Inactivation of an indigenous transglutaminase inhibitor in milk serum by means of UHT-treatment and membrane separation techniques

An indigenous inhibitor in raw milk inhibits cross-linking by transglutaminase (TG). The enzymatic cross-linking of micellar casein, compared with sodium caseinate, taking thermal inactivation of the TG inhibitor in the milk serum into consideration, was investigated. Inhibitor-free micellar casein was prepared by membrane separation combined with heat treatment of the UF permeate. The inhibitor permeated through MF (nominal pore size 0.1 μm) and UF (cutoff 25 kDa) membranes. TG-catalyzed cross-linking of casein micelles was clearly enhanced by UHT-treatment of UF permeate. Variation of the enzyme concentration showed that the inhibitory effect could not be compensated by higher enzyme concentrations when the casein micelles were suspended in unheated milk serum. Sodium caseinate, however, underwent high degrees of cross-linking even in unheated milk serum. By mixing an unheated milk serum and a UHT-treated milk serum at different ratios, the relative TG inhibitor activity was analysed. High inactivation (>80%) of the TG inhibitor is necessary to achieve high degrees of protein cross-linking.     

Effect of heat and transglutaminase on solubility of goat milk protein‐based films

The effect of heat, transglutaminase and combination of heat and transglutaminase treatments on the solubility of films prepared from goat milk casein, goat milk whey proteins and whole goat milk proteins was investigated. Goat milk casein films were less soluble when treated with transglutaminase and combination of heat with transglutaminase compare with heat‐treated caseins alone. Heat treatment was more effective at decreasing the solubility of whey protein films. SDS‐PAGE patterns demonstrated that goat milk caseins were better cross‐linked by transglutaminase, whereas whey proteins were better cross‐linked by heat. The extent of cross‐linking was further enhanced when a combination of heat and transglutaminase was used.     

Properties of casein micelles cross-linked by transglutaminase

Factors affecting the cross-linking of milk proteins by transglutaminase (TGase) were studied. Cross-linking of caseins in bovine skim milk was optimal over a very wide pH range. The role of micellar calcium phosphate (MCP) in maintaining the integrity of TGase-treated casein micelles was studied by incubating skim milk with 0.01% (w/v) TGase at 30°C for 1–24 h, followed by removal of MCP from untreated or TGase-treated milk by acidification and dialysis. The protein content and profile of the samples were determined by Kjeldahl and SDS-PAGE, respectively. Whey proteins in unheated milk were not susceptible to TGase-induced cross-linking. The higher level of sedimentable protein in MCP-free TGase-treated milk than in MCP-free control milk indicated that TGase treatment partially prevented disintegration of the micelle on removal of MCP, probably due to extensive intramicellar TGase-induced cross-linking of casein molecules which led to the formation of sedimentable covalently bonded casein aggregates.     

Effect of Ultra-high Temperature Treatment on the Enzymatic Cross-linking of Micellar Casein and Sodium Caseinate by Transglutaminase

ABSTRACT: It was found that ultra-high temperature (UHT) treatment of sodium caseinate and native whey protein-depleted micellar casein drastically increases the protein polymerization effect of an enzymatic treatment by microbial transglutaminase (TG). As a result the concentration of the isopeptide ε-(γ-glutamyl)lysine was increased significantly in UHT-treated micellar casein solutions after TG incubation compared with the unheated casein solution. Sodium caseinate was more susceptible to the cross-linking reaction as compared with the native casein micelles. The results demonstrate that the protein structure significantly affects the TG cross-linking reaction. The effect of an UHT treatment was considered to be related to a better TG accessibility due to a more open casein micelle structure and to the inactivation of a TG inhibitor substance. The results demonstrate that an unidentified component in the natural milk serum inhibits the TG reaction. The thermal inactivation of a TG inhibitor is the dominant effect explaining the improved cross-linking of UHT-treated casein micelles as well as sodium caseinate.     

Oxidative stability and antioxidant activity of bovine,caprine, ovine and asinine milk

This study compares the oxidative stability of fat isolated from bovine, caprine, ovine and asinine milk as well as the antioxidant activity of casein and whey from these milks. Fat from bovine and asinine milk showed the highest oxidative stability. The antioxidant activity of casein and whey was examined before and after an in vitro digestion process. Whey from asinine milk showed the highest antioxidant activity. The antioxidant activity of whey and casein increased after simulated intestinal digestion. In addition, the results of this study showed that asinine whey exhibited radical scavenging activity comparable with the strong synthetic antioxidants butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT).     

