Invited review: Microfiltration-derived casein and whey proteins from milk

Milk, a rich source of nutrients, can be fractionated into a wide range of components for use in foods and beverages. With advancements in filtration technologies, micellar caseins and milk-derived whey proteins are now produced from skim milk using microfiltration. Microfiltered ingredients offer unique functional and nutritional benefits that can be exploited in new product development. Microfiltration offers promise in cheesemaking, where microfiltered milk can be used for protein standardization to improve the yield and consistency of cheese and help with operation throughputs. Micellar casein concentrates and milk whey proteins could offer unique functional and flavor properties in various food applications. Consumer desires for safe, nutritious, and clean-label foods could be potential growth opportunities for these new ingredients. The application of micellar casein concentrates in protein standardization could offer a window of opportunity to US cheese makers by improving yields and throughputs in manufacturing plants.     

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.     

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。     

Comparative analysis of changes in whey proteins of goat milk throughout the lactation cycle using quantitative proteomics

The composition and content of goat milk proteins are affected by many factors and have been extensively studied. However, variation in whey protein composition in goat milk throughout the lactation cycle has not been clarified. In the current study, 15 dairy goats were selected, and milk samples were collected at 1, 3, 30, 90, 150, and 240 d after delivery. Whey proteins were separated and digested and then identified using data-independent acquisition (DIA) and data-dependent acquisition proteomics approaches. Protein profiles identified using DIA were consistent with those of the data-dependent acquisition proteomics approach according to clustering and principal component analyses. Significant differences in the abundance of 238 proteins around the lactation cycle were identified using the DIA approach. Developmental changes of the whey proteome corresponding to lactation stage were revealed: plasminogen, α-2-macroglobulin, and fibronectin levels decreased from d 1 to 240, whereas polymeric immunoglobulin receptor, nucleobindin 2, fatty acid-binding protein 3, and lactoperoxidase increased from d 1 to 240. Protein-protein interaction analysis showed that fibronectin with a higher degree of connectivity is a central node. The findings are of great significance to better understanding the potential role of specific proteins and the mechanism of protein biosynthesis or intercellular transport in the mammary glands related to the physiological changes of dairy goats.     

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.     

Physicochemical and functional properties of goat milk whey protein and casein obtained during different lactation stages

During lactation, goat milk contains colostrum, transitional milk, mature milk, and end milk. The protein present in goat milk during different lactation periods has different characteristics. This study aimed to characterize the protein profile of goat milk samples obtained at different lactation stages and to identify changes in the physicochemical and functional properties of whey protein and casein from goat milk collected at 1, 3, 15, 100, and 200 d after calving. The results demonstrated that the lactation period had a great influence on the physicochemical and functional properties of goat milk whey protein and casein, especially the protein properties of colostrum on the first day after delivery. The denaturation temperature, hydrophobicity, and turbidity of whey protein were significantly higher on the first day postpartum than at other lactation periods. Correspondingly, the colostrum whey protein also had better functional properties, such as emulsification, oil holding capacity, and foaming properties on the first day postpartum than at other lactation periods. For casein, the turbidity, particle size, water holding capacity, and foaming properties on the first day after delivery were significantly higher than those at other lactation periods, whereas the denaturation temperature, oil holding capacity, and emulsification followed the opposite trend. For both whey protein and casein, the 2 indicators of emulsifying properties, namely, emulsifying activity index and the emulsion stability, also followed an opposite trend relative to lactation stage, whereas the changes in foaming capacity with the lactation period were completely consistent with the change of foaming stability. These findings could provide useful information for the use of goat milk whey protein and casein obtained during different lactation stages in the dairy industry.     

