Rennet-induced Aggregation Research Articles

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.

Characterization of buttermilk serum fractions and their effect on rennet-induced coagulation of casein micelle dispersions

Upon centrifugation of milk and buttermilk (BM) the supernatant above the casein micelles pellet separates into three phases: a low-density opalescent fraction, a clear fraction, and a high-density opalescent (HDO) fraction. These fractions were collected and the concentrations of casein, whey and milk fat globule membrane (MFGM) proteins, fat and phospholipids determined. Reference casein micelles were pelleted from skim milk (SM) and re-suspended in the serum fractions. Rennet-induced aggregation, coagulation aptitude and gel syneresis were measured in these suspensions. Buttermilk serum fractions contained more MFGM components than SM fractions did. Overall, BM serum showed a negative impact on rennet-induced aggregation of casein micelles, gel formation and gel syneresis during cooking. This impact was greater for the HDO fraction, which contained much higher concentration of MFGM materials when isolated from buttermilk than from milk. The negative impact of buttermilk on rennet-induced coagulation of casein micelles was mainly attributed to the presence of tiny fat droplets and high density MFGM fragments.

Effect of carrageenan addition on the rennet-induced gelation of skim milk.

Carrageenan (CG) (κ-CG, ι-CG and λ-CG) was added to skim milk and the rennet-induced aggregation was studied. Caseinomacropeptide (CMP) release, diffusing wave spectroscopy (DWS) and rheology were used to follow the structural dynamics of casein micelles during gelation. The influence of carrageenan on the nature of protein interactions in the gels was investigated using a combination of ultracentrifugation and specific dissociating agents. For the recombined samples containing κ-CG and low concentrations of ι-CG and λ-CG, the CMP release was slowed down; however, the development of DWS and rheological parameters was similar to that of the control sample, and the increase in the incorporation of proteins through calcium bridges and hydrophobic interactions may be the most likely contributors. For the recombined samples containing high concentrations of ι-CG and λ-CG, other factors may impede the gel formation process. High concentrations of ι-CG and λ-CG strongly interfered with the rennet-induced aggregation, interrupted the interaction of caseins and therefore may contribute to good quality of low-fat cheese. © 2016 Society of Chemical Industry.

Rennet-Induced Aggregation of Heated pH-Adjusted Skim Milk

Heated (20-100 °C/0-30 min) skim milks (pH 6.5-7.1) were diluted in buffer (pH 7.0). Rennet was added, and the particle size with time was measured. For all samples, the size initially decreased (lag phase) and then increased (aggregation phase). Milks heated at ≤60 °C had short lag phases and rapid aggregation phases regardless of pH. Milks heated at >60 °C at pH 6.5 had long lag phases and slow aggregation phases. As the pH increased, the lag phase shortened and the aggregation phase accelerated. The aggregation time was correlated with the level of whey protein associated with the casein micelles and with the level of κ-casein dissociated from the micelles. Heated milks formed weak gels when renneted. It is proposed that the milks heated at low pH have whey proteins associated with the casein micelles and that these denatured whey proteins stabilize the micelles to aggregation by rennet and therefore inhibit gelation. In the milks heated at higher pH, the whey proteins associate with κ-casein in the serum and, on rennet treatment, the κ-casein-depleted micelles and the serum-phase whey protein/κ-casein complexes aggregate; however, the denatured whey proteins stabilize the aggregates so that gelation is still inhibited.

Rennet coagulation properties of milk in the presence of oil droplets stabilised by a combination of sodium caseinate and whey protein isolate

This manuscript describes the in situ investigation of the rennet-induced aggregation of skim milk recombined with emulsion droplets stabilised with whey protein isolate (WPI) and sodium caseinate (NaCas) in isolation or in combination. The experiments were conducted with two levels of rennet as well as with and without added CaCl2. Diffusing wave spectroscopy and small deformation rheology were used to follow the gelation behaviour. In addition, scanning electron microscopy was employed to observe changes in the aggregation state of the oil droplets. We report that the presence of WPI-stabilised droplets did not alter the gelation behaviour of the casein micelles in recombined milks, even in the presence of added CaCl2. In contrast, NaCas-stabilised droplets in solution impaired the rennetability of the recombined milk, even after the addition of CaCl2. The mixture of both proteins at the interface was also investigated. When the WPI covering the fat droplets was gradually and systematically substituted by NaCas, any amount over 30% also impaired the rennetability of the milks. It was speculated that the increased steric repulsion of the oil droplets in conjunction with an increase in NaCas present in the serum phase in the recombined milks were the most likely contributors to the impaired rennetability of the casein micelles.

