Dynamics of casein micelles in skim milk during and after high pressure treatment

The effect of high hydrostatic pressure on turbidity of skim milk was measured in situ together with casein micelle size distribution. High pressure (HP) treatment reduced the turbidity of milk with a stronger pressure dependency between 50 and 300 MPa when the temperature was decreased from 20 to 5 °C, while at 30 °C (50–150 MPa) turbidity exceeded that of untreated milk. At 250 and 300 MPa turbidity decreased extremely. During pressurization of milk at 250 and 300 MPa, the turbidity initially decreased, but treatments longer than 10 min increased the turbidity progressively, indicating that re-association followed dissociation of casein micelles. Especially at 40 °C and at 250 and 300 MPa, the turbidity increased beyond untreated milk. Dynamic light scattering was used to investigate casein micelle sizes in milk immediately after long time (up to 4 h) pressurization at 250 and 300 MPa and casein micelle size distributions were bimodal with micelle sizes markedly smaller and markedly larger than those of untreated milk. Pressure modified casein micelles present after treatment of milk at 250 and 300 MPa were concluded to be highly unstable, since the larger micelles induced by pressure showed marked changes toward smaller particle sizes in milk left at ambient pressure.     

Effects of pressurized thermal processing on native proteins of raw skim milk and its concentrate

Heating, pressurization, and shearing can modify native milk proteins. The effects of pressurized heating (0.5 vs. 10 MPa at 75 or 95°C) with shearing (1,000 s^?1) on proteins of raw bovine skim milk (SM, ～9% total solids) and concentrated raw skim milk (CSM, ～22% total solids) was investigated. The effects of evaporative concentration at 55°C and pressurized shearing (10 MPa, 1,000 s^?1) at 20°C were also examined. Evaporative concentration of SM resulted in destabilization of casein micelles and dissociation of α_S1- and β-casein, rendering CSM prone to further reactions. Treatment at 10 MPa and 1,000 s^?1 at 20°C caused substantial dissociation of α_S1- and β-casein in SM and CSM, with some dissociated caseins forming shear-induced soluble aggregates in CSM. The pressure applied at 10 MPa induced compression of the micelles and their dissociation in SM and CSM at 75 or 95°C, resulting in reduction of the micelle size. However, 10 MPa did not alter the mineral balance or whey proteins denaturation largely, except by reduction of some β-sheets and α-helices, due to heat-induced conformational changes at 75 and 95°C.     

酪蛋白胶束结构及其对牛乳稳定性的影响

论述了酪蛋白的组成及两类具有代表性的酪蛋白胶束模型—亚胶束模型和内部结构模型,这两种模型可以较好地解释酪蛋白胶束的稳定性。pH值、酶、添加成分、工艺过程等能够影响酪蛋白胶束的稳定性,并直接影响到牛乳的稳定。这一方面有利于乳制品的生产如制作酸奶、干酪,一方面影响乳制品的品质。     

定量分析加热后乳清蛋白与酪蛋白的结合

将复原脱脂乳在 70～ 90℃范围内加热 1 0～ 2 5min后 ,用超速离心分离出酪蛋白微粒 ,并用毛细管电泳法定量分析。结果显示 ,β-乳球蛋白更容易结合到酪蛋白微粒上。当加热条件为 70℃、1 0 min时就有相当多的 β-乳球蛋白发生了这种结合 ,这时酪蛋白微粒中没发现任何 α-乳清蛋白 ,只有当加热温度大于 75℃时才有少量 α-乳清蛋白与酪蛋白微粒结合。复原脱脂乳经 90℃、2 5min加热后几乎所有 β-乳球蛋白都已转入酪蛋白微粒部分 ,而只有近 50 %的α-乳清蛋白转入酪蛋白微粒。     

The association of lipophilic phospholipids with native bovine casein micelles in skim milk: Effect of lactation stage and casein micelle size

