Process scale chromatographic isolation, characterization and identification of tryptic bioactive casein phosphopeptides

International Dairy Journal 9 (1999) 639}652

Process scale chromatographic isolation, characterization and identi"cation of tryptic bioactive casein phosphopeptides Katrine H. Ellega> rd!,*, Claus Gammelga> rd-Larsen", Esben S. S+rensen#, Sergey Fedosov# !MD Foods R & D, Skanderborgvej 277, DK-8260 Viby J, Denmark "MD Foods, HOCO, Bu( lowsvej 9, DK-7500 Holstebro, Denmark #Protein Chemistry Laboratory, University of As rhus, Science Park, Gustav Wieds Vej 10c, DK-8000 As rhus C, Denmark Received 11 September 1998; accepted 10 August 1999

Abstract A process scale isolation of 40 kg tryptic Casein Phosphopeptides (CPP) was implemented using tryptic hydrolysis, acid precipitation, dia"ltration and anion exchange chromatography. The obtained yield corresponded to 20% (w/w) CPPs from caseinate. The CPP-product was obtained either as Ca-CPP (6.97% Ca, 0.23% Na) or Na-CPP (0.01% Ca, 8.53% Na). These products were completely soluble at pH as low as 0.5 and displayed no particular taste. All produced CPPs were phosphorylated peptides which exhibited calcium binding ability and inhibited calcium phosphate crystallization. The peptides were identi"ed by Mass Spectrometry and N-terminal sequencing as genuine truncated tryptic peptides. A high purity CPP product, available as Na-CPP and Ca-CPP, was obtained by this high yield process scale isolation of tryptic CPPs. ( 1999 Elsevier Science Ltd. All rights reserved. Keywords: Casein phosphopeptide; Bioactive ingredient; Calcium bioavailability; Crystallization inhibitor; Anticariogen; Functional foods

1. Introduction The primary biological function of milk is to supply the newborn with needed nutrients including calcium, a very important mineral for growth and maintenance of the skeleton and bone mineral mass. Bovine milk has a unique property of solvating calcium (1.2 g l~1) and inorganic phosphate (2.0 g l~1) in concentrations above the solubility of calcium phosphate by forming colloidal particles. These particles are casein micelles holding 66% of the calcium and 45% of the inorganic phosphate (Rollema, 1992). This colloidal calcium phosphate is retained in the micelles by the negatively charged phosphoseryl containing segments of a -, a - and b-caseins 41 42 (Holt, 1985). These phosphoseryl segments can be released enzymatically, e.g. by tryptic cleavage, resulting in general tryptic casein phosphopeptides; (a -CN 2P (f4341 58), a -CN 5P (f59-79), a -CN 1P (f106-119), a -CN 4P 41 41 42 (f2-21), a -CN 4P (f46-70), a -CN 2P (f126-136), a -CN 42 42 42 1P (f138-149), b-CN 4P (f2-25) and b-CN 1P (f33-48), all

* Corresponding author. Tel.: #45-8625-4455; fax: #45-8625-7786. E-mail address: [email protected] (K.H. Ellega> rd)

comprising the calcium binding capacity (Naito, 1990; Schlimme & Meisel, 1993) and including the characteristic inhibitory e!ect on calcium phosphate crystallization and precipitation (Schmidt, Both, Visser, Slangen & van Rooijen, 1987; Naito, 1990; Holt, 1996). CPPs are found in the small intestine of pigs (Meisel & Frister, 1988) and rats (Naito, Kawakami & Imamura, 1972; Naito & Suzuki, 1974) as an outcome of the in vivo digestion of ingested casein or after ingestion of a CPP supplemented whey protein diet (Hirayama, Toyota, Hidaka & Naito, 1992). Moreover, Kasai, Honda and Kiriyama (1992) found CPPs in the faeces of casein fed rats. This inertness of CPPs to further hydrolysis is probably due to the absence of proteolytic enzymes with speci"city for the phosphoserine residues, an absence also found in cheese where CPPs accumulate during cheese ripening (De Noni, Pellegrino, Resimi & Ferranti, 1997). The natural formation of CPPs in the small intestine and their property as calcium solubilisers has lead to the assumption that they play an important role in the calcium bioavailability. They are assumed to elevate the level of soluble and potentially available calcium for vitamin D-independent, paracellular transport of

0958-6946/99/$ - see front matter ( 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 9 5 8 - 6 9 4 6 ( 9 9 ) 0 0 1 3 5 - 1

