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Identification of casein peptides in plasma of subjects after a cheese-enriched diet
Food Research International 84 (2016) 108–112
Contents lists available at ScienceDirect
Food Research International journal homepage: www.elsevier.com/locate/foodres
Identification of casein peptides in plasma of subjects after a cheese-enriched diet Simonetta Caira a,⁎,1, Gabriella Pinto a,1, Paola Vitaglione b, Fabrizio Dal Piaz c, Pasquale Ferranti a,b, Francesco Addeo b a b c
Institute of Food Science, CNR, Via Roma 52, 83100 Avellino, Italy Department of Agricultural sciences, University of Naples “Federico II”, via Università 100, Parco Gussone, 80055 Portici, Italy Department of BioMedical and Pharmaceutical Sciences, Università degli Studi di Salerno, Fisciano, Campania, Italy
a r t i c l e
i n f o
Article history: Received 27 October 2015 Received in revised form 10 March 2016 Accepted 13 March 2016 Available online 15 March 2016 Keywords: Human blood Cheese phosphopeptides Hydroxyapatite chromatography Tandem mass spectrometry
a b s t r a c t Despite several studies support the functionality of casein peptides in vitro, a gap of knowledge still exist about their bioavailability. In this pilot study the bioavailability of phosphopeptides (CPPs) was investigated in four healthy subjects who consumed for one week 100 g/day of Parmigiano Reggiano cheese after one week of a dairy products-free diet. CPPs were detected in plasma samples from fasting subjects after the cheese-enriched diet and peptides were detected variously in almost all the 4 samples. Some αs1- and αs2-CN-derived CPPs, such as αs1-CN (f43–52 and f43–50) and αs2-CN (f8–12), (f7–12) and (f6–12) as well as four non-phosphorylated peptides belonging to the C-terminal end of β-CN (f193–209, f194–209, f200–209) were detected in plasma samples submitted to extraction and enrichment by hydroxyapatite (HA) chromatography followed by MALDI-TOF and nano LC-ESI/ MS/MS analysis. Data indicated that casein oligopeptides are bioavailable after a continued intake of cheese. Future studies are warranted to ascertain this finding on a wider population and to clarify the mechanisms behind CPPs bioaccessibility and absorption. © 2016 Elsevier Ltd. All rights reserved.
1. Introduction Almost 2000 records including the term “bioactive peptides” are present in PubMed topics since the 1970 and they mainly refer to articles published over the last 10 years. The elevated number of bioactive molecules released during the in vitro or in vivo digestion of dietary proteins by action of proteases has encouraged the isolation of bioactive molecules encrypted in different matrices. Several animal and plant proteins are sources of bioactive peptides (Udenigwe & Aluko, 2012), and can display beneficial effects on human health (Chakrabarti & Jahandideh, 2014; Erdmann, Cheung, & Schröder, 2008; Hernández-Ledesma, García-Nebot, Fernández-Tomé, Amigo, & Recio, 2014). In this context, casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) complexes were demonstrated to remineralize the tooth enamel subsurface lesions in situ and to be effective in the treatment of dental caries (Reynolds, 2014). In particular, CPPs are able to stabilize calcium and phosphate ions under neutral and alkaline conditions, forming metastable solutions that are supersaturated with respect to the basic calcium phosphate phases (Holt, ⁎ Corresponding author. E-mail address: [email protected] (S. Caira). 1 These authors contributed equally to this study.
http://dx.doi.org/10.1016/j.foodres.2016.03.023 0963-9969/© 2016 Elsevier Ltd. All rights reserved.
Wahlgren, & Drakenberg, 1996; Reynolds, 1998, 2007). Under these conditions, CPPs can bind calcium and phosphate making them bioavailable (Walsh, 2009). Although several studies support the functionality of bioactive peptides in vivo, the evidence of their bioavailability is still lacking in the scientific literature. The low abundance of peptides in bloodstream enormously limits their finding in plasma samples due to analytical issues. However, β-casein fragment (f134–138) and its shortened forms (f135–138) and (f134–137) were recently quantified in plasma samples of rats administered with 40 mg/kg body weight of the antihypertensive milk casein-derived pentapeptide HLPLP (Sánchez-Rivera, Ares, Miralles, Gómez-Ruiz, Recio, MartínezLarrañaga, Anadón and Martínez, 2014). Similarly, the lactotripeptide IPP and the dipeptide VY were demonstrated to be absorbed in humans following ingestion (Foltz, Meynen, Bianco, van Platerink, Koning and Kloek, 2007; Matsui, Tamaya, Seki, Osajima, Matsumoto and Kawasaki, 2002). Controversial opinions exist about resistance of CPPs to the gastro-intestinal enzymes, mainly phosphatases (Picariello, Mamone, Nitride, Addeo, & Ferranti, 2013) which would make questionable their effective passage in their intact forms into the bloodstream. In this pilot study, the hypothesis that soluble CPPs could be found in circulation of fasting subjects after consumption of a hard long ripened cheese-enriched diet for one-week was tested. A 30 month aged Parmigiano Reggiano (PR) cheese, containing ≈ 1% water-soluble
S. Caira et al. / Food Research International 84 (2016) 108–112
CPPs, was used for the experiments. By using mass spectrometric techniques, the presence of some CPPs and non-phosphorylated peptides in plasma samples of fasting subjects who ate the cheese every day for one week was confirmed.
2. Materials and methods 2.1. Materials PR cheese (30 month-ripened) was purchased from a local supermarket. Tris (hydroxymethyl) aminomethane hydrochloride (Tris–HCl), potassium chloride (KCl), urea, trifluoroacetic acid (TFA), acetonitrile (ACN) HPLC grade, 85% orthophosphoric acid (PA) and ethylenediaminetetraacetic acid (EDTA) were purchased from Carlo Erba (Milan, Italy). 2,5-dihydroxybenzoic acid (DHB) was obtained from Fluka (St. Louis, MO, USA), while hydroxyapatite (HA) (MacroPrep Ceramic Hydroxyapatite TYPE I) was purchased from Bio-Rad (Milan, Italy). All other reagents were of analytical grade and were used without further purification.
