4 h, (1.8-fold basal level)when hydrolysate concentrations of 0.1% and 1.0% were tested. Thetime of maximum expression, 4 h, was the same as that found forthe synthetic peptides AYFYPEL and YFYPEL.Whenmucin secretionwas determined, the highest response was found at 8 h with the0.1% hydrolysate (210% of controls, P < 0.001) and secretion at 24 hwas also significantly enhanced (172% of controls, P < 0.01). It wasnot possible to perform the mucin determination in the superna-tants containing 1% hydrolysate probably due to the interference ofpeptides or other components at high concentration with the ELLAreagents.

The quantification in the hydrolysate of the four containedpeptides with probable ability to interact with opioid receptors wasdetermined by RP-HPLC-MS/MS and a similar abundance for all ofthem was found (1.93 mg of RYLGY, 4.01 mg AYFYPEL, 2.61 mg ofYFYPEL, and 2.77 mg of YQKFPQY per g of hydrolysate). Thesevalues imply concentrations of the peptides in the 1% (w/v) hy-drolysate of 0.029mM RYLGY, 0.044mM AYFYPEL, 0.031mM YFYPEL,and 0.028 mM YQKFPQY, i.e., slightly lower than the lowest doseassayed for the synthetic peptides individually (0.05 mM).

4. Discussion

This work provides evidence that casein-derived peptidesdifferent from the b-casein fragments, previously described, havea stimulatory effect on mucin secretion and gene expression inhuman intestinal cells. All assayed synthetic peptides share acommon structure favourable to binding to opioid receptors, i.e.,

Fig. 4. Time-course effect of the casein hydrolysate in HT29-MTX cells. A) MUC5ACmRNA leconcentrations (-, 0.1%, w/v; , 1%, w/v). Data are expressed as relative MUC5AC expressdifferences of each concentration versus control were determined by one-way ANOVA applassay after addition of the casein hydrolysate at the concentration of 0.1% (w/v). Data are expEach point represents the mean � SE of three biological replicates in triplicate. Significant dapplying the Bonferroni test: (**) P < 0.01; (***) P < 0.001.

the presence of a Tyr in the first or second position from the N-terminal end and an aromatic residue at the third and/or fourthposition. The opioid activity of RYLGYL and RYLGYLE had beenpreviously demonstrated by Loukas et al. (1983) but, to the best ofour knowledge, the sequences here tested had not been previouslydescribed as opioid peptides. The possible interaction of these se-quences with m- or d-opioid receptors undoubtedly merits furtherresearch and experiments with guinea pig ileum preparations arealready in progress.

From the RT-PCR experiments, sequences AYFYPEL and YFYPELwere identified as those more active on MUC5AC expression andboth peptides reached similar expression values as those previouslydescribed for b-casomorphin 7 (Zoghbi et al., 2006) and a-lactor-phin (Martínez-Maqueda et al., 2012). Regarding the time at whichthe maximum effect was found, the time-course experimentsshowed a significant increase of MUC5AC mRNA levels at 4 h. Thistime is shorter than that previously reported for b-casormorphin-7,for which the maximal response was found after 24 h of treatment(Zoghbi et al., 2006). Previous results obtained in our laboratorywith a-lactorphin showed that mucin discharge preceded the in-crease of MUC5AC expression, probably to replenish the intracel-lular mucin pool in goblet cells (Martínez-Maqueda et al., 2012). Inthe present study, this behaviour could be observed for YFYPELwith an increased mucin concentration at 2 h and a maximumexpression level at 4 h. However for AYFYPEL, maximum mucinsecretion and MUC5AC expression occurred at 4 h, simultaneously.

Interestingly, peptide AYFYPEL was partially degraded to theactive form YFYPEL by the action of cellular peptidases although theamount released at 4 h does not allow anticipation of a significant

vel determined by quantitative RT-PCR after addition of the hydrolysate at two differention level of control (untreated cells), using cyclophillin as reference gene. Significantying the NewmaneKeuls test. B) Mucin secretion determined by enzyme-linked lectinressed as mucin-like glycoprotein secretion as a percentage of control (untreated cells).ifferences of each concentration versus control were determined by two-way ANOVA

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contribution of the peptide fragment to the observed activity.When testing the casein hydrolysate, the increased secretion wasfound later than the significant stimulation of the gene expressionat 4 h although the relative MUC5AC expression also increased at24 h without reaching significance. It is noteworthy that the hy-drolysate effect on mucin secretion was higher than that found forthe peptides AYFYPEL and YFYPEL separately (210% compared with160% of controls, approximately). Moreover, the times of maximumsecretion in the 0.1% hydrolysate treatment (8 and 24 h) weredelayed compared with those observed with AYFYPEL (4 h) andYFYPEL effect (2 and 4 h). Based on the peptide quantification, theconcentration of active peptides in the 0.1% hydrolysate was ten-fold lower than that of the synthetic peptides that have separatelyshown activity on mucin secretion.

