Commercial dairy products and ingredients with health or function claims based on CPP content (source: modified from ).
Nutraceuticals, a term combining the words “nutrition” and “pharmaceutical”, is
Many ingredients are included in the wide range of nutraceuticals, such as essential amino acids, conjugated fatty acids, vitamins, minerals and polyphenols. They have already been patented and incorporated in functional foods and nutritional beverages. Such components are believed to improve overall health and well-being, reducing the risk of specific diseases or minimizing the effects of other health concerns. However, milk is devoid of flavonoids, the most common group of vegetable polyphenolic compounds, which act as antioxidants and free radical scavengers. In contrast, the ingestion of soy and green tea extract may reduce the risk of developing prostate cancer and may protect against various other types of cancer [1-2]. An interesting patented invention has made available an extended-release form of polyphenols and riboflavin (vitamin B2) coated with methylcellulose . Coating slows down the release of polyphenols in the nutraceutical preparation . Possible applications of coating technology could be extended to all of the bioactive peptides susceptible to digestive enzymes. For example, glutathione can be maintained in human blood at normal levels by supplying it as dry-filled capsules . Nutrients and bioactive compounds may be microencapsulated by using mixtures of proteins or peptides and oils. Encapsulation of ω-3
Nutraceutical products comprising short bioactive peptides showing
2. Bioactive peptides released
in vitro by the hydrolysis of milk proteins
Peptides with various bioactivities can be produced according to two different methods: i)
(i) The proteolytic system of lactococci is able to degrade milk proteins using cell-wall-bound proteinases by releasing di-, tri-, and/or oligo-peptides and amino acids supporting the growth of bacteria. In addition, lactococcal peptidases released into the curd/cheese consequently to autolysis can further degrade the internalized peptides to amino acids . The higher the exopeptidase activity in cheeses, the greater the age of the cheese . Consistently, yogurt and other cultured dairy drinks have some of the highest counts of cells that actually survived and thus possessed the lowest number of peptides derived from the aminopeptidase activity. In a comprehensive review of literature, no enzyme with carboxypeptidase (CPase) activity has been reported for either lactococci or other LAB [19-20]. The bacterial peptidases have different and partly overlapping specificities (Figure 2).
Although the intracellular endopeptidases PepN and PepC are unable to hydrolyze casein molecules, the X-prolyl dipeptidyl aminopeptidases (PepX) are active on oligopeptides hydrolyzing the internal bonds of casein-derived peptides. Taken together, these enzymes are able to remove the N-terminal residues from peptides, with the specificity primarily depending on the nature of the N-terminal amino acid [20-21]. Di- and tripeptides generated by endopeptidases, general aminopeptidases and PepX are next subjected to additional cleavage by the tripeptidase, PepT, and dipeptidases, PepV and PepD (Figure 2). Other peptidases with more specific substrate specificities include PepA, which liberates N-terminal acidic residues from 3- to 9-residue long peptides; PepP, which prefers tripeptides carrying proline in the middle position; PepR and PepI, which act on dipeptides containing proline in the penultimate position; PepQ, which cleaves dipeptides carrying proline in the second position; and PepS, which shows preference for peptides containing two to five residues with Arg or aromatic amino acid residues in the N-terminal position [20-21,23] (Figure 2). This proteolytic system is able to support LAB growth to high cell densities (109–1010 cfu/mL) in milk containing only small amounts of hydrolytic products that are transportable into the cells for assimilation.
We also performed sequential milk protein (powder sample) digestions using various endoproteases facilitating a consistent partial hydrolysis. The high degree of specificity in terms of cleaving peptide bonds exhibited by a cocktail of enzymes (P, T and P432 from Biocatalysts, U.K) yielded a limited number of CPP. After fractionation on an HA column, both non-CPP and CPP were identified as shown in Table 3. Different oxidation rates of Met residues in the same protein resulted in formation of the peptides with different molecular mass. In most cases, a single Met-containing peptide and its oxidized counterpart were identified. However, in many cases, when proteins contained several consecutive endoprotease sensitive bonds, different long peptides containing the same Met-oxidation site(s) were identified. The data in Table 3 suggest that the cocktail enzymes containing amino- and CPases, in addition to P and T, progressively reduce the size of peptides without altering the degree of phosphorylation. Evaluation of protein/peptide quality can take advantage of the tandem MS for the detection of native, partly oxidized and partly dephosphorylated peptides.
By this means, phosphorylated peptides (2 and 3P/mole) of a precursor of lactophorin (LP) (28 kDa milk glycoprotein), proteose-peptone component 3, and glycosylation-dependent adhesion molecule 1 were detected. In addition, three low molecular weight non-CPP derived from LP were detected.
