Caseinophosphopeptides Research Articles

Identification of phosphorylation sites of equine β‐casein isoforms

AbstractEquine β‐casein is phosphorylated at variable degrees and isoforms carrying 3 to 7 phosphate groups (3P–7P) have been found in milk, but the phosphorylated amino acid residues of each isoform are not yet identified. In the present work, the different phosphorylation variants were first isolated by ion‐exchange chromatography and then hydrolysed by trypsin to generate caseinophosphopeptides (CPPs), each containing all the potential phosphorylation sites. The equine CPPs were prepared by metal oxide affinity chromatography, a method based on the affinity of phosphate groups towards titanium dioxide immobilized onto a micro‐column. This method turned out to be an efficient tool to separate the CPPs Arg1–Lys34 and Glu4–Lys34 from non‐phosphorylated peptides. Purification was achieved by reversed‐phase high‐performance liquid chromatography (RP‐HPLC) and each CPP was hydrolyzed by endoproteinase Glu‐C. Finally, the digests were analyzed by RP‐HPLC/electrospray ionization mass spectrometry (RP‐HPLC/ESI‐MS) and identified by nano‐electrospray ionization tandem mass spectrometry (nESI‐MS/MS) to locate the phosphorylated sites of the β‐casein isoforms 4P–7P with accuracy. Thus, the isoform 4P was found to be phosphorylated on residues Ser9, Ser23, Ser24, and Ser25. Addition of phosphate groups on Ser18, Thr12, and Ser10 led to the formation of the isoforms 5P–7P, respectively. The results indicated that the in vivo phosphorylation of the equine β‐casein follows a sequential way and is not randomly performed. Copyright © 2010 John Wiley & Sons, Ltd.

Effect of caseinophosphopeptides added to fruit beverages upon ferritin synthesis in Caco-2 cells

Ferritin synthesis was evaluated in iron sulphate solutions and in fruit beverages supplemented with iron, with/without zinc in the presence of caseinophosphopeptides (CPPs), based on a system combining simulated gastrointestinal digestion and the Caco-2 cell model. In iron sulphate solutions with/without zinc, the addition of CPPs had no effect upon ferritin synthesis. When CPPs were added to the bioaccessible fraction of fruit beverages supplemented with iron, ferritin synthesis increased in the same proportion as with the addition of milk. The addition of zinc to fruit beverages supplemented with iron and to iron sulphate solutions decreased ferritin synthesis. Nevertheless, in fruit beverages with milk, this decrease in ferritin synthesis was not observed when zinc was present.

Milk versus caseinophosphopeptides added to fruit beverage: Resistance and release from simulated gastrointestinal digestion

The influence of simulated gastrointestinal digestion on caseinophosphopeptides (CPPs) formation in milk-based fruit beverage was evaluated, together with resistance of a pool of CPPs added to fruit beverage. In milk-based fruit beverage, four CPPs were identified that can be justified by their presence in raw milk or due to processing. When it was subjected to simulated gastrointestinal digestion, 10 CPPs were identified, and only 1 presented the cluster (SpSpSpEE) (3 phosphoseryl group followed by 2 glutamic acid residues), which corresponded to α s2-CN(1–19)4P. CPPs added to fruit beverage are resistant to simulated gastrointestinal digestion, and 16 CPPs were identified originating from the fragmentation of added CPPs, and with a greater presence of the cluster compared with CPPs originating from milk-based fruit beverage. This could justify the use of CPPs as functional ingredients, and offer a good alternative to milk-based fruit beverage for improving mineral bioavailability.

Does the addition of caseinophosphopeptides or milk improve zinc in vitro bioavailability in fruit beverages?

Abstract The influence of caseinophosphopeptides (CPPs) added to fruit beverage versus milk based fruit beverage upon zinc retention, transport, and uptake, as well as the influence of Fe supplementation, were studied using a combined simulated gastrointestinal digestion/Caco-2 cell system. Zinc retention, transport, and uptake of milk based fruit beverage was 4- to 5-fold greater than that of fruit beverages with or without CPPs – no statistically significant differences being observed in relation to the presence or absence of CPPs. Possibly, a slow release of CPPs throughout the digestive tract, as can be expected to take place during the digestion of casein, has a more beneficial effect than the addition of preformed CPPs upon Zn availability. A significantly negative Fe × Zn interaction in relation to Zn retention and uptake was observed in fruit beverages. This should be taken into account in the formulation of such beverages, where the supplementing of both of these mineral elements is common practice.

