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International Dairy Journal 21 (2011) 129e134

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International Dairy Journal

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

Identification of caseinophosphopeptides generated throughin vitro gastro-intestinal digestion of Beaufort cheese

Isabelle Adt a, Coralie Dupas a, Rachel Boutrou b, Nadia Oulahal a, Claude Noel a,Daniel Mollé b, Thierry Jouvet c, Pascal Degraeve a,*

aUniversité de Lyon, Université Lyon 1, Laboratoire de Recherche en Génie Industriel Alimentaire (EA no 3733), Institut Universitaire de Technologie,Département de Génie Biologique, Technopole Alimentec, rue Henri de Boissieu, 01000 Bourg en Bresse, Franceb INRA, AgroCampus Ouest, UMR 1253 Sciences et Technologies du Lait et de l’Œuf, 35042 Rennes, FrancecActilait/ITFF, Institut Technique du Lait et des Produits Laitiers, Technopole Alimentec, rue Henri de Boissieu, 01000 Bourg en Bresse, France

a r t i c l e i n f o

Article history:Received 24 January 2010Received in revised form8 October 2010Accepted 19 October 2010

* Corresponding author. Tel.: þ33 474 455252; fax:E-mail address: pascal.degraeve@iut.univ-lyon1.fr

0958-6946/$ e see front matter � 2010 Elsevier Ltd.doi:10.1016/j.idairyj.2010.10.002

a b s t r a c t

Caseinophosphopeptides (CPPs) are bioactive peptides originating from proteolysis of caseins, the mainproteins of milk. In this study, the generation of CCPs during Beaufort cheese making and by in vitrosimulated gastro-intestinal hydrolysis using pepsin and pancreatin were assessed using selectiveprecipitation and liquid chromatographyeelectrospray ionisation tandem mass spectrometry. Seventy-two water-soluble CPPs, mainly originating from b-casein, were identified in Beaufort cheese, while 79CPPs, mainly generated from as1-caseins, were obtained from enzymatic hydrolysates. Most of thepeptides generated by the action of digestive enzymes were monophosphorylated; however, 17 out ofthe 23 polyphosphorylated CPPs identified from digested Beaufort cheese still contained the charac-teristic cluster sequence Ser(P)-Ser(P)-Ser(P)-Glu-Glu, providing calcium-binding properties to CPPs. Thecontent of CPPs in Beaufort cheese before and after the action of digestive enzymes was estimated to be230 and 275 mg per 100 g of fresh cheese, respectively. The action of proteolytic digestive enzymes onCPPs is discussed.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

Caseinophosphopeptides (CPPs), highly phosphorylated frag-ments of caseins, are bioactive peptides that have unique functionalproperties including calcium absorption and retention, and bonecalcification (Fitzgerald, 1998; Park & Allen, 1998a), anticariogenicity(Aimutis, 2004; Morgan et al., 2008) and immunostimulatory prop-erties (Otani, Kihara, & Park, 2000a;Otani et al., 2000b).Most of thesefunctionalities are due to the physico-chemical properties of CPPsthat enable the chelation of various divalent and trivalent cations andthereby enhance mineral solubility (Park & Allen, 1998a; Park,Swaisgood, & Allen, 1998b). As a consequence, the bioavailability ofcalciumand iron in the gut could be improved throughCPP-metal ioncomplexes in the distal small intestine that allow metal ions toremain in a soluble form and thus to be better absorbed (Peres et al.,1999). Although the stimulation of calcium absorption in the ileumafter the binding of calcium to CPPs is still questioned (Hartmann &Meisel, 2007), dietary supplementation with CPPs might still be

þ33 474 455253.(P. Degraeve).

All rights reserved.

relevant in particular populations, such as calcium-uptake-deficientsubjects or post-menopausal women, who are particularly suscep-tible to osteoporosis (Tsuchita, Goto, Shimizu, Yonehara, & Kuwata,1996).

