Caseinomacropeptide Research Articles

A novel approach to isolation of β-casein from micellar casein concentrate by cold microfiltration combined with chymosin treatment

Filtration is frequently employed in the dairy industry to tailor protein profiles and manipulate the nutri- and techno-functionality of ingredients. One milk protein of particular interest in infant formula is β-casein. However, during the enrichment of β-casein, co-fractionation of κ-casein, as 2–10% of the total protein, is common. In this study, isolation of β-casein from micellar casein using filtration, incorporating chymosin-treatment, was investigated. At 5 °C, casein curd formation can be avoided, allowing the fractionation of liquid streams. Three treatments with chymosin were applied: feed without treatment and feed or permeate treated with chymosin. An average β-casein enrichment of ∼90% (w/w) total protein was achieved. Treatment of feed with chymosin resulted in caseinomacropeptide (CMP)-enrichment. CMP-enriched β-casein showed a wider particle size distribution at 50 °C, and a reduced level of solubility. The novel materials produced may be relevant in applications such as encapsulation, emulsification, infant formula and cheese manufacture.

Comparison of Solid-Phase Extraction Sorbents for Monitoring the In Vivo Intestinal Survival and Digestion of Kappa-Casein-Derived Caseinomacropeptide.

Kappa-casein-derived caseinomacropeptide (CMP)-a 64-amino-acid peptide-is released from kappa-casein after rennet treatment and is one of the major peptides in whey protein isolate (WPI). CMP has anti-inflammatory and antibacterial activities. It also has two major amino acid sequences with different modifications, including glycosylation, phosphorylation, and oxidation. To understand the potential biological role of CMP within the human body, there is a need to examine the extent to which CMP and CMP-derived fragments survive across the digestive tract, where they can exert these functions. In this study, three solid-phase extraction (SPE) methods-porous graphitized carbon (PGC), hydrophilic interaction liquid chromatography (HILIC), and C18 chromatography-were evaluated to determine which SPE sorbent is the most efficient to extract intact CMP and CMP-derived peptides from WPI and intestinal digestive samples prior to LC-MS/MS acquisition. The C18 SPE sorbent was the most efficient in extracting intact CMP and CMP-derived peptides from WPI, whereas the PGC SPE sorbent was the most efficient in extracting CMP-derived peptides from intestinal digesta samples.

Loss of UHT milk quality: changes in compositional and physicochemical parameters triggered by different storage conditions

The objective of this study was to evaluate some compositional and physicochemical changes that occur during UHT (Ultra-high temperature) milk storage for up to four months. A total of 120 samples of UHT milk were collected from two dairy plants in the state of Minas Gerais, Brazil, and stored under two temperatures (20°C and 30°C) during five periods (0, 30, 60, 90 and 120 days). The experimental design was completely randomized, in a 2x2x5 factorial arrangement, with six replicates per treatment (six batches). Evaluated parameters included fat, total protein, casein, lactose, total solids, solids nonfat (SNF) and milk urea nitrogen (MUN) contents, as well as acidity, density, freezing point and indexes of lipolysis and caseinomacropeptide (CMP). When compared to the Brazilian requirements for UHT milk, the mean values ​​for acidity, fat and SNF contents were in accordance with the standards ​​until the last day of storage. The other parameters are still not covered by the specific UHT milk legislation in Brazil. There was no change of relative density and lactose content also remained constant, except for a decrease at the fourth month. A significant increase, over storage time, in acidity, MUN, lipolysis and CMP indexes occurred. On the other hand, there was a significant decrease in SNF and protein contents. The results showed that there was a gradual loss of quality during UHT milk storage, aggravated by the rise in temperature. These findings demonstrate that UHT milk legislation could be improved by adding parameters such as lipolysis and CMP indexes.