Addition of milk or caseinophosphopeptides to fruit beverages to improve iron bioavailability?

Abstract A study has been made of the influence of caseinophosphopeptides (CPPs) added to a fruit beverage versus milk based fruit beverages upon iron retention, transport and uptake, using a combined simulated gastrointestinal digestion/Caco-2 cell system. Grape concentrate, orange concentrate, and apricot puree were used for sample formulation. Eight samples were assayed with/without added Fe sulphate (3 mg/100 ml fruit beverage) and/or added Zn sulphate (1.6 mg/100 ml fruit beverage), with/without skimmed milk (11% v/v). The addition of milk to fruit beverages exerted a positive effect on iron retention, transport and uptake versus fruit beverages, and this effect was greater than that of CPPs added to soluble fractions of fruit beverages. The addition of CPPs to soluble fractions of fruit beverages improved iron transport. Iron supplementation increased Fe retention, transport and uptake – the effect being more notable in samples with milk. Zinc supplementation did not affect Fe retention, transport or uptake.

In vitro bioaccessibility of iron and zinc in fortified fruit beverages

SummaryIron and zinc bioaccessibility was estimated in the in vitro gastrointestinal digests of six different fortified fruit beverages (Fb) containing iron and/or zinc and/or skimmed milk (M). Solubility values can be used to establish trends in relative bioavailability of iron and zinc, as the first stage towards mineral bioavailability comprises solubility in the intestinal tract. FbFe, FbFeM and FbFeZnM samples showed iron bioaccessibility above 88%, differing (P < 0.05) from those of FbFeZn (53%). In turn, FbZn, FbFeZn and FbZnM samples presented higher zinc bioaccessibility (above 68%), differing (P < 0.05) from those of FbFeZnM (48%). The presence of milk‐derived caseinophosphopeptides (CPPs) formed during gastrointestinal digestion in dairy samples does not increase iron or zinc bioaccessibility in FbFeM or FbZnM vs. FbFe or FbZn, but it is hypothesised that the negative interacting effect of zinc upon iron bioaccessibility when co‐supplemented in these fruit beverages is overcome in the presence of CPPs, which favour iron solubility more than in the case of zinc.

The inhibitory effect of glycomacropeptide on dental erosion

Throughout the world, the consumption of acidic foods, soft drinks and fruit juices has increased and has been associated with a rise in dental problems such as caries and erosion. The aim of this study was to investigate the protective effect of glycomacropeptide (GMP) against dental erosion and to compare the effect with that of caseinophosphopeptides (CPP). To assess the contribution of the calcium content of these peptides and their protection, calcium-reduced GMP (GMP-ca) and calcium-reduced CPP (CPP-ca) were studied. Hydroxyapatite was used as the tooth model. At a pH of 3, the protective effect of GMP was superior to that of CPP; however, CPP performed better at a pH of 4.5. The effect of GMP-ca and CPP-ca was evident only at a pH of 4.5, when compared with native GMP and CPP. These findings indicate that the calcium content of the peptide may have a role in protection against mineral loss from teeth. However, the pH of the erosive solution was the most influential factor contributing to erosion.