Native milk proteins used as the substrate for digestion by enzymes did not form CPP. This suggests that denatured LP and other whey proteins could have the tendency to form low molecular-mass peptide aggregates characterized by a poor solubility. For this reason, the use of milk protein and/or any milk substrate powder must be discouraged to eliminate phosphates and salts from the substrate. The enzymatic hydrolysis of the casein implies the use of endoproteases. However, the protein hydrolysate with alcalase is used in infant formulae, dietetic foods, nutraceuticals, ice creams, dressings, fermented products, yogurts, and personal care products. CPP released by alcalase are truncated with respect to those released by trypsin. The identified peptides can be categorized into two groups, one containing multiphosphorylated peptides and the other tri-, di- and mono CPP. Each group contained a number of variously long peptides due to the broad specificity of alcalase cleaving peptide bonds mainly on the carboxyl side of Glu, Met, Leu, Tyr, Lys, and Gln. The exoproteases responsible for the hydrolysis are inactivated by heating for ~10 min to ~85 °C. The
The ingress of foreign material in general, such as CPP, across the mucosal brush-border into the enterocyte is conditioned by the efficient dephosphorylation of peptides by alkaline phosphatase. This aspect deserves more in-depth investigation.
In vivo digestion of casein, formation of CPP and their physiological importance
Among the biologically active peptides, CPP characterized by SerP and/or ThrP residues account for ~30% of monoesters of hydroxyl amino acids. They mainly occur in the Ser/Thr-Xaa-SerP/Glu/Asp sequence consensus, where Xaa is any amino acid residue but Pro. The three-phosphorylated motif -SerP-SerP-SerP-Glu-Glu- occurs in αs1-CN (f66-70), αs2-CN (f8-12), αs2-CN (f56-60), and β-CN (f17-21). According to the current CN nomenclature, bovine αs1-CN, αs2-CN, β-CN, and κ-CN possess 8-9, 11-13, 4-5, and 1-2 phosphate (P) residues, respectively, and the P number could change according to the casein variant . For example, β-CN D has one SerP residue less than the A counterpart due to the substitution Lys18 → Ser18.
Dental caries are initiated via the demineralization of tooth hard tissue by organic acids directly from the diet or produced from fermentable carbohydrate by dental plaque cariogenic bacteria. CPP can help to replace the minerals that were previously lost consequently to caries [66-67]. Hence, there is a great interest in developing CPP as nutraceutical ingredients for the formulation of functional foods.
4. CPP enrichment by different techniques
CPP preferably comprise components released by four different casein families, each having a molecular weight greater than 500 Da. Multiply and singly, tryptic CPP can be simultaneously detected using MALDI-TOF, and the location of phosphate groups by a combination of tandem mass spectrometry and computer-assisted database search programs, such as SEQUEST (Trademark, University of Washington, Seattle Wash) [68-69]. Nano-electrospray MS/MS has been used for phosphopeptide sequencing and exact determination of phosphorylation sites . However, mass spectrometric analysis of proteolytic digests of proteins rarely provides full coverage of the phosphorylated sequence, with parts of the sequence often going undetected. In addition, protein phosphorylation is often sub-stoichiometric, such that ionization of CPP present in lower abundance in complex hydrolysates is ordinarily suppressed by strongly ionizable non-phosphorylated peptides. The MALDI-MS desorption/ionization efficiency for phosphopeptides was reported to be an order of magnitude lower than that recorded for the non-phosphorylated counterpart, and ionization became more difficult as the number of phosphate groups increased . Direct analysis of phosphopeptides utilizes two orthogonal MS scanning techniques, both based on the production of phosphopeptide-specific marker ions at m/z 63 and/or 79 in the negative ion mode. These scanning methods combined with the liquid chromatography (LC)-electrospray mass spectrometry (ESI) and nano-electrospray MS/MS allow the selective detection and identification of phosphopeptides even in complex proteolytic digests. Thus, even when the signal of the phosphopeptide is indistinguishable from the background, as in the conventional MS scan, low-abundant and low-stoichiometric phosphorylated peptides can be selectively determined in the presence of a large excess of non-phosphorylated peptides. This strategy is particularly well suited to phosphoproteins that are phosphorylated to varying degrees of stoichiometry at multiple sites . However, the identification and characterization of phosphoproteins would be greatly improved using selective enrichment of CPP prior to MS analysis. An ancient technique for phosphoprotein enrichment consisted of the precipitation of phosphopeptides as insoluble barium salts and recovery by centrifugation, as according to the Manson & Annan method . High-throughput phosphoproteome technologies currently rely on combining pre-separation of proteins, most commonly by high-resolution two-dimensional polyacrylamide gel, in-gel tryptic cleavage of proteins, and subsequent MALDI-TOF or ESI-MS/MS mass spectrometry analysis of peptides . The high resolving power of 2-DE with the sensitive MS requires extensive manual manipulation of samples. Alternative methods are based on