Mass spectral profiling of caseinomacropeptide extracted from feeding material and jejunal fluid using three methods–ethanol precipitation, perchloric acid precipitation, and ultrafiltration

The ability of bovine κ-casein-derived caseinomacropeptide (CMP) to exert bioactivity in the human gut depends on its digestive survival. Sampling from the human jejunum after feeding CMP and top-down glycopeptidomics analysis facilitates the determination of CMP survival. To reduce interference from non-target molecules in mass spectrometric analysis, CMP must be isolated from digestive fluid. To identify an optimal extraction method, this study compared the profiles of CMP extracted from feeding material (commercial CMP in water) and digestive fluid by ethanol precipitation, perchloric acid (PCA) precipitation, and ultrafiltration. Ethanol precipitation yielded the highest ion abundances for aglycosylated CMP and glycosylated CMP in both feeding material and jejunal samples. Notably, PCA precipitation yielded the highest abundance of partially digested CMP-derived fragments in jejunal samples. Overall, ethanol precipitation was the most effective among the methods tested for intact CMP extraction from jejunal fluids, whereas PCA precipitation was optimal for extraction of CMP fragments.

Self-association of caseinomacropeptide in presence of CaCl2 at neutral pH: Calcium binding determination

The caseinomacropeptide (CMP) is a bioactive peptide produced during cheese making. It is found in abundance in whey. CMP aqueous solutions allow the incorporation of large amounts of CaCl2 but the mechanism of calcium-CMP interactions are unknown. In order to evaluate its calcium binding capacity, the following techniques were performed: Dynamic Light Scattering (DLS), Fourier Transform Infrared spectroscopy (FTIR), dialysis, conductivity, precipitation of CaCl2/CMP complex by ethanol, electrochemical Ca2+ binding isotherms, and inhibition of calcium phosphate precipitation. One mole of CMP can bind 9 mol of calcium, and the CMP self-assembles as a hexameric form. A model is proposed to explain the CMP self-association in presence of CaCl2.

Top-Down Glycopeptidomics Reveals Intact Glycomacropeptide Is Digested to a Wide Array of Peptides in Human Jejunum

BackgroundBovine milk κ-casein–derived caseinomacropeptide (CMP) is produced in large quantities during cheese-making and has various biological activities demonstrated via in vitro and in vivo experiments. Previous studies examined protein degradation and peptide release after casein or whey protein consumption. However, whether purified intact CMP that is partially glycosylated survives intact to its presumed site of bioactivity within the gut remains unknown. ObjectivesThe aim of this study was to determine the extent to which purified intact CMP (including glycosylated forms) is digested into peptide fragments within the jejunum of healthy human adults after consumption. MethodsJejunal fluids were collected from 3 adult participants (2 men and 1 woman, age: 27 ± 7 y; BMI: 23 ± 1 kg/m2) for 3 h after consuming 37.5 g of purified intact CMP. CMP and CMP-derived peptides were isolated from the collected jejunal fluids by ethanol precipitation and solid-phase extraction and identified by MS-based top-down glycopeptidomics. Relative abundances of CMP and CMP-derived peptides were compared qualitatively between the feed and the jejunal fluids. ResultsIntact CMP was dominant in feeding material, accounting for 90% of the total ion abundance of detected peptides, and in very low abundance (<2%) in the jejunal fluids. CMP-derived fragment peptides ranging from 11 to 20 amino acids in length were predominant (accounting for 68–88% of the total peptide ion abundance) in jejunal fluids during 1–3 h post consumption. ConclusionsThis study demonstrates that intact CMP (including glycosylated forms) is mostly digested in the human jejunum, releasing a wide array of CMP-derived peptide fragments. Some of the CMP-derived peptides with high homology to known bioactive peptides consistently survived across 3 h of digestion. Therefore, future research should examine the biological effects of the partially digested form—the CMP-derived fragments—rather than those of intact CMP.

Analysis of Bovine Kappa-Casein Glycomacropeptide by Liquid Chromatography-Tandem Mass Spectrometry.