Rennet-induced aggregation of milk containing homogenized fat globules. Effect of interacting and non-interacting fat globules observed using diffusing wave spectroscopy

The dynamics of the interactions between casein micelles and oil droplets during rennet-induced gelation were described using rheology and diffusing wave spectroscopy. Two different colloidal states were obtained by preparing WPI-stabilized oil droplets in either milk serum or low ionic strength buffer: flocculated and non-flocculated. These two colloidal states strongly affected the diffusion of the non-interacting droplets in the gel and the elasticity of the protein network. In homogenized milk, the droplets interact with the protein network and play a structuring role, causing an increased elastic modulus. However, after displacement of the protein from the interface using Tween 20, the interpretation of the rheological data would lead to incorrect conclusions without light scattering measurements. This work demonstrated that an in-depth understanding of the colloidal properties of the oil droplets is necessary to fine-tune the interactions between these particles and the casein matrix in dairy gels.

Addition of sodium caseinate to skim milk inhibits rennet-induced aggregation of casein micelles

The objective of this paper was to observe the rennet-induced aggregation behaviour of casein micelles in milk in the presence of additional sodium caseinate. Analysis of the centrifugal supernatants by size exclusion chromatography confirmed an increase in the soluble protein in the milk serum phase after addition of sodium caseinate. Although the total amount of κ-casein hydrolyzed over time was not affected, there was a significant effect of soluble casein on milk gelation, with a dose-dependent decrease of the gelation time as measured by rheology. Light scattering experiments also confirmed that the addition of soluble caseins inhibited the aggregation of casein micelles. Addition of 1 mM CaCl 2 prior to renneting increased the extent of rennet aggregation in samples containing additional sodium caseinate, but the inhibiting effect was still evident. The amount of soluble casein (as measured by chroma tography) significantly decreased after renneting, suggesting its association with the micellar fraction. Supporting experiments carried out with purified fractions of soluble caseins demonstrated that both α s-casein and β-casein played a role as protective colloids (increasing steric repulsion) during renneting. It was concluded that the inhibiting effect observed during gelation was caused by the adsorption of soluble casein molecules on the surface of rennet-altered casein micelles.

Rennet-induced aggregation of homogenized milk: Impact of the presence of fat globules on the structure of casein gels

The present study investigated the renneting behaviour of casein micelles as influenced by the presence of fat globules. The gelation of skim milk, homogenized milk and homogenized milk with Tween 20 added was observed using diffusing wave spectroscopy and rheology. By adding Tween 20 to homogenized milk, it was possible to displace most of the milk protein from the oil/water interface and create a system with the same colloidal attributes as homogenized milk but with oil droplets with a very different interfacial composition, and relatively inert during rennet-induced aggregation. The primary phase of gelation was faster in homogenized milk compared to skim milk. Casein micelles were altered by the homogenization process which spreads them at the interface, making κ-casein more readily available. The addition of Tween 20 did not further affect the enzymatic activity on the micelles. However, the onset of gelation occurred earlier in homogenized milk with Tween 20 added, as the micelles aggregated at a lower level of proteolysis compared to homogenized milk. This work clearly showed that the gels formed in homogenized milk with and without Tween 20 have different physico-chemical properties, because of the different colloidal state of the filler particles.

Diffusing wave spectroscopy and rheological studies of rennet-induced gelation of skim milk in the presence of pectin and κ-carrageenan

The effect of the presence of κ-carrageenan on casein micelle stability and their rennet-induced aggregation was studied using diffusing wave spectroscopy and rheology measurements. Different concentrations of κ-carrageenan, below the gelation regime, were added to skim milk at pH 6.7. No changes in casein stability and on the aggregation kinetics of casein micelles were observed at κ-carrageenan concentrations <0.015% (w/v). The effect of mixing different concentrations of high methoxyl pectin (HMP) (0.04, 0.12 and 0.18%, w/v) with 0.015% (w/v) κ-carrageenan in skim milk was then studied. It was observed that κ-carrageenan protects the system from HMP-induced destabilization. The study of the synergistic effect of κ-carrageenan and HMP on the structuring of casein micelles in milk opens new avenues for the development and control of processes where different mechanisms of aggregation and destabilization occur simultaneously.

Rennet-Induced Aggregation and Curd Formation from Skimmed Milk Powders Prepared under Different Sterilizing Conditions

Heat treatment during the production of skimmed milk powder causes denaturation of proteins, thereby affecting the physicochemical properties of the skimmed milk powder. To understand the effects of heat treatment on the sensitivity of the casein micelles in skimmed milk powders, low heating type (L), normal heating type (N), high heating type (H), and super-high heating type (SH), to reaction with rennet, rennet-induced curd formation was investigated. A well-developed network structure with wide spaces was observed only in the curd derived from the solution of type L skimmed milk powder. SDS-PAGE suggested that there was no difference in the amount of glycomacropeptide generated from kappa-casein in the four types of skimmed milk powder, but casein micelles in the solution of type L skimmed milk powder formed aggregates most effectively. These results are discussed with respect to the thermal denaturation of proteins in skimmed milk powder.