chemical derivatization. For example, for β-elimination, a strong base such as NaOH or Ba(OH)2 is used to cleave the phosphoester bonds of phosphoserine and phosphothreonine and form dehydroalanine or dehydroaminobutyric acid, respectively, each able to react with different nucleophiles, such as ethanedithiol (EDT) or dithiothreitol (DTT). This procedure provides a considerably simpler method to enrich CPP. By using cross-linking reagents with affinity tags, such as biotin, interfering non-cross-linked peptides are eliminated, and CPP are highly enriched . Although the chemical derivatization methods are highly selective, they are not widely applied in phosphoproteome studies due to sample loss by the multiple reaction steps and unavoidable side reactions . Immobilized metal-ion affinity chromatography (IMAC, with Fe3+, Ga3+, Ni2+ and Zr4+ metal ions) and metal oxide affinity chromatography (MOAC, with TiO2, ZrO2, Al2O3 and Nb2O5) have been widely used for the quantitative binding of CPP on resin or adsorbent. Iminodiacetic acid (IDA, a tridentate metal-chelator) or nitrilotriacetic acid (NTA, a quadradentate metal chelator) are often used as IMAC functional matrices reacting with multivalent metal ions to form chelated ions with positive charges useful for the purification of phosphopeptides. Usually Fe3+, Ga3+ and Al3+ are bound to a chelating support prior to fractionating the complex mixture of peptides before MS analysis [77-78]. Ga3+ showed selectivity for CPP higher than Fe3+ and Al3+ [77,79]. Phosphopeptides bound to IMAC resin are successively recovered in the column effluent by increasing either pH or the phosphate concentration in the buffer . The negatively charged CPP selectively interact with TiO2 microspheres via bidentate binding at the dioxide surface [81-83]. TiO2-MOAC showed higher specificity than immobilized gallium (Ga3+), immobilized iron (Fe3+), or zirconium dioxide (ZrO2) affinity chromatography for phosphopeptide enrichment. The main problem associated with the chelating resins is the metal-ion leaching, which leads to CPP loss during the enrichment procedure. The selectivity of these methods was somewhat compromised by the detection of several acidic non-CPP that were also retained by the TiO2 column . To overcome this drawback, the carboxyl groups are methyl esterified which eliminates the non-specific adsorption of acidic peptides on IMAC . Considerable efforts have been expended to remove acidic non-CPP by washing the resin with 2,5-dihydroxybenzoic acid (DHB)  or phthalic acid . It has been found that aliphatic hydroxyl acid modified metal oxide works more efficiently and more specifically than aromatic modifiers such as DHB and phthalic acid in titania and zirconia MOC . However, all affinity techniques developed for the current enrichment strategies of CPP gave reproducible but incomplete results due to
The more important advantages of the procedure are the possibility of (1) detecting phosphorylated proteins/peptides even in complex mixtures, (2) determining phosphorylated sites and those dephosphorylated by phosphatase, (3) attaining information regarding weakly and heavily phosphorylated peptides and (4) adding the HA-CPP complex directly to food, which is enabled by the use of an edible resin such HA . Moreover, use of available commercial CPP preparations by the food industry is difficult for three primary reasons: i) the matrix bound to CPP is often not edible and such products can be hazardous; ii) the preparation of CPP is a long and a laborious procedure that requires cumbersome and expensive manipulation; and iii) CPP have an unpleasant taste even in modest amounts, which disadvantageously limits their direct utilization as a human food ingredient. A novel HA-based method for food grade CPP preparation has been performed on tryptic digests of casein. HA captured all CPP free of non-CPP . There were approximately 32 HA bound CPP, and all non-CPP peptides were eluted . HA-based enrichment procedure has been successfully applied to phosphopeptide recovery from complex biological fluids such as human serum thus providing a great source of potential biomarkers of disease. Four primary phosphopeptides derived from fibrinogen were enriched from human serum (Figure 3a-b, Table 4). A similar set of phosphorylated peptides was previously obtained using a modified IMAC strategy coupled to iterative mass spectrometry-based scanning techniques , using the titanium ion-immobilized mesoporous silica particles and MALDI-TOF  and cerium ion-chelated magnetic silica microspheres .
Fibrinopeptide A (FPA) (f1-16)1P, (SerP3), a 16-residue long peptide (1615 Da) (Table 4), is the segment anchored on the thrombin surface . The other three phosphopeptides, (f1-15)1P, (f2-15)1P and (f2-16)1P (Table 4), are hydrolytic products of FPA. The serum level of fibrinogen and its hydrolytic products may reflect the expression and activation of enzymes including kinase, phosphatase, and protease . An altered ratio of FPA (f2-15) and FPA (f1-16) is detected in patients affected by hepatocellular carcinoma; the D2[pS]GEGDFLAEGGGV15 peptide is upregulated, and the A1D[pS]GEGDFLAEGGGVR16 peptide is down-regulated greatly. The other two peptides, A1D[pS]GEGDFLAEGGGV15 and D2[pS]GEGDFLAEGGGVR16, varied only slightly between the two groups . The proportions of fibrinogen and their phosphorylation products offer new opportunities for basic research in exploring new frontiers in bio-marker discovery.