A known biological role of casein micelles is to transport calcium from mother to young and provide amino acids for growth and development. Previous reports demonstrated that modified casein micelles can be used to transport and deliver hydrophobic probes. In this study, the distribution of lipid-soluble phospholipids, including sphingomyelins (SM) and phosphatidylcholines (PC), was quantified in whole raw milk, skim raw milk, and casein micelles of various sizes during early, mid, and late lactation stages. Low-pressure size exclusion chromatography was used to separate casein micelles by size, followed by hydrophobic extraction and liquid chromatography–mass spectrometry for the quantification of PC and SM. Results showed that the SM d18:1/23:0, d18:1/22:0, d18:1/16:0, d16:1/22:0, d16:1/23:0, and d18:1/24:0 and the PC 16:0/18:1, 18:0/18:2, and 16:0/16:0 were dominating candidates appearing in maximum concentration in whole raw milk obtained from late lactation, with 21 to 50% of total SM and 16 to 35% of total PC appearing in skim milk. Of the total SM and PC found in skim milk, 35 to 46% of SM and 22 to 29% of PC were associated with the casein micelle fraction. The highest concentrations of SM d18:1/22:0 (341 ± 17 µg/g of casein protein) and PC 16:0/18:1 (180 ± 20 µg/g of casein protein) were found to be associated with the largest casein micelles (diameter = 149 nm) isolated in milk from late lactation, followed by a decrease in concentration as the casein micelle size decreased.     

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.     

Rheological properties of concentrated skim milk: importance of soluble minerals in the changes in viscosity during storage

Properties of condensed milks prior to spray drying dictate to a large extent the functionality of the resulting milk powder. Rheological properties of concentrated skim milk, with total solids content of 45% but different mineral content, were studied as a function of shear rate and storage time at 50 degrees C. These milks are proposed as a model to study the effects of minerals on rheology and age gellation of condensed milk prior to drying. During storage of the concentrated milk, the apparent viscosity, particularly after 4 h, increased markedly at all shear rates studied. The yield stress also increased steeply after 4 h of storage at 50 degrees C. The changes in apparent viscosity of concentrated milk stored for up to 4 h were largely reversible under high shear, but irreversible in samples stored for longer time. The appearance of yield stress suggested the presence of reversible flocculation arising from weak attraction between casein micelles, with a transition from reversible to irreversible aggregation during storage. Particle size analysis confirmed irreversible aggregation and fusion of casein micelles during storage. Gradual reduction of mineral content of concentrated milks resulted in a marked decrease in the apparent viscosity and casein micelle aggregation during storage, while addition of minerals to milk had the opposite effect. The results demonstrated that the soluble mineral content is very important in controlling the storage-induced changes in the rheology of concentrated milks.     

Gelation mechanism of milk as influenced by temperature and pH; studied by the use of transglutaminase cross-linked casein micelles

Casein micelles in milk are colloidal particles consisting of four different caseins and calcium phosphate, each of which can be exchanged with the serum phase. The distribution of caseins and calcium between the serum and micellar phase is pH and temperature dependent. Furthermore, upon acidification casein micelles lose their colloidal stability and start to aggregate and gel. In this paper, we studied two methods of acid-induced gelation, i.e., 1) acidification of milk at temperatures of 20 to 50 degrees C and 2) decreasing the pH at 20 degrees C to just above the gelation pH and subsequently inducing gelation by increasing the temperature. These two routes are called T-pH and pH-T, respectively. The gelation kinetics and the properties of the final gels obtained are affected by the gelation route applied. The pH-T milks gel at higher pH and lower temperature and the gels formed are stronger and show less susceptibility to syneresis. By using intramicellar cross-linked casein micelles, in which release of serum caseins is prevented, we demonstrated that unheated milk serum caseins play a key role in gelation kinetics and characteristics of the final gels formed. This mechanism is presented in a model and is relevant for optimizing and controlling industrial processes in the dairy industry, such as pasteurization of acidified milk products.     

