Rennet-induced Gels Research Articles

Dry-heat treatment of low-heat skim milk powder improves rennet-induced gelation

The use of low-heat skim milk powder (SMP) in natural cheese production offers significant industrial advantages. However, SMP takes a long time to undergo rennet-induced gelation and forms a soft gel, which makes it difficult to use in cheese production. In this study, we investigated whether dry-heat treatment of low-heat SMP contributes to improving rennet-induced gel properties. We found that dry-heat treatment (65 °C; relative humidity, 40%; 2–6 h) accelerated rennet coagulation time (RCT), increased gel strength, and improved the water-holding capacity of the gel. Dry-heat treatment resulted in lactosylation, an increase in hydrophobicity, and a decrease in the zeta potential of casein micelles. These changes possibly contributed to the improvement in rennet-induced gelation. Additionally, the dry-heat treatment likely affected the surface properties of casein micelles by altering their secondary structure. This study offers valuable insights into SMP applications, particularly in the production of natural cheese in the dairy industry.

Structural dependence of concentrated skim milk curd on micellar restructuring

This study was conducted to establish an understanding of how milk concentration modulates the rennet curd structure. Rennet-induced gelation and renneting under slow acidification achieved using glucono-δ-lactone (GDL) and structural properties of reconstituted skim milk gels at two concentration levels (9 and 25 % total solids) were studied by measuring variations in (a) viscoelastic behaviour, (b) micellar size, charge density, diffusivity, and (c) hydrophobicity using dynamic rheometry, dynamic light scattering and fluorimetry, respectively. Concentrated milk showed a greater estimated hydrodynamic radius of casein micelles, lower zeta (ζ)-potential, ratio of serum to total Calcium (Ca) and charge density and increased surface hydrophobicity, all supporting the view that micellar restructuring particularly sub-particle transfer takes place and contributes to rapid gelation. Moreover, hydrophobic interactions occurred very quickly (within 5 min in combined gels, 10 min for renneting only), demonstrating their pivotal role during the flocculation stage. All gels exhibited a solid viscoelastic character as the elastic modulus (G′) was greater than loss modulus (G″) while both G′ and tan δ (G''/G′) were frequency-dependent. Frequency sweeps classified the concentrated gels into three stiffness categories caused by the level of rennet or GDL as rigid, hard and soft, whereas an increased flow-like behaviour (high tan δ), restricted diffusion and excessive water retention revealed limited structural rearrangements (contraction & macrosyneresis) during curd ageing. Acidification increased the diffusion rate in control curd, thus, enhanced contractive rearrangements, macrosyneresis and curd strength. Findings suggest that micellar restructuring induced by milk concentration is the principal modulator of the curd structure.

Cold ultrafiltered or microfiltered milk retentates: A systematic comparison of the effects of compositional differences on their gelation functionality

The colloidal stability of casein micelles suspensions prepared using ultrafiltration (UF) and microfiltration (MF) was studied by testing acid- and rennet-induced destabilization. Skim milk and 4× (based on volume reduction) concentrates were obtained by processing under similar conditions, at temperatures below 10°C. Concentrates were subjected to different levels of diafiltration (DF), resulting in samples with comparable casein volume fractions but different amounts of proteins and ions in the serum phase. The novelty of the work is the systematic comparison of MF and UF concentrates of similar history. More specifically, concentrates similar in ionic composition but with or without serum proteins were compared, to evaluate whether whey proteins and β-casein depletion from the micelles will play a role in the processing properties, or whether these are affected solely by the ionic balance. Microfiltered micelles' apparent diameter decreased by about 50 nm during the specific hydrolysis of κ-casein by chymosin, whereas those in skim milk control showed a decrease of about half that size. All concentrates subjected to extensive DF showed smaller hydrodynamic diameters, with reductions of ∼18 and 13 nm for MF and UF, respectively. Highly diafiltered UF retentates showed a delayed onset of rennet-induced gelation, due to low colloidal calcium, compared with other samples. Low-diafiltered samples showed weak storage modulus (∼1 Pa) after 60 min of onset of gelation. In addition, onset pH increased with diafiltration to ∼5.8 for UF and ∼6 for MF in high-diafiltered samples. These results clearly demonstrated that the functional properties of casein micelles change during membrane concentration, and this cannot be solely attributed to changes in ionic equilibrium.