Casein interference in bovine plasmin assays using a synthetic substrate.

Bovine plasmin (EC 3.4.21.7) activity on H-D-valyl-L-leucyl-L-lysyl-4-nitroanilide was measured by determining the change in absorbance at 405 nm. Initial rates of reactions were estimated at all combinations of the following substrate concentrations [.4, 4, and 40 times the substrate concentration at one-half maximum velocity (Vmax) (Km)] and casein concentrations [.068, .68, and 6.8 times the inhibitor constant for competitive inhibition (KI)]. By nonlinear least squares fitting of the data to an equation that described reversible enzyme kinetics, steady state kinetic parameters, maximum velocity (Vmax), substrate concentration at one-half maximum velocity (Vmax) (Km), inhibitor constant for competitive inhibition (KI), and inhibitor constant for uncompetitive inhibition (KI) were estimated. Casein fit the equation as a competitive inhibitor of bovine plasmin. This enzyme has a catalytic constant (Kcat) of .0158 change in absorbance at 405 nm/min per nM, substrate concentration at one-half maximum velocity (Vmax) (Km) of .107 mM substrate, and inhibitor constant for competitive inhibition (KI) of .86 mg/ml of casein. Bovine plasmin activity can be measured directly in bovine milk without interference from casein.     

P NMR spectroscopic investigations of caseins treated with microbial transglutaminase

Caseins - the main constituents of bovine milk proteins - self-assemble into large supramolecular aggregates, so-called casein micelles. The enhancement of the stability of casein micelles is advantageous with respect to technological milk processing. A promising approach to accomplish this goal is the cross-linking of caseins using microbial transglutaminase (mTG). The present paper describes the combined use of liquid- and solid-state ³¹P NMR spectroscopy as well as dynamic light scattering in order to characterize the influence of an mTG treatment upon the structure of micelles in ultrahigh temperature (UHT)-treated skim milk at a molecular level. Liquid-state ³¹P NMR spectroscopy was applied to characterize milk, milk serum and casein dispersions. A narrow SerP signal in the liquid-state ³¹P NMR spectra of UHT-treated milk is shown to be due to casein molecules in the milk serum whereas the casein molecules bound in the micelles give rise to broad signals. Most of the caseins contribute to a 3 kHz broad background signal which can be visualized in the spectrum of suspensions of re-dispersed micellar material derived from UHT-treated milk. Treatment with mTG results in a cross-linking of caseins, which could be followed by liquid-state ³¹P NMR spectroscopy. Especially, the cross-linking of β-casein was demonstrated by quantitative liquid-state ³¹P NMR experiments. Furthermore, the stability of cross-linked micellar aggregates against EDTA could be investigated by liquid-state ³¹P HR NMR in combination with dynamic light scattering (DLS). Solid-state ³¹P NMR was used to show that the motional state of the κ-caseins located at the outer surface of the micelles derived from UHT-treated milk is not significantly changed by the applied mTG treatment.     

Distribution of plasminogen and plasmin in fractions of bovine milk.

The relative amounts of immunoreactive plasminogen and active plasmin in different fractions of bovine milk were examined. Raw milk was centrifuged to separate skim, cream, and a somatic cell pellet. Skim milk was centrifuged to separate milk serum and casein micelles. Milk fat globule membranes were isolated from the cream fraction of bovine milk. Proteins from somatic cells were isolated following sonication of the cells. Western blot analysis showed the presence of several forms of plasminogen in bovine milk. The predominant forms of plasminogen identified following electrophoresis under nonreducing conditions were proteins with approximate molecular weights of 88,000, 152,000, and 160,000. The predominant forms of plasminogen identified after electrophoresis under reducing conditions were two proteins with approximate molecular weights of 88,000 and 50,000. The highest amount (82% of the total plasminogen), as determined by an ELISA, was associated with the casein fraction. Lower plasminogen concentrations were associated with the serum, cream fractions, and milk fat globule membranes. The SDS-PAGE of the cream and milk fat globule membranes indicated that some casein was present in both fractions. Thus, the low plasminogen concentrations in these fractions may be associated with the caseins there. No immunoreactive plasminogen was present in the somatic cells. Active plasmin was present in the same milk fractions in which plasminogen was detected: casein, serum, and cream.     