Importance of casein micelle size and milk composition for milk gelation

The economic output of the dairy industry is to a great extent dependent on the processing of milk into other milk-based products such as cheese. The yield and quality of cheese are dependent on both the composition and technological properties of milk. The objective of this study was to evaluate the importance and effects of casein (CN) micelle size and milk composition on milk gelation characteristics in order to evaluate the possibilities for enhancing gelation properties through breeding. Milk was collected on 4 sampling occasions at the farm level in winter and summer from dairy cows with high genetic merit, classified as elite dairy cows, of the Swedish Red and Swedish Holstein breeds. Comparisons were made with milk from a Swedish Red herd, a Swedish Holstein herd, and a Swedish dairy processor. Properties of CN micelles, such as their native and rennet-induced CN micelle size and their ζ-potential, were analyzed by photon correlation spectroscopy, and rennet-induced gelation characteristics, including gel strength, gelation time, and frequency sweeps, were determined. Milk parameters of the protein, lipid, and carbohydrate profiles as well as minerals were used to obtain correlations with native CN micelle size and gelation characteristics. Milk pH and protein, CN, and lactose contents were found to affect milk gelation. Smaller native CN micelles were shown to form stronger gels when poorly coagulating milk was excluded from the correlation analysis. In addition, milk pH correlated positively, whereas Mg and K correlated negatively with native CN micellar size. The milk from the elite dairy cows was shown to have good gelation characteristics. Furthermore, genetic progress in relation to CN micelle size was found for these cows as a correlated response to selection for the Swedish breeding objective if optimizing for milk gelation characteristics. The results indicate that selection for smaller native CN micelles and lower milk pH through breeding would enhance gelation properties and may thus improve the initial step in the processing of cheese.     

Heat and/or high-pressure treatment of skim milk: changes to the casein micelle size, whey proteins and the acid gelation properties of the milk

Skim milk was subjected to heat, pressure or combined processes. In general, higher levels of whey protein denaturation were observed for milk subjected to combined processes than those heat- or pressure-treated only. Heat treatment caused small changes to the casein micelle size. Pressure treatment decreased the casein micelle size; however, the effect was less marked when heat and pressure treatments were combined. Acidification of the skim milks produced gels with a range of firmness, yield stresses and yield strains depending on the treatments applied. These changes in acid gel properties were not related only to whey protein denaturation levels in the milks.     

Effect of alkalinization and ultra-high-pressure homogenization on casein micelles in raw and pasteurized skim milk

Mechanical and physicochemical treatments of milk induce structural modifications of the casein (CN) micelles, affecting their techno-functional properties in dairy processing. Here, we studied the effect of alkalinization and ultra-high-pressure homogenization (UHPH) on CN micelles in raw skim milk (rSM) and pasteurized skim milk (pSM). The pH of both skim milks (approximately 6.7) was adjusted to 8.5 and 10.5 before UHPH at 100, 200, and 300 MPa. The structural changes of the CN micelles during the treatments were assessed using laser diffraction, transmission electron microscopy, and turbidity measurements. Finally, ultracentrifugation (70,000 × g for 1 h at 20°C) was carried out to evaluate the protein's distribution between the supernatant (serum phase) and the pellet (colloidal phase) by gel electrophoresis and protein concentration measurement. Alkalinization of both skim milks induced a significant reduction in turbidity, whereas an increase of the average particle size was observed, the effect being more severe in pSM than rSM. At alkaline pH, more proteins were recovered in the serum phase, which suggested that the CN underwent major rearrangements into nonsedimentable CN forms of various sizes, as confirmed by transmission electron microscopy. The amount of CN found in the serum phase at pH 8.5 also increased with the UHPH pressure. Although UHPH did not influence the average CN micelle size at pH 6.7 and 8.5, a pressure-dependent decrease was observed at pH 10.5 for both skim milks. The structural changes of the CN micelles observed in this study throughout the combination of alkalinization and UHPH could be of interest for developing new dairy ingredients with improved functionality.     

pH-dependent sedimentation and protein interactions in ultra-high-temperature-treated sheep skim milk

Sheep milk is considered unstable to UHT processing, but the instability mechanism has not been investigated. This study assessed the effect of UHT treatment (140°C/5 s) and milk pH values from 6.6 to 7.0 on the physical properties of sheep skim milk (SSM), including heat coagulation time, particle size, sedimentation, ionic calcium level, and changes in protein composition. Significant amounts of sediment were found in UHT-treated SSM at the natural pH (～6.6) and pH 7.0, whereas lower amounts of sediment were observed at pH values of 6.7 to 6.9. The proteins in the sediment were mainly κ-casein (CN)–depleted casein micelles with low levels of whey proteins regardless of the pH. Both the pH and the ionic calcium level of the SSM at all pH values decreased after UHT treatment. The dissociation levels of κ-, β-, and α_S2-CN increased with increasing pH of the SSM before and after heating. The protein content, ionic calcium level, and dissociation level of κ-CN were higher in the SSM than values reported previously in cow skim milk. These differences may contribute to the high amounts of sediment in the UHT-treated SSM at natural pH (～6.6). Significantly higher levels of κ-, β-, and α_S2-CN were detected in the serum phase after heating the SSM at pH 7.0, suggesting that less κ-CN was attached to the casein micelles and that more internal structures of the casein micelles may have been exposed during heating. This could, in turn, have destabilized the casein micelles, resulting in the formation of protein aggregates and high amounts of sediment after UHT treatment of the SSM at pH 7.0.     