Effect of high hydrostatic pressure processing on the rennet coagulation kinetics and physicochemical properties of sheep milk rennet-induced gels

The effects of high hydrostatic pressure on the constituents and coagulation ability and their effect on cheese production of sheep milk have not been studied in detail. The objective of this work was to evaluate the effect of high hydrostatic pressure processing on the coagulation kinetics and physicochemical properties of sheep milk and to explore how such treatment could improve the cheesemaking process. Five batches of milk were tested: 1 untreated control batch and 4 batches each subjected to a different pressure (150, 300, 450, or 600 MPa) for 5 min at 10°C. As treatment pressure increased, values of electrical conductivity and oxidation-reduction potential were found to decrease. However, no significant reduction in pH was recorded. Treatment pressures >300 MPa produced milk with lower lightness (luminosity) and a more yellow and green hue. Pressures >150 MPa resulted in micellar fragmentation, as well as significant increases in particle size, viscosity, and water-holding capacity as a consequence of the denaturing of soluble proteins. High-pressure treatments increased the solubility of colloidal calcium phosphate, leading to a considerable increase in the concentration of minerals in the serum phase. The highest concentrations of calcium and phosphorus in the rennet whey of milk were reached at 300 MPa. Curd coagulation time was reduced by 28% at pressures >300 MPa, and an increase in the curd firming rate was observed. As treatment pressure increased to 450 MPa, the firmness, elasticity, and the percentage creep recovery of gels increased, whereas values of compliance and fracture strain were reduced. Thus, we can conclude that 300 MPa is the optimum treatment pressure for milk intended for cheesemaking by enzymatic coagulation. This pressure produced milk with optimal coagulation kinetics and water-holding properties with the least loss of fat and protein to the whey.

Acid and rennet gelation properties of sheep, goat, and cow milks: Effects of processing and seasonal variation

Gelation is an important functional property of milk that enables the manufacture of various dairy products. This study investigated the acid (with glucono-δ-lactone) and rennet gelation properties of differently processed sheep, goat, and cow milks using small-amplitude oscillatory rheological tests. The impacts of ruminant species, milk processing (homogenization and heat treatments), seasonality, and their interactions were studied. Acid gelation properties were improved (higher gelation pH, shorter gelation time, and higher storage modulus (G') by intense heat treatment (95°C for 5 min) to comparable extents for sheep and cow milks, both better than those for goat milk. Goat milk produced weak acid gels with low G' (<100 Pa) despite improvements induced by heat treatments. Seasonality had a marked impact on the acid gelation properties of sheep milk. The acid gels of late-season sheep milk had a lower gelation pH, no maximum in tan δ following gel formation, and 70% lower G' values than those from other seasons. We propose the potential key role of a critical acid gelation pH that induces structural rearrangements in determining the viscoelastic properties of the final gels. For rennet-induced gelation, compared with cow milk, the processing treatments of the goat and sheep milks had much smaller impacts on their gelation properties. Intense heat treatment (95°C for 5 min) prolonged the rennet gelation time of homogenized cow milk by 8.6 min (74% increase) and reduced the G' of the rennet gels by 81 Pa (85% decrease). For sheep and goat milks, the same treatment altered the rennet gelation time by only less than 3 min and the G' of the rennet gels by less than 14 Pa. This difference may have been caused by the different physicochemical properties of the milks, such as differences in their colloidal stability, proportion of serum-phase caseins, and ionic calcium concentration. The seasonal variations in the gelation properties (both acid and rennet induced) of goat milk could be explained by the minor variation in its protein and fat contents. This study provides new perspectives and understandings of milk gelation by demonstrating the interactive effects among ruminant species, processing, and seasonality.

Gastric devolution of transglutaminase-induced acid and rennet-induced casein gels using dynamic DIDGI® and static COST action INFOGEST protocols

Limited studies in the literature have compared in vitro dynamic and in vitro static protocols for modelling the gastric digestive process of food systems. This experiment explores the differences between two different in vitro approaches to the devolution of a transglutaminase-induced acid gel (TG, pH 5.1–5.3) and rennet-induced gel (RG, pH 6.5–6.7). Gels were exposed to a simulated oral phase, followed by either the dynamic DIDGI® or static COST action INFOGEST protocol to simulate gastric conditions. Protein hydrolysis was evident from 15 min onwards for TG exposed to the dynamic protocol where levels continued to increase at a steady rate. In contrast, RG exhibited a notable lag-phase before levels increased from around 60 min onwards. Under the static protocol, protein hydrolysis was observed for both TG and RG upon exposure to the gastric environment which continued to increase over time. Despite these differences, similar levels of protein hydrolysis were found for TG and RG at the gastric endpoint using either protocol demonstrating that both the dynamic DIDGI® and static COST action INFOGEST methods provide a suitable and comparable environment for the in vitro digestion of casein protein under simulated gastric conditions.

