Rennet Casein Research Articles

The effect of casein/whey protein ratio and minerals on the rheology of fresh cheese gels using a model system

Abstract An objective of the present study was to determine the effects of protein interactions on acid gelation behaviour at the higher concentrations encountered in fresh cheeses. A fresh cheese model was created by means of chemical acidification (glucono-δ-lactone) of 120 g L−1 protein solutions prepared from native phosphocasein, sodium caseinate and whey protein isolate (WPI) in lactose-free simulated milk ultrafiltrate. The effects of varying the ratios of (i) casein/whey protein, and (ii) phosphocasein/sodium caseinate (on a protein basis) on the rheology of the resulting acid gels were studied. The results showed that storage moduli of acid gels after 4 h (G′240) at 30°C increased as the amount of pre-denatured whey protein (78°C, 30 min) was increased to a maximum of 15 g L−1. At a constant protein content of 120 g L−1, the storage modulus increased from 250 Pa (no whey protein present) to 3.15 kPa following substitution with 15 g L−1 pre-denatured whey protein. At higher pre-denatured whey protein concentrations, the storage modulus decreased. Gelation also occurred more quickly and at a higher pH by increasing the amount of added pre-denatured whey protein up to the optimum level of inclusion. In the absence of whey protein, partial substitution of phosphocasein (40 g L−1) with sodium caseinate (80 g L−1) reduced Ca and P concentration to levels that typify fresh acid curd, and increased gel strength (G′240>1000 Pa). The inconsistent acid gelation behaviour arising from substitution (20–60 g L−1) with solubilised rennet casein in the form of sodium para-caseinate suggests that rennet addition may be unnecessary when protein concentration is high. The study demonstrates that the strong acid gels formed by recombination of novel and existing milk protein ingredients with defined functional characteristics have potential application in fresh cheese product developments.

Rennet caseins manufactured from seasonal milks: composition, hydration behaviour and functional performance in pilot‐scale manufacture of mozzarella cheese analogues

Compositional and hydration properties of edible rennet caseins prepared from Irish milks over three production seasons were studied. The ash content of the caseins declined during early to mid‐lactation and increased sharply in late‐lactation. Protein content was independent of season of manufacture, while moisture content showed no clear relationship to season of manufacture. The hydration properties of the rennet caseins on dispersion in a solution of a calcium‐sequestering salt were assessed in a simple model system relevant to mozzarella cheese analogue manufacture. The maximum viscosity index reached and the time taken to reach maximum viscosity index on dispersion of rennet casein both decreased as the season of manufacture progressed. Poor performance of rennet caseins in pilot‐scale mozzarella cheese analogue manufacture was observed for some caseins manufactured from both early lactation milks and late‐lactation milks, while caseins manufactured from mid‐lactational milks generally performed well in pilot‐scale mozzarella cheese analogue manufacture.

Influence of some physico-chemical characteristics of commercial rennet caseins on the performance of the casein in Mozzarella cheese analogue manufacture

Abstract The compositional and chromatographic characteristics of ten rennet caseins and their relationships to rennet casein performance in Mozzarella cheese analogue manufacture were determined. Some compositional parameters (moisture, ash and calcium) were weakly related to performance, while cation-exchange fast protein liquid chromatography did not indicate differences between the caseins which could be related to performance in Mozzarella cheese analogue manufacture. When the rennet caseins were hydrated at 8.0% (w/w) protein for 120 min in 0–1.0% (w/w) disodium orthophosphate (DSP), the level of protein and calcium solubilised varied depending on the DSP concentration and on the particular rennet casein. Differences were also observed in the rates of protein and calcium solubilisation when each rennet casein was hydrated at 4.0% (w/w) protein in 0.5% (w/w) DSP for up to 120 min. Levels of protein and calcium solubilised and rates of solubilisation were in some instances found to relate to rennet casein performance in Mozzarella cheese analogue manufacture. These results suggest that calcium mediated protein–protein interactions in the different caseins are disrupted at different rates and to different extents during hydration at similar concentrations of calcium sequestering salt. These differences may arise due to protein and/or mineral (ash, calcium) modifications during heat treatment of skim milk prior to rennet casein manufacture or on heat treatment during manufacture; the extent of modification appears to subsequently influence the hydration and solubilisation of a rennet casein in DSP and its performance in Mozzarella cheese analogue manufacture.

