Acid Casein Gels Research Articles

Cross-linking with microbial transglutaminase: Isopeptide bonds and polymer size as drivers for acid casein gel stiffness

Acid casein, cross-linked with microbial transglutaminase for different times, was mixed with uncross-linked reference to obtain samples with identical numbers of cross-links (expressed as N-ε-(γ-glutamyl)-lysine isopeptide content, IC). Mixtures from longer incubated casein solutions, where cross-links were also formed within and between already present polymers, resulted in a lower polymerisation degree because a higher proportion of the reference was necessary to achieve the target IC. For similar gel stiffness at the same isopeptide content, a particular size of the polymers was crucial; high dimer content resulted in lower gel stiffness at a given IC. Gels with high stiffness formed at high amounts of larger polymers. Stiffness at 30 °C was lower at identical IC when casein solutions cross-linked for more than 5 h were used for blending; this was not observed for gelation at 20 or 40 °C. Large deformation properties of the casein gels were clearly affected by cross-linking.

Cross-linking with microbial transglutaminase: Relationship between polymerisation degree and stiffness of acid casein gels

Acid casein in phosphate buffer was used as a simplified substrate for cross-linking experiments with microbial transglutaminase. The importance of the intensity of enzyme treatment on the rheology of acid gels was evaluated by preparing mixtures of casein that were cross-linked to a different extent and a native reference to obtain samples with a similar polymerisation degree. The stiffness of acid gels from caseins incubated longer then 3 h was always lower at identical polymerisation degree larger than approximately 30%. The quantification of isopeptides formed by the enzyme reveals a substantial increase of bonds at a stage of cross-linking where only 10% of monomers are available. This indicates that cross-linking must occur between and/or within already existing aggregates, which become larger and less flexible, and supports the hypothesis that, starting with a critical aggregate size the network formation is somewhat impaired by the size and reduced flexibility of the proteins.

Effect of gellan, alone and in combination with high-methoxy pectin, on the structure and stability of doogh, a yogurt-based Iranian drink

Doogh is a traditional Iranian drink prepared by fragmentation and dilution of yogurt, with addition of salt and flavouring. In the present work, we have used viscometry, microscopy, particle-size analysis and measurements of serum separation to explore the effect of very low concentrations of gellan gum (0.01, 0.03 and 0.05 wt %), alone or in combination with 0.25 wt % high-methoxy pectin (HMP), on its structure and stability. HMP is known to prevent association of casein particles in acidic milk drinks by steric stabilisation, forming a protective layer bound electrostatically to the surface of the particles. Doogh incorporating 0.25 wt % HMP alone showed satisfactory stability on storage for ∼10 days at 5 °C, but after 15 days there was obvious separation into a dense sediment and a much clearer upper layer that occupied more than 80% of the total volume, which we attribute to progressive sedimentation of individual sterically-stabilised particles. Samples incorporating gellan (with or without HMP), by contrast, showed rapid development of a clear serum phase, with little further separation at longer times (up to ∼1 month), suggesting expression of fluid by contraction of a gel network (i.e. syneresis rather than sedimentation). Particle size increased dramatically (more than 10-fold) with increasing concentration of gellan, and at the highest concentration studied (0.05 wt %) a continuous network of casein-rich strands was observed by phase-contrast microscopy. The concentration of NaCl used in the doogh samples (0.5 wt % ≈ 85 mM) is known to be sufficient to maintain gellan in its ordered (double-helix) conformation, which has higher charge density than individual molecules of HMP. We suggest that network structure is formed by electrostatic attachment of gellan to fragments of acid-casein gel, thus increasing particle size and inhibiting surface-coverage by HMP, with weaker associations between gellan helices allowing the samples to flow. Observed decrease in serum volume with increasing concentration of gellan is attributed to formation of progressively stronger coupled networks with greater resistance to syneresis. Stabilisation of doogh with 0.05 wt % gellan in combination with 0.25 wt % HMP had no adverse effect on organoleptic acceptability, and reduced serum volume on protracted storage to ∼10% (in comparison with over 80% for the same concentration of HMP alone), suggesting that gellan could be of practical value in extending the shelf-life of doogh (and related acidic milk drinks).

