Caseinomacropeptide Research Articles

Influence of preliminary treatments on structural properties of casein micelles affecting the rennetability

Summary - Different reconstituted skimmed milk powders, as weil as defatted pasteurized and raw milk, were used to probe the effects of preheating conditions on rennetability and molecular availability of casein to coagulate and to form a curd. The experiments for following coagulation and gel formation were carried out using the formagraph at pH 6.4, 35 C, and 3.8% total protein. Two new approaches to estimate the surface protein hydrophobicity (SPH) and to quantify the glycosylated and non-glycosylated caseinomacropeptide (CMP) were used to study the molecular state of casein micelles during the primary and secondary phases of renneting. The latter method was supported by ion-exchange fast-protein liquid chromatography (FPLC) using Mono-Q and Mono-S coJumns. Results with raw milk showed that the SPH of native micellar casein is about eight times higher than that of the individual casein components. In processed milks, the range of maximum SPH decreased as the severity of preheating increased. Il was found that a high SPH is correlated with an optimum micellar structure. The latter is progressively lost by association of denatured ~-Iactoglobulin (~-Lg) on the micellar surface, especially with the outer part of non-glycosylated K-casein (K-Cn). The extent of association is related to the severity of preliminary milk treatment, which may be measured by a reduced liberation of non-glycosylated CMP.

Microparticle-enhanced nephelometric immunoassay for caseinomacropeptide in milk

SummaryA microparticle-enhanced nephelometric immunoassay has been developed for the determination of caseinomacropeptide (CMP) in bovine milk. It is based on the nephelometric quantification of the competitive immunoagglutination of a microparticle–CMP conjugate with an anti-κ-casein (κ-CN) antiserum. This one step immunoassay was sensitive (detection limit in reaction mixture, 16μg/l), accurate (linear recovery of CMP in dilution overloading) and reproducible (CV 7–14% for within and between run precision). Because of the specificity of the polyclonal antiserum used, it was necessary to separate CMP from κ-CN by ultrafiltration before the quantification of bovine milk CMP. Under the conditions of milk ultrafiltration used, κ-CN was entirely retained (> 99·5%) but the concentration of CMP measured in milk ultrafiltrates was underestimated (by ∼25%) compared with its concentration in whole milk. Microparticle-enhanced nephelometric immunoassay of CMP, with a calibration range from 0·32 to 20 mg/1 for 20- fold diluted milk ultrafiltrate, allowed contamination of bovine milk by rennet whey as low as 5 ml/1 to be detected. Applied to ultrafiltrates from milk stored at 4 °C, this immunoassay also detected proteolysis of κ-CN not revealed by measurement of κ-CN concentration in milk. A statistical lower limit of 3·21 mg/1 was determined as the increase in CMP concentration in milk ultrafiltrates that indicated probable κ-CN proteolysis in the milk sample. Previously demonstrated to be an easy to perform method for assaying the main proteins of bovine milk, microparticle-enhanced nephelometric immunoassay thus also appeared to be appropriate to quantify CMP so as to detect slight contamination of milk by whey and to indicate the proteolysis of κ-CN during milk storage at low temperature.

Study of the Formation of Caseinomacropeptides in Stored Ultra-High-Temperature-Treated Milk by Capillary Electrophoresis

The proteolytic activity of enzymes of psychrotrophic bacteria on casein and raw and ultra-high-temperature (UHT)-treated milk was studied by capillary electrophoresis (CE) in order to characterize the degradation products and evaluate the possibility of distinguishing them from those due to the action of chymosin on K-casein. Casein hydrolysates with Pseudomonas fluorescens B52 proteinase, milk inoculated with P. fluorescens, and commercial UHT milks were studied. The degradation products included caseinomacropeptide (CMP), pseudo-caseinomacropeptide (pseudo-CMP; CMP lacking the 106 Met residue) and an unidentified third peak. These peaks were already present in some freshly prepared UHT milk samples, and a progressive increase in peak area was observed upon storage at 22 °C. This might give rise to false positive results when the presence of rennet whey is investigated by CE. However, low area ratios of pseudo-CMP to CMP might allow the presence of rennet whey solids to be suspected.