Effects of milk pH alteration on casein micelle size and gelation properties of milk

The effects of changes in pH above and below the natural pH of milk (ca 6.6) on the casein micelle size and the gelation properties of the pH adjusted and restored samples were investigated. The size of casein micelles increased at pH 7.0 and 7.5, then started to decrease above pH 8.5. It is postulated that at pH below 8.5 the casein micelles swell, while elevated pH causes their dissociation. Conversely, the size of casein micelles decreased on acidification to pH 5.5 and increased when the pH dropped below 5.5, due to the shrinkage of casein micelles at lower pH before their aggregation at pH below 5.5. In response to neutralising treated milk back to normal milk pH of 6.6, it was found that the casein micelle size of treated milk with a narrow range of pH change between 6.0 and 7.0 was reversible, while beyond this range the structure of casein micelles became irreversible. The restoration of casein micelle size was followed by the restoration of some parameters such as soluble calcium, ethanol stability, and milk whiteness. Acid-induced gelation did not change the elastic modulus, while rennet-induced gelation was affected by initial milk pH. In reference to the size of reversible range elastic modulus (G’) of acid or rennet gels made from restored milk, the sizes were similar to control milk after 6 h of gelation.     

Isolation of caseins from whey proteins by microfiltration modifying the mineral balance in skim milk

The objective of this work was to study the effect of different salts and salt concentration on the isolation of casein micelles from bovine raw skim milk by tangential flow microfiltration. Tangential flow microfiltration (0.22 μm) was conducted in a continuous process adding a modified buffer to maintain a constant initial sample volume. This buffer contained calcium chloride (CaCl₂), sodium phosphate (Na₂HPO₄), or potassium citrate (K₃C₆H₅O₇) in concentrations ranging from 0 to 100 mM. The concentrations of caseins and whey proteins retained were determined by sodium dodecyl sulfate-PAGE and analyzed using the Scion Image software (Scion Corporation, Frederick, MD). A complete isolation of caseins from whey proteins was achieved using sodium phosphate in the range of 10 to 50 mM and 20 times the initial volume of buffer added. No whey proteins were detected at 50 mM but this was at the expense of low caseins being retained. When lower sodium phosphate concentrations were used, the amount of caseins retained was higher but a small amount of whey proteins were still detected by sodium dodecyl sulfate-PAGE. Among the salts tested, calcium chloride at 50 mM and all volumes of buffer showed the higher retention of casein proteins. The highest casein:whey protein ratio was found at 30 mM CaCl₂, but no complete casein micelle isolation was achieved. Potassium citrate was the most ineffective salt because a rapid loss of caseins and whey proteins was observed at all concentrations and with low quantities of buffer added during the filtration process. Our results show the potential of altering the mineral balance in milk for isolation of casein micelles from whey proteins in a continuous tangential flow microfiltration system.     

Microstructural changes in casein supramolecules during acidification of skim milk

Pasteurized skim milk was acidified using different levels of glucono-δ-lactone at 10, 20, 30, and 40°C to give slow, medium, and fast rates of acidification. Milk coagulation was monitored by measuring turbidity and curd firmness, and microstructural changes during acidification were observed on glutaraldehyde-fixed, agar-solidified milk samples using transmission electron microscopy. Rate of acidification had little influence on changes observed during acidification, except at 10°C. At 40°C, the casein supramolecules were spherical throughout acidification, whereas at lower temperatures they became progressively more ragged in appearance. All of the milks gelled at the same pH (pH 4.8), as measured by curd firmness, whereas increases in turbidity, assumed to be brought about by an increase in number of light-scattering particles, were observed to start at about pH 5.2 to 5.4. As the milk was acidified, aggregates of loosely entangled proteins were observed, presumably originating from proteins that had dissociated from the casein supramolecules. These aggregates were often as large as the casein supramolecules, particularly as the pH of the milk approached the isoelectric point of the caseins. Larger aggregates were observed at 40°C than at the lower temperatures, suggesting the involvement of hydrophobic interactions between the proteins. A 3-phase model for acid-induced gelation of milk is proposed in which the first phase involves temperature-dependent dissociation of proteins from the casein supramolecules, with more dissociation occurring as temperature is decreased. Dissociation continues as milk pH is lowered, with the released proteins forming into loosely entangled aggregates, some as large as the casein supramolecules. The second phase of acid gelation of milk occurs between pH 5.3 and pH 4.9 and involves a reassociation of proteins with loosely entangled protein aggregates forming into more-compact colloidal particles or combining with any remaining casein supramolecules. The third and final phase involves rapid aggregation of the colloidal casein supramolecules into a gel network at about pH 4.8. Different gel structures were formed based on temperature of acidification, with a coarse-stranded gel network formed at 40°C and a fine-stranded gel network at 10°C.     