주정과 구연산 및 식이성 칼슘소재를 처리한 멸치분말이 흰쥐의 칼슘대사에 미치는 영향

본 연구는 주정과 구연산을 처리한 대멸분말과 함께 식이성 칼슘소재가 SD계 흰쥐의 칼슘대사에 미치는 영향을 조사하기 위하여 수행되었다. 실험식이는 대멸분말의 칼슘함량을 고려하여 식이중의 칼슘함량이 0.1%가 되도록 semipurified diet(AIN-diet, 1977)에 건조대멸분말을 첨가한 대조군(C), 주정-구연산처리군(EC), 주정-구연산＋CPPs처리군(ECC), 젖산칼슘첨가군(CL) 및 인산칼슘첨가군(CP)으로 처리하여 5주동안 실험한 결과는 다음과 같다. 증체량은 EC군이 ECC군과 일반적인 칼슘소재로 이용되는 젖산칼슘군 (CL)과 인산칼슘군(CP)에 비해 유의하게 높았고(p<0.05), 식이효율(FER)은 차이가 없었다. 생체내외(in vitro & invivo) 칼슘흡수율은 CPP를 처리한 ECC군이 각각 20.4%, 28.4%로 실험군중 가장 높았다(p<0.05). 혈당치는 CL군(105.7 mg/dL)이 대조군(89.5 mg/dL)보다 유의하게 높았고 (p<0.05), TC농도는 EC군(75.1 mg/dL)이 CP군(65.6 mg/dL)보다 높았으며, TG농도는 CP군(33.5 mg/dL)이 통계적으로 가장 낮았다(p<0.05). ALP활성과 OST농도는 실험군간 차이가 없이 CL군이 대조군보다 다소 높았다. 혈중 Ca농도는 칼슘흡수율이 가장 낮은 대조군(C)이 10.82 mg/dL로 유의하게 낮았고, EC군과 ECC군이 가장 높았다(p<0.05). 대퇴골무게는 CP군이 가장 낮았고(p<0.05), 길이는 ECC군이 가장 길었다(p<0.05). 골밀도는 CP군(0.1116 g/㎠)이 가장 낮았던 반면, ECC군(0.1149 g/㎠)이 가장 높았다. 이상의 결과에서 CPPs를 첨가한 ECC군이 생체내외 칼슘흡수율과 혈중 Ca농도 및 대퇴골의 길이와 밀도 등에 유의한 상승효과를 미친 것으로 나타났다. 향후 본 연구결과는 칼슘흡수율을 높이고 색택의 개선 및 관능이 향상된 기능성제품 개발의 기초자료로 활용될 수 있을 것으로 사료된다.

Calcium absorption is not increased by caseinophosphopeptides

One of the suggested health benefits of caseinophosphopeptides (CPPs) is their ability to enhance calcium absorption. This possibility is based on the assumption that they resist proteolysis in the upper gastrointestinal tract and maintain calcium in a soluble form at alkaline pH in the distal ileum. The effects of CPP-enriched preparations (containing candidate functional food ingredients) on calcium absorption from a calcium lactate drink were tested. A randomized crossover trial was undertaken in 15 adults in whom we measured the absorption of calcium from a calcium lactate drink (drink A: 400 mg Ca as lactate) and 2 preparations enriched with forms of CPP (1.7 g each; drinks B and C). Both drinks B and C contained 400 mg Ca as calcium lactate plus approximately 100 mg CPP-derived calcium). Each volunteer received the 3 drinks in random order. Absorption was measured by the dual-label calcium stable-isotope technique. The quantity of calcium absorbed was significantly lower from drink A (103 mg) than from drink B (117 mg; P = 0.012) or drink C (121 mg; P = 0.002), which indicated a positive effect of the CPPs. However, because the CPP preparations contributed additional calcium besides that found in the calcium lactate (drink A), fractional absorption of calcium from drink B (23%) was slightly but significantly (P = 0.015) lower than that from drink A (26%). The differences in calcium absorption are unlikely to have any biological significance. CPPs are unsuitable as candidate ingredients for functional foods that are designed to deliver improved calcium nutrition.