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calcium from the small intestine (for reviews see Naito & Sakata, 1986; Naito, 1990; West, 1990; Kitts & Yuan, 1992). Clinical tests have demonstrated enhanced absorption of calcium in rats after ingestion of casein, and this was related to the formation of CPPs found in the small intestine (Lee, Noguchi & Naito, 1980, 1983; Sato, Noguchi & Naito, 1986; Nagasawa, Yuan & Kitts, 1991; Kitts, Yuan, Nagasawa & Moriyama, 1992), especially the phosphoseryl residues of casein (Sato et al., 1983). A direct relationship between enhanced calcium absorption and CPPs was demonstrated by Hirayama et al. (1992) and Yuan and Kitts (1991) in long term rat studies, and by Sato et al. (1986) who injected CPP into a ligated loop of the small intestine of rats and found an enhanced calcium absorption. A few authors report no e!ect of CPP on calcium absorption in vivo (ScholzAhrens, Kopra & Barth, 1990; Brommage, Juillerat & Jost, 1991; Kopra, Scholz-Ahrens & Barth, 1992). However, as these authors point out, this could be an outcome of the high activity of alkaline phosphatase in rats or the application of low purity CPP. Recently, sodium-enriched CPP (Na-CPP) and calcium-enriched CPP (Ca-CPP) produced by MD Foods have been clinically tested in humans (Hansen, Sandstorm, Jenson & Sorensen, 1997a, b). The production and the characteristics of these CPP-products are reported in the present work. The human tests, when whole-body retention of a radioisotope (47Ca ore 65Zn) was measured, showed a signi"cant positive e!ect on calcium and zinc absorption from rice-based meals, but not from whole grain cereals (Hansen et al., 1997a) and breads (Hansen et al., 1997b). It was furthermore concluded that the CPP product signi"cantly enhanced the solubility of calcium and zinc in phytate-containing infant foods and that Ca-CPP addition to several types of high phytate meals signi"cantly enhanced calcium and zinc bioavailability in rat pups (Hansen et al., 1996). The positive e!ect of CPPs on bone mineralization has been demonstrated in important in vivo studies by Tsuchita, Goto, Shimizu, Yonehara and Kuwata (1996) and Asida, Nakajima, Hirabatashi, Saito, Matsui and Yano (1996) who concluded that ingested CPP inhibited bone loss in aged ovariectomized rats and in laying hens. Furthermore, Matsui, Yano, Awano, Harumoto and Saito (1994) showed that CPP supplementation mitigates the reduction of calcium content of implanted bone in rats fed a low calcium diet. CPPs are shown to increase calci"cation of the diaphyseal area of explanted rat bone rudiments in vitro (Gerber & Jost, 1986) and to improve eggshell quality of hens fed a low calcium diet. Reynolds (1991) and Reynolds et al., 1995 have demonstrated various positive e!ects of CPPs in dental research. Speci"cally peptides with a continuous sequences of anionic amino acids (SerP}SerP}SerP}Glu}Glu) in a -CN 5P (f59-79), a -CN 4P (f2-21), a -CN 4P (f4641 42 42 70) and b-CN 4P (f2-25), were shown to be anticariogenic

by increasing the plaque level of calcium phosphate. The a -CN 5P (f59-79) peptide was furthermore shown to 41 prevent the pH fall in plaque and to improve remineralization of enamel. The reported positive e!ects on mineral absorption, bone mineralization and its anticariogenic e!ects add CPPs to the group of bioactive peptides (Meisel, 1997). CPPs have many potential applications (FitzGerald, Smyth & Slattery, 1996) such as additives in calciumforti"ed foods, in tooth paste, as a bioactive ingredient in nutraceuticals and functional foods. In addition, CPPs can be used in pharmaceutical applications, for example to prevent osteoporosis or to enhance bone mineralization after fracture. All the reported tests have been carried out with various CPP preparations di!ering in purity regarding CPP and non-CPP content in the test sample. Previously, CPPs have been prepared with little focus on scalable methods in view of creating an economical method resulting in a high yield of pure CPPs. Traditionally, tryptic release of CPP form casein and isoelectric precipitation of residual casein was followed by scalable methods like selective precipitation (Peterson, Nauman & McMeekin, 1958; Hirayama et al., 1992) ore ultracentrifugation (Gagnaire, Pierre, Molle & LeH onil, 1996) however, yielding low purity products. Adamson and Reynolds (1995) succeeded in obtaining intermediate yield of high purity CPP by applying selective precipitation of CPPs using 100 mM CaCl and 50% (v/v) ethanol. The 2 most selective isolation procedures make use of chromatography as those reported by West (1977), Koide, Fukushima, Itoyama and Kuwata (1990), Juillerat, Baechler, Berrocal, Chanton, Scherz and Jost (1989). But these methods produced a fairly low yield of CPPs. The possible commercial potential of CPP, in the light of the in vivo observations, lead to the present work which was undertaken in order to develop a reproducible, industrial scale process for isolation of tryptic CPP. The requirements for the method were (1) high yield from caseinate, (2) high quality in terms of exclusively phosphorylated tryptic peptides and (3) good functionality, i.e. calcium binding, inhibition of calcium phosphate crystallization, as well as good taste and solubility. 2. Materials and methods 2.1. Process scale purixcation of casein phosphopeptides Ten production experiments were carried out. Process parameters were slightly adjusted in the "rst six experiments obtaining an optimized process which is reported in this work. 2.1.1. Trypsin hydrolysis of caseinate 200 kg of sodium caseinate (Miprodan 30; MD Foods Ingredients amba, Viby, Denmark) was dissolved in

K.H. Ellega> rd et al. / International Dairy Journal 9 (1999) 639}652

2000 l demineralized water (503C) to produce a 10% (w/v) solution. The pH of the solution was adjusted to 8.00 using 2 M sodium hydroxide (Natrium hydroxide Pure; NOVADAN A/S, Kolding, Denmark). 1 kg trypsin (EC 3.4.21.4, PTN 3.0, Type Special; Novo-Nordisk A/S, Bagsvvrd, Denmark) was dissolved in 3 l demineralized water immediately before addition to the caseinate solution in order to avoid extensive self hydrolysis. Hydrolysis was carried out under gentle agitation at constant temperature (503C) and constant pH (8.00), controlled by neutralization of the released hydrogen ions by addition of 5 M NaOH (49.0$1.6 l, n"4). The hydrolysis proceeded until the base consumption stopped and hydrolysis was considered completed. The trypsin was inactivated according to manufacturer's instructions by elevating the temperature in the crude hydrolysate to 803C and holding for 5 min. 2.1.2. Separation of hydrolysate by selective precipitation and diaxltration The crude hydrolysate was subjected to selective precipitation of hydrophobic peptides (LeH onil, MolleH , Bouhallab & Henry, 1994) by adjusting the temperature to 403C and the pH to 4.60 using concentrated hydrochloric acid (28.6$0.6 l, n"4) (NOVADAN A/S). After one to two hours of #occulation, the precipitate was separated from soluble peptides by ultra"ltration followed by dia"ltration with demineralized water at 453C in a SSPP Ultra Filtration Plant equipped with GR60PP membranes (approximate MW cut o!: 25,000 Da; APV