2.2. Bioavailability study It was a pilot study based on a two-week experimental protocol that was approved by the Ethic Committee of University of Naples. Subject selection was performed at the Department of Agricultural Sciences of University of Naples among people working in the research laboratories, having no symptom of gastrointestinal disorder or food allergies and being no pregnant or lactating. Four healthy volunteers, 2 males and 2 females, aged 24–49 years, with body weight ranging 55–92 kg participated in the study after signing an informed consent. Subjects were asked to avoid consumption of dairy products for one week and to eat 100 g/day of PR (that was provided by the experimenters) over the following week. After both weeks, fasting subjects were invited to reach at 9 am the nutritional laboratory of the Department and blood drawings were performed. The evenings before blood collection subjects were asked to eat the same dinner, avoiding alcohol, within the 22 pm. Blood was collected in 3 mL Vacutainer tubes containing EDTA that were immediately centrifuged at 4000 × g for 10 min at 4 °C. The obtained plasma samples were aliquoted and stored, within 20 min from collection, at −40 °C until analysis for the determination of CPPs. 2.3. Extraction of peptides from pH 4.6-soluble fractions of PR cheese The fractions of cheese soluble at pH 4.6 were prepared as previously described (Sforza, Aquino, Cavatorta, Galaverna, Mucchetti, Dossena and Marchelli, 2008). Briefly, 10 g of PR cheese were suspended in 45 mL of 0.1 N HCl and the suspension was homogenized for 1 min by an Ultra Turrax T50 (Janke & Kunkel IKA® Labortechnik, Staufen, Germany), adjusted to pH 4.6 with 1 M NaOH and then centrifuged at 4000 × g min-1 for 10 min at 4 °C. The thick fat layer formed on the surface was removed with the aid of spatula. The supernatant filtered through filter paper was adjusted to pH 7.0 with 1 M NaOH and freeze dried. Samples aliquots (1 mg) were dissolved in a solution of 50% ACN and 50% water containing 0.1% TFA (v/v).
2.4. Extraction of CPPs from plasma samples by HA-based enrichment Plasma samples (500 μL) were ultra-filtered with 10-kDa molecular weight cut-off membranes and the permeate was directly submitted to the CPP enrichment by in-batch hydroxyapatite (HA)-based chromatography (Pinto, Caira, Cuollo, Lilla, Fierro and F., 2010). CPPs immobilized on HA micro-granules were deposited onto a 96-well MALDI target for MS analysis.
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2.5. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis MALDI-TOF mass spectra were recorded using a Voyager DE PRO mass spectrometer (Applied Biosystems, Framingham, MA, USA). Mass spectra were acquired in positive linear mode due to the known instability of CPP in the reflector mode. This situation is amplified by the multiply phosphorylated peptides generating dephosphorylated peptide in the metastable decomposition (Annan & Carr, 1996; Schnölzer & Lehmann, 1997). In addition, the co-presence in high number of phosphorylated peptides would complicate the reflector spectrum. Instead, interpretation of the linear spectrum is straightforward, since it contains only the [M + H]+ ion for each species. The spectra were acquired in the range of 1–5 kDa with the following settings: an accelerating voltage of 20 kV and a grid voltage of 95% of the accelerating voltage, a guide wire of 0.05% and a delayed ion extraction time of 175 ns. The laser power was set just above the ion generation threshold to obtain peaks with the highest possible signal-to-noise ratio. All spectra were acquired with 200 shots in three replicates. Matrix solutions were freshly made each day from stocks of the required solvents and sonicated for 15 min in an ultrasonic water bath (Bransonic 220, Mazian Med Equip, Toronto, Canada) prior to use. The HAphosphopeptide microgranules (n = ∼1000) were deposited onto the MALDI plate, covered with 10 mg/mL of the DHB matrix solution in 50% ACN to promote analyte/matrix co-crystallization and air-dried at room temperature. For HA-CPP, 1% PA was included in the matrix. 2.6. NanoLC-ESI-MS/MS analysis Before nanoLC-ESI-MS/MS analysis, the HA-bound CPPs (10 mg) were solubilized by adding 120 μL of 5% aqueous PA solution. The CPP solution was analyzed by nanoLC-ESI-MS/MS using a Orbitrap XL instrument (Thermo Fisher) equipped by a nano-ESI source coupled with a nano-ACQUITY capillary UPLC (Waters): peptide separation was performed on a capillary BEH C18 column (0.075 mm × 100 mm, 1.7 μm, Waters) using aqueous 0.1% formic acid (A) and ACN containing 0.1% formic acid (B) as mobile phases. Peptides were eluted by means of a linear gradient from 5% to 50% of B in 45 min and a 300 nL/min flow rate. Mass spectra were acquired over m/z range from 400 to 1800; the ten most intense doubly-, triply- or quadruply-charged ions detected in each spectrum underwent CID fragmentation (dependent scan acquisition mode) and MS/MS spectra were acquired over a m/z range from 50 to 2000. MS and MS/MS data were used by Mascot (Matrix Science) to interrogate the Swiss-Prot protein database. Settings were as follows: taxonomy, other mammalia; enzyme, no cleave; mass accuracy window for parent ions, 10 ppm; mass accuracy window for fragment ions, 50 millimass units; no fixed modification; variable modifications, phosphorylation of serine, threonine and tyrosine, oxidation of methionine. 3. Results and discussion 3.1. MALDI-TOF analysis of hydroxyapatite-based enrichment of phosphopeptides from plasma samples The MALDI-TOF/MS profile of ultrafiltered plasma sample from the subject number four (A), the relative HA-enriched fraction before (B) and after one week of PR cheese-enriched diet (C) were shown in Fig. 1. The MALDI spectra of CPPs isolated by plasma samples from other participants are reported in the Supporting Information (Fig. S1, A,B,C). The characteristic peaks derived from fibrinogen α-chain phosphopeptides dominated MALDI spectra of the plasma samples before cheese intake. The mass signals at 1389.9, 1461.1, 1546.2 and 1616.6 Da corresponded to the fibrinopeptide A (FbA)-derived (f2– 15) P, (f1–15) P, (f2–16) P and (f1–16) P peptides respectively. These results confirmed the efficiency of HA to selectively capture the
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Fig. 1. MALDI-TOF/MS analysis of the ultrafiltrated (Cutoff 10 kDa) plasma sample of subject number four (A), the relative HA-enriched fraction before (B) and after the cheese-enriched diet (C). Peaks under brackets for A, B and C spectra grouped signals belonging to FbA. Note that all the spectra were recorded after covering the beds of HA-bound peptides spotted onto MALDI plate with DHB matrix (1% PA).