The application of microscopy and rheology to study the effect of milk salt concentration on the structure of acidified micellar casein systems

The microstructural and rheological properties of glucono-delta-lactone induced acid casein gels made from micellar casein dispersed in water and in lactose-free simulated milk ultrafiltrate (SMUF) containing 40, 70, 100 and 200% of the salts normally present in milk were studied. Confocal scanning laser microscopy (CSLM) and low amplitude oscillatory rheology (LAOR) were used to monitor structural development during acidification. Image analysis of CSLM micrographs was used for measurement of mean apparent equivalent circular diameter of pores, mean volume-weighted size (star volume) of pores and for measurement of homogeneity by grey-level pixel variance. Transmission electron microscopy (TEM) was used to determine mean particle diameter and star volume of protein aggregates within the gel networks. At low salt concentrations, gel networks formed more rapidly with consequently larger pores compared to more slowly forming, homogeneous gels being formed on acidification at higher salt concentrations. TEM analysis showed increased fusion of casein particles as salt concentration decreased, indicating significant destabilisation of micellar caseins. Dynamic LAOR indicated differences in storage modulus and phase angle depending on ionic strength. Results indicated an inverse relationship between pore or particle size and SMUF concentration. Some length-scale dependence was noted for pixel variance and apparent equivalent circular diameter.

Influence of selected factors on the viscosity of set style yogurt and acid casein gel at constant speed of spindle rotation

The influence of milk dry matter (DM) content (8.20%, 9.27% and 10.28%) and applied heat treatments (untreated milk and milk heat-treated at 90oC/10?) on the viscosity of set-style yogurt and acid casein gel gained by acidification with GDL (glucono-d-lactone) has been investigated. Viscosity was measured during the time of 3 minutes at constant speed of spindle rotation of 20 rpm. The results have shown that yogurt samples produced from untreated milk had higher viscosity values than samples produced from heat-treated milk. An increase of dry matter content influenced the increase of viscosity of yogurt samples produced from both untreated milk and heat-treated milk. Samples with 10.28% DM had the highest viscosity values. An increase of DM content in samples of acid casein gel produced by acidification with GDL had an opposite influence. The samples with smallest DM content had the highest viscosity values. There is no linear relationship between the period of storage of set-style yogurt produced from heat-treated milk and viscosity value.

Effects of low-methoxyl amidated pectin and ionic calcium on rheology and microstructure of acid-induced sodium caseinate gels

The influence of low-methoxyl amidated pectin on the rheological and microstructural properties of sodium caseinate gels (2%, w/v), made by acidifying with glucono-δ-lactone, has been investigated at 25 °C. In the absence of calcium ions, a substantial reduction in the time-dependent storage modulus was detected at pectin concentrations as low as 0.01–0.02% (w/v), and a significant increase in gelation time at pectin concentrations ≥0.05% (w/v). Complete inhibition of acid-induced caseinate gelation was noted at ≥0.8% (w/v) pectin. Confocal microscopy has confirmed that the presence of an increasing content of pectin prevents aggregation of caseinate particles into a coherent network. Consistent with the sensitivity of both these biopolymers to calcium ions, the addition of calcium chloride (2 mol Ca 2+/mol caseinate) has a major influence on the interactions in the caseinate+pectin system. Whereas for low pectin content the modulus was reduced (as for the calcium-free system), there was found to be a large enhancement of the time-dependent modulus at higher pectin contents (≥0.2%, w/v). The combination of a gradual release of calcium ions from caseinate aggregates as the pH is lowered and the electrostatic adsorption of pectin onto the casein particles influences the balance of casein–casein, pectin–pectin and casein–pectin interactions in the final gel structure. Strong syneresis was clearly evident in calcium-containing systems with 0.2–0.6% (w/v) pectin. Microscopy confirmed the total absence of the characteristic acid–casein gel network connections at high pectin content (0.8%, w/v).