641

Pasilac, Silkeborg, Denmark) resulting typically in 200 l retentate and 3700 l dia"ltrate. 2.1.3. Chromatographic purixcation of tryptic CPP Industrial scale chromatography was performed with a BPSS 1000/1000 process scale column (Phamacia, Uppsala, Sweden) containing 700 l PA Agarose anion-exchange resin (Up-Front Chromatography, Copenhagen, Denmark) at a #ow rate of 24 l min~1. The resin was regenerated with 315 l of 1 M hydrochloric acid, washed with 1100 l demineralized water before it was loaded with the 3700 l dia"ltrate. During loading, non-phosphorylated peptides were repelled from the resin by phosphorylated peptides of higher negative charge. These excess peptides were washed o! the resin with 2200 l demineralized water. This fraction of unbound peptides is referred to as the Casein Peptide Fraction (CPF). The bound CPPs were eluted by 1100 l 0.2 M sodium hydroxide and the resin was further washed with 1100 l water. During chromatography, the eluate pH (pH-Meter 761 Calmatic; Struers KEBO Lab A/S, Albertslund, Denmark), conductivity (CDM83 conductivity meter; Radiometer, R+dovre, Denmark) and absorbancy at 280 nm (Industrial Flow cell, UV1 monitor; Phamacia) were monitored (Fig. 1). A batch of 1100 l puri"ed casein phosphopeptide solution was collected and concentrated typically six times by Reverse Osmosis (RO) at 503C and 60 bars using a B1 module and AFC 30 membranes (Patterson Candy International Ltd., Hampshire, England) which, according to

Fig. 1. A chromatogram from industrial scale isolation of CPP showing the elution time versus eluent absorbance at 280 nm (*), pH (#) and conductivity (]) of eluent. The input of regenerating HCl, water, dia"ltrate and the eluting NaOH are indicated on the top of the chromatogram including times for starting and stopping the collection of CPP containing eluent.

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the manufacturer, expell ions and retain peptides. Peptides were pasteurized (853C, 15 s) and spray dried yielding sodium enriched CPP (Na-CPP). 2.1.4. Conversion of sodium enriched CPP into calcium enriched CPP In order to produce calcium enriched CPP, the ROconcentrate was mixed with 13.7 kg CaCl ) 6H O (E 2 2 509; Fvllesindk+bet, Kolding, Denmark) and dia"ltrated by RO until the conductivity of the "ltrate, containing excess ions, was less than 3.0 mS cm~1. The concentrate was spray dried and denoted calcium enriched CPP (Ca-CPP). 2.2. Characterization of casein phosphopeptides 2.2.1. Chemical analysis The protein and/or peptide concentration was calculated as protein%"nitrogen%]6.38, where nitrogen% was determined using a Nitrogen Analyzer (macro N; Foss Electrics A/S, Hiller+d, Denmark). Calcium, phosphorus and sodium were determined according to the Association of O$cial Analysis Chemists (AOAC, 1998) method 984.27 and amino acids by acid or oxidative hydrolysis according to AACC Method 07-01 (AACC, 1995) or AACC Method 07-11 (AACC, 1995) of the American Association of Cereal Chemists. 2.2.2. Capillary zone electrophoresis Capillary electrophoresis was performed on a Hewlett-Peckard HP3D CE capillary electrophoresis system controlled by ChemStation software (Hewlett-Packard Company, Waldbronn, Germany) mounted with a fused silica capillary of 72 cm e!ective length, internal diameter of 25 lm and extended light path (G1600-62132; Hewlett-Packard). Electrophoresis was performed at 503C and 30 kV starting with a voltage ramp of 0}30 kV for 1 min. The electrolyte was 70 mM borate bu!er adjusted to pH 9.20 by mixing 70 mM borax (B-9876; Sigma Chemical Company, St Louis, USA) and 70 mM boric acid (165; Merck, Darmstadt, Germany). Injection was 50 mbar in 25 s, analysis time 30 min and peptides were detected by their absorbancy at 210 nm. The repeatability of migration time (MT) was 0.085% relative standard derivation (RSD) and of area, 3.008% RSD (n"10). Analysis of Na-CPP and Ca-CPP resulted in analogous electrophoretograms. The peaks were tentatively identi"ed by trypsin (T-1426; Sigma) hydrolysis of 10 g l~1 a-casein (C-6780; Sigma), b-casein (C-6905; Sigma) or i-casein (C-0406; Sigma) dissolved in 0.1 M Na citrate, pH 8.00 using 0.2 g l~1 enzyme. The reaction 3 mixtures were dialysed against the electrolyte (as above) before analysis. 2.2.3. Fast protein liquid chromatography The purity of produced CPP was evaluated by Fast Protein Liquid Chromatography (FPLC; Phamacia)