Structure and Scaling Behavior of Aging Rennet-Induced Casein Gels Examined by Confocal Microscopy and Permeametry

A study is presented on the structure of rennet(-induced) casein or skim milk gels at three pH values (5.3, 6.0, and 6.65) and temperatures (20, 25, and 30 °C). The structure was examined by confocal scanning laser microscopy and permeametry. Deconvolution was applied to the microscopic images. A fractal scaling analysis has been applied to the images and permeametry results. In this analysis, the fractal dimensionality (Df), lower cutoff length (R0), and apparent pore size (P) of the linear scaling regime were calculated from the microscopical data. The Df and apparent pore size were also calculated from the permeametry data. During aging of the gels, a coarsening of the structure was observed; the pore size increased and the clusters became more compact. This was reflected in the fractal parameters: R0 and P increased during gel aging. Their values are generally high (0.5−1.5 and 5.0−15 μm, respectively) compared to data obtained by computer simulations. The Df value is also high (∼2.2−2.6), which is an indication of slow aggregation or rearrangements during aggregation. The gel aging effects are probably mainly due to rearrangements such as particle fusion and strand fracture, which rates increase with increasing temperature, and even more pronouncedly, with decreasing pH.

The effect of varying the calcium-sequestering salt cation on the hydration behaviour of rennet caseins in a simple model system

Di potassium orthophosphate and di-ammonium orthophosphate were used to replace di-sodium orthophosphate in a simple model of cheese analogue manufacture. Rheological changes that occurred as eight rennet caseins, possessing widely differing hydration behaviour, were dispersed in solutions of the different calcium-sequestering salts were determined. Dispersion in the potassium salt resulted in minor differences in hydration behaviour of rennet caseins when compared to the behaviour in di-sodium orthophosphate solution, while dispersion in the ammonium salt resulted in considerably modified hydration behaviours and rheological profiles.

Application of capillary electrophoresis to the characterization of processed cheeses.

Capillary electrophoresis using a hydrophilically coated capillary and a low pH buffer containing urea has been used to characterize processed cheeses. Different electrophoretic patterns were obtained depending on the ingredients used in the blend such as acid casein, rennet casein, sodium and calcium caseinates and skim milk powder. Isoelectric casein, and sodium and calcium caseinates were shown to contain intact non-glycosylated kappa-casein (kappa-CN), while rennet casein contained only trace amounts of kappa-CN and mainly para-kappa-CN. Therefore, the addition of casein or caseinate to processed cheeses has been detected by analysing the intact non-glycosylated kappa-CN. Quantitation of intact non-glycosylated kappa-CN in processed cheeses of known and unknown composition was carried out using a regression curve from standard mixtures of 150-550 g isoelectric casein/kg total rennet casein. This capillary electrophoresis method successfully confirmed the addition of isoelectric casein or caseinate to processed cheeses of known composition. The quantitative determination range was 0.605-3.688 mg kappa-CN/ml. This method cannot be used for measuring additions of rennet casein or any caseinates that have been exposed to chymosin.

Proteolysis in rennet casein-based cheese analogues

Abstract Rennet caseins manufactured from mid- or late-lactation milk were used to prepare pilot- and commercial-scale Mozzarella-type cheese analogues. Mid-lactation rennet casein and analogues made from it contained low levels of plasmin; late-lactation rennet caseins and analogues made from them had higher levels of plasmin while pilot-scale analogues had higher levels of plasmin than commercial-scale analogues. The cheese analogues were stored at 8°C for 32 weeks during which time total bacterial counts increased from ∼104 to 107–108 cfu g−1, with non-starter lactic acid bacteria dominating the microflora. Proteolysis, as reflected by pH 4.6-soluble nitrogen and free amino acids, increased on storage of all the analogues. The increase was greater in the analogues manufactured from late-lactation rennet caseins than in those manufactured from mid-lactation rennet casein and in pilot-scale analogues than in commercial-scale analogues, reflecting the differences in plasmin levels as well as slight differences in composition. Polyacrylamide gel electrophoretograms of the cheese analogues showed that β-casein was hydrolysed more extensively than αs1-casein and that the concentration of γ-caseins increased on storage. The degree of hydrolysis of β-casein was greater in the cheese analogues manufactured from late-lactation rennet caseins than in those manufactured from mid-lactation rennet casein and in the pilot-scale analogues than in the commercial-scale analogues reflecting the corresponding plasmin levels. Seventeen peptides in the pH 4.6-soluble extract of the cheeses were isolated and partially sequenced; all originated from the N-terminal region of β-casein; 9 peptides had Lys29 of β-casein as the N-terminal, 6 peptides had Arg1 of β-caesin as the N-terminal and the remaining two peptides had Ile30 and Phe33 of β-casein as the N-terminal, respectively. The pattern of casein hydrolysis in the cheese analogues suggests that plasmin was the primary proteolytic agent contributing to initial hydrolysis of the caseins while microbial proteinases and peptidases may have contributed to the high levels of free amino acids.

