Influence of enzymatic cross-linking on milk fat globules and emulsifying properties of milk proteins

The enzyme transglutaminase (TGase) can modify dairy protein functionality through cross-linking of proteins. This study examined the effects of TGase treatment on milk fat globules and the emulsifying properties of milk proteins. The extent of TGase-induced cross-linking of caseins increased with incubation time, with no differences between whole and skim milk. Extensive clustering of fat globules in extensively cross-linked raw whole milk occurred on homogenisation at 400 or 800 bar. Considerably less clustering of fat globules was observed when recombined milk (90 g fat L^–1) was prepared from TGase-treated skim milk and homogenised at 400 or 800 bar. TGase treatment did not affect fat globule size in cream, but prevented coalescence of fat globules therein, possibly through cross-linking of milk fat globule membrane components. TGase-induced cross-linking of milk proteins affected their emulsifying properties and may increase the stability of natural milk fat globules against coalescence.     

Influence of transglutaminase treatment on some physico-chemical properties of milk

Transglutaminase (TGase) is an enzyme that cross-links many proteins, including milk proteins. In this study, the effects of TGase on some physico-chemical properties of milk were studied. TGase-treated milk was not coagulable by rennet, which was due to failure of the primary (enzymic) stage of rennet action rather than the non-enzymic secondary phase. Dissociation of TGase-treated casein micelles by urea or sodium citrate or removal of colloidal calcium phosphate by acidification and dialysis was reduced, presumably due to the formation of cross-links between the caseins. Casein micelles in TGase-treated milks were also resistant to high pressure treatment and to hydrolysis by plasmin. Results of the present study show that milk proteins are fundamentally modified by the action of TGase, which may have applications in the manufacture of functional proteins for use as novel food ingredients.     

Use of micellar casein concentrate and milk protein concentrate treated with transglutaminase in imitation cheese products—Melt and stretch properties

Melt and stretch properties in dairy-based imitation mozzarella cheese (IMC) are affected by the amount of intact casein provided by dairy ingredients in the formulation. Rennet casein (RCN) is the preferred ingredient to provide intact casein in a formulation. Ingredients produced using membrane technology, such as milk protein concentrate (MPC) and micellar casein concentrate (MCC), are unable to provide the required functionality. However, the use of transglutaminase (TGase) has potential to modify the physical properties of MPC or MCC and may improve their functionality in IMC. The objective of this study was to determine the effect of TGase-treated MPC and MCC retentates on melt and stretch properties when they are used in IMC and to compare them with IMC made using RCN. The MCC and MPC retentates were produced using 3 different lots of pasteurized skim milk and treated with 3 levels of TGase enzyme: no TGase (control), low TGase: 0.3 units/g of protein, and high TGase: 3.0 units/g of protein. Each of the MCC and MPC treatments was heated to 72°C for 10 min to inactivate TGase and then spray dried. Each MCC, MPC, and RCN powder was then used in an IMC formulation that was standardized to 48% moisture, 21% fat, 20% protein, and 1% salt. The IMC were manufactured in a twin-screw cooker by blending, mixing, and heating various ingredients (4.0 kg). Due to extensive crosslinking, the IMC formulation with the highest TGase level (MCC or MPC) did not form an emulsion. The IMC made from MCC treatments had significantly higher stretchability on pizza compared with their respective MPC treatments. The IMC made from TGase-treated MCC and MPC had significantly lower melt area and significantly higher transition temperature (TT) and stretchability compared with their respective controls. Comparison of IMC made using TGase-treated MCC and MPC to the RCN IMC indicated no difference in TT or texture profile analysis-stretchability; however, the Schreiber melt test area was significantly lower. Our results demonstrated that TGase treatment modifies the melt and stretch characteristics of MCC and MPC in IMC applications, and TGase-treated MPC and MCC can be used to replace RCN in IMC formulations.     

