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.     

Partitioning of nitroxynil,oxyclozanide and levamisole residues from milk to cream,skim milk and skim milk powder

Veterinary drugs are necessary to control fluke in animals, and if not properly used, residues of these drugs may be found in milk. The aim of this study was to determine whether residues of nitroxynil, levamisole and oxyclozanide in milk partition into skim milk powder during processing. Milk targeted to contain high, medium or low levels of residue was obtained following treatment of trial animals. On separation of cream and skim milk, > 90% of the residue partitioned with the skim milk in all cases. During powder processing, the residues were not degraded with almost 90% of the residue detected in the powder.     

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.     

Relationship between physical properties of casein micelles and rheology of skim milk concentrate

The properties of casein micelles in milk concentrates are of interest for the use of ultrafiltered (UF) skim milk concentrates in dairy products, and for the general understanding of colloidal stability and behavior of the casein micelle. The rheological behavior of UF skim milk concentrate with a casein concentration of 19.5% (wt/wt) was investigated at different pH and NaCl concentrations by analyzing flow viscometry and small amplitude oscillatory shear measurements. Viscometric flow curves were fitted to the Carreau-Yasuda model with the aim of determining values for the viscosity at infinite high shear rates and thereby estimate the voluminosity of the casein micelles (ν_casein) in the UF concentrate. The voluminosity of the casein micelles increased with addition of NaCl and decreased when pH was decreased from 6.5 to 5.5. At pH 5.2, ν_casein increased because of acid-induced aggregation of the casein micelles. The changes in ν_casein could be interpreted from transmission electron microscopy of freeze-fractured samples of the UF concentrate and partly from dynamic light scattering measurements. Altered interactions between casein micelles due to different pH and NaCl concentrations are proposed to occur due to collapse of the κ-casein layer, changed ionic strength, and altered distance between casein micelles.     

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.     

葡萄糖对脱脂乳酪蛋白胶束稳定性的影响研究

研究了葡萄糖添加量、体系pH、热处理温度、热处理时间对脱脂牛乳酪蛋白胶束稳定性的影响。研究表明,在接近中性pH条件下,少量葡萄糖分子可降低脱脂乳酪蛋白胶束的稳定性,大量葡萄糖分子可增强其稳定性。添加葡萄糖后,在高温热处理的诱导下,酪蛋白沉淀的pH升高。随着热处理时间的延长,脱脂乳中酪蛋白胶束表现出聚集行为,致使粒径增大,浊度和沉淀率总体呈现上升趋势。该研究结论可为乳品加工提供参考依据。      

亲水作用色谱-串联质谱同时测定原料乳和液体乳中三聚氰胺和舒巴坦

建立了亲水作用色谱-串联质谱同时测定原料乳和液体乳中三聚氰胺和舒巴坦的分析方法。试样采用2%乙酸水溶液提取,经乙腈沉淀蛋白,以乙腈-乙酸铵溶液为流动相,经Acquity UPLC BEH HILIC色谱柱分离后,采用多反应监测（MRM）扫描,外标法定量。三聚氰胺定量下限为10μg/kg,加标回收率为85.4%¹02.5%,相对标准偏差为3.10%⁶.55%。舒巴坦检定量下限为1.0μg/kg,加标回收率为86.1%¹04.3%,相对标准偏差为2.35%⁵.37%。该方法前处理简便快捷,灵敏度高,回收率和重现性良好,适用于原料乳和液体乳中三聚氰胺和舒巴坦的测定。      

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

The objective of this paper was to observe the rennet-induced aggregation behaviour of casein micelles in milk in the presence of additional sodium caseinate. Analysis of the centrifugal supernatants by size exclusion chromatography confirmed an increase in the soluble protein in the milk serum phase after addition of sodium caseinate. Although the total amount of κ-casein hydrolyzed over time was not affected, there was a significant effect of soluble casein on milk gelation, with a dose-dependent decrease of the gelation time as measured by rheology. Light scattering experiments also confirmed that the addition of soluble caseins inhibited the aggregation of casein micelles. Addition of 1 mM CaCl₂ prior to renneting increased the extent of rennet aggregation in samples containing additional sodium caseinate, but the inhibiting effect was still evident. The amount of soluble casein (as measured by chroma tography) significantly decreased after renneting, suggesting its association with the micellar fraction. Supporting experiments carried out with purified fractions of soluble caseins demonstrated that both α_s-casein and β-casein played a role as protective colloids (increasing steric repulsion) during renneting. It was concluded that the inhibiting effect observed during gelation was caused by the adsorption of soluble casein molecules on the surface of rennet-altered casein micelles.     