Maillard reaction contributes to variability in hydration characteristics of rennet caseins in solutions of a calcium‐sequestering salt

The objective of this study was to assess the effect of protein modification by Maillard reactions on the functionality of rennet caseins. The extent of Maillard reactions in rennet caseins was determined by measuring furosine levels by a high‐performance liquid chromatography method following acid hydrolysis of the casein. An approximately 20‐fold range in furosine levels in commercially produced rennet caseins was observed. High furosine levels correlated significantly with increased time taken to reach maximum viscosity index and with decreased maximum viscosity index when the rennet caseins were dispersed in a solution of calcium‐sequestering salt, which suggests that modification of casein by Maillard reaction contributes to the variability in the hydration behaviour of the rennet caseins.

Factors Determining Large-Strain (Fracture) Rheological Properties of Model Processed Cheese

Abstract Torsional fracture was used to map changes in large-strain rheological properties of a model processed cheese that contained 20% protein (rennet casein), 27% anhydrous milk fat, 1.5% NaCl, and 1 to 3% Na 2 HPO 4 . Processing time (10, 20, or 30min), Na 2 HPO 4 (1, 2 or 3%), and pH of the Na 2 HPO 4 -NaCl solution (5.4, 5.6, or 5.8) were adjusted to alter the extent of casein micelle solubilization and heat-induced protein-protein interactions. A Box-Behnken experimental design was employed, and results were analyzed using response surface regression and ridge analysis techniques. Further investigation employed a single factorial design in which Na 2 HPO 4 increased from 1 to 4%. Model processed cheeses had fracture stress, fracture strain, and fracture modulus (fracture stress/fracture strain) values ranging from 25.1 to 79.7 kPa, 0.66 to 1.88 kPa, and 15.2 to 96.0 kPa, respectively. The properties of fracture modulus and fracture strain formed a master curve, independent of the processing variable. Conditions favoring increased protein solubilization and heat-induced protein-protein interactions increase fracture modulus and decreased fracture strain. This finding coincided with an increase in slope ratio (fracture modulus/modulus at 30% of the fracture strain), indicating a change in fracture mechanism from strain weakening to elastic fracture. These results suggested that large-strain (fracture) rheological properties could be used to characterize the effects of processing variables important to processed cheese quality.

Compositional characteristics of rennet caseins and hydration characteristics of the caseins in a model system as indicators of performance in Mozzarella cheese analogue manufacture

Rennet casein is widely used in the manufacture of Mozzarella cheese analogues. Wide variation in the functional performance of different rennet caseins during the manufacture of such cheese analogues is encountered, resulting in considerable variability in the functional properties of the cheese analogue. Samples of 44 batches of edible rennet caseins were studied for performance in Mozzarella cheese analogue manufacture in two pilot-scale cheese analogue manufacturing processes and scoring systems for performance in the two processes were devised. Rennet casein performance during cheese analogue manufacture was found to vary widely and also differed depending on the pilot-scale process used. Of the protein, moisture and ash contents of the caseins only the ash content showed significant correlation to the performance scores achieved in cheese analogue manufacture, but this was not a suitable parameter for predicting performance. The hydration characteristics of the rennet casein when dispersed in solutions of disodium orthophosphate (DSP) were determined using a rheological method and compared to the performance of the rennet caseins in the two pilot-scale cheese analogue production processes. Wide variations in the maximum viscosity index, in the time taken to reach the maximum viscosity index and in the viscosity stability of rennet caseins dispersed in 0.4% (w/w) DSP were observed. In a non-linear graphical model, the time taken to reach maximum viscosity and the maximum viscosity index reached in 0.4% (w/w) DSP were found to be good indicators of the performance of rennet caseins in both the pilot-scale cheese analogue manufacturing processes. The model, or a suitable development of the model, should prove useful to manufacturers and users of rennet casein alike in assessing the potential performance of different batches of rennet casein in the manufacture of cheese analogues.

