Nomenclature of the proteins of cows' milk--sixth revision

This report of the American Dairy Science Association Committee on the Nomenclature, Classification, and Methodology of Milk Proteins reviews changes in the nomenclature of milk proteins necessitated by recent advances of our knowledge of milk proteins. Identification of major caseins and whey proteins continues to be based upon their primary structures. Nomenclature of the immunoglobulins consistent with new international standards has been developed, and all bovine immunoglobulins have been characterized at the molecular level. Other significant findings related to nomenclature and protein methodology are elucidation of several new genetic variants of the major milk proteins, establishment by sequencing techniques and sequence alignment of the bovine caseins and whey proteins as the reference point for the nomenclature of all homologous milk proteins, completion of crystallographic studies for major whey proteins, and advances in the study of lactoferrin, allowing it to be added to the list of fully characterized milk proteins.     

Purification and characterization of rat alpha-lactalbumins: apparent genetic variants

Rat alpha-lactalbumin, from the milk of Fischer 344 (CDF) rats, was isolated and purified by a combination of gel filtration and diethylaminoethyl-cellulose ion exchange chromatography. Three electrophoretically distinct proteins had alpha-lactalbumin activity. Staining for carbohydrate indicated that at least two of the three forms were glycoproteins. The low molecular weight protein fraction from the wheys of two additional strains of laboratory rat were compared to ascertain whether the composition of this fraction was common in the divergent strains. Outbred Wistar and Long-Evans dams yielded wheys containing up to six forms of alpha-lactalbumin. Either one or both of two groups of three alpha-lactalbumins were in a given milk sample. The two groups of three alpha-lactalbumins appear to represent two genetic variants upon which is imposed a polymorphic character. All forms of alpha-lactalbumin, within and between strains, were immunologically identical.     

Colostrum and milk protein rankings and ratios of importance to neonatal calf health using a proteomics approach

Administration of colostrum to the newborn calf before gut closure is pivotal to its health, because of the transfer of passive immunity. Traditionally, passive immunity has been attributed to the transfer of immunoglobulins although it is increasingly clear that multiple other factors contribute, including innate immune proteins, developmental factors, immunomodulatory factors, and the presence of cellular immunity. The objective of this study was to produce a comprehensive comparison of the bovine colostrum proteome and the milk proteome by applying 2-dimensional liquid chromatography-tandem mass spectrometry. Further, the objectives were to rank proteins mutually and generate protein ratios from the spectral counts of the 2 proteomes and ELISA to gain insight into which proteins could be of most relevance to neonatal calf health. To obtain an in-depth picture of the bovine colostrum and milk proteome, we compared the contents of different fractions from bovine colostrum and milk from our 2 previous studies. A total of 140 colostrum fluid-phase proteins and 103 milk fluid-phase proteins were detected. In the cellular fraction, 324 and 310 proteins were detected in colostrum and milk, respectively. In total, 514 proteins were detected, of which 162 were in the fluid phase. Of these, 50 proteins were exclusively seen in colostrum, 13 were exclusively seen in milk, and 99 were common to colostrum and milk. Ranking of proteins mutually and calculating protein ratios based on spectral counts and ELISA resulted in new information on how proteins were associated with the fluid or cellular fraction of the samples. Interestingly, despite lower counts/concentrations than the classical proteins such as immunoglobulins, β-lactoglobulin, and lactotransferrin, several proteins appeared in higher or similar colostrum:milk spectral count ratios as these. Using this approach indicated, for example, that osteopontin, haptoglobin, milk amyloid A, and gelsolin may be interesting molecules to study in detail in their relation to calf health. Although the sensitivity, identification, and ranking of proteins varied between the 2 methods, and proteome analysis clearly suffers from low sensitivity, we believe that this idea and approach of generating ratios and ranking proteins can contribute new information and perspectives on how to prioritize the importance of multiple proteins, beyond immunoglobulins, in relation to neonatal calf health.     