Cooling causes changes in the distribution of lipoprotein lipase and milk fat globule membrane proteins between the skim milk and cream phase

Lipoprotein lipase (LPL) activity and free fatty acid levels were studied in freshly milked, uncooled milk from individual Danish Holstein or Jersey cows, or after storage for up to 24 h at either a cooling temperature (4°C) or at the milking temperature (31°C). Upon cooling for up to 24 h, LPL activity increased in the cream phase, whereas the activity in the skim milk was steady, as observed for Jersey cows, or increased, as seen for the Holsteins. Storage at 31°C decreased the LPL activity in both the cream phase and the skim milk phase. The increase in free fatty acid levels was found to depend on LPL activity, incubation temperature, substrate availability, and incubation time. Furthermore, the migration of milk proteins between the skim milk phase and the cream phase upon cooling of milk from Jersey cows or from Danish Holstein cows was studied using proteomic methods involving 2-dimensional gel electrophoresis and mass spectrometry. Proteins associated with the milk fat globules were isolated from all milk fractions and analyzed. Major changes in the distributions of proteins between the skim milk phase and the cream phase were observed after cooling at 4°C for 4 h, where a total of 29 proteins between the 2 breeds was found to change their association with the milk fat globule membrane (MFGM) significantly. Among these, the MFGM proteins adipophilin, fatty acid-binding protein, and lactadherin, as well as the non-MFGM proteins β-casein, lactoferrin, and heat shock protein-71, were identified. Adipophilin, lactadherin, and lactoferrin were quantitatively more associated with the MFGM upon cold storage at 4°C, whereas β-casein, fatty acid-binding protein, and heat shock protein-71 were found to be less associated with the MFGM upon cold storage.     

Addition of sodium caseinate to skim milk increases nonsedimentable casein and causes significant changes in rennet-induced gelation,heat stability,and ethanol stability

The protein content of skim milk was increased from 3.3 to 4.1% (wt/wt) by the addition of a blend of skim milk powder and sodium caseinate (NaCas), in which the weight ratio of skim milk powder to NaCas was varied from 0.8:0.0 to 0.0:0.8. Addition of NaCas increased the levels of nonsedimentable casein (from ～6 to 18% of total casein) and calcium (from ～36 to 43% of total calcium) and reduced the turbidity of the fortified milk, to a degree depending on level of NaCas added. Rennet gelation was adversely affected by the addition of NaCas at 0.2% (wt/wt) and completely inhibited at NaCas ≥0.4% (wt/wt). Rennet-induced hydrolysis was not affected by added NaCas. The proportion of total casein that was nonsedimentable on centrifugation (3,000 × g, 1 h, 25°C) of the rennet-treated milk after incubation for 1 h at 31°C increased significantly on addition of NaCas at ≥0.4% (wt/wt). Heat stability in the pH range 6.7 to 7.2 and ethanol stability at pH 6.4 were enhanced by the addition of NaCas. It is suggested that the negative effect of NaCas on rennet gelation is due to the increase in nonsedimentable casein, which upon hydrolysis by chymosin forms into small nonsedimentable particles that physically come between, and impede the aggregation of, rennet-altered para-casein micelles, and thereby inhibit the development of a gel network.     

Effect of seasonal variation on the properties of whipping cream,soft cheese and skim milk powder in the UK

Whipping cream, skim milk powder and soft cheese were produced throughout the year. Whipping cream manufactured in spring and winter produced significantly higher overrun and better serum stability, and whipping time was related to buffering capacity of raw milk. Heat stability of reconstituted skim milk powder (RSMP) at 9% total solids (TS) was greater in summer and autumn, and >25% TS throughout the year. It was positively related to the protein content of raw milk, but negatively with fat. In contrast to other dairy products, no significant effect of season on the properties of soft cheese was found.     