The balance between caseins and whey proteins in cow's milk determines its allergenicity

Cow's milk allergy is quite common in the first years of human life. Protein composition plays an important role in this pathology, particularly the casein/whey protein ratio. It is known that milks from different species have different sensitization capacities although their protein sources are quite similar. Thus, the objective of this work was to compare the allergenicity of native cow's milk and milk with a modified ratio of casein and whey proteins in a murine model of atopy. Twenty-four Balb/c mice were orally sensitized to native cow's milk or modified cow's milk with a casein/whey protein ratio of 40:60. During the sensitization period, the number of mice suffering from diarrhea was significantly higher in the native cow's milk-sensitized group than in the modified milk-sensitized group. Once mice were killed, plasma histamine levels were shown to be significantly higher in native cow's milk-sensitized mice. In addition, cow's milk proteins induced a higher lymphocyte sensitization in the native milk-sensitized mice, with a significant increase in the specific proliferation ratio of these cells.These results suggest that the balance between caseins and whey proteins plays an important role in the sensitization capacity of cow's milk, and its modification might be a way to reduce the allergenicity of cow's milk.     

Low‐fat Cheddar cheese made using microparticulated whey proteins: Effect on yield and cheese quality

Influence of different levels (0, 0.15, 0.35 or 0.50%) of microparticulated whey protein (MWP) on yield and quality of low‐fat (~7.3 g/100 g) Cheddar cheese was investigated. MWP improved cheese yield due to the water‐binding ability of denatured whey protein. MWP addition decreased meltability but improved the textural properties beneficial for shredding and slicing, by decreasing sensory firmness. The results emphasise the role of MWP as an inert filler within cheese matrix, in improving cheese yield and creating a softer texture without compromising the sensory or overall quality of cheese, even with moisture increases in 0.35 or 0.50% MWP cheeses.     

高通量快速检测母乳总蛋白、乳清蛋白和酪蛋白含量方法的比较研究

分别利用Lowry、BCA和Bradford三种方法对母乳中总蛋白、乳清蛋白和酪蛋白的含量进行了测定,并以凯氏定氮法为标准,对三种方法的检测结果进行了比较。结果表明Lowry和Bradford法适用于母乳中总蛋白含量的测定,Lowry法适于母乳中乳清蛋白含量的测定,Lowry、BCA和Bradford三种方法均适用于母乳中酪蛋白含量的测定。采用上述方法,只需要4 m L母乳样本,1.5 h内即可完成母乳总蛋白、乳清蛋白和酪蛋白的微量、高通量快速测定。上述方法适用于较大量母婴营养状况的调查研究,可为母乳化婴幼儿配方乳粉的研制以及促进婴幼儿生长发育提供科学依据。      

Effect of microparticulated whey proteins on milk coagulation properties

The enhancement of milk coagulation properties (MCP) and the reuse of whey produced by the dairy industry are of great interest to improve the efficiency of the cheese-making process. Native whey proteins (WP) can be aggregated and denatured to obtain colloidal microparticulated WP (MWP). The objective of this study was to assess the effect of MWP on MCP; namely, rennet coagulation time (RCT), curd-firming time, and curd firmness 30 min after rennet addition. Six concentrations of MWP (vol/vol; 1.5, 3.0, 4.5, 6.0, 7.5, and 9.0%) were added to 3 bulk milk samples (collected and analyzed during 3 d), and a sample without MWP was used as control. Within each day of analysis, 6 replicates of MCP for each treatment were obtained, changing the position of the treatment in the rack. For control samples, 2 replicates per day were performed. In addition to MCP, WP fractions were measured on each treatment during the 3 d of analysis. Milk coagulation properties were measured on 144 samples by using a Formagraph (Foss Electric, Hillerød, Denmark). Increasing the amount of MWP added to milk led to a longer RCT. In particular, significant differences were found between RCT of the control samples (13.5 min) and RCT of samples with 3.0% (14.6 min) or more MWP. A similar trend was observed for curd-firming time, which was shortest in the control samples and longest in samples with 9.0% MWP (21.4 min). No significant differences were detected for curd firmness at 30 min across concentrations of MWP. Adjustments in cheese processing should be made when recycling MWP, in particular during the coagulation process, by prolonging the time of rennet activity before cutting the curd.     