essentially as described by Juillerat (1989) utilizing a Mono Q HR 5/5 column (Phamacia), but using a linear gradient of 0 to 50% bu!er B (20 mM Tris HCl, 1 M NaCl, pH 8.00) from 8 to 68 min and injecting samples at 5 min. Bu!er A was 20 mM Tris HCl, pH 8.00. Phosphopeptides were dissolved to 10% (w/v) in demineralized water, diluted 1 : 3 in bu!er A, incubated 1 h at 373C, further diluted 1 : 3 in bu!er A and "ltered through a 0.22 lm "lter (Milex-GV; Millipore, Bedford, USA) before submission of 45 ll to the FPLC analysis. 2.2.4. Dephosphorylation and decalcixcation of phosphopeptides Dephosphorylated samples of CPP were prepared as described above, including addition of 0.125 g l~1 alkaline phosphatase (EC 3.1.3.1, analytical grade; Boehringer Mannheim, H+rsholm, Denmark) to the CPP solution before the incubation at 373C. Prior to submission to calcium binding determinations, the puri"ed CPPs were decalci"ed using the chelating ion exchange resin Chelex 100 (Bio-Rad Laboratories, Copenhagen, Denmark). The resin was equilibrated with 25 bed volumes of 0.5 M sodium acetate at pH 6.00 followed by 30 bed volumes of demineralized water in a batch process. Five gram of Na-CPP were disolved in 100 ml demineralized water followed by addition of 2.1 g resin. After gentle stirring for one hour, the resin was removed by "ltration and the decalci"cation of "ltrate was repeated. The second "ltrate was freeze dried. The protein concentration of this decalci"ed CPP was 76.70% (w/w). In order to detect remaining calcium, the decalci"ed CPP was "rst ashed (IDF, 1964) then subjected to calcium analysis according to application note 918-373-8511B (Radiometer, R+dovre, Denmark) using a titrator (Video Titrator, Vit 90; Radiometer, R+dovre, Denmark) mounted with a calcium ion selective electrode (M27AG-9; Radiometer, R+dovre, Denmark) and a calomel electrode (K401; Radiometer, R+dovre, Denmark). Calcium was not detectable in the decalci"ed CPP. 2.2.5. Calcium binding assay Titrations were performed in duplicate using the Radiometer equipment mentioned above, equilibrated as recommended by the manufacturer. A 100 mM acetatebu!er was prepared by mixing 100 mM sodium acetate (S-7670; Sigma) and 100 mM acetic acid (1.00063; Merck) to pH 5.60, or 100 mM Tris-bu!er (T-8524; Sigma) to pH 7.40 was used. Decalci"ed CPP was dissolved to peptide concentration 4.10 g l~1 in acetate bu!er and titrated at room temperature with 0.05 M CaCl ) 2H O 2 2 (2382; Merck) in acetate bu!er. The titrations were furthermore preformed in Tris bu!er and calibration curves were established for both bu!ers by titration of blanks. Calibration curves were the same for

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643

both bu!ers:

2.3. Identixcation of casein phosphopeptides

ER (mV)"12.6]Ln(free Ca)#39.6

2.3.1. Reverse phase high pressure liquid chromatography (RP-HPLC) and fraction collection Na-CPP was solubilized in 0.1 M trisodium citrate, pH 9.00 (1.06448; Merck) to 10 g l~1 and 150 ll peptide solution was analyzed by RP-HPLC using a C18-column (250/4.6 Nucleosil 100-5; Macherey-Nagel, DuK ren, Germany) by gradient elution (0% B at 2 min to 60% B at 92 min). Mobile phase A was 0.1% (v/v) Tri#ouro acetic acid (TFA) in water and B was 0.1% (v/v) TFA and 89.9% (v/v) methanol in water. All reagents were HPLC grade. Samples corresponding to the central portions of 13 major peaks were collected.

](r2"1.00, n"3, [Ca]"0.05 to 10.00 mM) (1) where ER is the eletrode response. On the basis of electrode responses during titrations, free calcium was calculated. Bound calcium was calculated as total calcium minus free calcium. Bound calcium was plotted versus free calcium and this curve was "tted according to the equation: [P@] ][Ca] 0 [P@!Ca]"[P@!Ca] # 0 K #[Ca] $

(2)

where [P@] is the molar concentration of peptides, [Ca] is the molar concentration of calcium, [P@] is the total 0 molar concentration of calcium binding sites on the peptides and K is the dissociation constant for the equilib$ rium P@!Ca H P@#Ca (M). [P@!Ca] represents the 0 error in determination of the zero point which was !0.11$0.01 mM for measurements at pH 5.60 and 0.06$0.01 mM at pH 7.40. 2.2.6. Inhibition of calcium phosphate crystallization The inhibition of calcium phosphate crystallization was measured essentially as described by Sikes, Yeung and Wheeler (1991), however, adding none and up to 200 mg l~1 Na-CPP (70.40% (w/w) protein, 0.01% (w/w) calcium) to a solution of 4.40 mM CaCl ) 2H O (1.02382; 2 2 Merck). Two sets of experiments were performed where 0.5 l of each CPP solution was equilibrated to 203C before adding 1 ml 1.5 M or 3.0 M NaH PO ) H O 2 4 2 (6346; Merck) "ring a solution of 3.0 mM or 6.0 mM in dissolved inorganic phosphate. The reaction mixtures were mixed and immediately adjusted to pH 7.40 by addition of 1 M NaOH. The pH of the reaction mixture was continously monitored using INGOLD electrodes (640340-73; Struers KEBO Lab A/S) and a data logger (PROLOG, version 1.10; OL Consult, As rhus, Denmark). The induction time for calcium phosphate crystallization was recorded when the pH decrease of reaction mixture was 0.2 pH units. 2.2.7. Solubility of CPP Aqueous solution of a 1% (w/v) Na-CPP was prepared and its protein content and pH was determined; then adjusted by addition of 1 M HCl or 1 M NaOH to values form pH 0.5 to 9.0. Samples were centrifuged at 7000]g for 20 min at ambient temperature and the protein content of the supernatant was measured. The solubility of the CPP product was calculated taking the dilution of samples during pH adjustment into account.