Production of skim milk powder by spray-drying from transglutaminase treated milk concentrates: Effects on physicochemical,powder flow,thermal and microstructural characteristics

The effects of transglutaminase (TGase) treatment of skim milk concentrates on physicochemical, powder flow, thermal, microstructural characteristics and protein profiles of skim milk powders (SMP) were investigated. Spray-dried SMP samples produced from milk concentrates were TGase-treated at enzyme concentrations of 0 (control), 0.020% (TG-20), 0.030% (TG-30) and 0.035% (TG-35). Lower particle size values were observed in TGase-treated samples. TGase treatment slightly decreased zeta potential and increased the a* value (redness) of SMPs. Up to a concentration of 0.030%, TGase treatment decreased caking characteristics and increased glass transition temperatures of samples. The TGase treatment above 0.035% significantly decreased cohesion index and compaction coefficient at different speeds. TG-35 showed free flowing flow behaviour depending on the cohesion index, whereas control, TG-20, and TG-30 showed easy flowing behaviour. SDS-PAGE analysis revealed that high molecular mass bands indicated cross-linking in TGase-treated milk powders.     

Stability of casein micelles cross-linked by transglutaminase

In this study, caseins micelles were internally cross-linked using the enzyme transglutaminase (TGase). The integrity of the micelles was examined on solubilization of micellar calcium phosphate (MCP) or on disruption of hydrophobic interactions and breakage of hydrogen bonds. The level of monomeric caseins, determined electrophoretically, decreased with increasing time of incubation with TGase at 30°C; after incubation for 24 h, no monomeric β- or κ-caseins were detected, whereas only a small level of monomeric α_S1-casein remained, suggesting near complete intramicellar cross-linking. The ability of casein micelles to maintain structural integrity on disruption of hydrophobic interactions (using urea, sodium dodecyl sulfate, or heating in the presence of ethanol), solubilization of MCP (using the calcium-chelating agent trisodium citrate) or high-pressure treatment was estimated by measurement of the L*-value of milk; i.e., the amount of back-scattered light. The amount of light scattered by casein micelles in noncross-linked milk was reduced by >95% on complete disruption of hydrophobic interactions or complete solubilization of MCP; treatment of milk with TGase increased the stability of casein micelles against disruption by all methods studied and stability increased progressively with incubation time. After 24 h of cross-linking, reductions in the extent of light scattering were still apparent in the presence of high levels of dissociating agents, possibly through citrate-induced removal of MCP nanoclusters from the micelles, or urea- or sodium dodecyl sulfate-induced increases in solvent refractive index, which reduce the extent of light-scattering.     

Ethanol stability of casein micelles cross-linked with transglutaminase

The influence of transglutaminase (TGase)-induced cross-linking on the ethanol stability of skimmed milk was investigated. The stability of milk against ethanol-induced coagulation increased in sigmoidal fashion with milk pH (5.0–7.5) for all samples; ethanol stability also increased upon incubation (0–24 h) with 0.05 g L⁻¹ TGase at 30 °C. In untreated milk, addition of ethanol induced a collapse of the polyelectrolyte brush of κ-casein on the micelle surface, thereby facilitating micellar aggregation. Dynamic and static light scattering measurements indicated that in TGase-treated milk, the ethanol-induced collapse of the polyelectrolyte brush was far less than in untreated milk, suggesting that the increased ethanol stability of TGase-treated casein micelles is caused by the cross-linking of the polyelectrolyte brush on the micellar surface.     

Use of micellar casein concentrate and milk protein concentrate treated with transglutaminase in imitation cheese products—Unmelted texture

The amount of intact casein provided by dairy ingredients is a critical parameter in dairy-based imitation mozzarella cheese (IMC) formulation because it has a significant effect on unmelted textural parameters such as hardness. From a functionality perspective, rennet casein (RCN) is the preferred ingredient. Milk protein concentrate (MPC) and micellar casein concentrate (MCC) cannot provide the required functionality due to the higher steric stability of casein micelle. However, the use of transglutaminase (TGase) has the potential to modify the surface properties of MPC and MCC and may improve their functionality in IMC. The objective of this study was to determine the effect of TGase-treated MPC and MCC powders on the unmelted textural properties of IMC and compare them with IMC made using commercially available RCN. Additionally, we studied the degree of crosslinking by TGase in MPC and MCC retentates using capillary gel electrophoresis. Three lots of MCC and MPC retentate were produced from pasteurized skim milk via microfiltration and ultrafiltration, respectively, and randomly assigned to 1 of 3 treatments: no TGase (control); low TGase: 0.3 units/g of protein; and high TGase: 3.0 units/g of protein, followed by inactivation of enzyme (72°C for 10 min), and spray drying. Each MCC, MPC, and RCN was then used to formulate IMC that was standardized to 21% fat, 1% salt, 48% moisture, and 20% protein. The IMC were manufactured by blending, mixing, and heating ingredients (4.0 kg) in a twin-screw cooker. The capillary gel electrophoresis analysis showed extensive inter- and intramolecular crosslinking. The IMC formulation using the highest TGase level in MCC or MPC did not form an emulsion because of extensive crosslinking. In MPC with a high level of TGase, whey protein and casein crosslinking were observed. In contrast, crosslinking and hydrolysis of proteins were observed in MCC. The IMC made from MCC powder had significantly higher texture profile analysis hardness compared with the corresponding MPC powder. Further, many-to-one (multiple) comparisons using the Dunnett test showed no significant differences between IMC made using RCN and treatment powders in hardness. Our results demonstrated that TGase treatment causes crosslinking hydrolysis of MCC and MPC at higher TGase levels, and MPC and MCC have the potential to be used as ingredients in IMC applications.     