The influence of dry matter, applied heat treatment and storage period on the viscosity of stirred yogurt

Skim milk powder reconstituted to 8.44% TS, 9.65% TS and 10.84% TS respectively was used for investigation. Untreated milk and milk heat treated at 85?C/20 min and 90?C/10 min, respectively, were used for the investigation. Milk was inoculated with 2.5% of yogurt culture (containing Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus in the ratio 1:1) at 43?C. Samples were incubated until pH 4.6 was reached. Samples were immediately cooled to 4?C and held at that temperature during 14 days. Acid casein gel was stirred after 1, 7 and 14 days of storage. Measurements were done at 30 rpm during 2 min, at 20?C. According to the investigation, it could be concluded that both applied heat treatment and dry matter content influence viscosity of stirred yogurt. Viscosity increases when dry matter content increases. The smallest viscosity had yogurt produced from untreated milk with 8.44% TS, while samples produced from milk with 10.84% TS had the highest viscosity. Applied heat treatments had significant influence on viscosity of yogurt gained by stirring of acid casein gels after 7 and 14 days of storage. Stirred yogurt produced from milk heat treated at 90?C/10 min had a higher viscosity than samples produced from milk heat treated at 85?C/20 min. Storage period influenced average viscosity of stirred yogurt. Samples of stirred yogurt produced from milk with 8.44% TS showed a decrease of average viscosity during storage regardless of the applied heat treatment of milk. The highest average viscosity had samples produced from milk with 10.84% TS.

The influence of investigated factors on viscosity of stirred yogurt

Skim milk was reconstituted to obtain milk with 8.44% DM, which was standardized with demineralized whey powder (DWP) to obtain milk sample A (9.71% DM) and milk sample B (10.75% DM). Milk samples were heat treated at 85?C/20 min and 90?C/10 min, respectively. Untreated milk was used as control. Milk samples were inoculated with 2.5% of commercial yogurt culture (containing Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus in the ratio 1:1) at 43?C. Samples were incubated until pH 4.6 was reached. Samples were immediately cooled to 4?C and held at that temperature until analyses. Samples of acid casein gels were stirred after 1, 7 and 14 days of storage. Measurements of viscosity were done with Brookfield DV-E Viscometer. Spindle No 3 at 30 rpm was used for all samples. Duration of fermentation decreased when DWP was used for standardization of milk dry matter content. Yogurt samples produced from milk heat treated at 85?C/20 min, obtained by stirring of gel 1 day after production had a higher viscosity than sample produced from milk heat treated at 90?C/10 min. On the other hand, samples produced from milk heat treated at 90?C/10 min had a greater viscosity after 7 and 14 days of storage, which indicates a greater hydrophilic properties and a more pronounced swelling of casein micelles.

Cross-linking casein micelles by a microbial transglutaminase: influence of cross-links in acid-induced gelation

Mechanical and structural properties of transglutaminase-induced casein gels are described here and compared with those of traditional acid casein gels. These gels were characterised by rheology and microscopy (confocal laser scanning microscopy and transmission electron microscopy). Unlike traditional casein gels produced by acidification and/or renneting which lead to gels cross-linked by weak physical interactions, gels formed using transglutaminase are covalently cross-linked. Gels with different characteristics can therefore be formed in this way and have some unusual and interesting features in terms of strength, kinetics of gelation, sensitivity to heat treatment and syneresis behaviour. The first steps of the transglutaminase-induced micellar aggregation were followed by turbidimetry and a mechanism for the aggregation process proposed.

Properties of acid casein gels made by acidification with glucono-δ-lactone. 2. Syneresis, permeability and microstructural properties

Syneresis of casein gels made by acidification with glucono-δ-lactone (GDL) was studied in relation to gel structure as derived from permeametry and confocal scanning laser microscopy (CSLM). Gels made at 40 °C exhibited ‘spontaneous syneresis’ with wheying-off almost immediately after gelation, while those formed at 30 °C exhibited little syneresis and only after it was initiated by wetting the surface. Syneresis decreased with a reduction in the pH of gels (increasing amount of GDL added). Gels cooled to 5 °C (before initiating syneresis) and low pH gels exhibited ‘negative syneresis’, i.e. an increase in the height of the gel after wetting. Addition of NaCl had little effect on syneresis, except at pH values > 4.6, where gels with added NaCl exhibited stronger syneresis than those made without added NaCl. Higher gelation temperatures resulted in a far greater permeability coefficient (B), indicating the presence of large pores in these gels. Gels formed at low temperatures had a very low B. Addition of NaCl, at all gelation temperatures, markedly reduced B. Confocal scanning micrographs showed that gels made at high gelation temperatures had large pores, many > 20 μm. At low gelation temperatures, the pores were small, mostly < 5 μm. Fractal aggregation theory was used to explain some of the results, especially the rearrangement of aggregated particles at an early stage of the gelation process, i.e. over relatively short distances. It was concluded that gelation temperature had a large effect on this rearrangement. At low temperatures (e.g. 20 °C), rearrangement did not occur, whereas it was already extensive at 30 °C, implying that the ‘building blocks’ of the fractal gel consisted of dense aggregates of, say, 25 casein particles. This resulted in increased permeability. It was also concluded that the syneresis occurring at 30 °C is primarily due to consolidation of the gel network under its own weight, which is soon counteracted by the stress induced by the deformation of this network. Gels formed at higher temperatures may be unstable and show spontaneous syneresis, presumably because some of the strands in the gel network were weak enough to break. No conclusive explanation could be given for the effects of NaCl concentration and pH on the properties of acid casein gels.