Effect of pH at heating on the acid-induced aggregation of casein micelles in reconstituted skim milk

Reconstituted skim milk samples at pH between 6.5 and 7.1 (heating pH) were heated at 80°C, 90°C or 100°C for 30 min (heating temperature). The particle size of the casein micelles was measured at pH 4.75-7.1 (measurement pH) and at temperatures of 10°C, 20°C and 30°C (measurement temperature) using photon correlation spectroscopy. The particle size of the casein micelles, at a measurement pH of 6.7 and a measurement temperature of 20°C, was dependent on the heating pH and heating temperature to which the milk was subjected. The casein micelle size in unheated milk was about 215 nm. At a heating pH of 6.5, the casein micelle size increased by about 15, 30 and 40 nm when the milk was heated at 80°C, 90°C or 100°C, respectively. As the heating pH of the milk was increased, the size of the casein micelles decreased so that, at pH 7.1, the casein micelles were ∼20 nm smaller than those from unheated milk. Larger effects were observed as the heating temperature was increased from 80°C to 100°C. The size differences as a consequence of the heating pH were maintained at all measurement temperatures and at all measurement pH down to the pH at which aggregation of the micelles was observed. For all samples, size measurements at 10°C showed no aggregation at all measurement pH. Aggregation occurred at progressively higher pH as the measurement temperature was increased. Aggregation also occurred at a progressively higher measurement pH as the heating pH was increased. The particle size changes on heating and the aggregation on subsequent acidification may be related to the pH dependence of the association of whey proteins with, and the dissociation of κ-casein from the casein micelles as milk is heated.     

Heat stability of reconstituted, protein-standardized skim milk powders

We determined the effects of standardization material, protein content, and pH on the heat stability of reconstituted milk made from low-heat (LH) and medium-heat (MH) nonfat dry milk (NDM). Low-heat and MH NDM were standardized downward from 35.5% to 34, 32, and 30% protein by adding either edible lactose powder (ELP) or permeate powder (PP) from skim milk ultrafiltration. These powders were called standardized skim milk powders (SSMP). The LH and MH NDM and SSMP were reconstituted to 9% total solids. Furthermore, subsamples of reconstituted NDM and SSMP samples were set aside to measure heat stability at native (unadjusted) pH, and the rest were adjusted to pH 6.3 to 7.0. Heat stability is defined as heat coagulation time at 140°C of the reconstituted LH or MH NDM and SSMP samples. The entire experiment was replicated 3 times at unadjusted pH values and 2 times at adjusted pH values. At an unadjusted pH, powder type, standardization material, and protein content influenced the heat stability of the samples. Heat stability for reconstituted LH NDM and SSMP was higher than reconstituted MH NDM and SSMP. Generally, decreased heat stability was observed in reconstituted LH or MH SSMP as protein content was decreased by standardization. However, adding ELP to MH SSMP did not significantly change its heat stability. When pH was adjusted to values between 6.3 and 7.0, powder type, standardization material, and pH had a significant effect on heat stability, whereas protein content did not. Maximum heat stability was noted at pH 6.7 for both reconstituted LH NDM and SSMP samples, and at pH 6.6 for both reconstituted MH NDM and SSMP samples. Furthermore, for samples with adjusted pH, higher heat stability was observed for reconstituted LH SSMP containing PP compared with reconstituted milk from LH SSMP containing ELP. However, no statistical difference was observed in the heat stability of reconstituted milk from MH NDM and MH SSMP samples. We conclude that powder type (LH or MH) and effect of standardization material (ELP or PP) can help explain differences in heat stability. The difference in the heat stability of powder type may be associated with the difference in the pH of maximum heat stability and compositional differences in the standardization material (ELP or PP).     

Dairy cow feeding system alters the characteristics of low-heat skim milk powder and processability of reconstituted skim milk

Low-heat skim milk powder (LHSMP) was manufactured on 3 separate occasions in mid lactation (ML, July 4–20) and late lactation (LL, September 27 to October 7) from bulk milk of 3 spring-calving dairy herds on different feeding systems: grazing on perennial ryegrass (Lolium perenne L.) pasture (GRO), grazing on perennial ryegrass and white clover (Trifolium repens L.) pasture (GRC), and housed indoors and offered total mixed ration (TMR). The resultant powders (GRO-SMP, GRC-SMP, and TMR-SMP) were evaluated for composition and color and for the compositional, physicochemical, and processing characteristics of the reconstituted skim milk (RSM) prepared by dispersing the powders to 10% (wt/wt) in water. Feeding system significantly affected the contents of protein and lactose, the elemental composition, and the color of the LHSMP, as well as the rennet gelation properties of the RSM. The GRO and GRC powders had a higher protein content; lower levels of lactose, iodine, and selenium; and a more yellow-green color (lower a* and higher b* color coordinates) than TMR powder. On reconstitution, the GRO-RSM had higher concentrations of protein, casein, and ionic calcium, and lower concentrations of lactose and nonprotein nitrogen (% of total N). It also produced rennet gels with a higher storage modulus (G′) than the corresponding TMR-RSM. These effects were observed over the combined ML and LL period but varied somewhat during the separate ML and LL periods. Otherwise, feeding system had little or no effect on proportions of individual caseins, concentration of serum casein, casein micelle size, casein hydration, heat coagulation time, or ethanol stability of the RSM at pH 6.2 to 7.2, or on the water-holding capacity, viscosity, and flow behavior of stirred yogurt prepared by starter-induced acidification of RSM. The differences in the functionality of the LHSMP may be of greater or lesser importance depending on the application and the conditions applied during the processing of the RSM.     