phosphopeptides in plasma samples without any specific sample pretreatment and the ability of MALDI-TOF to provide multiple CPP identification in a single mass spectrometric analysis. However, additional phosphopeptide signals with those still dominant of fibrinopeptide-
derived phosphopeptides (Fig. 1C) were detected in plasma samples from individual subjects after cheese intake. The mass signal at 777.4 Da was attributed to the Ser(P)-Ser(P)-Ser(P)-Glu-Glu cluster common to calcium sensitive αs1-, αs2- and β-CN. The ‘rugged end’
Fig. 2. MALDI-TOF/MS analysis of the pH 4.6 soluble fraction of PR cheese sample aged 30 months. Asterisks indicated putative precursor of the peptides identified in plasma samples shown in Table 1.
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signals (indicated with asterisks), corresponding to the parent αs2-CN (f6–12) 3P peptide and its N-terminally shortened CPPs, i.e. f7–12 and f8–12, fully (3P) and partially dephosphorylated (2P), indicated the co-presence of differently phosphorylated CPPs in plasma samples. Such peptides could be derived from the precursors, αs2-CN (f8–18) 3P and 4P (1435 and 1515 Da, respectively), identified in the sample of 30 hard long ripened Italian cheese used for the experiments (Fig. 2) and already detected in a previous study (Ferranti, Barone, Chianese, Addeo, Scaloni, Pellegrino and Resmini, 1997). 3.2. Structural characterization of peptide sequences detected by MS/MS analysis Plasma samples analyzed by MALDI-TOF/MS were also submitted to LC-ESI-MS/MS analysis to confirm the peptide identity established by MALDI-TOF. The use of two different ionization techniques together with the different sample treatment (see method section) did not provide identical results. In Table 1, the different sets of peptides identified using the two techniques were shown together with a partial list of CPPs identified in the PR cheese consumed by the subjects. NanoLC-ESI-MS/MS analysis allowed to unambiguously detect the mass signals corresponding to αs1-CN (f43–50) 2P (996.7 Da) and the αs1-CN (f43–52) 2P CPPs (1239.9 Da) in plasma samples of 3 out of 4 subjects after the consumption of PR cheese for one week (Table 1). It is likely that the retrieved peptides originated from the longer parent peptide αs1-CN (43–58) 2P (1927.8 Da) that was detected in the PR cheese consumed by the participants (Fig. 2). As an example, the complete MS/MS fragmentation pattern and the recorded signals for αs1-CN (f43–52) 2P CPPs (1239.9 Da) and β-CN (f193–209) (1881.26 Da) were reported in Fig. 3. MS/MS spectra of the peptides identified in the plasma samples of the four subjects are collected in the Supporting Informations (Fig. S2). Thus, non-phosphorylated peptides were also identified in the plasma samples collected after one-week consumption of cheese. They all originated from a common sequence from the β-CN C-terminal end, i.e. β-CN (f194–209) (1718.08 Da), and β-CN (f200–209) (1094.37 Da). This result may depend on two factors, the higher sensitivity of the LC-ESI-MS/MS with respect to MALDI-TOF and co-elution of very low amounts of non-phosphorylated peptides from the HA sorbent. This drawback was overcome by lowering the pH value of the wash buffers so that carboxyl groups were not ionized and non-phosphorylated peptides washed out (data not shown). However, the occurrence of non-phosphorylated β-CN peptides at different length in human plasma samples confirmed previous findings on the resistance of β-CN (f193–209) peptide to the hydrolysis by brush-border membrane peptidases, and its transport through the
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Caco-2 cell monolayer (Regazzo, Mollé, Gabai, Tomé, Dupont, Leonil and Boutrou, 2010; Sánchez-Rivera, Diezhandino, Gómez-Ruiz, Fresno, Miralles and Recio, 2014). The main route involved in trans-epithelial transport of the β-CN (f193–209) peptide was ascribed to the transcytosis via internalized vesicles, although the paracellular transport via tight-junctions could not be excluded. The in vivo transport of intact bioactive peptides in the intestinal epithelium is an important prerogative to prove their bioavailability (Regazzo et al., 2010). On the other hand, quantification of peptides in human plasma is necessary to transfer in humans the functional properties attributed to C-terminal peptides of β-CN by in vitro studies. Simply ingesting cheese, rather than separately casein and casein hydrolysates including CPPs, does not allow establishing the precise mechanism of the origin of the peptides found in the blood. They could be already present in the cheese or they could result from the gradual casein hydrolysis along all the gastro-intestinal tract including the gut microbiota metabolism. The ‘cluster sequence’ containing the motif Ser(P)-Ser(P)-Ser(P)-Glu-Glu, responsible for the ability to maintain calcium and phosphate ions in a soluble form (Gravaghi, Del Favero, Cantu', Donetti, Bedoni, Fiorilli, Tettamanti and Ferraretto, 2007) is common to αs1-, αs2- and β-CN. CPP-derived and non-phosphorylated peptides