Evaluation of cheese authenticity and proteolysis by HPLC and urea–polyacrylamide gel electrophoresis

Chromatographic and electrophoretic methods have been established as useful tools in characterising cheese ripening and in the detection of milk adulteration. The purpose of this work was to evaluate casein proteolysis of cheeses made from bovine, ovine or mixtures of bovine and ovine milks, as well as ovine cheese authenticity, for 30 days of ripening by HPLC and urea–polyacrylamide gel electrophoresis.Complementary information was obtained by both techniques when applied to the study of casein proteolysis during 30 days of ripening of ovine milk cheeses, ovine milk cheeses with 10% and 20% of bovine milk and bovine milk cheeses, manufactured according to the traditional Terrincho technology. For ovine cheeses, α-casein was the fraction that showed the higher degradation during cheese ripening. A similar behaviour was observed for ovine milk cheese with 10% of bovine milk. The profile for ovine milk cheese with 20% of bovine milk was more similar to that obtained for bovine cheese. Concerning bovine milk cheeses, electrophoresis was the most sensitive technique for the evaluation of proteolysis in these cheeses.Ten and 20% of bovine milk could be detected in ovine milk cheeses by urea–polyacrylamide gel electrophoresis and HPLC, respectively, even after 30 days of ripening.     

Evidence of calmodulin in bovine milk with high somatic cell counts

The objective was to determine whether calmodulin was present in bovine milk with high SCC. A highly specific antibody against calmodulin was developed in rabbits and affinity purified. Enzyme-linked immunoassays were used to determine the presence of calmodulin in the whey and casein fractions in milks with SCC ranging from less than 50,000 to greater than 1,000,000. Percentages of total protein and casein were determined. Calmodulin was detected in some casein fractions regardless of level of SCC. In the whey fraction, calmodulin was positively correlated with increased SCC. Calmodulin may be released into the milk as a result of the elevated proteolytic activity, which is evident in mastitis or as a result of leakage from the serum. This provides further information on the cellular and biochemical changes that occur in the diseased udder. Total protein increased with the increase in SCC while there was an inverse relationship of SCC to the percent casein.     

Natural plant enzyme inhibitors. Studies on a proteinase inhibitor from Italian millet (Setaria italica)

A proteinase inhibitor specific for trypsin was purified from Italian millet (Setaria italica) 170-fold by extraction with 0.02M HC1, ammonium sulphate fractionation, chromatography on CM-cellulose and trypsin-affigel chromatography. The inhibitor with an M_r of 14000 was found to be homogeneous by gel chromatography on Sephadex G-100 and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. It reacted with bovine trypsin in a 1:1 stoichiometric fashion. Inhibition of trypsin was nearly double when assayed with benzoyl DL -arginine p-nitroanilide as substrate compared to the activity with casein as substrate. The inhibitor affected the tryptic activity in human and bovine pancreatic preparations to an equal extent. Chemical modification studies showed that amino groups, disulphide bridges and sulphydryl groups are essential for the action of the inhibitor. Pretreatment with porcine pepsin or bovine α-chymotrypsin increased the antitryptic activity of the inhibitor when assayed with the low molecular weight substrate but not with casein.     

Microbial transglutaminase enhances antioxidant activity of yogurt through altering pattern of water‐soluble peptides and increasing release of amino acids

This study compared the antioxidant capacity of yogurt with and without microbial transglutaminase treatment after milk fermentation, and investigated the correlation between antioxidant property and the water‐soluble peptides and amino acids composition. Results showed that small molecular peptide fraction exhibited stronger antioxidant activity than large peptides. Microbial transglutaminase yogurt isolated fraction had stronger antioxidant capacity than that from the control. Microbial transglutaminase altered the peptides composition, and resulted in higher amount of small molecular peptides (<1.5 kDa) mainly consisting of κ‐casein‐derived peptides in yogurt. The dominant peptides sequences (IAKYI, IAKYIPI, IAKYIPIQY, PYYAK and NQFLPYPYYAK) in the treated yogurt were different from those in the control (PYPQ, VLPVPQKAVPYPQ, PVPQKAVPYPQ and VLSRYPSYGLN). Microbial transglutaminase improved the composition and concentration of amino acids in yogurt.     