Effect of skim milk treated with high hydrostatic pressure on permeate flux and fouling during ultrafiltration

Ultrafiltration (UF) is largely used in the dairy industry to generate milk and whey protein concentrate for standardization of milk or production of dairy ingredients. Recently, it was demonstrated that high hydrostatic pressure (HHP) extended the shelf life of milk and improved rennet coagulation and cheese yield. Pressurization also modified casein micelle size distribution and promoted aggregation of whey proteins. These changes are likely to affect UF performance. Consequently, this study determined the effect of skim milk pressurization (300 and 600 MPa, 5 min) on UF performance in terms of permeate flux decline and fouling. The effect of HHP on milk proteins was first studied and UF was performed in total recycle mode at different transmembrane pressures to determine optimal UF operational parameters and to evaluate the effect of pressurization on critical and limiting fluxes. Ultrafiltration was also performed in concentration mode at a transmembrane pressure of 345 kPa for 130 or 140 min to evaluate the decline of permeate flux and to determine fouling resistances. It was observed that average casein micelle size decreased by 32 and 38%, whereas β-lactoglobulin denaturation reached 30 and 70% at 300 and 600 MPa, respectively. These results were directly related to UF performance because initial permeate fluxes in total recycle mode decreased by 25% at 300 and 600 MPa compared with nonpressurized milk, critical flux, and limiting flux, which were lower during UF of milk treated with HHP. During UF in concentration mode, initial permeate fluxes were 30% lower at 300 and 600 MPa compared with the control, but the total flux decline was higher for nonpressurized milk (62%) compared with pressure-treated milk (30%). Fouling resistances were similar, whatever the treatment, except at 600 MPa where irreversible fouling was higher. Characterization of the fouling layer showed that caseins and β-lactoglobulin were mainly involved in membrane fouling after UF of pressure-treated milk. Our results demonstrate that HHP treatment of skim milk drastically decreased UF performance.     

Kinetics of heat-induced interactions among whey proteins and casein micelles in sheep skim milk and aggregation of the casein micelles

The interactions among the proteins in sheep skim milk (SSM) during heat treatments (67.5–90°C for 0.5–30 min) were characterized by the kinetics of the denaturation of the whey proteins and of the association of the denatured whey proteins with casein micelles, and changes in the size and structure of casein micelles. The relationship between the size of the casein micelles and the association of whey proteins with the casein micelles is discussed. The level of denaturation and association with the casein micelles for β-lactoglobulin (β-LG) and α-lactalbumin (α-LA) increased with increasing heating temperature and time; the rates of denaturation and association with the casein micelles were markedly higher for β-LG than for α-LA in the temperature range 80 to 90°C; the Arrhenius critical temperature was 80°C for the denaturation of both β-LG and α-LA. The casein micelle size increased by 7 to 120 nm, depending on the heating temperature and the holding time. For instance, the micelle size (about 293 nm) of SSM heated at 90°C for 30 min increased by about 70% compared with that (about 174.6 nm) of unheated SSM. The casein micelle size increased slowly by a maximum of about 65 nm until the level of association of the denatured whey proteins with casein micelles reached 95%, and then increased markedly by a maximum of about 120 nm when the association level was greater than about 95%. The marked increases in casein micelle size in heated SSM were due to aggregation of the casein micelles. Aggregation of the casein micelles and association of whey protein with the micelles occurred simultaneously in SSM during heating.     