2.3.2. Mass spectrometry and amino acid sequencing Peptides were identi"ed by amino acid sequence and mass spectrometric (MS) analyses essentially as described by S+rensen, H+jrup and Petersen (1995). Amino acid sequencing was carried out on a PE Applied Biosystem model 477A sequencer with on-line identi"cation of the phenylthiohydantoin derivatives. Mass spectra were acquired using a Bruker BIFLEX matrix-assisted laser desorption/ionization*time of #ight (MALDI-TOF) mass spectrometer equipped with a re#ector (BrukerFranzen, Bremen, Germany). Samples were prepared for analysis as described by Vorm, Roepstor! and Mann (1994). Thirty to one hundred calibrated mass spectra were averaged. Theoretical peptide masses were calculated using GPMAW programme (Lighthouse Data, Odense, Denmark). 2.4. Calculations According to the most frequent caseins genotypes, a -CN B 8P, a -CN A 11P and b-CN A1 5P (Swais41 42 good, 1992), the expected CPP to be obtained are a -CN 41 2P (f43-58), a -CN 5P (f59-79), a -CN 1P (f106-119), 41 41 a -CN 4P (f2-21), a -CN 4P (f46-70), a -CN 2P (f12642 42 42 139), a -CN 1P (f138-149), b-CN 4P (f2-25) and b-CN 1P 42 (f33-48). The molecular weight of these theoretical CPPs (MW k ) and caseins (MW k ) were calculated CPPi CN using GPMAW. On the basis of these and the typical concentration of caseins (C k ) the theoretical yield of CN CPP from casein (> ) and the theoretical average 5)%0. molecular weight of CPPs (MW ) were calculated as !7.5)%0. follows: Theoretical yield of CPP from casein (Y ): 5)%0. 3 > "+ 5)%0. k/1

A

A

] C

Ik + MW k /MW k ]100% CPPi CN ik /1

CNk

3 /+ C k CN k/1

BB

%(w/w)

(3)

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Theoretical average molecular weight of CPP (MW ): !7.5)%0. 3 Ik MW " + + MW k ]C k / !7.5)%0. CPPi CN k/1 ik /1 3 + I ]C k (g mol~1) (4) k CN k/1 where k (k"1,2, K) denotes the type of casein and i (i "1,2, I ) denotes CPP from type k casein. As k k k tryptic casein hydrolysis result in CPPs from three types of caseins, K"3. Furthermore, since the hydrolysis of a -casein results in three CPPs, of a -casein in four 41 42 CPPs and of b-casein in two CPPs the following is valid: For a -casein with k"1, i "i and I "I "3. For 41 k 1 k 1 a -casien with k"2, i "i and I "I "4. For b42 k 2 k 2 casein with k"3, i "i and I "I "2. k 3 k 3

A A

BB

2.4.1. Degree of hydrolysis (DH) The hydrolysis of every peptide bond was assumed to release a hydrogen ion which was neutralized by the addition of one hydroxide ion. On the basis of base consumption the degree of hydrolysis was calculated as the percentage hydrolysed peptide bonds of the total amount of peptide bonds. DH"(B]N ]100%)/(MP]a]h ) %, (5) B 505 where B is base consumption in ¸, N "5 M, MP" B N]6.38 in kg, a"0.88 at 503C, pH 8.00 and h " 505 8.2 meqv g~1 for casein (Adler-Nissen, 1982). 2.4.2. Solubility The peptide solubility was calculated as the concentration of soluble peptide in the supernatant after centrifugation, in percentage of initial peptide concentration in the sample. /C ]100%, (6) 461%3/!5!/5 4!.1-% where C is protein concentration in g l~1 (Haque & Moza!ar, 1992). Solubility%"C

3. Results and discussion 3.1. Process scale production of Na-CPP and Ca-CPP The overall process is outlined in Fig. 2 which includes information on the protein #ow showing a typical protein yield of 16% CPP from casein (calculated on the basis of the volume and protein content of RO-concentrate), which is somewhat less than the calculated theoretical protein yield of 23% (> "22.8%). However, 5)%0. our overall total gravimetric product yield of 20% CPP from caseinate is very high compared to other works (Juillerat et al., 1989; Adamson & Reynolds, 1995;

Fig. 2. Schematic representation of the process-scale isolation of tryptic CPP from caseinate showing the protein #ow through the process.

Groepfert & Meizel, 1996). The economy of the process strongly depends on this yield which in turn is mainly governed by the unit operations of hydrolysis and process chromatography. In our production experiments, the hydrolysis was exhaustive resulting in a DH of 18.8$0.6%. In similar hydrolysis experiments, Adamson and Reynolds (1997) found that this high DH, as a result of low enzyme/substrate ratio, implies an enhanced speci"c trypsin cleavage of casein, resulting in production of expected tryptic CPPs (see also Lorentzen, Fischer & Schlimme, 1994). The high product yield was further supported by the selective chromatographic isolation of phosphopeptides from all the non-phosphorylated peptides in the dia"ltrate as shown in Fig. 3. The process was carefully balanced with respect to the peptide load of the resin, because the highest CPP yield was achieved by matching the CPP content of the loaded dia"ltrate with the dynamic capacity of the resin. In order to ensure the high quality of the CPP-product, as solely phosphorylated peptides, the resin was slightly overloaded, resulting in non-binding of some of the CPPs during the process; these were found in the CPF. Due to the high dynamic capacity of the PA Agarose (&35 g peptides l~1) and the low pressure drop over the resin (&0.1 bar over 100 cm resin at a linear #ow rate of 183 cm l~1+24 l min~1) it

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Fig. 3. Electrophoretograms of MT versus absorbance as a result of Capillary Zone Electrophoresis analysis of 10 g l~1: (A) Dia"ltrate, (B) CPF and (C) CPP. a and b denotes the type of casein the tryptic CPPs originate from.

was possible to perform the continuous chromatography at a high #ow rate in a tall column in order to achieve low process time as well as a su$cient residence time of the components in the resin (&33 min). The concentration of eluting NaOH was selected to correspond to the dynamic capacity of the resin (Lihme, Aagesen, Gammelga> rd-Larsen & Ellega> rd, 1992, Example 7). The 0.2 M NaOH solution used was su$cient in order to elute all the phosphopeptides in a minimal volume without creating an excess of hydroxide in the eluate. As a consequence, the eluted pool of peptides showed a neutral pH of 7.03$0.05 (n"4). Thus, the load of the dia"ltrate, the process time, linear #ow rate and the concentration of eluting NaOH were optimized according to the dynamic capacity and selectivity of the resin ensuring a 20% overall yield of CPP from the caseinate. The entire process was found to be very reproducible and robust as ten production experiments (six preliminary and the four main experiments reported here) did not cause any technical problems or signi"cant variations in results, the amount and quality of the isolated CPP.