Properties of acid casein gels made by acidification with glucono-δ-lactone. 1. Rheological properties

The effects of gelation temperature (20, 30 or 40 °C), assay temperature, concentration of glucono-δ-lactone (GDL) added, and NaCl concentration on the rheological properties of acid casein gels were studied at small and large deformations. Gels prepared at a high incubation temperature had very low storage moduli ( G ′), whereas those made at a low incubation temperature had extremely high G ′ values. A higher concentration of GDL resulted in faster gelation but slightly lower G ′ values of aged gels. Addition of NaCl resulted in longer gelation times and a slower rate of increase of G ′. Cooling of gels prepared at 30 or 40 °C resulted in an increase in G ′. However, for gels formed at 20 °C, G ′ decreased initially on cooling but returned to its original value on holding at 5 °C. The loss tangent (tan δ) of gels formed at 20 or 30 °C was independent of frequency; however, for gels formed at 40 °C, tan δ was lower at low frequencies. As gels were cooled to 5 °C, tan δ increased. Fracture stress ( σ fr ) of gels formed at 20 °C was much greater than that of the gels formed at higher temperatures. Heating gels to temperatures higher than the gelation temperature resulted in a decrease in σ fr . At low gelation temperatures, young gels had very high ( σ fr values.

Rheology of Buildup, Breakdown, and Rebodying of Acid Casein Gels

Dynamic oscillations were measured during in situ buildup and breakdown of acid casein gels induced by glucono-δ-lactone at varying temperatures and skim milk solids concentrations. The gel structure was gradually broken down by continuous oscillation at high strains. Using high strains (up to .8), gel moduli were lowered to a few percentages of their original value with no wall slip problems. No equilibrium values were found for either buildup or breakdown. The time course of buildup and rebodying was complex, but different gels were affected by shear in the same way in relation to their initial values. Strain was the main rate determining parameter.

Structure of acid casein gels 2. Dynamic measurements and type of interaction forces

The time dependence of the mechanical behaviour and the type of interaction forces in acid casein gels were studied by dynamic rheological and permeability measurements. Gels were formed by quiescent heating of casein dispersions acidified in the cold to pH 4.6; they were investigated after an ageing time of 16 h or more. The dynamic moduli increased with frequency ω over an ω range of 10 −3 to 10 rad s −1 indicating that a considerable proportion of the bonds in an acid casein gel have a relaxation time between 0.1 and 10 3 s. The ratio of the loss to storage modulus was independent of ω at 30°C but increased with decreasing ω at lower temperatures and decreased at higher ones. This indicates that acid casein gels become relatively more viscous over longer time scales at lower temperatures. The dynamic moduli strongly decreased with increasing measuring temperature. Since most types of interaction forces, except hydrophobic interactions which increase in strength with temperature in the range considered, are not strongly temperature dependent this points to an indirect effect, involving reconformation of the protein molecules and particles. The dynamic moduli as a function of the pH showed a small but significant maximum near the isoelectric pH. For the formation of a casein gel the ionic strength had to be at least about 0.1 M. Further raising of the ionic strength by addition of NaCl resulted for acid skim milk gels at first in a small increase of the storage modulus and upon further addition in a decrease. These results point to (mainly attractive) electrostatic interactions being important in acid casein gels, besides hydrophobic interactions and, probably, the ever present Van der Waals attraction, as well as steric and entropic effects related to protein conformation. Experiments with sodium caseinate gels in which part of the NaCl was replaced by CaCl 2 point to a specific effect of Ca on gel formation and properties.