Diffusing Wave and Ultrasonic Spectroscopy of Rennet-Induced Gelation of Milk in the Presence of High-Methoxyl Pectin

Rennet-induced aggregation was studied in milk systems containing high-methoxyl pectin (HMP) using ultrasonic and diffusing wave spectroscopy. These two techniques allow for in situ measurements of sol–gel transitions without the need for dilution. At low HMP concentrations, the casein micelles aggregation behavior was similar to that of skim milk, although changes could be noted in the microstructure of the renneted gels. At HMP concentrations between 0.1 and 0.15%, phase-separation kinetics were slower than the rennet-induced aggregation, and different microstructures formed caused by different dynamics of interactions between casein micelles present in HMP-depleted flocs. Higher amounts of HMP failed to create a continuous gel, as phase separation occurred at a faster rate than rennet aggregation. These results highlight the importance of non-invasive techniques in the study of concentration-dependent phase separating and aggregating systems, as only with observations in situ is it possible to determine new ways to control the structuring of protein–polysaccharide mixed systems.

The impact of the concentration of casein micelles and whey protein-stabilized fat globules on the rennet-induced gelation of milk

The rennet-induced aggregation of skim milk recombined with whey protein-stabilized emulsion droplets was studied using diffusing wave spectroscopy (DSW) and small deformation rheology. The effect of different volume fractions of casein micelles and fat globules was investigated by observing changes in turbidity (1/ l*), apparent radius, elastic modulus and mean square displacement (MSD), in addition to confocal imaging of the gels. Skim milk containing different concentration of casein micelles showed comparable light-scattering profiles; a higher volume fraction of caseins led to the development of more elastic gels. By following the development of 1/ l* in recombined milks, it was possible to describe the behaviour of the fat globules during the initial stages of rennet coagulation. Increasing the volume fraction of fat globules showed a significant increase in gel elasticity, caused by flocculation of the oil droplets. The presence of flocculated oil globules within the gel structure was confirmed by confocal microscopy observations. Moreover, a lower degree of κ-casein hydrolysis was needed to initiate casein micelles aggregation in milk containing whey protein-stabilized oil droplets compared to skim milk. This study for the first time clearly describes the impact of a mixture of casein micelles and whey protein-stabilized fat globules on the pre-gelation stages of rennet coagulation, and further highlights the importance of the flocculation state of the emulsion droplets in affecting the structure formation of the gel.

Sodium caseinate-stabilized fat globules inhibition of the rennet-induced gelation of casein micelles studied by Diffusing Wave Spectroscopy

Sodium caseinate (NaCas)-stabilized oil-in-water emulsions were added to skim milk and the rennet-induced aggregation was observed in situ using light scattering and dynamic oscillatory rheology. The gelation of the recombined milk was greatly inhibited by the addition of the oil droplets, at volume fractions >0.025. The development of the turbidity parameter, 1/ l*, and the apparent hydrodynamic radius during renneting were determined using diffusing wave spectroscopy. Although the recombined milk samples contained two scattering particles, namely, casein micelles and fat globules, the latter overwhelmingly contributed to the overall light-scattering signal. This made possible to follow the behaviour of NaCas-stabilized fat globules during the gelation process. The enzymatic reaction associated with the hydrolysis of micellar κ-casein was not significantly affected by the presence of the NaCas-stabilized fat globules. However, the emulsion droplets impeded the aggregation of rennet-altered casein micelles preventing the formation of a gel network. The inability of renneted casein micelles to develop a gel network can be attributed in part to an altered equilibrium between soluble and micellar calcium phosphate, caused by the association of soluble Ca 2+ with casein molecules, but mostly can be attributed to the effect of non-adsorbed caseins on the surface of the casein micelles.

Determination of early stages of rennet-induced aggregation of casein micelles by diffusing wave spectroscopy and rheological measurements

Abstract Diffusing wave spectroscopy (DWS) and rheology have been used to study the aggregation of casein micelles dispersions induced by the addition of rennet, a proteolytic enzyme preparation. Aggregation times measured by DWS and by rheology were in good agreement. In addition, prior to the onset of aggregation DWS was able to detect the change in the casein micelle mobility due to the cleavage of κ-casein by the enzyme. We believe that, due to its the sensitivity to changes brought in particle size, in addition to being a non-invasive and a non-intrusive technique, DWS has a potential to be implemented and used in an industrial environment, such as manufacture of yogurts and cheeses.

Effect of denatured whey proteins on the rennet-induced aggregation of casein micelles

The effect of heat-denatured whey protein on the properties of rennet-treated casein micelles was investigated. Aggregation of renneted casein micelles was inhibited by heat-induced denaturation of whey proteins. The effect was partially reversed by increasing the temperature or the calcium ion concentration.