Saccharomyces cerevisiae improves rennet-induced gelation of ultra-high temperature milk

Heating milk at high temperatures impairs its renneting properties, but rennet-induced curds can be formed from ultra-high temperature (UHT) milk inoculated with Saccharomyces cerevisiae. Herein, we measured physicochemical indices of UHT milk inoculated with S. cerevisiae before rennet addition, monitored the kinetics of gel formation, and investigated the physicochemical properties and microstructure of rennet-induced curds to explore the mechanisms by which S. cerevisiae influenced rennet-induced gelation of UHT milk. Compared with untreated pasteurized cow milk and UHT milk, the ethanol content was increased, the pH was decreased, the particle size and ζ-potential were increased, the time points at which the elasticity index began to increase were advanced, and the maximum elasticity index was increased for UHT milk inoculated with S. cerevisiae. The number of S. cerevisiae cells affected the structure of rennet-induced curds; with few cells added, the protein network of curds was continuous and tight, the mean square displacement curves showed an asymptotic behavior, and the water retention capacity and curd yield were high; with more cells added, the loosely entangled proteins aggregated, the continuity of the network was destroyed, and the curd yield decreased. In summary, a low number of S. cerevisiae cells (<1.0 × 107 cfu/mL) can increase particle size, ζ-potential, and ethanol content, and decrease pH of S. cerevisiae-inoculated UHT milk, thereby accelerating the aggregation reactions after enzymatic reaction and improving the renneting properties.

Functional properties of skim milk concentrates produced by reverse osmosis filtration and reconstituted commercial powders

Substitution of commercial milk powders by fluid milk concentrates as ingredients in dairy products, may improve product quality and increase sustainability. This study compared the functional properties of skim milk concentrates made by reverse osmosis (SMC) with standard reconstituted high- and low-heat skim milk powders (POW-SMC). SMC had larger Z-average size values, higher viscosities, wider particle size distributions and lower extent of whey protein denaturation than POW-SMC. During storage, SMC showed no acid-induced gelation, but improved rennet-induced gelation compared with POW-SMC. Emulsification activity improved at 20 compared with 10% solids in all concentrates, while emulsion stability was lowest for SMC. Better foaming was observed for high-heat POW-SMC, except after 30 days, where SMC showed highest overrun. In certain dairy products, SMC can thus replace commercial powders to reduce processing costs and improve functionality. SMC showed to be especially advantageous for rennet-gelled products.

Development and evaluation of probiotic delivery systems using the rennet-induced gelation of milk proteins.

The aims of this study were to develop a milk protein-based probiotic delivery system using a modified rennet-induced gelation method and to determine how the skim milk powder concentration level and pH, which can affect the rennet-induced intra- and inter-molecular association of milk proteins, affect the physicochemical properties of the probiotic delivery systems, such as the particle size, size distribution, encapsulation efficiency, and viability of probiotics in simulated gastrointestinal tract. To prepare a milk protein-based delivery system, skim milk powder was used as a source of milk proteins with various concentration levels from 3 to 10% (w/w) and rennet was added to skim milk solutions followed by adjustment of pH from 5.4 or 6.2. Lactobacillus rhamnosus GG was used as a probiotic culture. In confocal laser scanning microscopic images, globular particles with a size ranging from 10 μm to 20 μm were observed, indicating that milk protein-based probiotic delivery systems were successfully created. When the skim milk powder concentration was increased from 3 to 10% (w/w), the size of the delivery system was significantly (p < 0.05) increased from 27.5 to 44.4 μm, while a significant (p < 0.05) increase in size from 26.3 to 34.5 μm was observed as the pH was increased from 5.4 to 6.4. An increase in skim milk powder concentration level and a decrease in pH led to a significant (p < 0.05) increase in the encapsulation efficiency of probiotics. The viability of probiotics in a simulated stomach condition was increased when probiotics were encapsulated in milk protein-based delivery systems. An increase in the skim milk powder concentration and a decrease in pH resulted in an increase in the viability of probiotics in simulated stomach conditions. It was concluded that the protein content by modulating skim milk powder concentration level and pH were the key manufacturing variables affecting the physicochemical properties of milk protein-based probiotic delivery systems.