3.2. Characterization of casein phosphopeptides 3.2.1. Product quality parameters The chemical analysis of the products revealed that the isolated CPPs were able to retain calcium and expel sodium during the conversion of Na-CPP (0.01% Ca, 8.53% Na) into Ca-CPP (6.97% Ca, 0.23% Na). As a consequence of this high mineral content, ash values were higher and protein content lower in the CPP products compared to the dia"ltrate and CPF (Table 1). Analysis of the amino acid composition demonstrated that only two of the "fteen amino acids we were able to compare were of higher concentration in the CPP products than in the caseinate (Table 2). These amino acids are isoleucine and serine whereas the concentration of the aromatic phenylalanine was quite low. The chemical data and the amino acid content of CPP products reveal a relationship between parameters describing the quality of the CPP (Table 3). The molar ratio of the phosphate/serine is 1 in both Na-CPP and CaCPP indicating that all serine residues of the CPPs are phosphorylated. These organic phosphate groups obviously have a higher a$nity for calcium than for sodium

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Table 1 Composition of caseinate, the intermediate products after dia"ltration and the "nal product casein peptide fraction (CPF), sodium enriched casein phosphopeptide (Na-CPP) and calcium enriched casein phosphopeptide (Ca-CPP) Chemical data % (w/w)

Caseinate!

Dial"ltrate"

CPF"

Na-CPP#

Ca-CPP#

Protein Ash Phosphorus Sodium Calcium

90.00 3.50 0.80 1.20 0.10

89.14 8.82 0.66 3.57 0.08

97.50 3.25 0.31 1.17 0.08

72.40$0.10 20.56$0.02 3.03$0.00 8.53$0.66 0.01$0.00

74.33$1.04 18.34$0.92 3.29$0.06 0.23$0.18 6.97$0.53

!Typical parameters for Miprodan 30 (MD Foods, Denmark). "Values of freeze dried product, n"1. #Mean$SD, n"2. Table 2 Amino acid composition of casein (as reference) and dia"ltrate, CPF and CPP products obtained in thin mark Amino acids % (w/w)

Casein!

Dia"ltrate" CPF#

Na-CPP" Ca-CPP"

Serine$ Isoleucine Leucine Lysine Methionine Threonine Valine Phenylalanine Tryptophan Tyrosine Asx% Glx% Arginine Histidine Glycine Cysteine Alanine Proline

6.10 5.36 10.16 8.44 3.02 4.64 6.85 5.47 1.31 6.04 7.71 24.00 3.71 2.97 2.00 0.47 3.30 11.72

5.52 4.04 7.46 7.62 * 3.87 6.55 4.35 * 4.60 6.55 19.30 3.57 2.11 1.68 * 2.87 8.58

10.80 7.09 3.78 3.64 * 3.41 4.91 0.36 * 1.06 7.01 30.20 2.71 0.78 1.36 * 2.09 2.62

4.74 4.97 8.15 8.61 2.25 4.22 6.52 5.18 * * 7.17 22.20 3.53 2.80 1.76 0.45 3.11 11.10

10.30 6.81 3.65 3.55 * 3.19 4.83 0.33 * 1.00 6.82 29.10 2.60 0.79 1.36 * 2.04 2.42

Note: For de"nition of CPF and CPP see Table 1. * means not determined. !Grappin and Ribadeau-Dumas (1992). "Determined by acid hydrolysis. #Determined by oxidative hydrolysis. $Serine is the sum of phosphoserine and non phosphorylated serine residues. %Asx is the sum of aspartic acid and asparagine and Glx is the sum of glutamic acid and glutamine.

as calcium was retained during dia"ltration during RO of Na-CPP with added CaCl . The CPPs produced were 2 able to retain 1.64 mol calcium per mole serine in the Ca-CPP product. On the basis of the theoretical average molecular weight of CPPs this corresponds to 5.24 mol of calcium per mole of peptide. 3.2.2. Chromatographic verixcation of product quality Analytical anion exchange chromatography resulted in a chromatogram revealing the elution pro"le of the

Table 3 Molar ratio of selected parameters demonstrating the quality of the produced Na-CPP! and Ca-CPP! as phosphorylated peptides binding large amounts of calcium when contacted calcium as in the case of Ca-CPP Molar ratio" (mol mol~1)

Na-CPP

Ca-CPP

P/Serine Ca/P Ca/Peptide!

1.09$0.03 0.00$0.00 0.01$0.00

1.06$0.03 1.64$0.10 5.24$0.47

!For de"nition see Table 1. "Calculated on the basis of an estimated average peptide MW of 2237 g mol~1.

CPP produced (Fig. 4, bold line). The elution pro"le of Na-CPP and Ca-CPP were similar as the peptides were eluted according to their net charge resulting in the longest retention time for the most negative peptides. Enzymatic dephosphorylation of the same CPPs resulted in hydrolysis of serine-organic phosphate bonds and, as a consequence, in loss of the negative phosphate groups from the peptides and thereby in lower retention times (Fig. 4, thin line). The almost 100% chromatographic distinction of the produced peptides from the same dephosphorylated peptides demonstrates the rich phosphorylation of produced CPP and suggests that all the produced peptides are phosphorylated. 3.2.3. Inhibition of calcium phosphate crystallization The reaction mixture forms a non-crystalline amorphous calcium phosphate (ACP) immediately after mixing of dissolved calcium and inorganic phosphate forming a supersatuated solution. Then, the ACP nucleates with a sudden signi"cant decrease of pH due to crystallization of calcium phosphate. The crystals formed are dicalcium phosphate (DCP, CaHPO ) 2H O) at low initial pH 4 2 ((6.1 at 253C) or octacalcium phosphate (OCH, Ca H(PO ) ) 21H O) at a higher initial pH ('6.7 at 4 43 2 2

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647

Fig. 4. Degree of CPP phosphorylation evaluated by FPLC-analysis using MonoQ anion exchange chromatography eluting samples with a linear salt gradient. The produced CPPs eluted at high salt concentrations ( ); the same but dephosphorylated CPPs (*), devoid of the negative organic phosphor, eluted "rst.

