Casein Macropeptide Research Articles

Targeted detection and quantification of casein macropeptide (variant A) by liquid chromatography-tandem mass spectrometry

Casein macropeptide (CMP) is an ideal ingredient for infant formula as well as special medical foods for patients suffering from phenylketonuria, but its large molecular mass and diversified glycosylation bring obstacles to its quantification in these products. Herein, we carried out enzymatic digestion simulation combined with database search and three proteotypic peptides with no glycosylation sites from CMP were screened out to represent CMPA isoform, one of the main variants of CMP, and avoid differences caused by glycosylation. The intensity of primary mass spectrometry, specificity (homology of peptide sequences), and degree of hydrolysis of three proteotypic peptides were evaluated, and target peptide 2 (147–152, TP2) was finally chosen as the quantitative peptide. A linear range of 2 ng/mL-2 μg/mL and a detection limit of 0.02 ng/mL for CMP was achieved by HPLC-MS/MS with isotope dilution. The recoveries of spiked TP2 in CMP hydrolysate were between 70% and 120%, and the strategy worked well in commercial formula samples.

Adsorption of flexible proteins in the ‘wrong side’ of the isoelectric point: Casein macropeptide as a model system

We analyze the conditions of the adsorption of a flexible peptide onto a charged substrate in the ‘wrong side’ of the isoelectric point (WSIP), i.e. when surface and peptide charges have the same sign. As a model system, we focus on the casein macropeptide (CMP), both in the aglycosylated (aCMP) and fully glycosydated (gCMP) forms. We model the substrate as a uniformly charged plane while CMP is treated as a bead-and-spring model including electrostatic interactions, excluded volume effects and acid/base equilibria. Adsorption coverage, aminoacid charges and concentration profiles are computed by means of Monte Carlo simulations at fixed pH and salt concentration. We conclude that for different reasons the CMP can be adsorbed to both positively and negatively charged surfaces in the WSIP. For negatively charged surfaces, WSIP adsorption is due to the patchy distribution of charges: the peptide is attached to the surface by the positively charged end of the chain, while the repulsion of the surface for the negatively charged tail is screened by the small ions of the added salt. This effect increases with salt concentration. Conversely, a positively charged substrate induces strong charge regulation of the peptide: the acidic groups are deprotonated, and the peptide becomes negatively charged. This effect is stronger at low salt concentrations and it is more intense for gCMP than for aCMP, due to the presence of the additional sialic groups in gCMP.

Functionality of MC88- and MPC85-Enriched Skim Milk: Impact of Shear Conditions in Rotor/Stator Systems and High-Pressure Homogenizers on Powder Solubility and Rennet Gelation Behavior.

Milk protein concentrate (MPC) and micellar casein (MC) powders are commonly used to increase the protein concentration of cheese milk. However, highly-concentrated milk protein powders are challenging in terms of solubility. The research question was whether and how incompletely dissolved agglomerates affect the protein functionality in terms of rennet gelation behavior. For the experiments, skim milk was enriched with either MC88 or MPC85 to a casein concentration of 4.5% (w/w) and sheared on a laboratory and pilot scale in rotor/stator systems (colloid mill and shear pump, respectively) and high-pressure homogenizers. The assessment criteria were on the one hand particle sizes as a function of shear rate, and on the other hand, the rennet gelation properties meaning gelling time, gel strength, structure loss upon deformation, and serum loss. Furthermore, the casein, whey protein, and casein macropeptide (CMP) recovery in the sweet whey was determined to evaluate the shear-, and hence, the particle size-dependent protein accessibility. We showed that insufficient powder rehydration prolongs the rennet gelation time, leading to softer, weaker gels, and to lower amounts of CMP and whey protein in the sweet whey.

The factors influencing rennet-induced coagulation properties of yak milk: The importance of micellar calcium during gelation

The objective of this study was to evaluate the importance and effects of age, hair color, lactation stage and number of yak, as well as physicochemical properties and composition of yak milk on rennet-induced coagulation characteristics. Results showed that casein micelle size distribution and total calcium content of milk from 68 yaks were the most important factors affecting the gelation characteristics, including gelling time and gel strength. The yak milk containing smaller casein micelles and higher calcium content formed stronger gels and had shorter incubation time. Moreover, casein micelle samples containing 20.6, 16.8, and 13.9 mmol/L micellar calcium were obtained through pH-induced dialysis method. The results clearly showed that although the removal of micellar calcium did not affect casein macropeptide (CMP) release, the gelation of samples containing lower micellar calcium value corresponded to lower aggregation rate of para-casein micelles and more CMP release. In addition, removal of micellar calcium may lead to longer incubation time and sparser gel structure. This study highlights that higher micellar calcium is indispensable to the aggregation of yak casein micelles and the formation of firm gel, because enough micellar calcium may promote the aggregation of para-casein and firmly links casein micelles.