While a lot of CPP-enriched products are commercially available,little is known about CPP contents of traditional foods. These mole-cules appearduringdairy processing throughout the actionof variousproteinases (from milk, milk-clotting preparations and microorgan-isms) that cleave the caseins into phosphorus-rich peptide fragments(Kyriakidis, Sakellaris, & Sotiroudis, 1993). Some studies also suggestthat indigenous phosphatases in milk play a role in breakdown ofCPPs. Thus, the degree of phosphorylation of caseins and products oftheir enzymatic hydrolysis may mainly depend on acid and alkalinephosphatase activity during cheese processing (Hynek, Zuzalkova,Sikac, & Kas, 2002; Ur-Rehman, Farkye, & Yim, 2006).

CPPs that accumulate during cheese ripening are ingested asfood components (Ellegard, Gammelgard-Larsen, Sorensen, &Fedosov, 1999; Hynek, Kozak, Drab, Sajdok, & Kas, 1999). Hence,when dairy products reach the gut, the CPPs already present in thefood product may be digested into even smaller molecules, whilenew CPPs may also appear from digestion of as1-, as2- and b-caseins(Fitzgerald, 1998).

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In the present study, the presence and the identification of CPPsin Beaufort cheese before and after the action of gastro-intestinalenzymes were investigated. This integrated approach includedboth an assessment of the quantity and the nature of CPPs in thecheese and an in vitro procedure simulating gastro-intestinaldigestion of the cheese. Beaufort cheese was considered as a potentsource of CPPs since this Swiss-type cheese has a high caseinconcentration. After selective precipitation of CPPs by ethanol inpresence of calcium, their concentration was estimated and theiridentification was performed by mass spectrometry. This alloweddiscussing the proteolysis throughout Swiss-type cheese ripening.Moreover, the effect of successive action of pepsin and pancreatinon formation and/or breakdown of CPPs from Beaufort cheese wasinvestigated. This procedure allowed estimation of whether, on onehand, the action of digestive proteases liberates CPPs encrypted incaseins and, on the other hand, if CPPs from cheese can be brokendown into inactive fragments by digestive enzymes.

2. Materials and methods

2.1. Materials

Six-month-old Beaufort cheese was kindly provided by theFrench Technical Institute for Cheese (Institut Technique Françaisdes Fromages, I.T.F.F., now Actilait). All chemicals were of analyticalgrade and Milli-Q water (Millipore, Bedford, MA, USA) was usedthroughout the study. Pepsin (from porcine stomach mucosa,activity 3000 to 4500 units per mg, EC 232-629-3), and pancreatin(from bovine pancreas, activity equivalent to 8� United StatesPharmacopeia specifications, EC 232-468-9) were purchased fromSigmaeAldrich (Saint Quentin Fallavier, France).

2.2. Preparation of Beaufort cheese homogenate andwater-soluble extraction

Grated Beaufort cheese (20 g) was homogenised in 40 mL ofwater for 10 min at room temperature at 20,500 rpm using an IKAUltra-Turrax T25 disperser-homogenizer (Janke & Kunkel, Staufen,Germany). This mixture was called “cheese homogenate”, anda water-soluble extract (WSE) was prepared from it as described byKuchroo and Fox (1982), with slight modifications as described byDupas et al. (2009). Thereafter, high-molecular-mass moleculeswere removed by ultrafiltration through polyethersulfonemembranes (10 kDa cut-off, Millipore, Saint-Quentin-en-Yvelines,France), and cheeseWSEswere kept at�20 �C until further analysis.

2.3. In vitro enzymatic digestion

In vitro enzymatic digestions (using pepsin and pancreatin)were carried out in triplicate, according to the conditions previ-ously described by Parrot, Degraeve, Curia, andMartial-Gros (2003)for the digestion of WSE of Emmental cheese, with minor modifi-cations: sample pH was adjusted to 2 using 1 mol L�1 HCl beforeaddition of pepsin solution to cheese homogenate; 30% (w/w)NaOH was used to inactivate pepsin. The in vitro enzymaticdigestion was applied to casein as a control. Controls withoutdigestive enzymes were prepared under the same conditions tocheck the potential contribution of proteases and phosphatises inBeaufort cheese.