Improvement of enzymatic cross-linking of casein micelles with transglutaminase by glutathione addition

The impact of glutathione (GSH) on cross-linking of micellar casein and sodium caseinate by microbial transglutaminase (TG) was investigated. Micellar casein was obtained by removing whey proteins from skim milk by means of membrane separation techniques. The addition of GSH (0.05–0.1 mm) was found to enhance the cross-linking of micellar casein suspended in milk serum. Cross-linking of sodium caseinate remained unaffected by GSH addition. Mixing TG and milk serum before addition of substrate proteins, however, resulted in an almost complete inhibition of the enzyme. When GSH and TG were present in the system before substrate addition, the reactivity of TG can be maintained. Hence, it was concluded that the addition of GSH mainly affected interactions between TG and an indigenous TG inhibitor present in milk serum. The addition of GSH allows cross-linking in milk products by TG without requiring a prior heat treatment of the milk beyond pasteurisation conditions.     

Plasminogen activation system in goat milk and its relation with composition and coagulation properties

The activity of plasmin (PL), plasminogen (PG), and plasminogen activator (PA) and their correlation with goat milk components and milk clotting parameters were investigated. Seven late-lactating Saanen goats were used to provide milk samples that were analyzed for PL, PG, and PA activity (colorimetric assay) fat, protein, noncasein nitrogen, nonprotein nitrogen, casein content, and somatic cell count (SCC). Milk clotting parameters (rennet coagulating time = coagulation time; K20 = firming rate of curd; A30 = curd firmness) were measured with a formagraph. Average milk yield and composition were similar to those previously observed in other studies. Plasmin, PG, and PA activity, expressed as units/ml, were, respectively, 20.04 +/- 0.94, 3.21 +/- 0.04, and 1154 +/- 57.61. Plasminogen activity was surprisingly low compared with other species (bovine, ovine), but it was consistent with the high activity of PA. A negative significant correlation was observed between PL and milk casein content. The correlation coefficients between PL and casein/protein ratio and PA and casein/protein ratio were negative and significant. A positive significant correlation was observed between PL and rennet clotting time and PA and rennet clotting time. Also positive was the correlation between PL and K20 and PA and K20. The plasmin activity was negatively correlated with A30. High plasmin and plasminogen activator activity in goat milk appeared to be negatively related with coagulating properties in late lactation, most probably via degradation of casein due to plasmin activity.     

Properties of proteases from milk somatic cells and blood leukocytes

Proteolytic activity of proteases associated with somatic cells isolated from high SCC milk and proteases associated with leukocytes isolated from bovine blood was assayed at pH 6.6 and 5.2 in a model system consisting of a beta-casein (.96%) substrate in Jenness/Koops buffer. Intact beta-casein and casein proteolysis products were measured by densitometric analysis of SDS-PAGE gels. Relative proteolytic activity was expressed as percentage degradation of beta-casein after 24 h at 37 degrees C per 1 x 10(6) cells/ml. Proteolytic activity associated with somatic cells isolated from bovine milk was 27.5 and 13.6% at pH 6.6 and 5.2, respectively. Proteolytic activity associated with leukocytes isolated from bovine blood was 16.0 and 8.4% at pH 6.6 and 5.2, respectively. Proteolytic activity at pH 6.6 was significantly higher for the somatic cells isolated from milk than for leukocytes isolated from blood. The reason for the difference in proteolytic activity of leukocytes isolated from blood of a healthy cow versus somatic cells isolated from milk produced by a cow with mastitis is not known. Further work is needed to determine whether the difference may be caused by a higher proportion of activated macrophages in somatic cells isolated from milk produced by cows with mastitis than in the leukocytes isolated from blood of healthy cows.     

Cross‐linking of bovine and caprine caseins by microbial transglutaminase and their use as microencapsulating agents for n‐3 fatty acids

Bovine and caprine caseins were cross‐linked with microbial transglutaminase (mTG). The mTG‐cross‐linked bovine or caprine casein dispersion, mixed with 14.5% maltodextrin (DE = 40), was used to prepare emulsions with 10.5% algae oil. Oxidative stability of emulsions was evaluated by peroxide values (PVs) and anisidine values. Adding liposoluble rosemary extract rich in carnosic acid and δ‐tocopherol lowered the formation of hydroperoxides and their subsequent decomposition products in emulsions. Emulsions stabilised with liposoluble rosemary extract rich in carnosic acid and δ‐tocopherol were spray‐dried at 180/95 °C. Algae oil microencapsulated with mTG‐cross‐linked bovine casein reduced PV by ≈ 34%, while the algae oil microencapsulated with mTG‐cross‐linked caprine casein with low levels of α_s1‐casein reduced PV by ≈ 42% at 4 weeks of storage at 30 °C. The investigation suggests that liposoluble rosemary extract rich in carnosic acid and δ‐tocopherol effectively protected algae oil during the coating process with mTG‐cross‐linked bovine and caprine caseins. The above results clearly indicated that the choice of milk caseins (bovine vs. caprine) cross‐linked with mTG impacts the oxidative stability of spray‐dried algae oil emulsions (microcapsules) enriched with n‐3 fatty acids.     