Microbiological, chemical, physical, and proteolytic activities of raw milk after thermal processing

ABSTRACT The aim was to evaluate the microbiological, chemical- physical, and shelf-life quality of milk samples after pasteurization (HTST) for 10 days or ultra-high temperature (UHT) treatment for 120 days. Raw milk counts of mesophilic aerobic microorganisms, Staphylococcus spp. and thermotolerant coliforms before HTST and UHT processing were 6.73 and 7.77; 2.84 and 4.30, and 4.68 and 4.37log10, respectively. Pseudomonas spp. were found in raw milk samples. No presence of any other microorganisms studied was detected and no microbial inhibitor was found. Processed samples met microbiological legal requirements. However, aerobic mesophilic counts for HTST pasteurized milk samples stored for 5 and 10 days increased to values comparable to those in raw milk. Composition chemical- physical of all samples were within legal limits. These results demonstrate that, although HTST and UHT processed milk comply with the microbiological standards required by Brazilian law, high microbial counts in raw milk are an issue, possibly due to failures in the early stages of the production chain. Increase in casein macropeptide (CMP), probably because of proteases psychrotrophic bacteria. It is concluded that the quality of raw milk directly influences the progressive increase of the CMP values.

Consumer profile and problems associated with uninspected raw milk consumption in western Paraná

ABSTRACT: The objectives of this study were to analyze consumer profile; to identify the main reasons for raw milk consumption; and to analyze in laboratory samples of uninspected raw milk from five towns in the western region of the state of Paraná, Brazil. The types of milk most frequently consumed were: 42.3% ultra-high temperature (UHT), 38.3% pasteurized milk, 17.6% uninspected raw milk, and 1.7% powered milk. The frequencies of households that preferred uninspected raw milk were, according to the town, 32.7% in Iporã, 29.2% in Marechal Cândido Rondon, 18.9% in Assis Chateaubriand, 17.6% in Palotina, and 10% in Toledo. Flavor was the main reason for uninspected raw milk consumption, and the purchase of this product was more frequent in households whose income was between one to four minimum wages. It was observed that the sales of uninspected milk are more financially advantageous to the producer than sales of inspected milk. All samples analyzed showed lack of compliance with at least one parameter, 60.9% for mesophilic counts, 56.6% for non-fat dry matter, 52.1% for freezing point, 43.5% for acidity, 23.9% for density, 23.9% for the casein macropeptide, 17.4% for fat content, 8.7% were reactors in the milk ring test, and 2.2% were reactors in microbial growth inhibitor test. Fraud by addition of water was observed in 20% of the samples. Uninspected raw milk analyzed in this study involved a low-quality product that is a financial hazard as it may be adulterated, and that poses risk to consumer health.

The Promising Efficacy of Probiotics, Casein Phosphopeptide and Casein Macropeptide as Dental Anticariogenic and Remineralizing Agents Part I; An In vitro Study

The Promising Efficacy of Probiotics, Casein Phosphopeptide and Casein Macropeptide as Dental Anticariogenic and Remineralizing Agents Part I; An In vitro Study

The inhibitory roles of native whey protein on the rennet gelation of bovine milk

Rennet gelation is used to produce many types of cheese. The effect of native whey protein on rennet gelation kinetics was investigated. Milks with a wide range of whey protein:casein (WP:CN) ratios (with standardised casein concentrations) were made from powders produced by microfiltration. Measurements of casein macro peptide release showed that native whey protein inhibited the enzymatic action of chymosin, which delayed the onset and reduced the subsequent rate of gelation. Experiments in which increased chymosin concentrations compensated for the inhibition, demonstrated that other factors also contributed to the reduced gelation rate. Neither an increase in viscosity nor a reduction in soluble calcium was responsible, leading to the conclusion that in addition to inhibiting chymosin, native whey proteins present a physical barrier to para-casein aggregation. This study demonstrates and explains how casein-enriched retentates from microfiltration gel faster than regular cheese milk that contains higher amounts of native whey protein.