2.4. Peptide analysis by reverse phase high performanceliquid chromatography

Peptide analysis was carried out by RP-HPLC/diode arraydetection, using the same materials, methods and conditions

previously described by Parrot et al. (2003), with the exception ofthe acquisition software, which was TotalChrom Navigator� soft-ware, and the column brand, which was Varian column (Les Ulis,France).

2.5. Nitrogen and phosphorus analysis

Total nitrogen (TN), water-soluble nitrogen (WSN), non-proteinnitrogen (12% trichloroacetic-acid-soluble nitrogen (TCA-SN)) or 5%(w/v) phosphotungstic-acid-soluble nitrogen (PTA-SN) of miner-alised samples were measured with the Kjeldahl method (AOAC,1984). The modified Cd-ninhydrin method C described by Doi,Shibata, and Matoba (1981) was used to estimate the free aminoacids concentration (FAAN). To compare data obtained by theKjeldahl method, values measured as equivalent mg of leucinewere transformed into mg nitrogen considering the relativeamount of N in the leucine molecule. Total phosphorus content ofmineralised samples was determined according to the AFNOR(Association Française de Normalisation) standard ISO 9874:1992(F). Inorganic phosphorus was determined by the Briggs methods(1922). Samples were adjusted to pH 2 with 1 mol L�1 HCl, beforedetermination of inorganic phosphorus (Pi). Organic phosphorus(Po) was calculated as the difference between total phosphorus andinorganic phosphorus.

2.6. Selective preparation of caseinophosphopeptides (CPPs)

Phosphopeptides were isolated from control and in vitrodigested Beaufort water-soluble fraction by a selective procedureinvolving precipitation with 50% (v/v) ethanol with the presence of0.1 mol L�1 CaCl2, as described by Reynolds, Riley, and Adamson(2004). After centrifugation (12,000 � g, 10 min), pellets contain-ing aggregates of phosphopeptides were dissolved in 20 mL ofwater and calcium chloride was removed using Chelex 100 beads(Biorad, U.S.A.). Samples were then dialysed with 500 Da dialysismembranes (Spectra/Por, Spectrum labs inc., U.S.A.) against waterto remove salts and then freeze-dried.

2.7. CPP analysis by gel filtration chromatography

To quantify CPPs in the samples, lyophilised fractions containingCPPs were dissolved in water, and a semi-quantitative gel-filtrationchromatographic procedure was used, as previously described inDupas et al. (2009).

2.8. Identification of peptides by liquid chromatographyeelectrospray ionisation tandem mass spectrometry

Peptides selectively precipitated from control and in vitrodigested samples were identified by reversed-phase nano-HPLC-MS/MS, as described by Dupas et al. (2009), with slight modifica-tions: after lyophilisation, peptides were dissolved into 0.1% (v/v)TFA and only 5 mL (i.e., 0.2, 1, and 5 mg of each sample) were injectedinto the system; peptides were then identified using the MascotMS/MS ion search V.2.2. from Matrix Science (Boston, MA, USA),using “STLO” data banks.

3. Results and discussion

Studies regarding CPPs released through the hydrolysis of casein(Adamson & Reynolds, 1997; Schmelzer et al., 2007; Su, Qi, He,Yuan, & Zhang, 2007), sodium caseinate (Ellegard et al., 1999) orcasein micelles (Chabance et al., 1998; Gagnaire, Pierre, Mollé, &Léonil, 1996; Ono, Takagi, & Kunishi, 1998) by digestive enzymeshave been performed. However, to our knowledge, no study dealing

I. Adt et al. / International Dairy Journal 21 (2011) 129e134 131

with release of CPPs following hydrolysis of cheese curd has beenachieved to date.