Effect of denatured whey protein concentrate and its fractions on cheese composition and rheological properties

The objectives of this study were (1) to assess the effect of a denatured whey protein concentrate (DWPC) and its fractions on cheese yield, composition, and rheological properties, and (2) to separate the direct effect of the DWPC or its fractions on cheese rheological properties from the effect of a concomitant increase in cheese moisture. Semihard cheeses were produced at a laboratory scale, and mechanical properties were characterized by dynamic rheometry. Centrifugation was used to induce a moisture gradient in cheese to separate the direct contribution of the DWPC from the contribution of moisture to cheese mechanical properties. Cheese yield increased and complex modulus (G*) decreased when the DWPC was substituted for milk proteins in milk. For cheeses with the same moisture content, the substitution of denatured whey proteins for milk proteins had no direct effect on rheological parameters. The DWPC was fractionated to evaluate the contribution of its different components (sedimentable aggregates, soluble component, and diffusible component) to cheese yield, composition, and rheological properties. The sedimentable aggregates were primarily responsible for the increase in cheese yield when DWPC was added. Overall, moisture content explained to a large extent the variation in cheese rheological properties depending on the DWPC fraction. However, when the effect of moisture was removed, the addition of the DWPC sedimentable fraction to milk increased cheese complex modulus. Whey protein aggregates were hypothesized to act as active fillers that physically interact with the casein matrix and confer rigidity after pressing.     

Comparative analysis of whey proteins in yak milk from different breeds in China using a data-independent acquisition proteomics method

Yak milk is rich in essential milk proteins of nutritional and therapeutic value. In this study, whey proteins of milk from 3 yak breeds (Gannan, GN; Huanhu, HH; Maiwa, MW) in China were comprehensively identified and compared using a data-independent acquisition quantitative proteomics approach. A total of 632 proteins were identified in yak milk whey samples, in which immune-related proteins were abundant. Compared with other milks, more proteins were involved in oxidation-reduction process and with ATP binding. In addition, we identified 96, 155, and 164 differentially expressed proteins (DEP) for GN versus HH, GN versus MW, and HH versus MW, respectively. “Phagosome” and “complement and coagulation cascades” were the most significant pathways for DEP of GN versus HH and GN or HH versus MW yak milk based on the Kyoto Encyclopedia of Genes and Genomes pathway analysis. Protein-protein interaction network analysis showed that DEP for the 3 comparisons had significant biological interactions but were associated with different functions. The results provide useful information on yak milk from different breeds in China, and elucidate the biological functions of yak milk proteins.     

Developmental changes in proteins of casein micelles in goat milk using data-independent acquisition-based proteomics methods during the lactation cycle

Casein micelles (CM) play an important role in milk secretion, stability, and processing. The composition and content of milk proteins are affected by physiological factors, which have been widely investigated. However, the variation in CM proteins in goat milk throughout the lactation cycle has yet to be fully clarified. In the current study, milk samples were collected at d 1, 3, 30, 90, 150, and 240 of lactation from 15 dairy goats. The size of CM was determined using laser light scattering, and CM proteins were separated, digested, and identified using data-independent acquisition (DIA) and data-dependent acquisition (DDA)-based proteomics approaches. According to clustering and principal component analysis, protein profiles identified using DIA were similar to those identified using the DDA approach. Significant differences in the abundance of 115 proteins during the lactation cycle were identified using the DIA approach. Developmental changes in the CM proteome corresponding to lactation stages were revealed: levels of lecithin cholesterol acyltransferase, folate receptor α, and prominin 2 increased from 1 to 240 d, whereas levels of growth/differentiation factor 8, peptidoglycan-recognition protein, and 45 kDa calcium-binding protein decreased in the same period. In addition, lipoprotein lipase, glycoprotein IIIb, and α-lactalbumin levels increased from 1 to 90 d and then decreased to 240 d, which is consistent with the change in CM size. Protein–protein interaction analysis showed that fibronectin, albumin, and apolipoprotein E interacted more with other proteins at the central node. These findings indicate that changes in the CM proteome during lactation could be related to requirements of newborn development, as well as mammary gland development, and may thus contribute to elucidating the physical and chemical properties of CM.     