Mechanical and Barrier Properties of Lactic Acid and Rennet Precipitated Casein‐Based Edible Films

ABSTRACTTensile strength (TS), percent elongation (%E), oxygen permeability (OP), and water vapor permeability (WVP) of lactic acid and rennet precipitated casein‐based edible films were studied to determine the effect of protein to plasticizer ratio (0.6:1, 1:1, 1.4:1) and plasticizer type (sorbitol, glycerol) on these properties. TS increased (p<0.05) with increase in protein to plasticizer ratio. Sorbitol plasticized films were stronger (p<0.05) than glycerol plasticized films. However, films plasticized with glycerol were more extensible (p<0.05). Film %E decreased with increase in protein/plasticizer ratio for lactic acid casein films, whereas it increased for rennet casein films. Films plasticized with sorbitol were more effective (p<0.05) moisture and oxygen barriers than glycerol plasticized films. Overall, lactic acid casein films plasticized with sorbitol had the most effective mechanical and barrier properties.

The hydration behaviour of rennet caseins in calcium chelating salt solution as determined using a rheological approach

Rennet casein is used in the manufacture of Mozzarella cheese analogues. Hot solutions of calcium chelating salts are used to facilitate hydration of the protein which subsequently acts as an emulsifier for the oil phase dispersed in the protein/aqueous phase of the cheese analogue. On cooling, a matrix resembling cheese in texture and other rheological properties is produced. Considerable variations in the functional performance of different rennet caseins during cheese analogue manufacture have been observed. Commercial rennet caseins have been obtained and are being studied to establish the nature and causes of these variations in performance. A simplified model system to study rennet casein hydration was developed. The method involves dispersing rennet caseins in solutions of chelating salt at 55°C with stirring. The changes in viscosity index that occur as the rennet casein hydrated were followed using a controlled shear rate rheometer equipped with a custom designed paddle stirrer. Hydration of the rennet casein particles was found to occur in a number of stages including particle swelling, clumping, network formation and subsequent breakdown of the network. Considerable variations in the times taken for different rennet caseins to swell, clump and reach maximum viscosity index and in the stability of the hydrated protein dispersions were found. The rheological properties of the rennet caseins during hydration were highly dependent on the concentration of the chelating salt in the hydrating solution.

The Effect of Milk Fat, the Ratio of Casein to Water, and Temperature on the Viscoelastic Properties of Rennet Casein Gels

The objective of this study was to evaluate the effects of milk fat, the ratio of casein to water, and temperature on the viscoelastic properties of a model system for cheese based on dried rennet casein power. Casein gels were formed by mixing rennet casein, butter (as a source of milk fat), water, and some minor ingredients at 0.89, 0.58, and 0.38 ratios of casein to water; milk fat was added at 0, 12, and 24% at each ratio. Strain sweep, frequency sweep, melting behavior, and stress relaxation were used to characterize the viscoelastic properties of casein gels. The addition of milk fat reduced the linear viscoelastic region of casein gels. The complex modulus increased as milk fat content increased at temperatures ≤20°C, decreased at temperatures ≥30°C, and increased at all temperatures as the ratio of casein to water increased. Any particular value for the complex modulus could be obtained by a number of different combinations of the milk fat content, the ratio of casein to water, and temperature. The casein gels showed three distinctive zones of viscoelastic behavior over a temperature range of 5 to 70°C: rubbery solid, transition, and melt. The melting temperature and relaxation time of the casein gels increased as the ratio of casein to water increased; milk fat content had less effect on these gel properties. The casein matrix alone (0% added milk fat) showed a gradual softening with temperature starting at about 10°C. Thus, these composite gels represent a rather unique system by which the dispersed filler phase (milk fat) melts over the same temperature range as the underlying polymer matrix dissociates.