3.1. Recovery of CPPs from non-digested and digestedBeaufort cheese

Total nitrogen (TN), total phosphorus and inorganic phosphorus(and so organic phosphorus (Po)) contents of digested and non-digested Beaufort cheese homogenates and of correspondingselectively precipitated peptides were determined. This allowedcalculation of the Po/TN ratio to estimate purification of CPPs. Thisratio was 0.04 � 0.01 and 0.03 � 0.01 mol organic bound phos-phorus per mol of nitrogen in control and digested Beaufort cheesehomogenates, respectively. After subsequent ultrafiltration toremove intact soluble caseins and large fragments, and selectiveprecipitation of CPPs, this ratio increased 6.75-fold and 10-fold forcontrol and digested Beaufort cheese, respectively, indicating theselectivity of the purification procedure. This selectivity of theprecipitation procedure for recovery of CPPs was also demonstratedthrough the results of CPP identification since 72 of the 74 peptidesidentified were CPPs (Table 1).

3.2. Estimation of CPP content of non-digested anddigested Beaufort cheese

Since the smallest identified peptide presenting the phospho-serine cluster Ser(P)-Ser(P)-Ser(P)-Glu-Glu of functional CPPs hasamolecular mass of 600 Da, selectively precipitatedmolecules witha molecular mass >600 Da were considered as functional CPPs. Onthis basis, the estimated CPP content of the Beaufort cheese samplebefore and after its enzymatic hydrolysis by digestive proteolyticenzymes were 230 and 275 mg per 100 g of fresh cheese, respec-tively. These CPPs concentrations correspond to 8.8 and 10mg per gof protein for Beaufort cheese and its hydrolysate, respectively. TheCPP content of Beaufort cheese reported here is thus significantlylower than that reported by Dupas et al. (2009) (1.9 g per 100 g ofcheese): this difference likely results from the selectivity differenceof the respective CPPs purification procedures used. Dupas et al.(2009) purified CPPs by immobilized metal (FeIII) affinity chro-matography; following this procedure, 37 of 48 CPPs identifiedwere monophosphorylated, while only 6 of 72 were mono-phosphorylated in the present study (Table 1). The fact that most ofpurified CPPs were polyphosphorylated is consistent with thew0.3 mol of organic bound phosphorus per mol of nitrogen of theprecipitated peptides. This highlights the necessity of taking intoaccount the selectivity of the purification procedure for CPPs whenestimating the CPP content of dairy products.

3.3. Effect of the successive action of pepsin and pancreatinon the nitrogenous fractions of Beaufort cheese

The impact of in vitro enzymatic digestion on the profiles of theproteins and peptides present in Beaufort cheese was assessed by

Table 1Number of mono-, di- and poly-phosphorylated peptides identified from Beaufortcheese homogenates before and after in vitro digestion by pepsin and pancreatin,using nano-RP-LCeESI-MS/MS.

Peptides Beaufortcheese

Digested Beaufortcheese

Total of identified peptides 74 177Phosphorylated peptides 72 79Mono-phosphorylated peptides 6 35Di-phosphorylated peptides 27 21Poly-phosphorylated peptides 39 23

nitrogen analysis using the Kjeldahl method and RP-HPLC analysis(Fig. 1). The trichloroacetic-acid-soluble nitrogen/total nitrogen(TCA-SN/TN) ratio increased from 17.0 � 0.5% to 40.0 � 1.3%following the successive action of pepsin and pancreatin mimickingthe gastric and the duodenal phases of digestion, respectively. Theenzymatic hydrolysis of casein under the same conditions wasreported by Parrot et al. (2003) to increase TCA-SN/TN from 5% to32%. The 12% TCA-SN corresponds to casein fragments of less than3.5 kDa (Rohm et al., 1996); increasing TCA-SN/TN ratio thusmainlyresults from hydrolysis of intact casein or large casein fragmentsinto smaller fragments (<3.5 kDa) by pepsin and pancreatin.