Effects of protein and fat levels in milk on cheese and whey compositions

Bulk tank milk was standardised to six levels of fat (3·0, 3·2, 3·4, 3·6, 3·8, 4·0%) and similarly to six levels of protein, thus giving a total of 36 combinations in composition. Milk was analyzed for total solids, fat, protein, casein, lactose and somatic cell count and was used to make laboratory-scale cheese. Cheese samples from each batch were assayed for total solids, fat, protein and salt. Losses of milk components in the whey were also determined. Least squares analysis of data indicated that higher protein level in milk was associated with higher protein and lower fat contents in cheese. This was accompanied by lower total solids (higher moisture) in cheese. Inversely, higher fat level in milk gave higher fat and lower protein and moisture contents in cheese. Higher fat level in milk resulted in lower retention of fat in cheese and more fat losses in the whey. Higher protein level in milk gave higher fat retention in cheese and less fat losses in the whey. Regression analysis showed that cheese fat increased by 4·22%, while cheese protein decreased by 2·61% for every percentage increase in milk fat. Cheese protein increased by 2·35%, while cheese fat decreased by 6·14% per percentage increase in milk protein. Milk with protein to fat ratio close to 0·9 would produce a minimum of 50% fat in the dry matter of cheese.     

Comparison of micellar casein isolate and nonfat dry milk for use in the production of high-protein cultured milk products

High protein levels in yogurt, as well as the presence of denatured whey proteins in the milk, lead to the development of firm gels that can make it difficult to formulate a fluid beverage. We wanted to prepare high-protein yogurts and explore the effects of using micellar casein isolate (MCI), which was significantly depleted in whey protein by microfiltration. Little is known about the use of whey protein-depleted milk protein powders for high-protein yogurt products. Microfiltration also depletes soluble ions, in addition to whey proteins, and so alterations to the ionic strength of rehydrated MCI dispersions were also explored, to understand their effects on a high-protein yogurt gel system. Yogurts were prepared at 8% protein (wt/wt) from MCI or nonfat dry milk (NDM). The NDM was dispersed in water, and MCI powders were dispersed in water (with either low levels of added lactose to allow fermentation to achieve the target pH, or a high level to match the lactose content of the NDM sample) or in ultrafiltered (UF) milk permeate to align its ionic strength with that of the NDM dispersion. Dispersions were then heated at 85°C for 30 min while stirring, cooled to 40°C in an ice bath, and fermented with yogurt cultures to a final pH of 4.3. The stiffness of set-style yogurt gels, as determined by the storage modulus, was lowest in whey protein-depleted milk (i.e., MCI) prepared with a high ionic strength (UF permeate). Confocal laser scanning microscopy and permeability measurements revealed no large differences in the gel microstructure of MCI samples prepared in various dispersants. Stirred yogurt made from MCI that was prepared with low ionic strength showed slow rates of elastic bond reformation after stirring, as well as slower increases in cluster particle size throughout the ambient storage period. Both the presence of denatured whey proteins and the ionic strength of milk dispersions significantly affected the properties of set and stirred-style yogurt gels. Results from this study showed that the ionic strength of the heated milk dispersion before fermentation had a large influence on the gelation pH and strength of acid milk gels, but only when prepared at high (8%) protein levels. Results also showed that depleting milk of whey proteins before fermentation led to the development of weak yogurt gels, which were slow to rebody and may be better suited for preparing cultured milk beverages where low viscosities are desirable.     