Detection of meso-scale heterogeneity in the rennet-induced gelation process via image analysis technique

Detection of meso-scale heterogeneity in the rennet-induced gelation process via image analysis technique

Quantification of the rennet-induced gelation kinetics using image analysis technique

Quantification of the rennet-induced gelation kinetics using image analysis technique

Evaluation of rennet-induced gelation under different conditions as a potential method for 3D food printing of dairy-based high-protein formulations

The rennet-induced gelation of milk proteins was evaluated as a potential method for the formation of 3D printed food structures. The effects of pH, [Ca2+], and temperature on the rennet gelation properties of milk protein dispersion with 15% (w/w) protein content were assessed using low amplitude strain oscillation rheometry. A cycled-temperature ramp (heating-holding-cooling) during rheological measurements was suitable to evaluate gel firmness development, as an imitation of the temperature profile in the 3D printing process. A factorial design considering two levels of pH (6.0 and 6.4), [Ca2+] (1.5 and 5.7 mM), and temperature (31 and 40 °C), showed that the pH, temperature, and its interaction were the main factors enhancing gel formation and the strength of the resultant gel. At pH 6.0 and temperature ramped to 40 °C followed by cooling to 15 °C, a very high gel strength (~8–9 kPa) was obtained. These results showed that rennet-induced gelation could be manipulated for developing printable dairy formulations.

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.

Ultrasound improves the rheological properties and microstructure of rennet-induced gel from goat milk

Goat milk was treated by ultrasound (US) at 20 kHz and 12.1 ± 0.89 W from 15 to 30 min. The influence of the treatments on the rheological properties and microstructure of the rennet-induced gel was measured. The particle size, zeta potential, hydrophobicity, and secondary structures affected by the treatments were also measured. Sonication time >20 min resulted in a significant (p ≤ 0.05) increase in the storage modulus (G′) compared with that of the controls (raw goat milk and pasteurised goat milk), while the rennet coagulation time was significantly decreased. Scanning electron microscopy showed the gel microstructures of the sonicated samples were more interconnected. US treatments reduced the particle size and changed the secondary structures of the protein which were significantly increased in β-sheets and random coils. However, there was significant decrease in α-helix and β-turn. The β-sheet increase showed a positive correlation (r = 0.74) with hydrophobicity.

Evaluation of the effect of Gleditsia amorphoides gum on the properties of rennet-induced milk protein gels

The study and characterisation of food gels obtained from phase-separated systems has gained interest since a wide variety of gel structures and textures can be developed. In this study, the phase and rheological behaviour of milk protein/espina corona gum (MP/ECG) mixtures were evaluated. These mixtures presented a segregative phase separation and a rheological behaviour proportional to the ECG concentration. Microstructural analysis, textural parameters and water-holding capacity of gels obtained from MP/ECG mixed systems using rennet as gelling agent were determined. At high ECG concentrations (≥0.05%, w/v), the gel microstructure changed from a coarse strand to a bicontinous microstructure. Such microstructural changes affected the textural parameters, firmness and break point, and the water-holding capacity of the gels. The results obtained in this work could be explained by the interplay between the segregative interaction of the biopolymers and the rennet-induced gelation rate.

Effect of Sheep's Milk Composition on Strength and Syneresis of Rennet-Induced Milk Gel During Lactation.

SUMMARYThis study investigates the effects of raw sheep’s milk composition on the strength and syneresis of obtained gels throughout the lactation. Casein, fat and total solids content, as well as ionic calcium mass fraction significantly (p<0.05) increased during lactation. As lactation progressed, milk formed firmer gel with higher syneresis ability. Increasing casein to fat ratio in sheep’s milk significantly increased (p<0.05) gel strength and syneresis. On the other hand, gel strength and syneresis were significantly reduced as a result of increased fat content in sheep’s milk. Ionic calcium mass fraction affected gel strength but not syneresis. Neither gel strength nor syneresis were affected by casein and urea content or by somatic cell count in sheep’s milk. Correlation coefficients between milk components and gel strength, as well as syneresis, were significant (p<0.01, p<0.05) but never higher than 0.35.

Effect of Enzyme Modified Soymilk on Rennet Induced Gelation of Skim Milk

In this study, soymilk was hydrolyzed to different degrees with flavourzyme, and then soymilk and enzyme modified soymilk at various levels were added to skim milk respectively, to generate a mixed gel using rennet. Rheological properties, scanning electron microscopy imaging, and physical and chemical indexes were examined to reveal the effect of enzyme modified soymilk on rennet induced gelation of skim milk. Results showed that soymilk inhibited the aggregation of skim milk, led to a decrease in storage modulus (G’), significantly increased moisture content and curd yield, and the resulting network was coarse. Enzyme modified soymilk with a molecular weight below 20 kDa led to a more uniform curd distribution, which counteracted the reduction of G’ and allowed for the formation of a stronger gel. Both the moisture content and the curd yield increased with the addition of soymilk and enzyme modified soymilk, and overall the effect of adding a high degree of hydrolysis of enzyme modified soymilk was superior. Compared to untreated soymilk, the addition of a certain amount of enzyme modified soymilk resulted in a new protein structure, which would improve the texture of blend cheese.