253C) (Schmidt et al., 1987). The OCP slowly transforms into hydroxyapatite (HAP, Ca OH(PO ) ) under hy5 43 drolysis with further release of hydrogen ions (Holt, 1985). The crystallization can be delayed and even prevented by the presence of crystallization inhibitors in the reaction mixture. The e!ectivenness of the inhibitor can be estimated from the time interval between mixing and calcium phosphate crystallization, seen as a rapid decrease of pH of the reaction mixture. This interval was called the &induction time' and was measured by a drop from pH 7.40 to 7.20. CPPs are excellent calcium phosphate crystallization inhibitors (Fig. 5). Table 4 summarizes the experimental data revealing that a reaction mixture of 4.4 mM calcium and 3.0 mM phosphate crystallized in 27$3 min (n"2) at 203C, and that this induction time was prolonged at even very low concentrations of Na-CPP (5 to 30 mg l~1). The crystallization was fully inhibited by the presence of 40 mg l~1 Na-CPP corresponding to 0.02 mM peptides (MW "2237 g mol~1) and a Ca/ !7.5)%0. Peptide ratio of 246 moles of Ca per mole of peptide. At 6 mM phosphate (Table 4B) the crystallization was completely inhibited in 24 h at Na-CPP concentrations above 135 mg l~1. These "ndings are in agreement with the work of Holt (1996, 1998) who demonstrated the stabilization of amorphous dicalcium phosphate in a spherical particle by the CPP peptide b-CN 4P (f1-25). In his model system, Holt found that the peptide was linked to the calcium phosphate through its phosphorylated residues and that these stabilized nanometer-size particles were stable for years at ambient temperature.

Fig. 5. The inhibition of calcium phosphate crystallization by CPP evaluated by the time delay of pH decrease after CPP addition. CPPs were added in concentrations 0, 5, 10, 20, 30 and 40 mg l~1.

3.2.4. Calcium binding of CPP In agreement with the previous results, calcium titrations experiments showed the calcium binding ability of the produced CPP at pH 7.40 and at pH 5.60 (Fig. 6). K for the calcium binding to CPPs was 1.86$0.07 mM $ at pH 5.60 and 2.24$0.11 at pH 7.40 (n"2) (Table 5). At pH 5.60, the number of potential calcium binding sites was found to be 1.71$0.02 M which corresponds to 0.93$0.01 mol calcium per mole peptides, whereas the peptides can bind 2.44$0.02 mol calcium per mole

648

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Table 4 The e!ect of CPP as a calcium phosphate crystallization inhibitor A

B

Na-CPP (mg l~1)

Induction time! (min)

Na-CPP (mg l~1)

Induction time! (min)

0 5 10 20 30 40 80 120

23$3 30$1 45$6 50$1 64$8 * * *

0 50 100 135 170 200

18$4 44$8 163$4 960 (n"1) * *

Note: The 203C reaction mixture contained either 4.4 mM calcium and 3.0 mM phosphate (A) or 4.4 mM calcium and 6.0 mM phosphate (B) at initial pH of 7.40. pH was continuously followed over a period of 24 h and the induction time was measured as interval for a 0.2 pH unit decrease in the reaction mixture. * Represents a slow minor decrease in pH as seen in Fig. 5. !Mean$SD, n"2.

6.57}7.10 (Baumy, Guenot, Sinbandhit & BruleH , 1989). Our "ndings are in agreement with the work of Berrocal, Chanton, Juillerat, Pavillard, Scherz and Jost (1989) who also found an increase in calcium binding capacity with increasing pH. However, we noticed a decrease in K with decreasing pH which indicates a higher stability $ of the calcium-peptide complex at a lower pH. 3.2.5. Solubility The solubility of the CPP product is an important functional parameter with respect to applications of the product. Both Na-CPP and Ca-CPP were found to be completely soluble in the tested range of pH from 0.5 to 9.0 (results not shown). 3.3. Identixcation of casein phosphopeptides The chromatogram resulting from RP-HPLC analysis of the CPP produced is depicted in Fig. 7. MS and N-terminal sequence analysis of these fractions resulted in identi"cation of the phosphorylated peptides a -CN 41 2P (f43-58), a -CN 1P (f1-21), a -CN 4P (f2-21), a -CN 42 42 42 4P (f46-70), a -CN 3P (f46-70), b-CN 4P (f1-25), b-CN 42 3P (f1-25), b-CN 1P (f30-48) and b-CN 1P (f33-48) (Table 6). Furthermore, the peptide a -CN (f59-d) with 41 an unknown number of phosphorylations was present in fraction 3. However, we did not detect the somewhat smaller and low phosphorylated peptides a -CN 1P 41 (f106-119), a -CN 2P (f126-136) and a -CN 1P (f138s2 42 149). This is due either to their escaping from fraction collection, MS detection or N-terminal sequencing or, more likely, to the di$culties isolating these with the other phosphopeptides during production (see Fig. 3B

Fig. 6. Concentrations of free versus bound calcium during titration of CPP at pH 7.40 (L) and pH 5.60 (n), showing Ca binding by CPP.

Table 5 The kinetic parameters of calcium dissociation constant (K ) and $ amount of calcium binding sides ([P@] ) for the CPP and the calculated 0 molar ratio of calcium bound per mole of peptide pH

K $ (mM)

[P@] 0 (mM)

Ca/Peptide! (mol mol~1)

5.60 7.40

1.86$0.07 2.24$0.11

1.71$0.02 4.48$0.07

0.93$0.01 2.44$0.02

!Calculated using MW of 2237 g mol~1. !7.5)%0.

peptides at pH 7.40. This higher calcium binding capacity at higher pH can be ascribed to the e!ect of partial protonation of the phosphoseryl residues in the phosphopeptides at lower pH; pK values of the residues in ! the b-casein 4P (f1-25) peptide were determined as

Fig. 7. A chromatogram from RP-HPLC analysis of CPP. Fractions 1 to 13 are described in Table 6.