Investigation of the microstructure of milk protein concentrate powders during rehydration: Alterations during storage

The aim of this work was to use scanning electron microscopy to investigate the microstructure of rehydrated milk protein concentrate powder (MPC) particles. A sample preparation method for scanning electron microscopy analysis of rehydrated MPC particles is described and used to characterize the time course of dissolution and the effects of prior storage on the dissolution process. The results show that a combination of different types of interactions (e.g., bridges, direct contact) between casein micelles results in a porous, gel-like structure that restrains the dispersion of individual micelles into the surrounding liquid phase without preventing water penetration and solubilization of nonmicellar components. During storage of the powder, increased interactions occur between and within micelles, leading to compaction of micelles and the formation of a monolayer skin of casein micelles packed close together, the combination of which are proposed to be responsible for the slow dissolution of stored MPC powders.     

Interactive effects of casein micelle size and calcium and citrate content on rennet‐induced coagulation in bovine milk

Interactive effects of casein micelle size and milk calcium and citrate content on rennet‐induced coagulation were investigated. Milk samples containing small (SM) and large (LM) micelles, obtained from individual Holstein cows, were modified by addition of calcium and/or citrate and milk coagulation properties were evaluated in a full factorial design. The results showed that LM milk had a higher relative proportion of casein, coagulated faster, and resulted in a stronger gel than SM milk. Addition of calcium slightly decreased casein micelle size, while addition of citrate slightly increased micelle size. Calcium addition resulted in a shorter coagulation time and the strongest gels, while citrate addition increased the coagulation time and resulted in the weakest gels. Addition of calcium and citrate in combination resulted in intermediate coagulation properties. The interactive effect of micelle size and citrate was significant for gel strength. Microstructural differences between the milk gels were consistent with the rheological properties, for example, the micrographs revealed that a more homogeneous network was formed when calcium was added, resulting in a stronger gel. A more inhomogeneous network structure was formed when citrate was added, resulting in a weaker gel. Thus, variations in casein micelle size and in calcium and citrate content influence rennet‐induced coagulation in bovine milk. The calcium and citrate contents in Swedish milk have changed over time, whereby calcium content has increased and citrate content has decreased. In practical cheese making, calcium is added to cheese milk, most likely altering the role of inherent citrate and possibly influencing casein micelle size. The observed interaction effect between casein micelle size and citrate in this study, suggests that larger micelles with moderate citrate level will result in firmer gels, whereas a higher citrate content reduced gel strength more in case of large than SM. Since firmer gels are likely to retain more protein and fat than less firmer gels, this interaction effect could have implications in practical cheese production.     

Effects of varying casein and pectin concentrations on the rheology of high-protein cultured milk beverages stored at ambient temperature

Shelf-stable cultured milk beverages that have high protein levels can be difficult to successfully manufacture. With increasing protein level, rapid phase separation and gel formation occur in cultured beverages, which may not be prevented even with the inclusion of stabilizers such as high methoxy (HM) pectin. To limit protein aggregation in cultured milk beverages we investigated micellar casein as an interesting alternative to milk, due to the absence of whey proteins, which can contribute to increased gel strength in cultured products. In this study, micellar casein dispersed in ultrafiltered milk permeate was fermented to pH 4.1, blended with HM pectin, homogenized, thermally processed, and bottled for storage at ambient temperature for 6 mo. Utilizing response surface methodology with a central composite rotatable design, the protein and pectin contents were varied between 5 and 9% and 0.0 and 1.0%, respectively. The elastic modulus, loss tangent, and yield stress of these beverages were measured during storage to observe the extent of bond restructuring, whereas particle size and visual phase separation were measured to determine stability. Response variables were measured initially after thermally processing the beverages, and after 1 and 6 mo of storage at ambient temperature. All samples quickly formed gels after homogenizing, regardless of the pectin level. The stiffness (elastic modulus) of all samples increased throughout storage and was determined mainly by the protein content; however, the growth of elastic bonds over time was slowed with high levels of pectin. At 6 mo of storage, yield stress values were significantly lower for beverages with <7.5% protein when they were stabilized with ≥0.85% pectin. Prediction models for visual phase separation in beverages stored for 6 mo were significantly affected by the protein content, with increasing instability at lower protein levels. Models were used to identify optimal protein (<7.5%) and pectin (≥0.85%) concentrations to minimize the stiffness of gels during ambient storage. Samples in this optimized region were predicted to have low yield stress values and were easily fluidized by gentle shaking of the bottle at 6 mo.