occurred in vivo, display different ability to elicit the [Ca2+] increase in the blood. The two classes of molecules act towards calcium aggregation in a completely different manner, with CPPs exhibiting the highest level of Ca2+ coordination with respect to non-phosphorylated. The presence of three phosphorylated serine and two glutamic acid residues in the cluster sequence accounts for such a strong negative charge that attractive interactions towards Ca+2 prevail on aggregation, thus maintaining the calcium, in a soluble form and absorbable state. The cluster sequence does not exhibit any aggregation in solution, regardless of the presence of Ca2+, as the intensity of the light scattered by CPPs is not dissimilar to that of pure solvent (Gravaghi et al., 2007). 4. Conclusions The present data constitute an evidence that specific casein peptides and CPPs are present in circulation in humans. For the first time in this pilot study, the bioavailability of the specific casein peptides was demonstrated in humans consuming 100 g of PR cheese (1 g CPPs), every day for one week. As expected, a large inter-individual variation was found since not all the subject plasma samples contained the same peptides and future study with a larger sample size is needed. However, these findings open also new research perspectives focused on investigation of physiological effects of peptides in vivo, such as calcium transport and delivery as well as antimicrobial or immunomodulating properties. These goals may be achieved by quantitation of peptides in
Table 1 CPPs identified in human plasma samples after MS-based studies and pH 4.6 soluble counterparts identified in the 30-month old PR cheese as shown in Fig. 2. Molecular mass (Da) Analytical technique
MALDI TOF/MS Human serum HA-CPPs
NanoLC-ESI/MSMS
PR cheese HA-CPPs from pH 4.6 soluble fraction a
MALDI TOF/MS
Theoretical
Measured
1013.7 876.2 796.6 777.4 697.4 1094.4 1240.0 1240.0 996.8 1881.3 1718.1 1094.4 1435.1 1515.1 1927.8
1013.7 877.0 797.1 778.4 698.5 1095.3 1240.5 1240.5 997.1 1881.5 1718.3 1095.3 1435.1 1515.2 1927.8
1,2,3,4 indicates the subject plasma sample where the cheese peptides were detected.
Peptide identification
Aminoacidic sequence (a)
αs2-CN (f6–12)3P αs2-CN (f7–12)3P αs2-CN (f7–12)2P αs2-CN (f8–12)3P αs2-CN (f8–12)2P β-CN (f200–209) αs1-CN (f43–52)2P αs1-CN (f43–52)2P αs1-CN (f43–50)2P β-CN (f193–209) β-CN (f194–209) β-CN (f200–209) αs2-CN (f8–18)3P αs2-CN (f8–18)4P αs1-CN (f43–58)2P
HVSSSEE (2,3) VSSSEE (1,3,4) VSSSEE (4) SSSEE (4) SSSEE (2,4) PVRGPFPIIV (1,2,3,4) DIGSESTEDQ (1,2,4) DIGSESTEDQ (1,3,4) DIGSESTE (4) YQEPVLGPVRGPFPIIV (1,3,4) QEPVLGPVRGPFPIIV (1,2,4) PVRGPFPIIV (1,2,3,4) SSSEESIISQE SSSEESIISQE DIGSESTEDQAMEDIK
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Fig. 3. MS/MS spectrum for confirmation of the identity of αs1-CN (f43–52) 2P and β-CN (f193–209) peptides found in plasma samples of subjects consuming a PR cheese-enriched diet for one week.
plasma, through MS-based-omic techniques and use of labeled peptides or peptide analogs. Using an appropriate analytical methodology, bioactivity of cheese peptides could be also investigated in vivo, thus possibly clarifying mechanisms behind food component bioavailability and food allergy and intolerance. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.foodres.2016.03.023. References Annan, R. S., & Carr, S. A. (1996). Phosphopeptide analysis by matrix-assisted laser desorption time-of-flight mass spectrometry. Analytical Chemistry, 68, 3413–3421. Chakrabarti, S., Jahandideh, F., & J., Wu (2014). Food-derived bioactive peptides on inflammation and oxidative stress. BioMed Research International, 608979. Erdmann, K., Cheung, B. W., & Schröder, H. (2008). The possible roles of food-derived bioactive peptides in reducing the risk of cardiovascular disease. The Journal of Nutritional Biochemistry, 19, 643–654. Ferranti, P., Barone, F., Chianese, L., Addeo, F., Scaloni, A., Pellegrino, L., & Resmini, P. (1997). Phosphopeptides from Grana Padano cheese: Nature, origin and changes during ripening. Journal of Dairy Research, 64, 601–615. Foltz, M., Meynen, E. E., Bianco, V., van Platerink, C., Koning, T. M., & Kloek, J. (2007). Angiotensin converting enzyme inhibitory peptides from a lactotripeptide-enriched milk beverage are absorbed intact into the circulation. Journal of Nutrition, 137, 953–958. Gravaghi, C., Del Favero, E., Cantu', L., Donetti, E., Bedoni, M., Fiorilli, A., ... Ferraretto, A. (2007). Casein phosphopeptide promotion of calcium uptake in HT-29 cells — Relationship between biological activity and supramolecular structure. FEBS Journal, 274, 4999–5011. Hernández-Ledesma, B., García-Nebot, M. J., Fernández-Tomé, S., Amigo, L., & Recio, I. (2014). Dairy protein hydrolysates: Peptides for health benefits. International Dairy Journal, 38, 82–100. Holt, C., Wahlgren, N. M., & Drakenberg, T. (1996). Ability of a beta-casein phosphopeptide to modulate the precipitation of calcium phosphate by forming amorphous dicalcium phosphate nanoclusters. Biochemistry Journal, 314, 1035–1039.