Structure of acid casein gels 1. Formation and model of gel network

The formation and the resulting structure of acid casein gels was studied by rheology, permeametry and electron microscopy. Most gels were formed by quiescent heating of casein solutions acidified in the cold at 0–2°C. After acidification of a skim milk powder or a sodium caseinate dispersion to pH 4.6 at low temperature a physically stable suspension of casein particles was obtained. Gelation results from the aggregation of these casein particles at temperatures above about 10°C. These particles have a complex structure due to the association of numerous different casein molecules. Lowering of the temperature after gel formation to 4°C shows that the gel for mation is irreversible in this respect. It is shown that the formation of acid casein gels is subject to an activation Gibbs energy, which markedly decreases with increasing temperature and depends on at least two factors. At ageing temperatures above 10°C, the dynamic moduli G′ and G linearly increase with the logarithm of time over at least a week. Their absolute values depend, primarily, on the heteroge neity of the network. From the strong dependence of the dynamic moduli on the casein concentration, and from permeability measurements and electron microscopy it follows that the gel network is very heterogeneous. It consists of rather big conglomerates and holes each with a diameter of 1–10 μm. The big conglomerates are thought to be built of smaller ones, which in turn consist of casein particles aggregated in strands and nodes. This picture implies that acid casein gels, although they are basically built of macromolecules, have a particulate structure. The strands and nodes themselves can be regarded as concentrated (∼25%) protein gels with a modulus of about 10 5N m -2.

On the fractal nature of the structure of acid casein gels

Acid casein gels are formed by aggregation of casein particles in an acid environment. Two different types of gels were formed and studied by rheological measurements at small (< 2%) and large deformations (like creep measurements). Gel type 1 was made by quiescent warming of a sodium caseinate dispersion of pH 4.6 from 4 to 30°C and gel type 2 was made by acidification of a sodium caseinate dispersion with glucono-δ-lactone (GDL) at 30°C. For small deformations, the dynamic moduli of type 1 gels were much higher than those of type 2 gels. The relative difference between the moduli of the two gel types was greater at lower casein concentrations, probably caused by a difference in the way the big aggregates of casein particles are linked together. The difference supposedly stems from the difference in voluminosity of casein particles in the cold (3.5–4.5 ml g −1) and at 30°C (about 2.5 ml g −1). For gel type 1 the gelation occurred at a high voluminosity; after the gel was formed the voluminosity of the casein particles decreased due to temperature increase, and this led to a stretching of the strands in the gel. The structure of gel type 2 did not change significantly after the gel was formed. The proposed explanation agreed with the results of creep measurements, showing the deformation at which fracture occurred to range between 0.5 and 0.6 for type 1 and between 1 and 1.2 for type 2 gels. Little difference was observed between the shear stresses at which fracture occurred for either type of gel and the relative difference was independent of the casein concentration. The rheological behaviour of both gels could be explained quantitatively using the concept of fractal geometry.

Rheological properties of casein gels

SummaryThe rheological properties of rennet-induced skim milk gels at 30 °C are compared with casein gels formed by acidification to pH 4·6 at 2 °C and subsequent heating to 30 °C. Both types of gels are viscoelastic. However, the gels formed by rennet action are relatively more viscous over time scales longer than 1 s. This implies that on average the protein-protein bonds in these gels are relatively more mobile than in the acid casein gels. It is thought that this is primarily due to differences in the structure of the casein particles and in the type of the main interaction forces in and between the casein molecules and particles.

Theoretical and experimental study of the fractal nature of the structure of casein gels

Casein gels consist of particulate disordered structures, formed by aggregation of casein micelles. The aggregation leads to flocs of a fractal nature, which form a gel as soon as the occupy the total volume. A model was developed to describe gel formation and structure usnig the concept of fractal geometry. Parameters derived from this model were compared with experimental results concerning the structure and related properties.The model was in good agreement with the dependence of the permeability or rheological properties, such as the storage modulus of the gels, on the volume fraction of casein, and with the dependence of the turbidity of the gel on wavelength.It is shown that acid casein gels can be described very well as a collection of fractal clusters with a fractal dimensionality, D, of 2.3. Rennet-induced casein gels appeared to lose their fractal behaviour due to microsyneresis. Consequently, the model was not fully valid for the latter gels.