Heat stability of transglutaminase-treated milk

Heat-induced coagulation of unconcentrated (9%, w/w) and concentrated (18%, w/w) reconstituted skim milk was determined after incubation with transglutaminase (TGase). Cross-linking ∼20% of κ-casein strongly increased the heat stability of unconcentrated milk at pH >6.9, presumably by preventing heat-induced dissociation of κ-casein, whereas increased heat stability of unconcentrated milk at pH 6.6–6.8 was only observed when >80% of casein was cross-linked. Treatment with TGase reduced heat stability of unconcentrated milk at pH <6.6, presumably due to the increased susceptibility of partially cross-linked casein micelles to coagulation arising from heat-induced acidification. A low degree of cross-linking increased the heat stability of concentrated milk at pH >6.8, but more extensive cross-linking progressively reduced heat stability. The degree of cross-linking studied did not increase the heat-stability of concentrated milk at its natural pH. The outcomes of this study substantiate the crucial roles of heat-induced acidification and casein dissociation in heat stability of milk.     

Use of Electrodialysis to Improve the Protein Stability of Frozen Skim Milks and Milk Concentrates

Removal of calcium from skim milk by electrodialysis was logarithmically related to the extent of demineralization (log% calcium removed = 0.18% ash removed + 0.53). This removal of calcium caused a logarithmic dissociation of micellar to serum (sub-micellar) casein (log% total casein in serum = 0.020% calcium removed + 0.70). Increases in protein stability for skim milk and concentrated skim milk were related to this dissociation of micellar casein, and the initial degree of dissociation persisted throughout frozen storage. Protein stability was > 53 wk at –8°C when 80% of the casein in unconcentrated skim milk was dissociated. Concentrated skim milk was stable for > 30 wk at –8°C when 45% of the casein was dissociated. Control samples (7–9% casein dissociated) were stable for only 4–8 wk at –8°C.     

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

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

Fortification of milk protein content with different dairy protein powders alters its compositional,rennet gelation,heat stability and ethanol stability characteristics

Skim milk (SM) was fortified from 3.3 to 4.1% protein using different milk protein powders: skim milk powder (SMP), native phosphocasein (NPC), calcium-reduced phosphocasein (CaRPC), sodium caseinate (NaCas) or calcium caseinate (CaCas). Compared with SMP or NPC, fortification with NaCas and CaRPC, and to a lesser extent CaCas, resulted in milk samples having higher proportions of non-sedimentable casein and calcium, and lower- and higher-levels of κ- and α_S1-casein, respectively, as a proportion of non-sedimentable casein. These changes coincided in milk samples fortified with NaCas, CaRPC or CaCas failing to undergo rennet-induced gelation, and having higher heat stability in the region 6.7–7.2 and ethanol stability at pH 6.4. The study demonstrates that the aggregation behaviour of protein-fortified milk samples is strongly influenced by the degree of mineralisation of the protein powder used in fortification, which affects the partitioning of casein and calcium in the sedimentable and non-sedimentable phases.     