Matsui, T., Tamaya, K., Seki, E., Osajima, K., Matsumoto, K., & Kawasaki, T. (2002). Val-Tyr as a natural antihypertensive dipeptide can be absorbed into the human circulatory blood system. Clinical and Experimental Pharmacology and Physiology, 29, 204–208. Picariello, G., Mamone, G., Nitride, C., Addeo, F., & Ferranti, P. (2013). Protein digestomics: Integrated platforms to study food-protein digestion and derived functional and active peptides. TrAC Trends in Analytical Chemistry, 52, 120–134. Pinto, G., Caira, S., Cuollo, M., Lilla, S., Fierro, O., & F., Addeo (2010). Hydroxyapatite as a concentrating probe for phosphoproteomic analyses. Journal of Chromatography B, Analytical Technologies in the Biomedical and Life Sciences, 878, 2669–2678. Regazzo, D., Mollé, D., Gabai, G., Tomé, D., Dupont, D., Leonil, J., & Boutrou, R. (2010). The (193–209) 17-residues peptide of bovine β-casein is transported through Caco-2 monolayer”. Molecular Nutrition & Food Research, 54, 1428–1435. Reynolds E. (2007). Calcium phosphopeptide complexes. US7312193. Reynolds, E. C. (1998). Anticariogenic complexes of amorphous calcium phosphate stabilized by casein phosphopeptides: A review. Special Care in Dentistry, 18, 8–16. Reynolds, E. C. (2014). Dental mineralization, https://www.google.com.ar/patents/ US8673363, 2014/03/18/Google Patents Sánchez-Rivera, L., Ares, I., Miralles, B., Gómez-Ruiz, J.Á., Recio, I., Martínez-Larrañaga, M. R., ... Martínez, M. A. (2014b). Bioavailability and kinetics of the antihypertensive casein-derived peptide HLPLP in rats. Journal of Agricultural and Food Chemistry, 62, 11869–11875. Sánchez-Rivera, L., Diezhandino, I., Gómez-Ruiz, J.Á., Fresno, J. M., Miralles, B., & Recio, I. (2014a). Peptidomic study of Spanish blue cheese (Valdeón) and changes after simulated gastrointestinal digestion. Electrophoresis, 35, 1627–1636. Schnölzer, M., & Lehmann, W. D. (1997). Identification of modified peptides by metastable fragmentation in MALDI mass spectrometry. International Journal of Mass Spectrometry and Ion Processes, 169/170, 263–271. Sforza, S., Aquino, G., Cavatorta, V., Galaverna, G., Mucchetti, G., Dossena, A., & Marchelli, R. (2008). Proteolytic oligopeptides as molecular markers for the presence of cows' milk in fresh cheeses derived from sheep milk. International Dairy Journal, 18, 1072–1076. Udenigwe, C. C., & Aluko, R. E. (2012). Food protein-derived bioactive peptides: Production, processing, and potential health benefits. Journal of Food Science, 77, R11–R24. Walsh, L. J. (2009). Contemporary technologies for remineralization therapies: A review. International Dentistry SA, 11, 6–16.
Contents lists available at ScienceDirect
Food Research International journal homepage: www.elsevier.com/locate/foodres
Identification of casein peptides in plasma of subjects after a cheese-enriched diet Simonetta Caira a,⁎,1, Gabriella Pinto a,1, Paola Vitaglione b, Fabrizio Dal Piaz c, Pasquale Ferranti a,b, Francesco Addeo b a b c
Institute of Food Science, CNR, Via Roma 52, 83100 Avellino, Italy Department of Agricultural sciences, University of Naples “Federico II”, via Università 100, Parco Gussone, 80055 Portici, Italy Department of BioMedical and Pharmaceutical Sciences, Università degli Studi di Salerno, Fisciano, Campania, Italy
a r t i c l e
i n f o
Article history: Received 27 October 2015 Received in revised form 10 March 2016 Accepted 13 March 2016 Available online 15 March 2016 Keywords: Human blood Cheese phosphopeptides Hydroxyapatite chromatography Tandem mass spectrometry
a b s t r a c t Despite several studies support the functionality of casein peptides in vitro, a gap of knowledge still exist about their bioavailability. In this pilot study the bioavailability of phosphopeptides (CPPs) was investigated in four healthy subjects who consumed for one week 100 g/day of Parmigiano Reggiano cheese after one week of a dairy products-free diet. CPPs were detected in plasma samples from fasting subjects after the cheese-enriched diet and peptides were detected variously in almost all the 4 samples. Some αs1- and αs2-CN-derived CPPs, such as αs1-CN (f43–52 and f43–50) and αs2-CN (f8–12), (f7–12) and (f6–12) as well as four non-phosphorylated peptides belonging to the C-terminal end of β-CN (f193–209, f194–209, f200–209) were detected in plasma samples submitted to extraction and enrichment by hydroxyapatite (HA) chromatography followed by MALDI-TOF and nano LC-ESI/ MS/MS analysis. Data indicated that casein oligopeptides are bioavailable after a continued intake of cheese. Future studies are warranted to ascertain this finding on a wider population and to clarify the mechanisms behind CPPs bioaccessibility and absorption. © 2016 Elsevier Ltd. All rights reserved.
1. Introduction Almost 2000 records including the term “bioactive peptides” are present in PubMed topics since the 1970 and they mainly refer to articles published over the last 10 years. The elevated number of bioactive molecules released during the in vitro or in vivo digestion of dietary proteins by action of proteases has encouraged the isolation of bioactive molecules encrypted in different matrices. Several animal and plant proteins are sources of bioactive peptides (Udenigwe & Aluko, 2012), and can display beneficial effects on human health (Chakrabarti & Jahandideh, 2014; Erdmann, Cheung, & Schröder, 2008; Hernández-Ledesma, García-Nebot, Fernández-Tomé, Amigo, & Recio, 2014). In this context, casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) complexes were demonstrated to remineralize the tooth enamel subsurface lesions in situ and to be effective in the treatment of dental caries (Reynolds, 2014). In particular, CPPs are able to stabilize calcium and phosphate ions under neutral and alkaline conditions, forming metastable solutions that are supersaturated with respect to the basic calcium phosphate phases (Holt, ⁎ Corresponding author. E-mail address: [email protected] (S. Caira). 1 These authors contributed equally to this study.
http://dx.doi.org/10.1016/j.foodres.2016.03.023 0963-9969/© 2016 Elsevier Ltd. All rights reserved.