Caseinomacropeptide (CMP) is released from bovine kappa-casein after rennet treatment and is one of the major peptides in whey protein isolate. CMP has in vitro anti-inflammatory and antibacterial activities. CMP has two major amino acid sequences with different modifications, including glycosylation, phosphorylation and oxidation. However, no previous work has provided a comprehensive profile of intact CMP. Full characterization of CMP composition and structure is essential to understand the bioactivity of CMP. In this study, we developed a top-down glycopeptidomics-based analytical method to profile CMP and CMP-derived peptides using Orbitrap mass spectrometry combined with nano-liquid chromatography with electron-transfer/higher-energy collision dissociation. The liquid chromatography–tandem mass spectrometry (LC–MS/MS) spectra of CMPs were annotated to confirm peptide sequence, glycan composition and other post-translational modifications using automatic data processing. Fifty-one intact CMPs and 159 CMP-derived peptides were identified in four samples (one CMP standard, two commercial CMP products and one whey protein isolate). Overall, this novel approach provides comprehensive characterization of CMP and CMP-derived peptides and glycopeptides, and it can be applied in future studies of product quality, digestive survival and bioactivity.

Formulation of a low-protein rice drink fortified with caseinomacropeptide concentrate

A rice drink with low protein and fat contents was developed by response surface methodology. Effects of three factors, levels of white rice flour (3–8 % w/v) and xanthan gum (0.01−0.05% w/v) and process temperature (80−90 °C) on physical stability of the drink were evaluated. Viscosity, phase separation, sedimentation, color, particle size and zeta potential of the samples were measured as response variables. Models were developed from the data to predict response variables from the factors evaluated. Models for viscosity, particle size, color, zeta potential and phase separation of the drink were found significant while that of sedimentation was not significant. Significant models were then used in optimization to achieve selected response variables, minimum phase separation, particle size and viscosity and zeta potential in range of values measured in commercial samples of milk-like drinks. Optimum conditions were found to be 3% rice flour and 0.05% xanthan gum in formula and process temperature of 80 °C applied for 15 min. Sucrose addition at a level of (2.5% w/v) to the formula increased acceptability of the drink in sensory analysis but it had no significant effect on physical properties of the rice drink. The drink was fortified with a caseinomacropeptide (CMP) concentrate for development of a milk-like drink for consumers with phenylketonuria. Fortification with CMP concentrate resulted in partial neutralization of zeta potential, slight sedimentation and an increase in particle size of the drink. Color and zeta potential of the drink became closer to those of low fat milk by the addition of CMP concentrate.

The influence of yak casein micelle size on rennet-induced coagulation properties.

Yak milk formed stronger rennet-induced gels if the milk contained smalled casein micelles and a higher concentration of calcium. Also casein gels could formed after a shorter incubation time if the milk contained smalled casein micelles. The objective of this study was to estimate the importance of yak casein micelle size on rennet-induced coagulation properties. Three fractions of different-sized, undamaged casein micelles (Ф112.17 ± 0.83 nm, Ф207.13 ± 0.59 nm and Ф269.37 ± 2.89 nm) were obtained by ultracentrifugation. The smallest casein micelles had the highest concentrations of calcium (803.21 ± 8.49 mM), phosphate (445.52 ± 10.66 mM), and κ-casein/total casein (19.45%). Rheological analyses determined the optimal gelation times of small, medium, and large casein micelles to be 9.5 ± 0.5, 10.8 ± 0.5, and 13.3 ± 0.2 min, respectively. Higher κ-casein concentration in the small casein micelles appeared to facilitate their shorter incubation time. Both the faster caseinomacropeptide (CMP) release rate and rennet-induced aggregation rate of small casein micelles contributed to a faster change in turbidity. Furthermore, small casein micelles had the highest elastic modulus (G', 73.21 ± 4.5 Pa) 60 min after the addition of rennet. This was consistent with micro-photographs, which showed that small casein micelles could form a more homogeneous gel, which had smaller pore sizes. Trial cheese manufacture verified that yak cheese containing small casein micelles, formed curd faster and the cheese had higher texture profile analysis (TPA) values for hardness, cohesiveness, and springiness. This is important information for the optimization of yak cheese industrial production. © 2020 Society of Chemical Industry.

Influence of chaperone-like activity of caseinomacropeptide on the gelation behaviour of whey proteins at pH 6.4 and 7.2.