全脂牛乳粉中糖基化酪蛋白分析

研究全脂牛乳粉中糖基化酪蛋白,探讨其酶解特性。重点考察了不同pH、酸及提取方法对酪蛋白提取质量百分数的影响,并通过SDS-PAGE电泳分析酪蛋白,高碘酸—希夫碱染色法及莫氏试验鉴定糖基化酪蛋白,木瓜蛋白酶水解酪蛋白,测定其氨基酸含量。结果表明,pH 4.6时提取酪蛋白的质量百分数显著高于其他组(P0.05);质量百分数为2%的乙酸(pH 3.58)提取酪蛋白的质量百分数为(40.45±0.66)%,显著高于其他组(P0.05);鞣酸提取酪蛋白的质量百分数为(46.13±0.46)%,显著高于其他方法(P0.05);酪蛋白SDS-PAGE电泳结果表明,不同酸、不同方法提取的酪蛋白均有3条带,其分子量均分别为34,24,60ku;高碘酸—希夫碱染色结果表明,分子量为34,24ku的酪蛋白是糖基化酪蛋白;莫氏试验结果表明,乳粉酪蛋白中存在糖基化酪蛋白;酶解结果表明,乳粉酪蛋白经木瓜蛋白酶水解后游离氨基酸含量显著高于鲜乳酪蛋白(P0.05)。说明全脂牛乳粉中有糖基化酪蛋白,其分子量分别为34,24ku,易于水解。     

Development and shelf-life determination of pasteurized,microfiltered, lactose hydrolyzed skim milk

The segment of the world population showing permanent or temporary lactose intolerance is quite significant. Because milk is a widely consumed food with an high nutritional value, technological alternatives have been sought to overcome this dilemma. Microfiltration combined with pasteurization can not only extend the shelf life of milk but can also maintain the sensory, functional, and nutritional properties of the product. This studied developed a pasteurized, microfiltered, lactose hydrolyzed (delactosed) skim milk (PMLHSM). Hydrolysis was performed using β-galactosidase at a concentration of 0.4 mL/L and incubation for approximately 21 h at 10 ± 1°C. During these procedures, the degree of hydrolysis obtained (>90%) was accompanied by evaluation of freezing point depression, and the remaining quantity of lactose was confirmed by HPLC. Milk was processed using a microfiltration pilot unit equipped with uniform transmembrane pressure (UTP) ceramic membranes with a mean pore size of 1.4 μm and UTP of 60 kPa. The product was submitted to physicochemical, microbiological, and sensory evaluations, and its shelf life was estimated. Microfiltration reduced the aerobic mesophilic count by more than 4 log cycles. We were able to produce high-quality PMLHSM with a shelf life of 21 to 27 d when stored at 5 ± 1°C in terms of sensory analysis and proteolysis index and a shelf life of 50 d in regard to total aerobic mesophile count and titratable acidity.     

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.     

Isolation and partial characterization of CD36 from skim milk

CD36, a common milk fat globule membrane glycoprotein, was isolated from skim milk by methods similar to those previously utilized for the isolation of sulfhydryl oxidase. Two separate methods that were employed, gave similar purity as observed by electrophoresis. The first was based on differential centrifugation and size-exclusion chromatography, whereas the second combined ultrafiltration and affinity chromatography. After significant purification, the protein was identified by Western blotting and sequence analysis. Deglycosylation decreased the apparent molecular mass from approximately 85 to 57 kDa. These results suggested tissue-specific glycosylation. The purified fractions also exhibited low levels of sulfhydryl oxidase activity, the significance of which will require further study.