Wahlgren, & Drakenberg, 1996; Reynolds, 1998, 2007). Under these conditions, CPPs can bind calcium and phosphate making them bioavailable (Walsh, 2009). Although several studies support the functionality of bioactive peptides in vivo, the evidence of their bioavailability is still lacking in the scientific literature. The low abundance of peptides in bloodstream enormously limits their finding in plasma samples due to analytical issues. However, β-casein fragment (f134–138) and its shortened forms (f135–138) and (f134–137) were recently quantified in plasma samples of rats administered with 40 mg/kg body weight of the antihypertensive milk casein-derived pentapeptide HLPLP (Sánchez-Rivera, Ares, Miralles, Gómez-Ruiz, Recio, MartínezLarrañaga, Anadón and Martínez, 2014). Similarly, the lactotripeptide IPP and the dipeptide VY were demonstrated to be absorbed in humans following ingestion (Foltz, Meynen, Bianco, van Platerink, Koning and Kloek, 2007; Matsui, Tamaya, Seki, Osajima, Matsumoto and Kawasaki, 2002). Controversial opinions exist about resistance of CPPs to the gastro-intestinal enzymes, mainly phosphatases (Picariello, Mamone, Nitride, Addeo, & Ferranti, 2013) which would make questionable their effective passage in their intact forms into the bloodstream. In this pilot study, the hypothesis that soluble CPPs could be found in circulation of fasting subjects after consumption of a hard long ripened cheese-enriched diet for one-week was tested. A 30 month aged Parmigiano Reggiano (PR) cheese, containing ≈ 1% water-soluble
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CPPs, was used for the experiments. By using mass spectrometric techniques, the presence of some CPPs and non-phosphorylated peptides in plasma samples of fasting subjects who ate the cheese every day for one week was confirmed.
2. Materials and methods 2.1. Materials PR cheese (30 month-ripened) was purchased from a local supermarket. Tris (hydroxymethyl) aminomethane hydrochloride (Tris–HCl), potassium chloride (KCl), urea, trifluoroacetic acid (TFA), acetonitrile (ACN) HPLC grade, 85% orthophosphoric acid (PA) and ethylenediaminetetraacetic acid (EDTA) were purchased from Carlo Erba (Milan, Italy). 2,5-dihydroxybenzoic acid (DHB) was obtained from Fluka (St. Louis, MO, USA), while hydroxyapatite (HA) (MacroPrep Ceramic Hydroxyapatite TYPE I) was purchased from Bio-Rad (Milan, Italy). All other reagents were of analytical grade and were used without further purification.
2.2. Bioavailability study It was a pilot study based on a two-week experimental protocol that was approved by the Ethic Committee of University of Naples. Subject selection was performed at the Department of Agricultural Sciences of University of Naples among people working in the research laboratories, having no symptom of gastrointestinal disorder or food allergies and being no pregnant or lactating. Four healthy volunteers, 2 males and 2 females, aged 24–49 years, with body weight ranging 55–92 kg participated in the study after signing an informed consent. Subjects were asked to avoid consumption of dairy products for one week and to eat 100 g/day of PR (that was provided by the experimenters) over the following week. After both weeks, fasting subjects were invited to reach at 9 am the nutritional laboratory of the Department and blood drawings were performed. The evenings before blood collection subjects were asked to eat the same dinner, avoiding alcohol, within the 22 pm. Blood was collected in 3 mL Vacutainer tubes containing EDTA that were immediately centrifuged at 4000 × g for 10 min at 4 °C. The obtained plasma samples were aliquoted and stored, within 20 min from collection, at −40 °C until analysis for the determination of CPPs. 2.3. Extraction of peptides from pH 4.6-soluble fractions of PR cheese The fractions of cheese soluble at pH 4.6 were prepared as previously described (Sforza, Aquino, Cavatorta, Galaverna, Mucchetti, Dossena and Marchelli, 2008). Briefly, 10 g of PR cheese were suspended in 45 mL of 0.1 N HCl and the suspension was homogenized for 1 min by an Ultra Turrax T50 (Janke & Kunkel IKA® Labortechnik, Staufen, Germany), adjusted to pH 4.6 with 1 M NaOH and then centrifuged at 4000 × g min-1 for 10 min at 4 °C. The thick fat layer formed on the surface was removed with the aid of spatula. The supernatant filtered through filter paper was adjusted to pH 7.0 with 1 M NaOH and freeze dried. Samples aliquots (1 mg) were dissolved in a solution of 50% ACN and 50% water containing 0.1% TFA (v/v).
2.4. Extraction of CPPs from plasma samples by HA-based enrichment Plasma samples (500 μL) were ultra-filtered with 10-kDa molecular weight cut-off membranes and the permeate was directly submitted to the CPP enrichment by in-batch hydroxyapatite (HA)-based chromatography (Pinto, Caira, Cuollo, Lilla, Fierro and F., 2010). CPPs immobilized on HA micro-granules were deposited onto a 96-well MALDI target for MS analysis.