Influence of protein concentration and coagulation temperature on rennet-induced gelation characteristics and curd microstructure

This study characterized the coagulation properties and defined the cutting window (CW; time between storage modulus values of 35 and 70 Pa) using rheometry for milk standardized to 4, 5, or 6% protein and set at 28, 32, or 36°C. Milks were standardized to a protein-to-fat ratio of approximately 1 by blending ultrafiltration retentate, skim milk, and whole milk. The internal curd microstructure for selected curd samples was analyzed with transmission electron microscopy and scanning electron microscopy. Lowering the coagulation temperature caused longer rennet coagulation time and time to reach storage modulus of 35 Pa, translating into a wider CW. It also led to a lower maximum curd-firming rate (MCFR) with lower firmness at 40 min at a given protein level. Increasing protein levels resulted in the opposite effect, although without an effect on rennet coagulation time at a given temperature. On coagulation at 28°C, milk with 5% protein resulted in a similar MCFR (∼4 Pa/min) and CW (∼8.25 min) compared with milk with 4% protein at 32°C, which reflects more standard conditions, whereas increasing milk to 6% protein resulted in more than doubling of the curd-firming rate (MCFR = 9.20 Pa/min) and a shorter CW (4.60 min). Gels set at 28°C had lower levels of rearrangement of protein network after 40 min compared with those set at 36°C. Protein levels, on the other hand, had no influence on the levels of protein network rearrangement, as indicated by loss tangent values. The internal structure of curd particles, as investigated by both scanning electron microscopy and transmission electron microscopy, appeared to have less cross-linking and smaller casein aggregates when coagulated at 28°C compared with 36°C, whereas varying protein levels did not show a marked effect on aggregate formation. Overall, this study showed a marked interactive effect between coagulation temperature and protein standardization of milk on coagulation properties, which subsequently requires adjustment of the CW during cheesemaking. Lowering of the coagulation temperature greatly altered the curd microstructure, with a tendency for less syneresis during cutting. Further research is required to quantify the changes in syneresis and in fat and protein losses to whey due to changes in the microstructure of curd particles arising from the different coagulation conditions applied to the protein-fortified milk.

Effect of transglutaminase on rennet-induced gelation of skim milk and soymilk mixtures.

Protein gels made from cow milk and soymilk can yield products of exceptional value. Transglutaminase (TG) affect rennet-induced gelation of proteins, and improves the functionality of the final products. In this paper, TG and rennet were added to skim milk and soymilk mixtures simultaneously, and the rennet-induced coagulation was studied. Diffusing wave spectroscopy and rheology measurements were used to access the structural changes of the mixtures during renneting. Syneresis analysis and microscopy can give more information for understanding the system. Soymilk and TG have synergetic effects and inhibit rennet-induced gelation to a certain degree. With increasing soymilk and TG, elastic index and storage modulus decreased, gelation time was delayed, and curd yield and moisture content increased. At excess soymilk and TG, no curds can be formed. There were significant effects of soymilk and TG on curd microstructure. Soymilk inhibited the aggregation of casein micelles and contributed to more coarse and heterogeneous networks. TG limited reorganization of the proteins, leading to more homogenous networks with small pores. The use of soymilk and TG simultaneously impair rennet-induced gelation and curd syneresis, and consequently lead to a higher yield of high-moisture curd. © 2018 Society of Chemical Industry.

Rheological and structural properties of coagulated milks reconstituted in D2O: Comparison between rennet and a tamarillo enzyme (tamarillin)

The physicochemical properties and structural characteristics of milk gels in deuterium oxide (D2O) induced by rennet and tamarillo enzyme (tamarillin) were investigated. SDS-PAGE showed that both proteinases hydrolysed κ-casein while tamarillin also exhibited a broader specificity on α- and β-casein. Small and large deformation rheological measurement showed that rennet-induced milk gels at low milk solid content (10% w/w) displayed higher elasticity than the gels induced by tamarillin. In high concentration milk gels (20% w/w), the aggregation time was sharply decreased and the elasticity was increased for both rennet and tamarillin induced gels. In addition, large deformation experiments indicated that rennet-induced gels are more brittle than those made with tamarillin for both 10 and 20% (w/w) milk gels. The microstructures of the milk gels displayed more porosity in the gels made with tamarillin, compared to those made with rennet, likely due to the broader caseinolytic activity of the tamarillin. Ultra-small angle neutron scattering (USANS) showed that there are no differences in the large-scale structure of tamarillin and rennet induced gels. However, small angle X-ray scattering (SAXS) revealed that the milk gel fine structures are different.