4.1. CPP in commercial milk as specific indicators of heated milks
Because of the lower value, the addition of UHT and milk powder to raw or pasteurized milk is prohibited (EU Directives 92/46 CEE and 94/71 CEE) for cheese milk. The intensity of heat treatment was found to correlate with the furosine content. Glycated proteins and peptides formed during the initial stages of the Maillard reaction are indirectly evaluated through the furosine content [100-101]. The Amadori compound formed upon the reaction of lysine residue with a lactose molecule will prevent the digestive enzymes from reaching the binding sites. Native and lactosylated forms of β-CN (f1-28)4P, (f1-27)4P and αs2-CN (f1-24)4P, although typical of UHT milk and milk powder, are missing in raw, pasteurized milk (71.7 °C for 15 s) and intensely pasteurized milk. The lactosylated peptides that varied with heat treatment characterize UHT milk added in amounts not lower than 10% to raw and pasteurized milk . Milk delactosed with microbial β-galactosidase did not suppress the Maillard reaction; indeed, the furosine concentration increased to 35-400 mg/100 g of protein . As expected, a lactose-reduced UHT milk had β-CN (f1-28)4P glycated mainly by its monosaccharides (Figure 4a).
Therefore, the nonenzymatically glycated CPP derived from the reaction of one molecule of glucose or galactose with a lysine residue (m/z 3641) can be considered to be the signature peptide of lactose-reduced milk (Figure 4).
4.2. CPP in yogurt
Yogurt is a fermented milk defined as the “food produced by culturing one or more of the optional dairy ingredients (cream, milk, partially skimmed milk, and skim milk) with a characteristic bacteria culture that contains the lactic acid-producing bacteria,
For example, β-CN (f1-28)4P, a peptide representing 100% intensity (assumed as base peak) of the signals in MALDI spectra, was reduced by approximately 30%; this loss was associated with the transformation of pasteurized milk into yogurt. One can deduce that the original peptide undergoes degradation even considering the higher number of formed CN peptides. In yogurt, β-CN (f1-28)4P, the most common CPP in pasteurized milk, was hydrolyzed into the peptide β-CN (f15-28)4P, which thus becomes the most abundant CPP.
β-CN (f1-29)4P, β-CN (f1-28)4P, β-CN (f1-25)4P, β-CN (f1-24)4P, αs2-CN (f1-24)4P, αs2-CN (f1-21)4P, resulting from the CN hydrolysis by plasmin and enriched on HA, were also found as C-terminally shortened peptides. During fermentation and storage, αs2-CN (f115-150)3P and αs2-CN (f115-149)3P derived by plasmin action did not react further to produce shorter peptides, most likely because of the absence of proteolytic enzymes. In the yogurt fraction recovered by centrifugation, only five multi-phosphorylated αs1-CN and two low-phosphorylated κ-CN, κ-CN (f106-163)1P and κ-CN (f106-169)1P, were identified. Few CPP were less phosphorylated than the native peptides due to the presence of milk phosphatase, which was denatured in all pasteurized cheese-milks. The presence of lactosylated β-CN (f1-28)4P CPP was indicative of yogurt made with high-heat treated or milk fortified with milk protein powder . Proteolysis of milk proteins in model yogurt systems has shown a similar set of primary CPP (Table 5). Therefore, the question is raised how CPP, derived from the enzymatic hydrolysis of yogurt CN are digested and absorbed in adult humans. For this reason, it is important to know the gastrointestinal resistance of CPP if used as a functional ingredient for fruit beverages. In the various stages of human digestion, a large quantity of CPP is produced in the stomach by partial hydrolysis of CN through pepsin action and in the small intestine by trypsin; these peptides are successively refined by endoproteases/exopeptidases. Although analysis of the intestinal contents of milk and yogurt ingestion has revealed the presence of CPP , their further resistance to gastrointestinal enzymes is poorly documented. Fragment β-CN (f1-24)4P has been previously identiﬁed in the lumen contents of rats after 60 min of digestion as a β-CN (f1-25)4P derived peptide . Moreover, after yogurt ingestion, β-CN (f1-32)4P CPP was released in the human stomach  and β-CN (f1-31)4P was found in a yogurt sample. The fragment β-CN (f1-28)4P constitutes a clear example of the multi-functionality of milk-derived peptides because some regions in the primary structure of caseins contain overlapping peptide sequences that exert different biological effects, in this case both mineral binding and immunostimulatory action . Even with the difference in the peptide pattern, it is evident that CPP binding iron (or other metal ions) remains soluble in the digestive tract, where they escape further enzyme digestion . The authors have studied in depth the simulated digestion of CPP from peptide precursors. These studies greatly benefit from the knowledge of enzyme specificities and degradation mechanisms. CPase and chymotryptic activity of pancreatin exhibits broad speciﬁcity, cleaving bonds on the carboxyl side of several amino acid residues of CPP. The latter, which are in the mass range 960-7087 Da (Table 5), are good candidates for intestinal absorption and for playing a possible physiological role in mineral bioavailability. However, there are conflicting results on the lack of αs1-CN (f43-52)2P and αs2-CN (f1-19)4P identified by other authors after CN hydrolysis with pancreatin, an enzyme used during the intestinal step of simulated physiological digestion . Generally, the physiological effects of CPP may not always be extended to precursor peptides, although they are structurally similar. There are not enough data concerning the effects of CPP addition to probiotic acid fermented milks. However, probiotic bacteria such as