The effect of caseinomacropeptide (CMP) on the heat-induced denaturation and gelation of whey proteins (2.5–10%, w/v) at pH 6.4 and 7.2, at a whey protein:CMP ratio of 1:0.9 (w/w), was investigated using differential scanning calorimetry (DSC), oscillatory rheology (90 °C for 20 min) and confocal microscopy. Greater frequency-dependence in the presence of CMP suggested that the repulsive interactions between CMP and the whey proteins affected the network generated by the non-heated whey protein samples. At pH 6.4 or 7.2, CMP increased the temperature of denaturation of β-lactoglobulin by up to 3 °C and increased the gelation temperature by up to 7 °C. The inclusion of CMP strongly affected the structure of the heat-induced whey protein gels, resulting in a finer stranded structure at pH 6.4 and 7.2. The presence of CMP combined with a lower heating rate (2 °C/min) prevented the formation of a solid gel of whey proteins after heating for 20 min at 90 °C and at pH 7.2. These results show the potential of CMP for control of whey protein denaturation and gelation.

Influence of desialylation of caseinomacropeptide on the denaturation and aggregation of whey proteins

The effect of the addition of caseinomacropeptide (CMP) or desialylated CMP on the heat-induced denaturation and aggregation of whey proteins was investigated in the pH range 3 to 7 after heating at 80°C for 30 min. The rate and temperature of denaturation, the extent of aggregation, and the changes in secondary structure of the whey proteins heated in presence of CMP or desialylated CMP were measured. The sialic acid bound to CMP favored the denaturation and aggregation of whey proteins when the whey proteins were oppositely charged to CMP at pH 4. A transition occurred at pH 6, below which the removal of sialic acid enhanced the stabilizing properties of CMP against the denaturation and aggregation of the whey proteins. At pH >6, the interactions between desialylated CMP and the whey proteins led to more extensive denaturation and aggregation. Sialic acid bound to CMP influenced the denaturation and aggregation behavior of whey proteins in a pH-dependent manner, and this should be considered in future studies on the heat stability of such systems containing CMP.

Ultrafiltration isolation and characterization of caseinomacropeptide

Biologically active peptides from milk whey, such as caseinomacropeptide (CMP), are of great interest for food technology and for the development of new functional foods. CMP is the soluble derivative of k-casein, generated by the cleavage of the enzyme chymosin enzyme in manufacture of cheese. The objective of this work was to isolate the CMP from whey and characterize. The method applied was the ultrafiltration membrane 50, 30 and 5 kDa. The fraction of CMP isolated consisted of 73.7% protein content, 12% lactose and 2.5% sialic acid. The fractions R30, R5R30 and SPD presented chromatographic profiles compatible with CMP by high performance liquid chromatography (HPLC). Process parameters and characteristics of the raw material can change the efficiency of CMP insolation.

Short communication: Application of proteomics for characterization of caseinomacropeptide isoforms before and after desialidation

Caseinomacropeptide (CMP) is an important polypeptide found in cheese whey that has various biological activities and functional properties. Because sialylations play an important role in the functional properties of CMP, the aim of the present work was to characterize CMP isoform heterogeneity in a commercial glycosylated CMP (gCMP) isolate using liquid chromatography- and gel-based proteomics before and after desialidation. Using 2-dimensional gel electrophoresis (2-DGE), we observed a shift in isoelectric point (pI) after enzymatic desialidation, indicating that sialylated gCMP were located at pI 3.0 to 3.1, but desialylated gCMP had repositioned to pI 3.7 to 3.9. We used liquid chromatography/mass spectroscopy (LC-ESI/MS) for further analysis of the glycan chains of gCMP. In total, we identified 19 CMP isoforms, of which 13 were glycosylated and 6 were nonglycosylated. We also detected 3 genetic variants (A, B, and E), with up to 3 glycosylations attached per gCMP. Further, we identified up to 4 isomers, reflecting different sites of glycosylation in the peptide backbone of these isoforms. The dominating gCMP isoform of genetic variant E appeared to contain 4 N-acetyl-neuraminic acid (NeuAc) residues, whereas the dominating gCMP isoforms of variants A and B appeared to contain 2 NeuAc. The identification revealed conversions of isoforms containing different combinations of genetic variants and degrees of glycosylation, sialylation, and phosphorylation to various desialylated counter-isoforms. The content of sialylated gCMP relative to the total CMP content was 37% (wt/wt), which decreased to 1.5% after sialidase treatment, indicating 96% effectivity of the desialidation. This approach can be valuable for future functionality studies specifically addressing the importance of NeuAc.