Effect of soluble calcium on the renneting properties of casein micelles as measured by rheology and diffusing wave spectroscopy

Addition of calcium chloride to milk has positive effects on cheese-making because it decreases coagulation time, creates firmer gels, and increases curd yield. Although addition of calcium chloride is a widely used industrial practice, the effect of soluble calcium on the preliminary stages of gelation is not fully understood. In addition, it is not known whether the manner of addition and equilibration of the soluble calcium would affect the rennetability of the casein micelles. Therefore, the aim of this paper was to study the details of the coagulation behavior of casein micelles in the presence of additional calcium, and to elucidate whether the manner in which this cation is added (directly as calcium chloride or by gradual exchange through dialysis) affects the functionality of the micelles. Calcium was added as CaCl2 (1mM final added concentration) directly to skim milk or indirectly using dialysis against 50vol.mes of milk. Additional soluble calcium did not affect the primary phase of the renneting reaction, as demonstrated by the analysis of the casein macropeptide (CMP) released in solution; however, it shortened the coagulation time of the micelles and increased the firmness of the gel. The turbidity parameter of samples with or without calcium showed that similar amounts of CMP were needed for particle interactions to commence. However, the amount of CMP released at the point of gelation, as indicated by rheology, was lesser for samples with added calcium, which can be attributed to a greater extent of calcium bridging on the surface or between micelles. The results also showed that the manner in which calcium was presented to the micelles did not influence the mechanism of gelation.

New insights into the immunological effects of food bioactive peptides in animal models of intestinal inflammation

Bioactive peptides have proven to be active in several conditions, including inflammatory bowel disease (IBD). This is a chronic and relapsing condition of unknown aetiology that comprises chiefly ulcerative colitis and Crohn's disease. Although there are treatments for IBD, they have frequent side effects and they are not always effective; therefore there is a need for new therapies that could alleviate this condition. Two bioactive peptides present in milk (transforming growth factor-beta (TGF-beta) and casein macropeptide, also named glycomacropeptide) have been shown to have intestinal anti-inflammatory activities. In fact, TGF-beta is currently added to formulas intended for patients with IBD, and several studies indicate that these formulas could induce clinical remission. In this paper, evidence supporting the anti-inflammatory effect of TGF-beta and bovine glycomacropeptide, as well as their mechanisms of action, is reviewed, focusing on the evidence obtained in animal models.

Rheological Properties of Rennet Gels Containing Milk Protein Concentrates

Different milk protein concentrates (MPC), with protein concentrations of 56, 70, and 90%, were dispersed in water under different treatments (hydration, shear, heat, and overnight storage at 4°C), as well as in a combination of all the treatments in a factorial design. The particle size distribution of the dispersions was then measured to determine the optimal conditions for the dispersion. Heating at 60°C for 30min with 5min of shear was chosen as the best condition to dissolve MPC powders. The samples were also characterized for composition, presence of protein aggregates, and ratio of calcium to protein. The total calcium present in MPC increased with increasing concentration of protein; however, the total calcium-to-protein ratio was lower in MPC90 than in MPC56 and MPC70. The level of whey protein denaturation, the presence of κ-casein-whey protein aggregates in the supernatant after centrifugation, and the amount of caseins dissociated from the micelle increased as the protein concentration in the powder increased. The total amount of casein macropeptide released was lower in samples from powders with a higher protein concentration than for MPC56 or the skim milk control. The gelation behavior of reconstituted MPC was tested in systems dispersed in water (5% protein) as well as in systems dispersed in skim milk (6% protein). The gelation time of MPC dispersions was considerably lower and the gel modulus was higher than those of reconstituted skim milk with the same protein concentration. When MPC dispersions were dialyzed against skim milk, a significant decrease in the gelation time and modulus were shown, with a complete loss of gelling functionality in MPC90 dispersed in water. This demonstrated that the ionic equilibrium was key to the functionality of MPC.

Rennet-induced coagulation of enzymatically cross-linked casein micelles

The enzymatic cross-linking of casein micelles with transglutaminase had an adverse influence on rennet-induced coagulation. Incubation with transglutaminase at 30 °C progressively reduced the levels of monomeric caseins and increased rennet flocculation time (RFT) in a Berridge test. For incubation up to 3 h at 30 °C, the reciprocal of the RFT was linearly correlated with the level of residual monomeric κ-casein, indicating that at complete cross-linking flocculation is absent. After treatment for 4–24 h at 30 °C, no residual monomeric κ-casein was detected and no rennet-induced flocculation of the casein micelle suspension was observed. Monitoring rennet-induced coagulation by diffusing wave spectroscopy revealed that transglutaminase-induced inhibition of rennet-induced coagulation of casein micelles is primarily due to an inhibition of the secondary phase of rennet coagulation, i.e., the gelation and gel-firming phase of the casein micelle coagulation. The gelation and fusion of κ-casein-depleted para-casein micelles as in normal milk appears to be absent if the casein macropeptide remains attached to the casein micelle.