4.3. CPP in a few cheese varieties
Whole milk contains a variety of endogenous plasmin-mediated CN peptides. In addition, CPP were released following cell lysis and release of intracellular LAB enzymes. This phenomenon was observed especially at the end of ripening in long-ripened cheeses, such as Comté , Grana Padano , Parmigiano-Reggiano and semi-hard Herrgard cheese . In Grana Padano cheese, 45 CPP were identified, of which 24 originated from β-casein, 16 from αs1-casein and 5 from αs2-casein. These CPP formally derive from three parent peptides, namely β-CN (f7-28)4P, αs1-CN (f61-79)4P and αs2-CN (f7-21)4P . By comparing CPP of Grana Padano and Herrgard cheese, it was clear that CPP were all progressively shortened and dephosphorylated during ripening. CPP were very resistant to enzymatic degradation, especially when SerP residue was at the N-terminal end. The number of CPP identified according to the different procedures was comparable for Grana Padano  and Herrgard cheese . In both the cheeses, CPP were progressively shortened and dephosphorylated during ripening, with both cheeses constituting heterogeneous mixtures of peptides phosphorylated at various sites sharing N- and C-terminally truncated CPP. However, some peptides proved very resistant to enzymatic degradation, especially when SerP residue was present at the N-terminal end of CPP . The only SerP residue located at the N-terminus of CPP was subjected to dephosphorylation, exposing the dephosphorylated residue to aminopeptidase action. A heterogeneous CPP pattern also differentiated the cheese samples within a given form because of the phosphatase gradient amongst peripheral and central parts of the Grana Padano cheese form (3 105
It is likely that ingested cheese carries a concentrated pH 4.6 soluble CPP fraction and a variable number of CPP according to the cheese variety. Above all, the presence and integrity of plasmin-mediated products of CN is a function of the milk, whether raw or pasteurized. Pasteurization reduces the milk plasmin activity only by ~15 percent, whereas plasmin activity increases during milk storage. UHT does not inactivate the plasmin in milk, and proteolytic activity will continue to damage milk. Heat treatments modify the peptide profile by increasing the content of larger peptides. The CN breakdown occurring during the ripening of PR cheese proceeded more slowly in PM cheese.. This means that eating PR cheese increases the quota of the co-ingested mineral bound CPP. In contrast, GR cheese show a different CPP level such as that of β-CN (f1-27)4P (3350.2 Da), resulting the most abundant CPP, when compared with hard cheeses (Figure 5a and 6); β-CN (f1-27)4P was further hydrolyzed into the shorter β-CN (f7-27)4P (2580.4 Da), β-CN (f10-27)4P (2270.0 Da), β- CN (f12-27)4P (2083.8 Da) and β-CN (f15-27)4 (1742.4 Da). The most abundant CPP in all three cheeses derived from the peptide β-CN (f1-28)4P, but long-ripened PR cheese was dissimilar from the other cheeses in its content of αs1-CN (f62-79)5P (2332.9 Da) (Figure 5d and 6).
β-CN (f1-28)4P (3478.4 Da) in AA cheese was the most abundant signal of the MALDI spectra and it was partially hydrolyzed into the shorter peptides β-CN (f7-28)4P (2708.5 Da), β-CN (f15-28)4P (1869.7 Da) and β-CN (f17-28)3P (1589.6 Da) (Figure 5b and 6). Considering exclusively the CPP molecular mass in the 3-3.5 kDa range of AA and GR cheese, the intensity of a high number of peptides transformed into a number of progressively lower molecular weight CPP, with accompanying liberation of peptides (Figure 5). The most common group of CPP occurred in the mass range of 1.7-2.9, reaching the maximum intensity for β-CN (f12-28)4P (2212.0 Da) in PM and αs1-CN (f62-79)5P (2332.9 Da) in PR cheese (Figure 5c-d and 6). The presence of β-CN (f16-22)3P (977.7 Da) was discovered in both AA and PR cheeses and was not detected in the PM and GR cheeses (Figure 5). Our results show that longer plasmin-mediated peptides degraded into shorter CPP. These peptides became more evident when the chymosin retained in the cheese was largely inactivated by cooking the curd at high temperatures (~55 °C). The hydrolysis of CN by chymosin was covered by that of plasmin, which became the principal proteolytic enzyme in the cheese. This phenomenon is particularly evident in PM raw milk cheese for which the plasmin-mediated β-CN (f1-28)4P peptide, representing ~0.1% of the CPP, was almost completely hydrolyzed into the shorter peptides β-CN (f11-28)4P (2341.1 Da), β-CN (f12-28)4P (2212.0 Da), β-CN (f11-25)4P (1985.7 Da) and β-CN (f12-25)4P (1856.6 Da) (Figure 5c). When comparing the PR and PM cheese, the former had a high extent of β-CN (f1-28)4P as judged by the higher levels of the peptide. This demonstrates that CPP of PR cheese are progressively transformed into a number of lower-molecular-weight peptides. In contrast, the quasi-total absence in the PM cheese of β-CN (f1-28)4P and relatively few of the various sizes β-CN-derived CPP (Figure 6) could be the effect of the enzyme decline from the optimum level of activity to zero enzyme activity.