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2.5. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis MALDI-TOF mass spectra were recorded using a Voyager DE PRO mass spectrometer (Applied Biosystems, Framingham, MA, USA). Mass spectra were acquired in positive linear mode due to the known instability of CPP in the reflector mode. This situation is amplified by the multiply phosphorylated peptides generating dephosphorylated peptide in the metastable decomposition (Annan & Carr, 1996; Schnölzer & Lehmann, 1997). In addition, the co-presence in high number of phosphorylated peptides would complicate the reflector spectrum. Instead, interpretation of the linear spectrum is straightforward, since it contains only the [M + H]+ ion for each species. The spectra were acquired in the range of 1–5 kDa with the following settings: an accelerating voltage of 20 kV and a grid voltage of 95% of the accelerating voltage, a guide wire of 0.05% and a delayed ion extraction time of 175 ns. The laser power was set just above the ion generation threshold to obtain peaks with the highest possible signal-to-noise ratio. All spectra were acquired with 200 shots in three replicates. Matrix solutions were freshly made each day from stocks of the required solvents and sonicated for 15 min in an ultrasonic water bath (Bransonic 220, Mazian Med Equip, Toronto, Canada) prior to use. The HAphosphopeptide microgranules (n = ∼1000) were deposited onto the MALDI plate, covered with 10 mg/mL of the DHB matrix solution in 50% ACN to promote analyte/matrix co-crystallization and air-dried at room temperature. For HA-CPP, 1% PA was included in the matrix. 2.6. NanoLC-ESI-MS/MS analysis Before nanoLC-ESI-MS/MS analysis, the HA-bound CPPs (10 mg) were solubilized by adding 120 μL of 5% aqueous PA solution. The CPP solution was analyzed by nanoLC-ESI-MS/MS using a Orbitrap XL instrument (Thermo Fisher) equipped by a nano-ESI source coupled with a nano-ACQUITY capillary UPLC (Waters): peptide separation was performed on a capillary BEH C18 column (0.075 mm × 100 mm, 1.7 μm, Waters) using aqueous 0.1% formic acid (A) and ACN containing 0.1% formic acid (B) as mobile phases. Peptides were eluted by means of a linear gradient from 5% to 50% of B in 45 min and a 300 nL/min flow rate. Mass spectra were acquired over m/z range from 400 to 1800; the ten most intense doubly-, triply- or quadruply-charged ions detected in each spectrum underwent CID fragmentation (dependent scan acquisition mode) and MS/MS spectra were acquired over a m/z range from 50 to 2000. MS and MS/MS data were used by Mascot (Matrix Science) to interrogate the Swiss-Prot protein database. Settings were as follows: taxonomy, other mammalia; enzyme, no cleave; mass accuracy window for parent ions, 10 ppm; mass accuracy window for fragment ions, 50 millimass units; no fixed modification; variable modifications, phosphorylation of serine, threonine and tyrosine, oxidation of methionine. 3. Results and discussion 3.1. MALDI-TOF analysis of hydroxyapatite-based enrichment of phosphopeptides from plasma samples The MALDI-TOF/MS profile of ultrafiltered plasma sample from the subject number four (A), the relative HA-enriched fraction before (B) and after one week of PR cheese-enriched diet (C) were shown in Fig. 1. The MALDI spectra of CPPs isolated by plasma samples from other participants are reported in the Supporting Information (Fig. S1, A,B,C). The characteristic peaks derived from fibrinogen α-chain phosphopeptides dominated MALDI spectra of the plasma samples before cheese intake. The mass signals at 1389.9, 1461.1, 1546.2 and 1616.6 Da corresponded to the fibrinopeptide A (FbA)-derived (f2– 15) P, (f1–15) P, (f2–16) P and (f1–16) P peptides respectively. These results confirmed the efficiency of HA to selectively capture the
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Fig. 1. MALDI-TOF/MS analysis of the ultrafiltrated (Cutoff 10 kDa) plasma sample of subject number four (A), the relative HA-enriched fraction before (B) and after the cheese-enriched diet (C). Peaks under brackets for A, B and C spectra grouped signals belonging to FbA. Note that all the spectra were recorded after covering the beds of HA-bound peptides spotted onto MALDI plate with DHB matrix (1% PA).
phosphopeptides in plasma samples without any specific sample pretreatment and the ability of MALDI-TOF to provide multiple CPP identification in a single mass spectrometric analysis. However, additional phosphopeptide signals with those still dominant of fibrinopeptide-
derived phosphopeptides (Fig. 1C) were detected in plasma samples from individual subjects after cheese intake. The mass signal at 777.4 Da was attributed to the Ser(P)-Ser(P)-Ser(P)-Glu-Glu cluster common to calcium sensitive αs1-, αs2- and β-CN. The ‘rugged end’
Fig. 2. MALDI-TOF/MS analysis of the pH 4.6 soluble fraction of PR cheese sample aged 30 months. Asterisks indicated putative precursor of the peptides identified in plasma samples shown in Table 1.