Milk Proteins and Iron Absorption: Contrasting Effects of Different Caseinophosphopeptides

Clusters of phosphoserine residues in cow milk caseins bind iron (Fe) with high affinity. Casein inhibits Fe absorption in humans, but protein hydrolysis lessens this effect. Phosphopeptides from different caseins gave conflicting results on Fe absorption; release of phosphate residues by intestinal alkaline phosphatase could be a key point of that metabolism. The objectives of this study were to compare the absorption of Fe complexed to caseinophosphopeptides (CPP) of the main cow milk caseins beta-casein (beta-CPP) and alpha(s)-caseins (alpha(s1)-CPP) and to assess the role of alkaline phosphatase on this absorption. Two experimental models were used: an in vivo perfused rat intestinal loop and an in vitro Caco-2 cell culture model. In addition, we determined the effect of an intestinal phosphatase inhibitor on these various forms of Fe. Gluconate Fe was used as control. In both models, uptake and net absorption of Fe complexed to CPP from alpha(S1)-caseins were significantly lower than from Fe complexed to beta-CPP. Inhibition of the intestinal phosphatase significantly increased the uptake and the absorption of Fe complexed to beta-CPP without effect on the other forms of Fe. These results confirm the enhancing effect of beta-casein and its CPP on Fe absorption. The differences between CPP could be explained by their structural and/or conformational features: binding Fe to alpha(S1)-CPP could impair access to digestive enzymes, whereas beta-CPP-bound Fe is better absorbed than its free form. The differences in protein composition between cow and breast milk, which does not contain alpha-casein, could explain some of their differences in Fe bioavailability.

Improved absorption of caseinophosphopeptide-bound iron: role of alkaline phosphatase

Hydrolysis of proteins could lessen their inhibiting effect on the poor absorption of cow's milk iron (Fe), which is responsible for the high incidence of Fe deficiency worldwide. When bound to Fe, caseinophosphopeptides (CPP) derived from milk proteins resist luminal digestion, enhance Fe solubility and could improve its bioavailability; brush border enzyme alkaline phosphatase activity could influence iron absorption by releasing free Fe; this study assessed its role in the absorption of CPP-bound Fe. Rat duodenal loops were perfused with Fe gluconate or Fe bound to the CPP of β casein [β-CN (1–25)], with or without the addition of an inhibitor of alkaline phosphatase, Na 2WO 4. The uptake of Fe–β-CN (1–25) was greater than Fe gluconate. Na 2WO 4 enhanced the uptake of Fe–β-CN (1–25) and not of Fe gluconate. So the release of free, insoluble Fe, by alkaline phosphatase seems to be prevented by providing Fe in the Fe–β-CN (1–25) complex form. Its good disappearance rate makes β-CN (1–25)-bound Fe a candidate for food fortification.

Antioxidant Mechanisms of Caseinophosphopeptides and Casein Hydrolysates and Their Application in Ground Beef

Caseinophosphopeptides (CPP) and casein hydrolysates have been shown to bind prooxidant metals such as iron, but their effectiveness as metal chelators to inhibit lipid oxidation in foods has still not been fully investigated. Thus, the antioxidant activity of CPP and casein hydrolysates was studied in phosphatidylcholine liposome model systems. CPP (< 1.0 mg/mL) and casein hydrolysates (0.3-1.7 mg/mL) were effective inhibitors of TBARS development when oxidation was promoted by ferric/ascorbate. High amounts of CPP (> 1.0 mg/mL) were prooxidant, whereas casein hydrolysates were observed to be only antioxidative. In the presence of peroxyl radicals, casein hydrolysates were more effective scavengers than enriched CPP (3-15 mM). In cooked ground beef, TBARS formation was inhibited 75, 39, and 17% by 0.5% enriched CPP, casein hydrolysates, and low molecular weight casein hydrolysates, respectively, after 4 days of storage. The results show that CPP and casein hydrolysates are promising sources of natural antioxidants for foods.