K.H. Ellega> rd et al. / International Dairy Journal 9 (1999) 639}652

649

Table 6 Identi"cation of individual phosphopeptides in RP-HPLC fractions from mass spectrometry and N-terminal amino acid sequencing Fraction No.!

N-terminal sequence"

Protonated mass Experimental

1 1 1 2 3 3 3 3 4 5 5 6 6 6 7 7 7 8 8 8 9 9 10 11 12 13

Peptide identi"cation# Calculated

NANE DIGS FQAEEQQ

QMEA NANE NANE VNEL NANE NANEEEY NTMEHVAAA NTME KNTM NANE DIGA NMTE NANE RELE DIGA DIGJEAT RELE RELEELNVP RELE

1964.9 2062.9 2064.0 2434.5

1963.8 2063.0 2063.0 2433.5

3009.4

3009.7

2931.0 2508.8

2929.7 2508.6

2635.2

2636.5

1929.1

1928.8

3121.7

3122.4

1944.1 3043.9 3043.9 3025.5

1944.8 3044.0 3044.0 3026.0

a -CN dP (f46-d) 42 a -CN dP (f43-d) 41 b-CN 1P (f33-48) b-CN 1P (f33-48) b-CN 1P (f30-48) a CN dP (f59-d) 41 a -CN dP (f46-d) 42 a -CN 4P (f46-70) 42 a -CN dP (f37-d) 41 a -CN dP (f46-d) 42 a -CN 3P (f46-70) 42 a -CN 1P (f1-21) 42 a -CN dP (f2-d) 42 a -CN 4P (f2-21)$ 42 a -CN dP (f1-d) 42 a -CN dP (f46-d) 42 a -CN 2P (f43-58) 41 a -CN dP (f2-d) 42 a -CN dP (f46-d) 42 b-CN 4P (f1-25) a -CN dP (f43-d) 41 a -CN 2P (f43-58)$ 41 b-CN 3P (f1-25) b-CN 3P (f1-25) b-CN 3P (f1-25)%

!Numbers refer to numbering of HPLC fractions in Fig. 7. "Amino acids are in conventional one letter code and phosphoseryl residues are symbolized by A. #Unknown numbers of phosphate groups or unknown number of amino acid are denoted by d. $Contains an oxidized methionine. %Short of one water molecule.

showing that some phosphorylated peptides were found in the CPF-product). An important "nding is that all the identi"ed peptides were phosphorylated and that these were tryptic peptides, properly cleaved by trypsin (EC 3.4.21.4). No chymotryptic peptides were found indicating the low amount of chymotrypsin (EC 3.4.21.1) in the PTN 3.0 Trypsin did not interfere with alleged integrity of the products as genuine tryptic phosphopeptides. 4. Conclusions It was possible to establish a robust process scale production of tryptic casein phosphopeptides from caseinate with an excellent yield of 20% (w/w) product and a process output of 40 kg sodium enriched CPP (Na-CPP) or calcium enriched CPP (Ca-CPP). The unit operation process scale chromatography permitted eminent control over the process in terms of producing exclusively phosphorylated peptides in order to ensure the high quality of the product (best illustrated in Fig. 3)

with a molar serine/phosphate ratio of 1. The unique complete solubility of the isolated CPP in the tested pH range pH 0.5 to pH 9.0, the slight sodium taste of NaCPP and the neutral taste of Ca-CPP gives the product great potential in various applications. As a bioactive ingredient, the most important property of the produced CPP is its calcium binding ability. This was demonstrated by chemical data, by calcium binding kinetics (K and [P@] ), and by its capacity as an e!ective $ 0 calcium phosphate crystallization inhibitor. These abilities are crucial for the status of CPPs as bioactive peptides in terms of enhancing the solubility of minerals, indicating an enhanced bioavailability of minerals, as documented for our CPP products by Hansen, SandstoK rm and LoK nnerdal (1996), Hansen et al. (1997a, b) in clinical tests on rat pups and humans. Abbreviations B C

base consumption concentration

650

Ca-CPP CN CPF CPP DH ER FPLC Glu h 505 i k k K $ MP MS MT MW MW !7.5)%0. n N B Na-CPP P [P@] [P@] 0 Pser RO RP-HPLC TFA > 5)%0. a a -CN 2P (f43-58) 41 A

K.H. Ellega> rd et al. / International Dairy Journal 9 (1999) 639}652

calcium enriched casein phosphopeptide casein casein peptide fraction casein phosphopeptides degree of hydrolysis, percentage peptide bonds cleaved electrode responce fast protein liquid chromatography glutamic acid total number of peptide bonds in a protein casein phosphopeptide from type k casein casein type dissociation constant mass of protein mass spectrometry migration time molecular weight theoretical average molecular weight number of experiments normality of base sodium enriched casein phosphopeptide phosphate peptide concentration total concentration of peptide binding sides phosphoserine reverse osmosis reverse phase high pressure liquid chromatography tri#uoro acetic acid theoretical yield of CPP form casein average degree of dissociation of the a-NH group 2 a -casein peptide fraction of 41 amino acid number 43 to 58 with two organic phosphate groups phosphoseryl residue

Acknowledgements The authors are grateful to laboratory technicians Elin Hanberg and Lene Prip Buhelt, MD Foods Research and Development Centre, for excellent assistance in the laboratory and to dairy technician, Lars Christensen, for excellent work and assistance in the Pilot Plant at MD Foods Research and Development Centre.

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