Glycation of caseinmacropeptide

Glycation of caseinmacropeptide (CMP) during storage with lactose at 40 and 50 °C and water activity 0.33–0.65 was studied by measurement of furosine. At pH 8.0 and a w0.44, maximum levels of furosine up to 1.9 mg/100 mg CMP were obtained within five days at 50 °C, while this value had not been reached at 40 °C after 13 days of storage. Increasing pH up to 11 caused considerable increase in the rate of furosine formation and maximum values were obtained after 9 h at 50 °C. The rate of furosine formation was also enhanced with increasing a w up to 0.65. These results showed that lactosylated CMP can be efficiently prepared during short time storage of CMP–lactose mixtures under appropriate pH, water activity and temperature conditions.

Impaired Rennetability of Heated Milk; Study of Enzymatic Hydrolysis and Gelation Kinetics

Casein micelles in milk are stable colloidal particles with a stabilizing hairy brush of κ-casein. During cheese production rennet cleaves κ-casein into casein macropeptide and para-κ-casein, thereby destabilizing the casein micelle and resulting in aggregation and gel formation of the micelles. Heat treatment of milk causes impaired clotting properties, which makes heated milk unsuitable for cheese production. In this paper we compared five different techniques, often described in the literature, for their suitability to quantify the enzymatic hydrolysis of κ-casein. It was found that the technique is crucial for the yield of casein macropeptide and this yield then affects the calculated enzymatic inhibition caused by heat treatment, ranging from 5 to 30%. The technique, which we found to be the most reliable, demonstrates that heat-induced calcium phosphate precipitation does not affect the enzymatic cleavage, while whey protein denaturation causes a very slight reduction of enzyme activity. By using diffusing wave spectroscopy, a very sensitive technique to monitor gelation processes, we demonstrated that heat-induced calcium phosphate precipitation does not affect the clotting. Whey protein denaturation does not affect the start of flocculation but has a clear effect on the clotting process. This work adds to a better understanding of the processes causing the impaired clotting properties of heated milk.

Isolation and characterisation of caseinmacropeptide from bovine, ovine, and caprine cheese whey

Based on the thermostability of caseinmacropeptide (CMP) and on the differences in molecular weight of its polymeric and monomeric forms, we have developed a method of isolating CMP from whey protein concentrate (WPC) and from liquid sweet cheese whey, particularly suited to large-scale industrial production. This procedure includes acidification and heating and ultrafiltration of cheese whey to give a CMP powder with a protein content from 75 to 79%. CMP obtained from WPC and from pure bovine, ovine, and caprine cheese whey were characterized. The CMP recovery was close to 71–76% and the purity determined by RP-HPLC ranged from 75 to 90%.

Characterization of peptides produced by the action of psychrotrophic proteinases on kappa-casein.

Psychrotrophic bacteria, capable of growing in milk during refrigerated storage before processing, produce thermostable proteinases that strongly influence the keeping quality of long life dairy products (Cousin, 1982). This is the case with UHT milk, where the activity of residual or reactivated bacterial proteolytic enzymes is linked to flavour deterioration and gelation. Furthermore, since most of these enzymes are acid proteinases with a broad specificity and particularly active against κ-casein (Fairbairn & Law, 1986), their hydrolysis products can interfere with detecting the fraudulent addition of rennet whey (López-Fandiño et al. 1993a, b; Recio et al. 1996, 2000). This is usually based upon detecting, by reversed-phase (RP-) HPLC (Olieman & van Riel, 1989; Van Riel & Olieman, 1995a) or capillary electrophoresis (CE) (Van Riel & Olieman, 1995b), caseinmacropeptide (CMP), the 106–169 fragment of κ-casein released by chymosin during the initial stages of cheesemaking (for review, see López-Fandiño & Olano, 1999). Nevertheless, it is not yet clear whether degradation of κ-casein by psychrotrophic enzymes gives rise to hydrolysis products that are identical to those produced by chymosin or just indistinguishable by CE (Van Riel & Olieman, 1995b; Recio et al. 1996).In the present investigation, we studied CMP-like degradation products produced by the action of extracellular psychrotrophic proteinases on κ-casein by RP-HPLC and CE. Electrospray ionization–mass spectrometry (ESI–MS) was used for peptide identification. Characterization of breakdown products specific to psychrotrophic enzymes may help the investigation of their residual activity remaining in milk after processing, as well as provide an indication of the suitability of CMP as an indicator of adulteration of UHT milk with whey solids.