Continuous hydrolysis of caseinomacropeptide in a membrane reactor: kinetic study and gram-scale production of antithrombotic peptides

Summary - A recycle ultrafiltration membrane reactor was used for continuous production of antithrombotic peptides from enzymatic digestion of caseinomacropeptide (CMP). The kinetic parameters of the continuous process were determined on hydrolysis of various substrate concentrations and compared to those of the batch reactor. On the basis of peptide Iiberation, enzymatic activity was 10-fold lower during the continuous process (according to kcatvalues) while the Km value was not modified. Taking into account these data, an experiment for bioactive peptide production from continuous hydrolysis of 34 9 of CMP was carried out. Alter 6 h of continuous operation, permeate was concentrated by nanofiltration (volume concentration ratio = 12.5), freeze-dried, and the productivity as weil as peptide yield determined. Although desired peptides were partially rejected by the ultrafiltration membrane du ring the continuous process, 2.65 9 of these small molecules (500 to 780 Da) were obtained in the final powder.

Detection of proteolysis in raw milk stored at low temperature by an inhibition ELISA

SummaryAn inhibition ELISA (enzyme-linked immunosorbent assay) was developed for the determination of caseinomacropeptide (CMP) in order to estimate the proteolysis of κ-casein due to the enzymes of psychrotrophic bacteria in bulk raw milk. The CMP present in milk was quantified specifically by an antibody. The limit of detection was ∽ 0·1 μg/ml and the CV was < 10%. This method was used to study the proteolytic activity of three strains of psychrotrophic Pseudomonas fluorescens in raw milk and to analyse different raw milk samples supplied by four dairy plants. The proteolytic activity for different strains of psychrotrophs and for different milk samples varied considerably, but no correlation was established between the level of microbial flora and κ-casein proteolysis. It is thus not possible to determine the extent of proteolysis from the bacterial count alone. However, by CMP determination in bulk raw milk samples after 6 d storage at 4°C, the mean κ-casein proteolysis was ∽ 4%. Among the milk samples analysed that contained < 107 cfu psychrotrophs/ml, 30% exhibited a proteolysis of κ-casein < 0·5%, i.e. < 5μg CMP/ml.

Effects of caseinomacropeptide (CMP) on digestion regulation.

Caseinomacropeptide (CMP) is a 64-amino-acid-residue peptide which is released from kappa-casein by gastric proteinases. This review sums up the knowledge concerning its effects on the digestive function. Part 1 recalls the origin and heterogeneity of CMP. Here we underline that there are various forms of CMP which differ by their glycosylation level and genetic mutation. Consequently the forms used for studying biological activities need to be defined accurately. Part 2 summarizes the effects of CMP on digestive secretions. The major effect is an inhibitory effect on acid gastric secretions. Simultaneously, the blood concentration of regulatory digestive peptides is modified. In part 3 we try to clarify the mechanisms of action of CMP. A slightly glycosylated form of CMP, the A variant, appears to be responsible for the biological activity. Evidence suggests that CMP triggers stimuli from intestinal receptors without being absorbed. The signal would be then transmitted to organs through regulatory digestive peptides.

Effect of caseinomacropeptide (CMP) on cholecystokinin (CCK) release in rat

Effect of caseinomacropeptide (CMP) on cholecystokinin (CCK) release in rat

Effect of caseinomacropeptide (CMP) on gastric secretion and plasma gut regulatory peptides in preruminant calves

Effect of caseinomacropeptide (CMP) on gastric secretion and plasma gut regulatory peptides in preruminant calves

Specificity and kinetics of the milk-clotting enzyme from cardoon (Cynara cardunculus L.) toward bovine .kappa.-casein