Rennet coagulation and calcium distribution of raw milk reverse osmosis retentate

Reverse osmosis (RO) filtration at the farm is a method for reducing transport costs; this study investigated cheese making properties of RO concentrated milk. In this study, several combinations of concentration factor and rennet concentration were examined based on caseino-macropeptide (CMP) release and rheological attributes of the coagulum. Furthermore, the calcium distribution of retentate compared with raw milk was included in the study, as a possible explanation of observed differences. The results showed a clear influence of the ratio between rennet concentration and retentate dry matter on CMP release rate and coagulation onset. The calcium distribution shifted to a larger fraction of colloidal calcium in the retentate samples compared with raw milk.

CHEMOMETRIC TOOLS AND FTIR-ATR SPECTROSCOPY APPLIED IN MILK ADULTERATED WITH CHEESE WHEY

Brazilian law forbids the addition of cheese whey in milk. However, adulteration with cheese whey is one of the most applied fraud due to its low cost. The detection of this fraud is the quantification of Caseinomacropeptide (CMP). The CMP is a constituent of the whey that can be used as adulteration marker. Thus, an analytical method capable of identifying CMP by Fourier Transform Infrared Spectra (FTIR) was developed using chemometrics methods. Firstly, we attempted to develop an exploratory analysis model by Hierarchical Grouping Analysis (HCA) and Principal Component Analysis (PCA) that indicated similarity between samples of raw milk and semi-skimmed milk. Moreover, in the PCA scores, it was possible to observe a tendency of separation between samples with different concentrations of CMP. Afterwards, multivariate regression models were used for Partial Least Squares (PLS), Partial Least Square with Interval Synergism (siPLS) and Supporting Machines with Least Squares (LS-SVM) to quantify the adulteration in different types of milk by Cheese serum through the CMP. All the models were then compared to each other and the results of the official method with Liquid Chromatography Tandem mass spectrometry (LCMS/MS) analysis used by the Ministry of Livestock and Supply (MAPA). The model LS-SVM, employing the full spectrum, obtained the best result compared to the other models (PLS and siPLS) to quantify the CMP in the milk samples.

Influence of calcium and sodium chloride on caseinomacropeptide self-assembly and flow behaviour at neutral pH

The effect of adding NaCl or CaCl2 on particle size distribution and flow behaviour of caseinomacropeptide (CMP) aqueous solutions was investigated over a wide range of concentrations (0–200 mmol L−1), temperatures (5–60 °C) and during 14 days of storage. In the absence of salts, CMP mainly presented a monomeric form at pH 7.0. If sodium or calcium chloride is added, hydrophobic associations of CMP is promoted due to screening of electric charges. Calcium chloride had a bigger impact than NaCl. Indeed, small variations in CaCl2 concentration induced major changes in size distributions and increased the viscosity and the cloudiness of CMP solutions upon storage. The largest aggregates and highest viscosities were obtained at concentrated regime (CMP concentration > 8 g/100 g), high CaCl2 concentration (>1.2 mmol g−1 CMP) and upon storage.

Flow properties of caseinomacropeptide aqueous solutions: Effect of particle size distribution, concentration, pH and temperature

The purpose of this research was to evaluate the influence of particle size distribution, pH (5.0–9.0), concentration (1–15 g/100 g.) and temperature (5–60 °C) on the steady shear flow properties of caseinomacropeptide (CMP) aqueous solutions. These measurements were carried out by using a controlled stress rheometer. Flow curves were satisfactorily fitted by the Herschel-Bulkley model. CMP solutions exhibited Newtonian flow dependence, particularly at pH values 5.0–6.0. Non-Newtonian shear thinning behaviour was observed at pH 7.0–9.0. The concentration dependence on viscosity showed two different regimes of viscosity increase (dilute and concentrated). The overlap concentration was 8 g/100 g. The temperature dependent behaviour of CMP solutions fitted to the Arrhenius model regardless pH and concentration, and the calculated activation energy was 20 kJ/mol. The flow behaviour of CMP is explained in terms of peptide-peptide and peptide-water interactions. Based on these results, CMP molecules would form spontaneously micelles at pH > 4.5 in ultrapure water.