Detection of rennet whey solids in UHT milk by capillary electrophoresis

The fraudulent presence of rennet whey solids in UHT milk was studied by capillary electrophoresis (CE). Commercial UHT samples of different origins, genuine milk samples and milk samples adulterated with rennet whey were analysed. Linear discriminant functions using ratios of peak areas of caseinmacropeptide (CMP) and two other CMP-like degradation products were defined. The interference of proteolysis in the detection was estimated in samples adulterated on purpose with rennet whey, UHT treated in a pilot plant, and stored at 10, 20 and 30°C for up to 150 days. The application of the classification functions obtained allowed the detection of rennet whey solids added to milk. Only interferences due to very severe proteolysis, occurring after very long storage periods and/or at storage temperatures above room temperature, were observed.

Apparent chemical composition of nine commercial or semi‐commercial whey protein concentrates, isolates and fractions

Summary Analytical results are given for whey powders prepared on a commercial or semi‐commercial scale by three companies. Altogether, five preparations enriched in β‐lactoglobulin, four whey protein isolates and a fraction enriched in α‐lactalbumin were analyzed for protein composition, including %β‐lactoglobulin, α‐lactalbumin, bovine serum albumin, casein (glyco) macropeptide and the main triglycerides. Protein composition was determined by high pressure gel permeation and reversed phase liquid chromatography and by capillary zone electrophoresis. The extent of modification of the native β‐lactoglobulin structure was also measured through the degree of lactosylation and the fraction of accessible free sulphydryl groups. One significant finding was that the calculated recovery of protein following quantitation of the chromatogram or electropherogram was seldom above 90% and occasionally below 60% of that loaded onto the column or capillary, raising doubts as to the reliability of the analytical results. Extrapolation by linear regression to 100% recovery allowed estimates to be made of the true β‐lactoglobulin composition of the samples. The nine samples could be placed into three distinct groups with estimated true β‐lactoglobulin weight % of 70.9 ± 1.1, 62.0 ± 3.4 and 39.5 ± 4.9. Physico‐chemical properties of the group of samples are reported elsewhere (Holt et al., 1999).

Characteristics and potential uses of the casein macropeptide

The casein macropeptide (CMP) is a heterogeneous fraction of peptides formed by the action of rennet on casein and more specifically κ-casein. This fraction has unique composition and characteristics. Several methods have been developed for the determination of CMP in order to follow the action of chymosin on milk and κ-casein or to detect the presence of whey solids in milk powders. The CMP or its tryptic digest was reported to have diverse biological activities and some of these have been used as a base for medicinal preparations. Also, the unique amino acid composition and functional properties of the CMP suggest that it can be incorporated in some dietetic foods. Methods based on ion exchange chromatography and ultrafiltration have been used for large-scale preparation of CMP with either chymosin-treated casein, caseinates or rennet whey as a starting material.

Hydrolysis of κ-casein in solution by chymosin, plasmin, trypsin and Lactobacillus -proteinases

The aim of this study was to examine the enzymatic hydrolysis of κ-casein by isolating and identifying the released peptides. The enzymes employed in the study were chymosin, plasmin and trypsin, as well as a cell-free extract from three Lactobacillus helveticus and nine Lactobacillus casei strains. The findings showed that the bond most sensitive to the proteolytic activity of chymosin was the Phe 105-Met 106. After 24 hours of hydrolysis a few other bonds in the casein macropeptide were also cleaved. Plasmin was found to have weak proteolytic activity under the conditions of this study. When the enzyme-substrate ratio was raised from 1:200 to 1:50, a few peptides were released from the N-terminal region. Trypsin was found to hydrolyze several κ-casein bonds, and peptides were released from almost all regions of the protein. The proteases of Lactobacillus had less effect than chymosin, plasmin or trypsin. The strains could be divided into three categories. L. helveticus strains had activity on bonds in the mid-section and C-terminal region, L. casei strains EB, P3, P8 and A 1 had activity on bonds in the N- and C-terminal regions, while L. casei A5 and M9 had activity only on bonds in the C-terminal region.