Preparation and characterization of micellar calcium phosphate-casein phosphopeptide complex.

Micellar calcium phosphate (MCP) in bovine milk was separated as the complex with casein phosphopeptide (CPP) by the following procedures. Rennet curd obtained from skim milk was suspended in water, the pH was adjusted to 4.6, and the suspension was centrifuged at 1,000 x g. CPP was separated from the precipitated casein by tryptic hydrolysis and ethanol precipitation. The supernatant, which contained calcium and inorganic phosphate liberated from casein micelles by acidification, and CPP were mixed; the pH was adjusted to 6.7; and then the solution was lyophilized. From 1 L of skim milk, 3.16 g of the MCP-CPP complex was obtained. The dried MCP-CPP complex was easily dissolved in water and contained 12.7% calcium, 0.3% magnesium, 3.4% inorganic phosphorous, and 2.2% organic phosphorous. No crystal structure of hydroxyapatite was shown in the MCP-CPP complex by the X-ray diffraction analysis, although the pattern of NaCl crystal was observed. The X-ray diffraction pattern of commercial whey mineral, which was prepared by precipitation at alkaline pH from rennet whey, was similar to that of hydroxyapatite. It was confirmed by high-performance gel chromatographic analysis that the form of calcium phosphate in the MCP-CPP complex was similar to that of casein micelles. The MCP-CPP complex was also separated from commercial rennet casein. The method for the separation of MCP-CPP complex described above can be applied to the large-scale preparation.

Variation of water-holding capacity and mechanical properties between the particle size fractions of mineral acid, lactic and rennet casein curds

Mineral acid casein, lactic casein and rennet casein curds were prepared either in the laboratory or on pilot-scale equipment. The water-holding capacities of mineral acid casein curd particles prepared in the laboratory were relatively constant for particle sizes >4 mm both after sieving and after centrifugation; however, they increased dramatically at smaller particle sizes to a maximum of 7·2 g water/g dry curd after sieving and 4·3 g water/g dry curd after centrifugation at 4060 g for the size fraction that passed a 0·85 mm screen. The mechanical strength of the centrifugally compressed curd as assessed by a penetrometer probe also decreased dramatically with decrease in particle size. This variation of mineral acid casein curd properties with particle size was attributed partly to variation in particle pH at the instant of curd formation because of imperfect mixing of acid and skim milk, i.e. localized pH gradients. Lactic casein and rennet casein curds and curd formed by acidification of skim milk with glucono-δ-lactone also produced similar but smaller variations in curd properties with particle size. These curd particles are all formed by break up of a quiescent gel and the variation in curd properties with particle size was attributed to variations in mechanical strength in the original milk gel, i.e. weak areas of the gel break up more easily, resulting in small, weak curd particles.

Effects of pH and temperature on the water-holding capacity of casein curds and whey protein gels

SummaryThe water-holding capacities of mineral acid casein (MAC) curd, rennet casein curd and whey protein concentrate (WPC) gel were determined as a function of pH and temperature by drainage of liquid on a stationary, horizontal screen. As precipitation pH was varied over the range 4·3–5·55, the water-holding capacity of MAC curd ranged from 2·4 to 5·2 g water/g dry casein with minimum water-holding capacity at precipitation pH 5·3. Samples of dried MAC curd over the same range of precipitation pH did not vary significantly in water-holding capacity, and all absorbed ∼2·0 g water/g dry casein. Both rennet casein curd and WPC (170 g total solids/kg) gels increased in water-holding capacity as the pH of the suspending liquid increased in the range 3·5–10, but the changes with pH were much smaller than for MAC curd. For both types of casein curd and for WPC gel, water-holding capacity decreased with increase in temperature in the range 5–90 °C. The results are interpreted largely in terms of changes in electrostatic interactions with pH and changes in hydrophobic interactions with temperature.