Milk lipoprotein lipase distribution in the major fractions of bovine milk

Raw, bovine bulk tank milk and milks from selected cows were separated by ultracentrifugation into four major fractions: casein, sloughed membrane material, serum, and milk fat globule membrane. Milk lipoprotein lipase activity was measured by the pH stat method and protein determinations were made by the Lowry procedure for each of the four fractions in order to calculate specific activity (units per milligram of protein). In six farm-cooled bulk milk samples stored less than or equal to 24 h, casein had a significantly higher milk lipoprotein lipase total activity, 35.66 units/ml of milk, than all of the fractions. Serum had the next highest activity with 11.69 units/ml of milk. Fluff and milk fat globule membrane had activities of .80 and .41 units/ml of milk, respectively. The specific activity of the fluff was 3.3 milk lipoprotein lipase units/mg of protein, which was significantly higher than the casein and serum fractions in pooled milk. Milks from five cows in midlactation were assayed individually for milk lipoprotein lipase activity and protein content immediately after milking and after 12, 24, 48 and 72 h of cold (4 degrees C) storage. Fresh warm milk was characterized by the absence of fluff. Casein had the highest mean activity (29.91 units/ml), followed by serum (10.25 units/ml) and milk fat globule membrane (.26 units/ml) in the warm milk from the individual cows. Upon cooling to 4 degrees C, significant increases in enzyme activity in the fluff and milk fat globule membrane fractions were observed at 12 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of pH on retention of camel chymosin in curd

Retained coagulant in cheese initiates casein breakdown and influences cheese structure and flavour formation. This study investigated the influence of milk pH on retention of camel chymosin in curd and compared it with bovine chymosin. Milk at five different pH levels was coagulated with same coagulant activity of each chymosin and centrifuged. Chymosin activity in whey was determined using the synthetic peptide Pro-Thr-Glu-Phe-(NO₂-Phe)-Arg-Leu as substrate and HPLC analysis of the resulting product. Camel chymosin had 2.7 times lower activity in milk than bovine chymosin at the same coagulation activity. The retention of camel chymosin in curd was rather constant at ∼20% between pH 6.65 and 6.00, while it increased almost linear from 2 to 21% for bovine chymosin. The lower pH dependence for retention of camel chymosin than of bovine chymosin may be explained by a lower negative charge of the camel chymosin molecule.     

The behaviour of Arabian donkey milk during acidification compared to bovine milk

To better understand the fermentation kinetic of Arabian donkey milk, its physicochemical properties, conductivity and viscosity were assessed during acidification, and compared to that of the bovine milk. Donkey milk showed a shorter latency phase and slightly lower acidification rate than bovine milk. Measurement of electric conductivity during acidification showed that maximum demineralisation of casein micelles occurred at around pH_I 5.44 for donkey milk and pH_I 5.16 for bovine milk. Donkey milk was also found to be less viscous. The technological characteristics of donkey milk were different from those of bovine milk due to intrinsic physicochemical properties of both milks.     

Update on optimized purification and characterization of natural milk allergens

Highly purified allergens namely cow's milk alpha-lactalbumin (ALA). (Bos d 4), beta-lactoglobulin (BLG) (Bos d 5) and casein (Bos d 8) and goat's milk casein were prepared from the raw milk from a single animal with a known genetic background. Consequently the natural isoforms are limited, constant and characterized. Purification included selective precipitations and chromatographical steps. Characterization of structure and allergenic activity assessment of milk allergens were carried out using physicochemical and immunochemical methods. Taken together data demonstrated the absence of impurities and of contamination by other milk allergens in each preparation. NMR and circular dichroism analyses confirmed the native conformation and proper folding of ALA and BLG and the expected absence of folding of bovine and caprine casein. Enzyme immuno assays confirmed the native conformation of BLG and the purity and immunoreactivity of all the proteins. The allergenic activity, e. g. the IgE binding capacity, of purified proteins was identical as that of those proteins when present in milk. The purified proteins also demonstrated the ability to provoke the degranulation of humanized rat basophilic leukaemia cells. All the data thus confirm the purity, identity, structural conformation and functionality of the prepared milk allergens.