In addition to the contribution of AYFYPEL and YFYPEL, theseresults suggest that peptides or other components of the hydroly-sate could participate in a synergistic manner or through a non-opioid mechanism. Alternatively, the action of cellular peptidasesmay produce the release of new peptides, although supernatantanalysis of synthetic peptides shows that synthetic peptidesremained stable up to 8 h. To clarify if opioid receptors are involvedin the mucin production activity, experiments using an opioidantagonist (ciprodime) are being conducted. In any case, the cleareffect of the casein hydrolysate on mucin secretion and MUC5ACexpression is consistent with a previous report by Han, Deglaire,Sengupta, and Moughan (2008). Here it was shown that a caseinhydrolysate produced a significant increase of Muc3mRNA levels inthe small intestine tissue and Muc4 gene expression in the colontissue of rats fed with the hydrolysate for 8 days. In contrast, noeffect was found with a free L-amino acid diet simulating the hy-drolysate (Han et al., 2008).

There are obvious limitations with the use of a cell culturemodel in this study. Although intestinal cell lines constitute asuitable approximation to the in vivo environment, certain short-comings may be present as non-identical natural responses orphysiology in comparison with the intestine (Langerholc,Maragkoudakis, Wollgast, Gradisnik, & Cencic, 2011). General lim-itations of cancer-derived lines are non-specificity, altered glyco-sylation and unresponsiveness to hormones or cytokines(Peracaula, Barrabes, Sarrats, Rudd, & de Llorens, 2008). The HT29-MTX cell line is a mucin-secreting cell line that forms a homoge-neous monolayer of polarised goblet cells that exhibit a discreteapical brush border. This cell culture has proven to be a reliable toolfor the study of gastrointestinal mucin secretion (Lesuffleur et al.,1993; Zoghbi et al., 2006) but results should be confirmed byanimal testing.

It is important to highlight that some of the peptides here testedhad been previously found in the human gastric and intestinalcontents after milk ingestion or in vitro gastrointestinal simulationsof dairy products. This is the case for AYFYPEL, which is released inthe human stomach after milk or yoghurt ingestion and the shorterform, YFYPEL that was detected in duodenal samples after milkingestion (Chabance et al., 1998). Similarly, these two peptides havealso been found in simulated gastrointestinal digestions of infantformulae, milk and yoghurt by different authors (Dupont et al.,2010; Hernández-Ledesma, Quirós, Amigo, & Recio, 2007). Thedoses of synthetic peptides used in our study are within the con-centration range theoretically expected in the intestinal contentsafter milk ingestion.

5. Conclusions

Some casein-derived peptides whose sequences might antici-pate interaction with opioid receptors have been assayed in HT29-MTX cells to study their influence on mucin production. Two of

them, AYFYPEL and YFYPEL, have proven to significantly increaseMUC5AC gene expression and mucin secretion. A casein hydroly-sate, containing these peptides among the most abundant species,is able to stimulate MUC5AC expression and mucin secretion at ahigher rate than the individual peptides. Thus, a casein hydrolysateand two contained peptides, AYFYPEL and YFYPEL with probableability to bind opioid receptors, have demonstrated their ability toaffect mucin secretion and MUC5AC expression in human HT29-MTX cells. Such knowledge may help the development of func-tional foods that promote the strengthening of the mucus barrierand its protective role in gastrointestinal diseases.

Acknowledgements

This work was supported by projects AGL2011-24643 andConsolider-Ingenio FUN-C-Food CSD 2007-063 from Ministerio deEconomía y Competitividad, and project P2009/AGR-1469 fromComunidad de Madrid. The authors are participants in the FA1005COST Action INFOGEST on food digestion. D. Martínez-Maquedawants to acknowledge to CSIC for a JAE Program fellowship. We aredeeply grateful to T. Lessuffleur for generously providing HT29-MTX cells, and Innaves S.A. for preparing the casein hydrolysate.

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