The action of Cynara cardunculus L. protease on whole bovine K-casein, over a 3-h period at pH 6.4, was investigated. RpHPLC of the 3 % trichloroacetic acid (TCA)-soluble fraction of the K-casein digestion mixture showed three peptide peaks, which were identified by amino acid analysis and N-terminal analysis as the 106-169 fragment [caseinomacropeptide (CMP)] . Upon selective precipitation with 12% TCA, one glycosylated and two nonglycosylated forms of CMP were distinguished. Analysis of the whole digestion mixture showed no additional peptides. The kinetics of hydrolysis of the PhelO5Met106 bond was studied by spectrofluorometry, using fluorescein isothiocyanate-labeled K-casein (FTCK-casein). The values obtained for kat, k,, and ka&, were 1.04 s-l, 0.16 pM, and 6.5 pM-l s-l, respectively. The proteolytic coefficient is of the same order of magnitude as those obtained for other milk-clotting enzymes, but the k, is significantly lower, which reflects the higher affinity of Cynara protease to K-casein.

Characterization of caseinomacropeptides released from renneted raw and UHT treated milks

The kinetics of release of the glycosylated and carbohydrate-free forms of caseinomacropeptide (CMP) was studied in renneted rawand UHT (140 oCfor 10 s) milks. The new chromatographic method utilized allowed quantitative determinations of both molecular torrns. UHT treatment leads to a 40% decrease of the final content in glycosylated forms compared to the value determined in raw milk. The results obtained show that carbohydrate-free ic-casein constitutes 52% of whole x-cassin. When milk is UHT treated, ~-Iactoglobulin only seems to influence the release of the glycosylated form. Nevertheless, no conclusion can be drawn concerning the localization in the micellar structure of both molecular forms of x-caseln.

SOLUBILITY AND EMULSIFYING PROPERTIES OF KAPPA CASEIN AND ITS CASEINOMACROPEPTIDE

Kappa-casein A was treated with chymosin in order to isolate the caseino-macropeptide corresponding to the C-terminal 106–169 residues of K-casein. Whole casein, K-casein and the caseinomacropeptide (CMP) were studied for their water solubility and emulsifying activity. The CMP was soluble over the range of pH from 1 to 10, with a “minimum” solubility (88%) in the range of pH 1–5 and a “maximum” solubility (98%) in the range of pH 5–10. For whole casein and K-casein, at pH values above 5.5, the emulsifying activity increased when pH increased and the maximum value was obtained for very alkaline solutions; for pH values below 4.5, the increase in emulsifying activity was much more pronounced at pH 2.5; below pH 2.5, emulsifying activity decreased. For CMP, the increase in emulsifying activity was much more pronounced in the acidic range than in the alkaline range. After 24 h storage and heating of the emulsion, a large pH-dependant decrease of emulsifying activity (22–60%) was observed for CMP for pH values below 4.0; under the same conditions, the emulsifying activity of whole casein and K-casein showed a 5–19% and a 1–21% decrease, respectively. For pH values above 6.0, a 22–59% decrease was observed for CMP as compared to a 1–12% and a 4–17% decrease with whole casein and K-casein, respectively.

Kinetic study of the action of bovine chymosin and pepsin A on bovine κ-casein

A study was carried out to determine the Michaelian parameters relative to the action of chymosin and pepsin A on bond Phe105-Met106 of bovine κ 0-casein (carbohydratefree fraction in micellar state). The reaction was performed in citrate buffer, pH 6.2, at 30 °C. The reaction mixture was analysed by reverse phase HPLC. Dosages of peptide 106–169 (caseino macropeptide) at different reaction times from recordings of its absorbance at 220 nm gave the initial rates of reaction at each substrate concentration. From these values the following parameters were determined: k cat = 68.5 s−1, K m = 0.048 mM, k cat K m = 1,413 mM −1 s −1 for chymosin, and k cat = 45 s −1, K m = 0.018 mM, k cat K m = 2,439 mM −1 s −1 for pepsin A. For chymosin they are similar to those obtained previously in dimethyl glutarate buffer, pH 6.6, at 30 °C, using fragment 98–111 of κ-casein as substrate. It can thus be concluded that neither the micellar state nor the presence of the whole peptide chain of κ-casein (our conditions) significantly affect the action of chymosin on fragment 98–111, which seems to contain all information that makes bond 105–106 highly sensitive to chymosin. For pepsin A, only the information contained in fragment 103–108 appears to be required.