Comparison of casein micelles in raw and reconstituted skim milk

During the manufacture of skim milk powder, many important alterations to the casein micelles occur. This study investigates the nature and cause of these alterations and their reversibility upon reconstitution of the powders in water. Samples of skim milk and powder were taken at different stages of commercial production of low-, medium-, and high-heat powders. The nature and composition of the casein micelles were analyzed using a variety of analytical techniques including photon correlation spectroscopy, transmission electron microscopy, turbidity, and protein electrophoresis. It was found that during heat treatment, whey proteins are denatured and become attached to the casein micelles, resulting in larger micelles and more turbid milk. The extent of whey protein attachment to the micelles is directly related to the severity of the heat treatment. It also appeared that whey proteins denatured during heat treatment may continue to attach to casein micelles during water removal (evaporation and spray-drying). The process of water removal causes casein and Ca in the serum to become increasingly associated with the micelles. This results in much larger, denser micelles, increasing the turbidity while decreasing the viscosity of the milk. During reconstitution, the native equilibrium between colloidal Ca and serum Ca is slowly reestablished. The reequilibration of the caseins and detachment of the whey proteins occur even more slowly. The rate of reequilibration does not appear to be influenced by shear or temperature in the range of 4 to 40°C.     

Gelation and Water Binding Properties of Transglutaminase-treated Skim Milk Powder

Transglutaminase (TGase)-treated skim milk powder (TG-SMP) was prepared by freeze-drying skim milk after TGase treatment (10 U/g milk protein, 40°C for 3 h), followed by TGase inactivation at 85°C for 5 min. TGase modification resulted in significant increases in hardness and water holding capacity (WHC) of heat-induced gels (10% protein, w/v). A marked increase in storage modulus (G') of TG-SMP upon heating suggests that TG-SMP has a greater gelling ability than control-SMP (C-SMP) prepared with predenatured TGase. Acid gels prepared from TG-SMP had a significantly higher WHC at all solid levels (12%, 14%, and 16%) tested and formed a more elastic network than C-SMP.     

热处理及转谷氨酰胺酶对糖基化燕麦蛋白凝胶性质的影响

对燕麦蛋白及其乳糖糖基化燕麦蛋白进行热诱导和转谷氨酰胺酶诱导凝胶改性,探究2 种不同诱导方式对燕麦蛋白及其糖基化产物凝胶性质及结构的影响。结果表明：与燕麦蛋白热诱导凝胶相比,其他3 种燕麦蛋白凝胶的弹性、硬度和持水性均显著提高（P＜0.05）;酶诱导凝胶的质构性质（弹性、硬度、胶黏性）与持水性优于热诱导凝胶;糖基化改性蛋白凝胶的质构性质与持水性显著高于未糖基化改性蛋白凝胶（P＜0.05）。酶诱导蛋白凝胶的表面疏水性分别高于两种热诱导蛋白凝胶;而糖基化改性蛋白凝胶的表面疏水性分别低于未糖基化改性蛋白凝胶。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、傅里叶变换红外光谱、荧光光谱分析、微观结构分析进一步证明了转谷氨酰胺酶能引起燕麦蛋白发生分子间或分子内交联而形成大分子质量的交联产物,转谷氨酰胺酶诱导的燕麦蛋白凝胶具有更为致密的微观三维网络结构。研究表明,与热处理相比,转谷氨酰胺酶更能诱导燕麦蛋白及糖基化产物形成良好的凝胶,为燕麦粉的多元开发提供了一定理论基础。     

Films Based on Egg White Protein and Succinylated Casein Cross-Linked with Transglutaminase

Transglutaminase (TGase) and succinylation could modify the physicochemical characteristics and the functional properties of proteins. The aim of this work was to cross-link egg white protein (EWP) and succinylated casein with TGase and to assess the feasibility of making a novel composite protein film. The effects of succinylated casein content, reaction time, and TGase concentration on the mechanical properties, physical characteristics (thermal property and degree of crystallinity), and structure properties (secondary structure and surface micrograph structure) of the film were investigated in this study. The results revealed that the susceptibility of EWP to TGase-mediated cross-linking modification was enhanced by succinylated casein. Meanwhile, the films with TGase were more homogeneous and smoother and possessed better water resistance and thermal stability. The content of α-helix, β-turn structures were increased whereas β-sheet structure was decreased. The spatial conformation and degree of crystallinity of composite protein film were also affected by TGase. The increase of the degree of crystallinity of the composite film further proved the improvement of film mechanical properties induced by TGase and succinylated casein. Based on the above results, this study provides relevant data and insights for the TGase/chemical modification of protein-based edible films.     