Impact of the order of acid and heat treatments on the composition of caseinomacropeptide isolate

Effect of the order of acidification and heat treatment on recovery, purity and stability of caseinomacropeptide (CMP) from sweet whey was investigated. In acid-heat treatment, sweet whey was first acidified to pH values in the range of 3–5 and then heat treated at 90 °C for 1 h. In heat-acid treatment, applying the same treatment parameters, sweet whey was first heated and then acidified. Supernatants from both treatments were separated by centrifugation and analysed as CMP isolates. Reducing the pH of sweet whey from 5 to 3 resulted in a loss of CMP and retention of most of whey proteins in the isolates obtained by both treatments. Recovery and purity of CMP were highest at pH 5 for both treatments and at pH 4.5 for heat-acid treatment. Heat-acid treatment at pH 5 preserved more of bound sialic acid compared with other treatment combinations; this treatment is recommended for isolation of CMP.

Rennet-induced coagulation properties of yak casein micelles: A comparison with cow casein micelles

It is essential for yak cheese processing to understand the rennet-induced coagulation properties of gel formation from casein micelles. We have previously discovered that yak milk requires a longer incubation time but forms stronger gels compared with cow milk. In this study, we are aiming to understand the rennet-induced coagulation properties of yak casein micelles comparing with cow casein micelles. Rheological analyses revealed that the gelling times of yak and cow casein micelles were 11.6±0.5 and 8.7±0.4min (P<0.05) respectively, but yak casein gel had a higher elastic modulus G′ (6.5±0.2Pa) than cow casein gel (2.5±0.2Pa; P<0.05). This is consistent with the results obtained by micro-rheology. Confocal laser scanning microscopic images (CLSM) and cryo-scanning electron microscopic images (cryo-SEM) showed that yak casein gel was more homogeneous and had smaller pore size than cow casein gels. Yak casein micelles had higher calcium (26.00mM), phosphate (19.90mM) and β-casein (relative 32%) concentrations. In addition, yak casein micelles were larger (Z-average 218.6nm) than cow casein micelles, and contained lower κ-casein (relative 13%). By comparison with cow casein micelles, yak casein micelle composition corresponding to their micellar calcium phosphate and κ-casein content may greatly contribute to the longer coagulation time and denser gel structure. An initial slower caseinomacropeptide (CMP) release rate and the slower rate of aggregation between para-casein micelles contributed to a more homogeneous yak gel network. Higher colloidal calcium phosphate is crucial for yak casein micelle aggregation and gel firmness because sufficient colloidal calcium phosphates can firmly glue sub-micelles and links casein micelles. This study provides valuable information for yak cheese production.

Inulin increases Bifidobacterium animalis Bb-12 in vitro gastrointestinal resistance in margarine

This study aimed to investigate the effect of inulin, whey protein concentrate (WPC), and/or caseinomacropeptide (CMP), in different proportions up to 3%, on the viability and resistance to simulated gastric and enteric conditions of Bifidobacterium animalis Bb-12 added in probiotic and synbiotic margarine, during 35 days of storage at 5 °C. Supplementation was important, since the control margarine presented very low Bb-12 populations. Inulin at 3% resulted in higher Bb-12 counts (8 log cfu g−1). CMP contributed for higher Bb-12 counts, compared to WPC. Inulin increased Bb-12 in vitro survival significantly after 6 h (P<0.05). In margarines with WPC at 3%, Bb-12 populations decreased drastically during the in vitro assays for all storage periods. For the other formulations, Bb-12 populations decreased 2 log cfu g−1 after 2 h. Taken together, results on viability of B. animalis Bb-12 and on in vitro GI resistance of the strain incorporated in margarine produced with inulin were unique, but the addition of inulin was necessary.