Bioactive Properties of Milk Proteins with Particular Focus on Anticariogenesis

Beyond nutrition, there is an increasing amount of data and information to demonstrate a bioactive role for dairy components in adults including a role in prevention of dental caries. Specifically, the casein fraction and hydrolysates thereof have been the focus of researchers investigating cariogenicity prevention. Tooth enamel is a polymeric substance consisting of crystalline calcium phosphate embedded in a protein matrix. Dental caries develop by acidic demineralization (calcium and phosphorus solubilization) of tooth enamel. Demineralization occurs directly (acidic food consumption) or indirectly (by fermentation products of dental plaque odontopathogenic bacteria growing on residual food particles between teeth or adhering to the plaque). Research efforts with milk derived bioactive peptides have focused on inhibition of cariogenic, plaque-forming bacteria, inhibition of tooth enamel demineralization, and subsequent enamel remineralization. Caseinophosphopeptides (CPP) and glycomacropeptide (GMP) have been patented for use in common personal hygiene products to prevent dental caries. Research has shown CPP and GMP to be growth inhibitory to the cariogenic bacteria Streptococcus mutans and other species. Additionally, CPP forms nanoclusters with amorphous calcium phosphate (AMP) at the tooth surface to provide a reservoir of calcium and phosphate ions to maintain a state of super saturation with respect to tooth enamel. This would buffer plaque pH, and also provide ions for tooth enamel remineralization. Glycosidic structures attached to GMP are important to numerous bioactive properties of the peptide including anticariogenicity. Like CPP, GMP has shown inhibitory activity to enamel demineralization and promotes tooth enamel remineralization.

Caseinophosphopeptides and their cell modulating potential

In the context of the EU research project FAIR-CT 98-3077, studies were carried out to investigate caseinophosphopeptides (CPP) as potential ingredients for functional food and pharmaceutical applications. CPP preparations were produced by enrichment of CPP from hydrolytic casein digests. Enzyme preparations used for hydrolysis were PTN 3.0 S, Alcalase, Bioprotease P conc, Proteinase DS. Cytochemical studies were carried out to examine the cytotoxic potential or cell modulating activities of CPP using human cancer cell lines (HL-60, Caco-2) and non-malignant polymorphonuclear leukocytes (PML) from oral cavity. PML cells isolated by magnetic cell sorting using CD-15-antibody-labelled paramagnetic beads were used for the first time for testing food-derived peptides. Cell activity was measured by formazan dye formation. Effects on enterocytic differentiation properties of Caco-2 cells were examined by transepithelial membrane resistance of Caco-2 cell monolayers and brush border associated alkaline phosphatase activity. In conclusion, (1) no deleterious cytochemical consequences (apoptotic, antiproliferative or general cytotoxic effects) were observed on presenting a range of CPP preparations to various human cell systems, indicating that these compounds can be rated harmless at a cellular level, (2) stimulation of IgG-secretion into culture supernatant of PBL points to possible immunoenhancing properties of CPP preparations.

Detection of caseinophosphopeptides in the distal ileostomy fluid of human subjects

Caseinophosphopeptides (CPP) were detected for the first time in ileostomy fluid, collected at 2 h intervals for 10 h post milk and CPP ingestion, from human volunteers with an ileostomy. The level of CPP present in ileostomy fluid obtained from milk-fed volunteers was markedly higher than that from volunteers fed with selected CPP preparations. The findings are based on HPLC analysis in combination with peptide-bound P determination, thin-layer electrophoresis and amino acid analysis, together with ELISA studies using polyclonal antibodies raised against a set of CPP to detect immunoreactive CPP in ileostomy fluid. These procedures allowed the detection of nm concentrations of CPP. CPP, which can be released during intestinal digestion, may function as bioactive constituents and carriers for different minerals, especially Ca, and may be used as ingredients in functional foods or pharmaceutical preparations.

Product distribution of casein tryptic hydrolysis based on HPSEC analysis and molecular mechanism

Tryptic hydrolysis of whole casein was analyzed by high performance size exclusion chromatography (HPSEC) in combination with the degree of hydrolysis (DH). In terms of chromatograms obtained at different DH values and mass percentage calculated equations established by the normalization method, the complex process of enzymatic reaction and the molecular mass distribution of multiple hydrolysates were quantitatively characterized via 2-D plot. Based on the information of casein micelle structure, the possible reaction mechanism was deduced from a series of chromatographic results and experimental analysis. Taking into account the primary structure of whole casein and the target amino acid of trypsin, the distribution of theoretical peptides was accurately calculated by determining the split sites of complete enzymatic hydrolysis. According to the relationship between retention time and molecular mass, the corresponding chromatogram absorption peaks of active peptides in hydrolysates were identified, and caseinophosphopeptides (CPPs) sequence was also characterized.