αs1-CN CPP originated for the greater parts from the internal regions of the amino acid sequence, namely αs1-CN (f61-79)5P and αs1-CN (f33-60)3P.
In PR cheese, 23 casein-derived CPP were found to derive from the internal region of αs1-CN,
The profile of the CPP depicts the mechanisms of both the proteolysis and dephosphorylation in a long ripened cheese. Peptide αs1-CN (f61-79)5P, most likely arising from the parent peptide αs1-CN (f1-79)7P through cleavage at Met60-Glu61, was dephosphorylated and concurrently hydrolyzed into shorter peptides. Alkaline and/or acid phosphatases acting on SerP residue dephosphorylated CPP. N-terminal Ser was then exposed to aminopeptidase and released as a free amino acid. Bacterial CPase or exopeptidase such as cathepsin D/chymosin release αs1-CN (f61-74)4P and other derived CPP through cleavage at Asn74-SerP75 (Figure 8). In PR aged 30 months, αs1-CN (f61-79)5P (2462.1 Da), αs1-CN (f62-79)5P (2332.9 Da), αs1-CN (f63-79)5P (2261.9 Da), αs1-CN (f64-79)5P (2132.8 Da), αs1-CN (f64-79)4P (2052.8 Da), αs1-CN (f65-79)4P (1965.7 Da), αs1-CN (f62-74)4P (1681.3 Da), αs1-CN (f64-74)4P (1481.2 Da), αs1-CN (f64-74)3P (1401.2 Da) and αs1-CN (f65-74)3P (1314.1 Da) were the dominant CPP (Figure 5d and 8). Indeed, only 7 CPP for PM cheese and 11 CPP for GR were derived from αs1-CN (f59-79)5P, namely αs1-CN (f61-79)5P (2462.1 Da) and αs1-CN (f61-74)4P (1810.5 Da) (Figure 5a and c and 7). Considering the αs2-CN peptide, the αs2-CN (f1-24)4P CPP were similar in number but significantly different in the case of the four cheese varieties (Figure 6). The dominant αs2-CN-derived CPP were αs2-CN (f1-24)4P (3132.9 Da) and αs2-CN (f1-21)4P (2747.6 Da) in AA cheese (Figure 5b). The most abundant αs2-CN-derived CPP were αs2-CN (f1-18)4P (2355.1 Da) for PM and αs2-CN (f6-18)4P (1751.4 Da), a shortened form of the primary CPP αs2-CN (f1-18)4P, for GR cheese (Figure 5a and c). αs2-CN (f7-18)4P (1614.3 Da) and αs2-CN (f8-18)4P (1515.1 Da) characteristically accumulated in PR cheese (Figure 5d). Similar and discrete phosphorylated CPP derived species for αs2-CN (f1-24) (3P and 4P) and β-CN (f1-28) (3P and 4P) occurred in all cheeses. For the other casein fractions, primary CPP are fully phosphorylated, such as αs1-CN (f61-79)5P, whereas the derived peptides show a level of phosphorylation less than native form as observed in PM and GR. A higher dephosphorylation level characterized the CPP profile of PR cheese (Figure 8). The different profile of CPP could derive from the different length of ripening and from the cheese variety.