Rheological properties and microstructure of model processed cheese containing low molecular weight emulsifiers

AbstractThe influence of low molecular weight emulsifiers (SDS, CTAB, lecithin, mono‐diglycerides) on rheological properties and the microstructure of model processed cheese, made using rennet casein, at three different pH values was investigated. Interactions between the low molecular weight emulsifiers and rennet caseins in model processed cheese are consistent with those found in model food emulsions of liquid continuous phase. Compared with the control, the addition of CTAB (cationic) resulted in the hardest and most elastic processed cheese, while the incorporation of SDS (anionic) produced the softest and least elastic cheese. Processed cheese with added lecithin or mono‐diglycerides behaved much like the controls, but with an increase in syneresis level. No syneresis was observed with the SDS cheeses. In general, low pH cheeses (pH 5.45) were harder than high pH cheeses (pH 6.05). Rheologically, all the processed cheese samples can be described as [weak gels]. The protein matrix of the high pH processed cheese containing CTAB consisted mainly of protein particles linked into a chain‐like form, while those containing lecithin and SDS showed a mixture of individual as well as short chained protein aggregates, and the control had protein aggregates of intermediate size. The added low molecular weight emulsifiers resulted in a finer dispersion of the fat in the protein matrix. Protein‐emulsifier charge interactions seem to be the prime determinant of the rheological properties of these model processed cheeses.

Non-Size Exclusion Effects During Gel Permeation Chromatography of Milk Protein Hydrolysates on an FPLC Superose 12 Column

Abstract Hydrophobic peptides and aromatic amino acids adsorbed to the matrix of a Superose 12 FPLC column during gel permeation chromatography (GPC) of milk protein hydrolysates. The adsorption phenomenon was most obvious when hydrolysates were prepared using enzyme mixtures which contained exopeptidases. The elution areas of peaks for tryptophan and tyrosine from a Superose 12 column was linear with concentration in the range 0 to 1 μmol. ml-1. Amino acid analysis confirmed that a strongly adsorbed peak in the elution profile, measured by absorbance at 280 nm, of an extensively hydrolysed rennet casein contained tryptophan. Individual hydrophobic peptides, used as molecular weight markers, also interacted with the column, eluting later than expected. Nitrate in a general water supply used for the production of a whey protein hydrolysate on a pilot-scale was responsible for the appearance of an additional peak in the elution profiles from a Superose 12 column, as measured by absorbance at 214 nm.

Proteolytic and rheological changes during ageing of cheese analogues made from rennet caseins

On storage of cheese analogues at 4°C for up to 51 weeks, proteolysis, as measured by pH-4·6-soluble nitrogen, increased, whereas cheese hardness, stress at 15% compression, gumminess, elasticity, and chewiness decreased. Polyacrylamide-gel electrophoretograms of the cheese analogues showed that β-casein was hydrolyzed more extensively than αs1-casein and that the concentration of γ-caseins increased on storage. The pattern of casein proteolysis was similar to that reported for the hydrolysis of caseins by the indigenous alkaline milk proteinase, plasmin. Changes in the rheological properties of the cheese analogues were statistically significantly correlated with cheese age, pH 4·6-soluble nitrogen and αs1- and β-casein contents; it is suggested that plasmin is responsible for proteolysis and rheological changes during cheese-analogue storage.

Relationships between plasmin levels in rennet caseins and proteolytic and rheological changes on storage of cheese analogues made from these caseins

SummarySixteen rennet casein samples and the corresponding sixteen cheese analogues made from these caseins were obtained from a local processor. Following storage at 4°C for 6 months, pH 4·6-soluble N contents of the cheese analogues increased, while αs- and β-casein contents, elastic moduli and apparent relaxation times decreased. The β-casein and pH 4·6-soluble N contents of the 6 month old products were significantly correlated, both were significantly correlated with the plasmin (EC 3.4.21.7) contents of the corresponding rennet caseins from which the analogues were made and all three were significantly correlated with the rheological properties of the stored analogues. Of the compositional characteristics, only percent moisture in non-fat substance and percent fat in dry matter were significantly correlated (P < 0·05) with any of the proteolytic or rheological properties. The sixteen cheese analogues were also assayed at one time, when they ranged in age from 1 to 35 weeks, and highly significant interrelationships between plasmin activity, proteolysis and rheological properties were established. The results indicate that the plasmin content of rennet caseins had a significant influence on the proteolytic and rheological changes that occurred during storage of cheese analogues made from these caseins.