Production of Caseinophosphopeptides from Na-Caseinates Prepared from the Milk of Several Species by a Proteinase of Lactobacillus helveticus PR4

The aim of this work was to produce caseinophosphopeptides (CPPs) from Na-caseinates prepared from the milk of six species (bovine, sheep, goat, pig, buffalo, human) hydrolyzed by a partially purified proteinase from Lactobacillus helveticus PR4 and to characterize the CPP preparations by RP-FPLC (reversed-phase fast-protein liquid chromatography) and for calcium solubilizing/binding activity. The yield of CPPs ranged from 0.60 to 1.86% of the original proteins. The calcium-solubilizing activity varied from 2.5 (human CPPs) to 11.4 (buffalo CPPs) mg Ca2+ mg−1 CPP. Moreover, minor differences in calcium-binding ability between the CPP from different species were observed. This work showed that CPPs with different calcium-solubilizing activity can be produced from various Na-caseinates by a Lb. helveticus proteinase. Milks having casein-fractions with similar amino acid sequences (e.g., sheep and goat), showed CPPs with the same activity. The data generated in this study show the potential of different caseins to yield CPPs and may help in the selection of milk for the production of functional foods enriched in CPPs.

Bioavailability of caseinophosphopeptide-bound iron

Iron deficiency, one of the main worldwide nutritional deficiencies, results from the low bioavailability of most dietary iron, including cow milk. Hydrolysis of the cow milk protein casein produces low molecular weight caseinophosphopeptides (CPPs). Binding of iron to CPPs keeps it soluble in the digestive tract and prevents the formation of high molecular weight ferric hydroxides, which are poorly absorbed. Previous experimental studies have shown that iron bound to the phosphopeptide containing the first 25 amino acids of β-casein, or β-CN (1-25), is well absorbed and corrects efficiently iron deficiency. We sought to assess in vivo iron absorption and uptake by tissues involved in iron metabolism and storage (liver, spleen, bone marrow), using radiolabeled iron. β-CN (1-25)-Fe displayed better absorption and tissue uptake by the vascularized rat loop model compared with a control substance, ferric ascorbate. The metabolism of β-CN (1-25)-Fe labeled with iron 59, added to cow milk, was also studied in young women. Although the absorption of β-CN (1-25)-Fe was not significantly higher than that of ferrous sulfate, it displayed significantly higher tissue uptake. This increase was transient and had disappeared by the 14th day of the study, suggesting that iron was used for metabolic purposes. (J Lab Clin Med 2002;140:290-4)

Selective separation of caseinophosphopeptides through immobilized metal ion affinity chromatography: effects of pH values, salt concentrations and degree of phosphorylation

A method based on immobilized metal ion affinity chromatography (IMAC) using a CS-IDA-Cu(II) gel is described for the separation of caseinophosphopeptides (CPPs) from a casein hydrolysate. According to the amino acid sequence and the degree of phosphorylation, operating parameters, such as pH values, salt concentrations for selective purification of different CPPs can be designed.

Iron tissue storage and hemoglobin levels of deficient rats repleted with iron bound to the caseinophosphopeptide 1-25 of beta-casein.

Caseinophosphopeptides (CPP) issued from enzyme digestion of caseins bind cations and keep them soluble in the digestive tract. They could be used as ligands to improve iron (Fe) bioavailability. Fe-deficient young rats were repleted with Fe (40 or 200 mg/kg of diet) bound either to the beta-CN (1-25) of beta-casein or to whole beta-casein or as FeSO(4). A control pair-fed group was given 200 mg of Fe (FeSO(4))/kg of diet for 6 weeks. After repletion, hemoglobin concentration of the control group was reached only by the ) animals fed 200 mg of Fe/kg; beta-CN (1-25) bound Fe (40 and 200 mg) produced higher Fe liver and spleen stores than FeSO(4). Binding Fe to the whole, nonhydrolyzed beta-casein gave results intermediate between the other experimental groups. Binding Fe to phosphoserine residues of low molecular weight CPP improved its ability to cure anemia and to restore iron tissue stores, as compared to Fe bound to the whole casein and to inorganic salts.