CPP. in ovine cheeses
Cheeses that contain CPP are also manufactured from ovine milk. Proteolytic enzymes in Pecorino cheese originate from chymosin, pepsin and other clotting preparations such as paste rennet. These enzymatic activities are complemented by those secreted by the vegetative spores of
In PR cheese of different ages, the released CPP were progressively degraded at C- and N-terminal ends. CPases work from the C-terminal end and aminopeptidases from the N-terminal end, both removing the terminal amino acid residues incrementally. LAB does not produce CPases; thus, the ability to liberate the carboxyterminal amino acid and peptides is typical of the mold. The N-terminal amino acid seemed to be released faster than the C-terminal residues because of the lower activity of CPases. There are negligible differences in the CPP level of different cheese lots primarily because of the action of the enzymes from
5. Anticariogenic effect of CPP in yogurt and cheese
Due to proteolytic activity, a great number of CPP are formed in raw milk cheese. In contrast, the enzymatic digestion of proteins to peptides can be reduced by milk pasteurization. Notwithstanding this, yogurt remains a consistent source of bioavailable CPP even if milk is heated to high temperatures (90 °C, 30 min) to create an inoculation medium in which the bacteria can grow and produce lactic acid. LAB provide plenty of CPP in the form of soluble complexes with Ca2+ that are effective in avoiding the life-threatening calcium phosphate precipitation, enhancing the intestinal absorption of minerals and retention in the human body [34,118]. The mineral-binding power of CPP also depends on the number of binding sites and their relative accessibility . Dairy products such as cheese and yogurt are both rich in multi-phosphorylated peptides capable of interacting with colloidal calcium phosphate to manifest anticariogenic properties in human and animal [120-121,13]. The mechanism of anticariogenicity might be due to a direct chemical effect of casein and calcium phosphate components . Tooth enamel is a polymeric substance consisting of crystalline calcium phosphate, embedded in a protein matrix. Thus, CPP can significantly enhance localization of ACP at the tooth surface, inhibiting enamel demineralization and promoting remineralization. In the development of teeth and bone, CPP act as hydroxyapatite nucleator and control the growth of the crystals, resulting in unique crystal morphology. A new calcium phosphate remineralization technology has been recently developed based on the complex CPP-ACP [RecaldentTM CASRN691364-49-5] . This preparation is claimed to stabilize calcium and phosphate ions in high concentrations by binding ACP to pellicle and plaque of the tooth surface. Moreover, CPP-ACP inhibited the adhesion of
6. CPP as nutraceutical ingredients in functional foods
6.1. Examples of commercial CPP preparation
Some commercial products have been developed containing moderately hydrolyzed milk proteins as the sole protein source. Hypoallergenic formulas are based on partially or extensively hydrolyzed proteins. Both formulae are better tolerated by small premature infants than native cow milk protein. Ultrafiltered micellar casein and microfiltered whey protein concentrate are known to slow down the digestive process. Typical composition data of commercial phosphopeptide preparations (m/m) include 91.3% (TN x 6.47) or 94.8 (dry basis) protein, 16.0% CPP, and 6.0% free amino acids. Compared with the expected CPP composition, the commercial preparation contained ~5% undigested protein, ~30% peptides in the intermediate molecular mass 5000-20000 Da range, and ~48% of molecular mass in the 500-5000 Da range (Tatua, New Zealand). The preparation is generally very complex and dependent on the procedure used to perform casein hydrolysis. Therefore, commercial products can be considered to be an enriched-CCP preparation containing 16% CPP without specification of the peptide size and phosphorylation degree. Preliminary analysis performed by MALDI-TOF analysis indicated that the signal in the mass spectra originated exclusively from the β-CN digestion. The intensity of non-CPP such as β-CN (f191-209), β-CN (f184-209), β-CN (f177-209) and β-CN (f170-209) was sufficiently high to obscure other CN peptides (MALDI spectrum not shown). As reported above, a number of available techniques allow the separation of CPP and non-CPP. To reduce the large dynamic range of non-CPP, HA was used for CPP enrichment [90,94,102]. CPP included multiply phosphorylated peptides (up to 4 phosphorylation residues). CPP β-CN (f1-28)4P and 3P, β-CN (f1-25)4P, 3P and 2P and β-CN (f2-25)4P, 3P and 2P were found and may be indicative of the progressive CPP dephosphorylation (MALDI spectrum not shown). Interestingly, in addition to β-CN, the commercial CPP (Tatua, NZ) also displayed αs1- or αs2-CN-derived CPP, invisible or weakly visible before the sample was treated with HA (Table 6).
Finally, a method for reducing the complexity of peptide mixtures was the separation of non-CPP and CPP by trapping CPP on HA under neutral conditions. This could be the principle for an industrially based production of CPP.
6.2. Examples of industrial methods for CPP preparation
Starting from CN, the overall preparation process gave 16% CPP (Figure 10), less than the theoretical yield of 23% (Ytheor =22.8%), which means approximately 20% yield on the basis of weight, a yield higher than that obtained by other researchers [138-140]. In their production experiments, the authors obtained a CPP preparation as high as 18.8% degree of hydrolysis (DH).