Peptides were assessed by RP-HPLC analysis of water-solubleextracts of digested and non-digested (control) Beaufort cheese(Fig. 1). The peaks eluted during the first 10 min of the analysis areclassically attributed to hydrophilic, or small and hydrophilicpeptides. As many peaks are eluted in this part of the chromato-gram, this confirms that the soluble nitrogen fraction of BeaufortWSE is mainly due to small peptides, as previously stated. Threemajor peaks were then identified by scans and retention time ofstandards in the 20e50 min interval, as the aromatic amino acidstyrosine (Tyr), phenylalanine (Phe) and tryptophan (Trp). No peakswith a significant area were eluted after 50 min, in the elution zoneof large and/or hydrophobic peptides. This is a likely consequence ofthe intense secondary proteolysis preventing the accumulation oflarge peptides resulting from primary proteolysis in mature Beau-fort cheese curd. Digestive hydrolysis of Beaufort cheese modifiedits peptide profile (Fig. 2b). The number of peaks eluting between25 min and 80 min increased markedly. These new peaks after50 min probably result from the liberation of large and/or hydro-phobic peptides from large casein fragments by endoproteases such

Fig. 1. Effect of in vitro hydrolysis by digestive enzymes on RP-HPLC peptide patternsof Beaufort cheese: (a) water-soluble fraction of Beaufort cheese homogenate (control),(b) water-soluble fraction of Beaufort cheese homogenate after 30 min digestion bypepsin followed by 4 h digestion by pancreatin. mUA; milliunits of absorbance at215 nm.

Fig. 2. Primary sequences of caseinophosphopeptides in Beaufort samples, in vitrodigested or not, and identified using LC/ESI/MS: (a) from as1-casein; (b) from as2-casein;(c) from b-casein. Some sequences correspond to peptides with different molecularmasses due to differences in phosphorylation. Solid black line, present in controlsamples; dotted line, present in digested samples; Solid grey line, present in bothsample. Y indicates some enzymatic cleavage sites close to sequences of interest; ?indicates sites that may be phosphorylated; T: trypsin, C: chymotrypsin, P: pepsin; Pl:plasmin.

I. Adt et al. / International Dairy Journal 21 (2011) 129e134132

as pepsin, trypsin or a-chymotrypsin. This is consistent with theincrease from 17% to 40% in TCA-SN/TN. Tryptophan, and to a lesserextent, phenylalanine, and tyrosine concentrations in the water-soluble fraction increased following the successive action of pepsinand pancreatin. A similar trendwas observed following the action ofpancreatin on Emmental cheese (Parrot et al., 2003). These authorsproposed that activity of a-chymotrypsin leads to release ofpeptideswith an aromatic amino acid at their C-terminus. Then, theincrease of the concentrations of the free amino acids Trp, Phe, andTyr observed on chromatograms would result from the action ofcarboxypeptidase A on the C-terminus of these peptides (Lee &Yamamoto, 1990).

A slight decrease from 65 � 5% to 60 � 5% of the ratio of organicphosphorus to total phosphorus in the cheese homogenate was

observed following incubation for 30 min at pH 2 and for 4 h at pH7.5 with or without (control, data not shown) gastro-intestinalproteases. This may be due to dephosphorylation of organicallybound phosphate groups by phosphatases present in Beaufortcheese curd during the 30 min incubation at pH 2 and/or the 4 hincubation at pH 7.5. However, RP-HPLC chromatograms of thecontrol incubated without digestive enzymes were identical tothose of non-digested Beaufort cheese (data not shown). Moreover,no change in the NPN/TN ratio was observed; taken together, thesedata indicate that the action of proteolytic enzymes present inBeaufort cheese could be neglected during the 30min incubation atpH 2 and the 4 h incubation at pH 7.5. Proteolysis can thus beunambiguously attributed to the successive actions of pepsin andpancreatin (no significant differences were observed before andafter water-soluble extraction).