High-pressure treatment of milk in industrial and pilot-scale equipments: effect of the treatment conditions on the protein distribution in different milk fractions

The effects of two different high-pressure (HP) equipments, operating at industrial- and pilot scales, and of the HP-release rate on the contents of non-sedimentable proteins and denatured whey proteins were investigated after treatments of skim milk—from 250 to 650 MPa. Non-sedimentable caseins and denatured whey proteins significantly increased with the pressure level. The industrial-scale equipment produced lower micellar disintegration than the pilot-scale equipment with similar degrees of whey protein denaturation. Ultracentrifugation supernatants obtained from skim milk at 100,000×g and 20 °C for 1 h were also HP-treated for comparative purposes, showing that, in skim milk, the presence of casein promoted the denaturation of whey proteins, although the extent of whey protein denaturation did not influence the release of casein to the soluble phase. Furthermore, most denatured whey proteins remained soluble after treatment in both equipments. In the pilot-scale equipment, the pressure-release rate influenced casein solubilization and whey protein denaturation.     

大豆分离蛋白膜的DSC分析

采用差示扫描量热法(DSC),研究不同相对湿度(RH)条件下,大豆分离蛋白(SPI)膜的热特性以及谷氨酰胺转移酶(TGase)改性对热特性的影响。对于SPI膜,无论是对照膜还是TGase改性膜,蛋白膜的热稳定性随着RH的增高而下降。TGase改性改变了SPI膜吸热峰出现的位置与数量。在同一RH条件下,TGase改性膜更易失去水分。TGase改性使SPI膜的耐热特性基本未变。     

转谷氨酰胺酶催化对不同大豆蛋白凝胶性的影响

研究转谷氨酰胺酶对大豆分离蛋白和7S、11S球蛋白凝胶特性的影响,采用TA-XT plus物性测定仪、荧光分光光度计对各参数进行测定。结果表明：转谷氨酰胺酶能够显著提高大豆蛋白凝胶的凝胶强度,最佳工艺条件为酶添加量40U/g、温度40℃、pH7.5、作用时间2.5h,但此时凝胶表面疏水性和保水性有所下降。经转谷氨酰胺酶催化后,不同蛋白形成热处理凝胶的凝胶特性均发生显著变化,凝胶强度均显著增加,转谷氨酰胺酶催化后大豆蛋白凝胶强度的顺序为11S＞7S＞SPI。     

Selenium content of goat milk and its distribution in protein fractions.

This study reports on selenium distribution in goat milk. Skim milk was found to contain the major part (94%) of total milk selenium. The selenium distribution over casein and whey protein fractions depends on the separation method used, but irrespective of these methods, skim milk selenium is mainly associated with the casein fraction (greater than 69%). Approximately 9%, 7% and 24% of selenium is removed by dialysis (molecular cutoff 10-12 kDa) from skim milk, casein and whey respectively, indicating a major association of selenium with milk proteins. This observation is confirmed by selenium analysis of individual caseins and whey proteins isolated through ion-exchange chromatography and gel filtration. Selenium concentrations of the different isolated milk proteins show considerable variation (caseins: 294-550 ng Se/g; whey proteins: 217-457 ng Se/g).     

Cryo-transmission electron tomography of native casein micelles from bovine milk

Caseins are the principal protein components in milk and an important ingredient in the food industry. In liquid milk, caseins are found as micelles of casein proteins and colloidal calcium nanoclusters. Casein micelles were isolated from raw skim milk by size exclusion chromatography and suspended in milk protein-free serum produced by ultrafiltration (molecular weight cut-off of 3 kDa) of raw skim milk. The micelles were imaged by cryo-electron microscopy and subjected to tomographic reconstruction methods to visualize the 3-dimensional and internal organization of native casein micelles. This provided new insights into the internal architecture of the casein micelle that had not been apparent from prior cryo-transmission electron microscopy studies. This analysis demonstrated the presence of water-filled cavities (∼20 to 30 nm in diameter), channels (diameter greater than ∼5 nm), and several hundred high-density nanoclusters (6 to 12 nm in diameter) within the interior of the micelles. No spherical protein submicellar structures were observed.