The example of 2000 L Na-caseinate solution containing 180 kg protein and 1 kg trypsin yielded 29 kg of calcium-enriched CPP, corresponding to a yield of ~16% (w/w) . A variety of raw materials such as acid casein, sodium caseinate, and calcium caseinate, as well as skimmed raw milk, milk concentrate by ultrafiltration, pasteurized and UHT milk near the limit date for consumption, may be used as the substrate for CPP production. The optimal parameters for the hydrolysis with trypsin were 37 °C and pH between 7.5 and 8.5. The casein hydrolysate solution was roughly fractioned by ultrafiltration with appropriate membranes to obtain soluble CPP both in the “permeate” and “retentate”. Lower-molecular peptides/phosphopeptides occurred in the “permeate” and larger in the “retentate”. The diafiltrate containing the tryptic casein digest was loaded on the ion exchange resin, and the non-CPP flowed through and were recovered for further use,
6.3. Traditional and new processes for the use of CPP in alimentary products
A Ca2+/ethanol selective precipitation procedure was used to produce a CPP and non-CPP concurrently from an alcalase digest of whole casein in which the traditional and new processes for CPP production were reported . CN is trypsinized, and the pH of the solution is adjusted to 4.6 to separate the non-peptide material. The CPP-Ca2+ aggregation was induced by ethanol addition in the supernatant and recovered as precipitate for freeze drying. In the novel process, the step of non-peptide material removal was omitted, and non-CPP (CNPP) was recovered as supernatant for use in alimentary products. For casein, the use of alcalase, a cheap enzyme suitable for industrial application, for hydrolysis was suggested . The CN hydrolysates were separated into the two types of peptides using combined treatment with CaCl2 and ethanol. CPP and non-CPP comprised components with molecular weight lower than 2509 Da and 2254 Da, respectively, as determined using size exclusion HPLC. A DH of 20% for the CN hydrolysate was achieved. At the end, the recovery of CPP reached 24%. The phosphorus component of CPP was 3.08%, and nitrogen recovery was approximately 76% . CPP generally had an improved solubility and transparency even under acid conditions and could be used as ingredient for beverages such as sport drinks, soft drinks, health drinks, fermented products, vitamin concentrates, fruit or fruit fractions.
6.4. Patented methods for CPP production as ingredients for alimentary products
A method for the preparation of selected anticariogenic CPP comprised the steps of complete digestion of CN as soluble monovalent cation salt with a proteolytic enzyme: the addition of a mineral acid to the solution to adjust the pH to approximately 4.7; the removal of any produced precipitate; the addition of CaCl2 to a concentration of approximately 1.0% (w/v) to cause the aggregation of CPP; the separation of the aggregated CPP from the solution through a filter with a molecular weight exclusion limit within the range 10000 to 20000 while passing the bulk of the remaining CPP in solution; the diafiltration of the separated CPP with water through a filter; the concentration of the solution; and the drying the retentate . Peptides not included in the aggregation were removed by ultrafiltration/diafiltration. By this means, anticariogenic CPP at purity greater than 90% were obtained . CPP including calcium, magnesium or both salts (or zinc, ferric or other salts) are produced by submitting CN to proteolytic enzyme hydrolysis, ultrafiltering the resulting hydrolyzate to produce a permeate containing CPP, adding a bivalent cation salt to the peptides to form CPP aggregates, and separating by ultrafiltration the CPP aggregates and non-CPP . When CPP salts need to be converted to free phosphopeptides, they can be restored by acidification with HCl; the solution is then diafiltered extensively through a 1000 molecular weight cut-off membrane to remove excess calcium chloride.
6.5. Example of CPP applications as nutraceuticals in functional foods
The CPP-salts complex can be added to different foods. A stable acidic beverage or other alimentary products can be obtained by digesting casein with trypsin, precipitating the insoluble components at acid pH, adjusting the pH of the obtained supernatant to approximately 6.0, then adding calcium chloride and ethanol to recover an acid-soluble calcium complex of CPP and the reaction product and adding them to a soft drink . The acid-soluble calcium complex of CPP enhances calcium absorption from food because calcium may be absorbed by the body in the form of soluble calcium. Acid-soluble CPP is a mixture of αs1-, αs2- and β-CPP, forming essentially no turbidity in solution at pH of 3.0 or less, and having purity greater than 90%, molecular weights between approximately 2500 and 4600 Da, and the ability to solubilize at least 100 ppm calcium at a concentration of 0.5 mg/ml CPP. The acid-soluble CPP produced
These inventions create functional foods with undoubted beneficial effects on human health, possibly promoting recalcification of bones, protecting the tooth enamel from decay and other possible health benefits.
Proteins are no longer considered merely nutritional components because they possess encrypted peptides with possible biological properties [149-153]. The cited literature has highlighted that bioactive peptides may be released
Publication in partial fulfilment of the requirements for PhD in Sciences and Technologies of Food Productions - XXV Cycle, University of Naples ‘Federico II’. The Authors gratefully acknowledge the American Journal Experts Association for the text revision (http://www.journalexperts.com/). This work was partly supported by the financial aid to C.L. from MIUR, Program PRIN-2008 HNHAT7-004.
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