3.4. Identification of Beaufort cheese CPPs before andafter in vitro digestion

To gain further insight into the nature of the peptides purifiedfrom Beaufort cheese by selective precipitation in the presence ofcalcium, freeze-dried CPP preparations were analysed using LCeESI-MS/MS. To assess the effect of digestive enzymes on formation and/orbreakdown of CCPs in Beaufort cheese, the degrees of phosphoryla-tion and the origins (parent proteins) of the CPPs identified beforeand after in vitro digestion were compared.

Most of CPPs isolated from Beaufort cheese were poly- (i.e.,bearing 3 or 4 phosphorylated sites) ordi-phosphorylated (39 and 27,respectively) (Table1). Interestingly, thephosphoserineclusterwhichhas mineral-binding sites is contained in 28 CPPs (Fig. 2), makingthem potential candidates for calcium absorption (Ferraretto,Gravaghi, Fiorilli, & Tettamanti, 2003). However, some identifiedCPPs in Beaufort cheese were partially dephosphorylated and con-tained only two to three of the four native phosphate groups (forexample: b-CN f 7e28 2P and 3P, b-CN f 11e28 2P, b-CN 12e25 2P, b-CN 13e27 2P and 3P and b-CN 14e25 2P and 3P). Dephosphorylationof CPPs corresponding to as1- and b-CN fragmentswere also reportedby Addeo et al. (1992) and Roudot-Algaron, Le Bars, Kerhoas, Einhorn,and Gripon (1994) in ParmigianoeReggiano cheese and Comtécheese, respectively. This suggests thatphosphatase is active inSwiss-type cheese. Two indigenous phosphatases are present in milk:alkaline and acid phosphatases. Due to its acidic optimal pH (w4),milk acid phosphatase has been suggested to hydrolyse phosphategroupsbound to serine residues (Akuzawa&Fox,2004). Furthermore,an acid phosphatase activity was detected in lactic acid bacteria(Larsen & Parada, 1988) and could also be involved in dephosphory-lation of CPPs. After digestion of Beaufort cheese, 79 out of the 177peptides identified were caseinophosphopeptides. Among these 79CPPs, 35, 21 and 23 were mono-, di- and poly-phosphorylated,respectively (Table 1). The numberof sites of phosphorylation of CPPsfound in non-digested and digested samples differed greatly: (i)mostof the new CPPs identified following successive action of pepsin andpancreatin were monophosphorylated; (ii) while 39 poly-phosphorylated peptides of 72 phosphopeptides (54%) were identi-fied in the non-digested sample, only 23 polyphosphorylatedpeptides of 79 CPPs (29%) were identified in the digested sample.Interestingly, 17 out of 23 polyphosphorylated CPPs identified fromdigested Beaufort cheese still contained the characteristic clustersequence Ser(P)-Ser(P)-Ser(P)-Glu-Glu.

With respect to the origin of the CPPs identified in Beaufortcheese, 1, 13 and 58 CPPs arose from as1-, as2- and b-casein, respec-tively (Table 2). Bovine caseins are composed of as1-, as2-, b- and k-casein in a ratio of 3:0.8:3:1 (Swaisgood, 1992), and as1-, as2-, b- andk-casein contain 8e9, 10e13, 5 and 1 phosphoseryl residues,respectively. Unsurprisingly no CPPs arose from k-casein because the

Table 2Number of phosphorylated peptides generated from the different caseins andidentified from Beaufort cheese homogenates before and after in vitro digestion bypepsin and pancreatin using nano-RP-LCeESI-MS/MS.