Influence of proteolysis of milk on the whey protein to total protein ratio as determined by capillary electrophoresis

Capillary electrophoresis (CE) was used to determine the whey protein to total protein ratio in raw and UHT milk samples with different degrees of proteolysis caused by storage. In raw milks, the analysis of samples taken at regular times demonstrated the influence of proteolysis in the whey protein to total protein determination, which was overestimated after 4 d of storage. In UHT milks, the overestimation of the whey protein to total protein ratio took place after 30 or 60 d of storage. However, the ratios alphaS1-CN/beta-CN and alphas1-CN/kappa-CN permitted detection of the samples of raw or UHT milk with degraded proteins. The distorted capillary electrophoretic pattern obtained for UHT milks made necessary an integration of the electropherograms in a "valley-to-valley" way. Results for raw milk samples were identical when "valley-to-valley" was compared to standard integration techniques. This CE method could be considered an alternative method to derivative spectroscopy for the determination of the whey protein to total protein of milk and could be used to detect samples with proteolysis.     

Effect of goat milk composition on cheesemaking traits and daily cheese production

Cheese yield is strongly influenced by the composition of milk, especially fat and protein contents, and by the efficiency of the recovery of each milk component in the curd. The real effect of milk composition on cheesemaking ability of goat milk is still unknown. The aims of this study were to quantify the effects of milk composition; namely, fat, protein, and casein contents, on milk nutrient recovery in the curd, cheese yield, and average daily yield. Individual milk samples were collected from 560 goats of 6 different breeds. Each sample was analyzed in duplicate using the 9-laboratory milk cheesemaking assessment, a laboratory method that mimicked cheesemaking procedures, with milk heating, rennet addition, coagulation, curd cutting, and draining. Data were submitted to statistical analysis; results showed that the increase of milk fat content was associated with a large improvement of cheese yield because of the higher recovery of all milk nutrients in the curd, and thus a higher individual daily cheese yield. The increase of milk protein content affected the recovery of fat, total solids, and energy in the curd. Casein number, calculated as casein-to-protein ratio, did not affect protein recovery but strongly influenced the recovery of fat, showing a curvilinear pattern and the most favorable data for the intermediate values of casein number. In conclusion, increased fat and protein contents in the milk had an effect on cheese yield not only for the greater quantity of nutrients available but also for the improved efficiency of the recovery in the curd of all nutrients. These results are useful to improve knowledge on cheesemaking processes in the caprine dairy industry.     

Effects of protein and fat levels in milk on Cheddar cheese yield

Over a 14-month period, bulk tank milk was collected twice a week and was adjusted with cream and skim milk powder to provide six levels each of fat and protein varying from 3·0 to 4·0%. Milk samples were analyzed for total solids, fat, protein, casein, lactose and somatic cell count and were used for laboratory-scale cheesemaking. Data obtained from the milk input and the cheese output were used to determine actual, moisture adjusted, theoretical yield, and efficiency of yield. Least squares analyses of data indicated that higher cheese yields were obtained from higher fat and protein contents in milk. Higher yield efficiency was associated with higher ratios of protein to fat and casein to fat. Regression analysis indicated that a percentage increase in fat content in milk resulted in an increase of 1·23–1·37% in moisture adjusted yield in the different protein levels. For a similar increase of protein in milk, there were 1·80–2·04% increase in moisture adjusted yields in different fat levels.     

乳清蛋白对直接超高温乳贮存稳定性的影响研究进展

直接超高温（direct ultra-high temperature,dUHT）牛乳是蒸汽直接接触牛乳快速加热,然后通过闪蒸迅速蒸发水分,具有热负荷能低、最大程度保留牛乳营养和风味等优点,有望成为继UHT乳的新一代高品质液态乳制品。目前我国dUHT乳产业尚处于起步阶段。虽然dUHT乳的营养全面,但其贮存过程中有时出现沉淀、老化凝胶等品质问题,影响了产品货架期。dUHT乳的贮存稳定性影响因素非常复杂,乳清蛋白的热不稳定性是影响dUHT乳贮存稳定性的主要原因之一。本文综述了dUHT乳生产基本原理,探讨了乳清蛋白分子间、乳清蛋白与酪蛋白和纤溶酶等物质之间的交联反应机制及其对dUHT乳贮存稳定性的影响,以期为dUHT乳的加工技术优化升级和我国dUHT乳的快速发展提供参考。