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signals (indicated with asterisks), corresponding to the parent αs2-CN (f6–12) 3P peptide and its N-terminally shortened CPPs, i.e. f7–12 and f8–12, fully (3P) and partially dephosphorylated (2P), indicated the co-presence of differently phosphorylated CPPs in plasma samples. Such peptides could be derived from the precursors, αs2-CN (f8–18) 3P and 4P (1435 and 1515 Da, respectively), identified in the sample of 30 hard long ripened Italian cheese used for the experiments (Fig. 2) and already detected in a previous study (Ferranti, Barone, Chianese, Addeo, Scaloni, Pellegrino and Resmini, 1997). 3.2. Structural characterization of peptide sequences detected by MS/MS analysis Plasma samples analyzed by MALDI-TOF/MS were also submitted to LC-ESI-MS/MS analysis to confirm the peptide identity established by MALDI-TOF. The use of two different ionization techniques together with the different sample treatment (see method section) did not provide identical results. In Table 1, the different sets of peptides identified using the two techniques were shown together with a partial list of CPPs identified in the PR cheese consumed by the subjects. NanoLC-ESI-MS/MS analysis allowed to unambiguously detect the mass signals corresponding to αs1-CN (f43–50) 2P (996.7 Da) and the αs1-CN (f43–52) 2P CPPs (1239.9 Da) in plasma samples of 3 out of 4 subjects after the consumption of PR cheese for one week (Table 1). It is likely that the retrieved peptides originated from the longer parent peptide αs1-CN (43–58) 2P (1927.8 Da) that was detected in the PR cheese consumed by the participants (Fig. 2). As an example, the complete MS/MS fragmentation pattern and the recorded signals for αs1-CN (f43–52) 2P CPPs (1239.9 Da) and β-CN (f193–209) (1881.26 Da) were reported in Fig. 3. MS/MS spectra of the peptides identified in the plasma samples of the four subjects are collected in the Supporting Informations (Fig. S2). Thus, non-phosphorylated peptides were also identified in the plasma samples collected after one-week consumption of cheese. They all originated from a common sequence from the β-CN C-terminal end, i.e. β-CN (f194–209) (1718.08 Da), and β-CN (f200–209) (1094.37 Da). This result may depend on two factors, the higher sensitivity of the LC-ESI-MS/MS with respect to MALDI-TOF and co-elution of very low amounts of non-phosphorylated peptides from the HA sorbent. This drawback was overcome by lowering the pH value of the wash buffers so that carboxyl groups were not ionized and non-phosphorylated peptides washed out (data not shown). However, the occurrence of non-phosphorylated β-CN peptides at different length in human plasma samples confirmed previous findings on the resistance of β-CN (f193–209) peptide to the hydrolysis by brush-border membrane peptidases, and its transport through the
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Caco-2 cell monolayer (Regazzo, Mollé, Gabai, Tomé, Dupont, Leonil and Boutrou, 2010; Sánchez-Rivera, Diezhandino, Gómez-Ruiz, Fresno, Miralles and Recio, 2014). The main route involved in trans-epithelial transport of the β-CN (f193–209) peptide was ascribed to the transcytosis via internalized vesicles, although the paracellular transport via tight-junctions could not be excluded. The in vivo transport of intact bioactive peptides in the intestinal epithelium is an important prerogative to prove their bioavailability (Regazzo et al., 2010). On the other hand, quantification of peptides in human plasma is necessary to transfer in humans the functional properties attributed to C-terminal peptides of β-CN by in vitro studies. Simply ingesting cheese, rather than separately casein and casein hydrolysates including CPPs, does not allow establishing the precise mechanism of the origin of the peptides found in the blood. They could be already present in the cheese or they could result from the gradual casein hydrolysis along all the gastro-intestinal tract including the gut microbiota metabolism. The ‘cluster sequence’ containing the motif Ser(P)-Ser(P)-Ser(P)-Glu-Glu, responsible for the ability to maintain calcium and phosphate ions in a soluble form (Gravaghi, Del Favero, Cantu', Donetti, Bedoni, Fiorilli, Tettamanti and Ferraretto, 2007) is common to αs1-, αs2- and β-CN. CPP-derived and non-phosphorylated peptides occurred in vivo, display different ability to elicit the [Ca2+] increase in the blood. The two classes of molecules act towards calcium aggregation in a completely different manner, with CPPs exhibiting the highest level of Ca2+ coordination with respect to non-phosphorylated. The presence of three phosphorylated serine and two glutamic acid residues in the cluster sequence accounts for such a strong negative charge that attractive interactions towards Ca+2 prevail on aggregation, thus maintaining the calcium, in a soluble form and absorbable state. The cluster sequence does not exhibit any aggregation in solution, regardless of the presence of Ca2+, as the intensity of the light scattered by CPPs is not dissimilar to that of pure solvent (Gravaghi et al., 2007). 4. Conclusions The present data constitute an evidence that specific casein peptides and CPPs are present in circulation in humans. For the first time in this pilot study, the bioavailability of the specific casein peptides was demonstrated in humans consuming 100 g of PR cheese (1 g CPPs), every day for one week. As expected, a large inter-individual variation was found since not all the subject plasma samples contained the same peptides and future study with a larger sample size is needed. However, these findings open also new research perspectives focused on investigation of physiological effects of peptides in vivo, such as calcium transport and delivery as well as antimicrobial or immunomodulating properties. These goals may be achieved by quantitation of peptides in
Table 1 CPPs identified in human plasma samples after MS-based studies and pH 4.6 soluble counterparts identified in the 30-month old PR cheese as shown in Fig. 2. Molecular mass (Da) Analytical technique
MALDI TOF/MS Human serum HA-CPPs
NanoLC-ESI/MSMS
PR cheese HA-CPPs from pH 4.6 soluble fraction a
MALDI TOF/MS
Theoretical
Measured
1013.7 876.2 796.6 777.4 697.4 1094.4 1240.0 1240.0 996.8 1881.3 1718.1 1094.4 1435.1 1515.1 1927.8
1013.7 877.0 797.1 778.4 698.5 1095.3 1240.5 1240.5 997.1 1881.5 1718.3 1095.3 1435.1 1515.2 1927.8
1,2,3,4 indicates the subject plasma sample where the cheese peptides were detected.
Peptide identification
Aminoacidic sequence (a)
αs2-CN (f6–12)3P αs2-CN (f7–12)3P αs2-CN (f7–12)2P αs2-CN (f8–12)3P αs2-CN (f8–12)2P β-CN (f200–209) αs1-CN (f43–52)2P αs1-CN (f43–52)2P αs1-CN (f43–50)2P β-CN (f193–209) β-CN (f194–209) β-CN (f200–209) αs2-CN (f8–18)3P αs2-CN (f8–18)4P αs1-CN (f43–58)2P
HVSSSEE (2,3) VSSSEE (1,3,4) VSSSEE (4) SSSEE (4) SSSEE (2,4) PVRGPFPIIV (1,2,3,4) DIGSESTEDQ (1,2,4) DIGSESTEDQ (1,3,4) DIGSESTE (4) YQEPVLGPVRGPFPIIV (1,3,4) QEPVLGPVRGPFPIIV (1,2,4) PVRGPFPIIV (1,2,3,4) SSSEESIISQE SSSEESIISQE DIGSESTEDQAMEDIK
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Fig. 3. MS/MS spectrum for confirmation of the identity of αs1-CN (f43–52) 2P and β-CN (f193–209) peptides found in plasma samples of subjects consuming a PR cheese-enriched diet for one week.
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