Protein Beaufortcheese

DigestedBeaufortcheese

Commonpeptides

Peptides generatedby the action ofenzymatichydrolysis

as1-casein 1 17 0 17as2-casein 13 23 3 20b-casein 58 39 16 23

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k-CN f 106e169 fragment (glycomacropeptide) including the onlyphosphoseryl residue, Ser149, is eliminated in whey during drainingand pressing of Beaufort cheese. CPPs originated from b-CN (58 of 72(Table 2), i.e., 80%) were over-represented in Beaufort cheese. Thisresults from manufacture conditions of Beaufort cheese that inducea high plasmin and a low chymosin activity (Grappin, Beuvier,Bouton, & Pochet, 1999). The CPPs originated from b-CN covered allphosphorylation sites of b-CN, while CPPs containing either phos-phoserines Ser64, Ser66, Ser67, Ser68, Ser75 and Ser115 from as1-CN orSer31, Ser56, Ser57, Ser58 and Ser61 from as2-CN were not identified(Fig. 2). Similarly, although theas1-CN sequence f 60e80 containsfiveclose phosphorylated residues, no fragment generated from thisregion was identified within this study. However, two fragments ofthis region (as1-CN f 67e74 (2P) and as1-CN f 75e83 (1P)) have beenidentified following purification of CPPs from the same Beaufortcheese sample by immobilized metal (FeIII) affinity chromatography(IMAC) instead of selective precipitation in presence of calcium(Dupas et al., 2009). This difference results from differences inselectivity between these two techniques for CPP purification: moremono-phosphorylated peptides (41 compared with 52) and less di-and poly-phosphorylated peptides (11 compared with 52) wereidentified following IMAC purification.

Seventeen, 23 and 39 CPPs identified in digested Beaufortcheese arose from as1-, as2- and b-CN, respectively (Table 2).Nineteen of these peptides (3 as2-CN and 16 b-CN fragments) werealso isolated from the non-digested Beaufort cheese sample. Asa consequence, 60 new CPPs were generated under pepsin andpancreatin successive action, of which 17, 20, and 23 originatedfrom as1-, as2- and b-CN, respectively. Since CPPs identified fromBeaufort cheese covered all phosphorylation sites of b-CN, no newphosphoserine residue was present in their sequences and 16 of 39CPPs identified following enzymatic hydrolysis were also identifiedin non-digested Beaufort cheese. Twelve from these 16 peptideswere generated from the f 1e28 region of b-CN, which contains 4phosphoserine residues, while only 4 of the 23 new CPPs identifiedwere fragments from this N-terminal region. Most of the new CPPsidentified (14 of 23) originated from the f 26e56 region of b-CN,including only one phosphoserine residue (Ser35). Consideringpeptides sequences, only one CPP originating from as1-CN (as1-CN f39e52) was identified before action of digestive enzymes (Fig. 2a).From the 17 new as1-casein derived CPPs identified following theiraction, five diphosphorylated peptides were originating from as1-CN f 40e60, while 12 mono-phosphorylated peptides were origi-nating from as1-CN f 103e124, which includes one phosphoserylresidue (Ser115). However, as was found before action of digestiveenzymes, no fragment of the polyphosphorylated region of as1-casein containing the phosphoseryl cluster (as1-CN f 60e70) wasidentified. With respect to as2-casein, 12 out of 13 CPPs containedfour phosphoserine residues before enzymatic hydrolysis and wereoriginated from the N-terminal region of as2-CN f 3e21 (exceptionwas the as2-CN f 123e136mono-phosphorylated peptide), and onlyone triphosphorylated peptide, as2-CN f 123e136 (3P), was iden-tified. After in vitro digestion, 5 CPPs originating from this region

and 18 CPPs from the as2-CN f 123e150 region were identified.Eight of the 13 new CPPs identified contained the phosphoserineresidue (Ser143) in their sequence. As for the as1-CN f 60e80sequence, no CPPs arose from the f 50e70 region of as2 CN con-taining a phosphoseryl cluster, either before or after hydrolysis ofBeaufort cheese by pepsin and pancreatin.

The pattern of CPPs identified from Beaufort cheese (Table 2 andFig. 2) strongly suggests that most of them were generated byplasmin (or a bacterial protease with a similar substrate specificity)and subsequently by the action of other endopeptidases orexopeptidases from starter bacteria, as suggested for Emmentalcheese (Gagnaire, Mollé, Herrouin, & Léonil, 2001).

Twenty-six out of the 40 CPPs identified from Beaufort cheeseshown in Fig. 2 had lysine at their C-terminal end, indicating thatthey were likely released by plasmin. The CPPs arising from b-CNoften differed from each other by one or two N-terminal orC-terminal residues; these differences might have resulted fromthe action of bacterial aminopeptidases and possibly carboxypep-tidases or dipeptidyl peptidases, as suggested by Addeo et al.(1992). The CPPs identified after successive hydrolysis by pepsinand pancreatin contained, at their C-terminus, Lys (30), Arg (13),Asn (7), Glu (6), Phe (5), Gln (5), Met (4), Leu (3), Asp (3), Thr (2),and His (1) (Fig. 2b). About half of C-terminal residues of theidentified peptides were either Lys or Arg. This kind of cleavage isnot very specific, as it may arise either from the tryptic activity ofpancreatin during digestion, or from plasmin action during cheeseripening. The occurrence of Glu, Leu and Asp can be attributed tothe action of chymotrypsin. However, this enzyme is classicallyknown to be more specific for aromatic amino acids (Phe, Trp, Tyr)than of these residues. Pepsin is also known to have a similar actionon Phe. As a consequence, the presence of Phe as C-terminal resi-dues in the present study might be attributed either to chymo-tryptic activity of pancreatin, or to pepsin activity. Finally,carboxypeptidase A activity has been suggested to be responsiblefor the cleavage of Thr on its C-terminus, since these residues arelocated penultimate to bonds cleaved by trypsin or chymotrypsin(Adamson & Reynolds, 1995). In conclusion, most of the C-terminalresidues from CPPs isolated from digested Beaufort cheese prob-ably result from the activity of trypsin and chymotrypsin, and toa lesser extent from the action of pepsin and carboxypeptidase A.

The absence of CPPs from the region of as2-CN containing phos-phoseryl cluster in digested cheese might also result from theirextensive hydrolysis as shown using pure as2 casein hydrolysed bypancreatin (Su et al., 2007) or their non-hydrolysis because of theirnon accessibility within cheese matrix. The structure of caseins inBeaufort cheese following enzymatic coagulation, acidification (topH w 5.5), cheese pressing and ripening is modified and availabledata regarding their structure is too limited to predict accessibility ofdigestive enzymes to the different caseins involved in this structure.However this question of the accessibility of digestive enzymes tothe different caseins only concerns the as2-CN f 55e65 (4P) frag-ment, since as1-CN f 60e70 (4P) fragments were identified fromBeaufort cheese in another study (Dupas et al., 2009) and fromItalian hard-type cheeses (Addeo et al., 1992; Ferranti et al., 1997).

Nevertheless, the question of bioavaiblility of CPPs after diges-tion is still unclear, since enzymes such as those from intestinebrush border membrane could hydrolyse CPPs into inactive frag-ments (Boutrou., Coirre, Jardin, & Léonil, 2010); further work is nowneeded in order to estimate their bioavailability and their potentialinterest in different nutritional situations.

4. Conclusions

Thiswork allowed a semi-quantitative and qualitative assessmentof in vitro two-step hydrolysis by pepsin and pancreatin on Beaufort

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cheese CPPs for the first time. The quantity of CPPs increased w1.2fold. Seventy-four peptides, of which 72 were CPPs, were identifiedby LCeESI-MS/MS from Beaufort cheese, and 177, of which 79 wereCPPs, from its enzymatic digest. Most of the CPPs generatedthroughout in vitro digestion were monophosphorylated. However,17 out of 23 polyphosphorylated CPPs identified from digestedBeaufort cheese still contained the characteristic cluster sequence Ser(P)-Ser(P)-Ser(P)-Glu-Glu with its mineral-binding sites.

Acknowledgements

The authors would like to thank Région Rhône-Alpes, ConseilGénéral de l’Ain and Communauté d’Agglomération de Bourg enBresse for their financial support, Samia Dhahri and Roselyne Rouxfor their precious technical contribution.

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