Effect of fluorescent light on amino acid composition of serum proteins from homogenized milk.

The serum proteins isolated from homegenized milk by centrifugation and exposed to 2152lx of fluorescent light for 72 h were fractionated into the major protein components by gel filtration chromatography. Amino acid composition of the major serum proteins, beta-lactoglobulin and alpha-lactalbumin which accounted for 80% of the total serum protein, did not change substantially following light exposure. Changes in amino acid composition associated with fluorescent light exposure occurred primarily in the high molecular weight fraction of serum proteins. This fraction amounts to at most 10% of the serum proteins.     

Immunochemical quantification of heat denaturation of camel ( Camelus dromedarius ) whey proteins

The major whey proteins IgG, serum albumin and alpha-lactalbumin were purified from camel milk using gel permeation and ion-exchange chromatography. Specific antisera against each of them were raised and used to quantify their heat denaturation in early or mature milk over a range of 60-90 degrees C for 10-60 min using the single radial immunodiffusion technique. The heat denaturation midpoints for the mature milk heated 30 min were 67.2, 73.0 and 77.5 degrees C for IgG, albumin and alpha-lactalbumin respectively. The early milk was more sensitive to heat treatment with coagulation at low temperature and heat denaturation midpoints of 64.8, 71.6 and 72.6 degrees C respectively. This difference was related to the high IgG content of the early milk (12.6 mg/ml v. 0.5 mg/ml for the mature milk) and stresses the importance of monitoring the IgG level of milk to assess the absence of colostrum.     

牛乳蛋白过敏原改性的研究

对牛乳中的主要过敏原酪蛋白、β-乳球蛋白（β-LG）及α-乳白蛋白（α-LA）进行了介绍。重点论述了热处理、蛋白水解、糖基化作用和乳酸发酵等技术对乳蛋白过敏改性的研究现状，提出了牛乳中过敏蛋白原改性的研究方向。     

Cow's milk allergens identification by two-dimensional immunoblotting and mass spectrometry

Cow's milk allergy (CMA) has become a common disease in early childhood, its prevalence ranging from 1.6% to 2.8% among children younger than 2 years of age. The role of different cow's milk protein (CMP) in the pathogenesis of CMA is still controversial. Even if the proteins most frequently and most intensively recognized by immunoglobulin E (IgE) seem to be the most abundant in milk (caseins and beta-lactoglobulin), with an although great variability all milk proteins appear to be potential allergens, even those that are present in trace amounts (i.e., lactoferrin, IgG, and BSA). In this work proteomics techniques have been applied for CMP allergens analysis. Allergens have been identified by immunoblotting following resolution of CMP components by two-dimensional electrophoresis. Sera from 20 milk-allergic subjects, as proven by oral provocation test, CAP-RAST and skin prick test, have been used for cow's milk major allergen identification. Cow's milk proteins and their isoforms were identified by matrix assisted laser desorption/ionization-time of flight (MALDI-TOF)-mass spectrometry. In our group of patients, the prevalence of CMP allergens, i.e., the total number of subjects sensitized to CMP divided by the total number of the subjects enrolled in the study, was: 55% alpha(s1)-casein, 90% alpha(s2)-casein, 15% beta-casein, 50% kappa-casein, 45% beta-lactoglobulin, 45% BSA, 95% IgG-heavy chain, 50% lactoferrin, and 0% alpha-lactalbumin.     

Short communication: effects of increased expression of alpha-lactalbumin in transgenic mice on milk yield and pup growth

Lactose synthase (a complex of beta1,4-galactosyltransferase and alpha-lactalbumin) forms lactose in the Golgi complex of mammary epithelial cells. To determine whether alpha-lactalbumin is a limiting component in this complex, transgenic mice that expressed bovine alpha-lactalbumin were studied. Transgenic mice produced 0.5 to 1.5 mg/ml of bovine alpha-lactalbumin in their milk, 5- to 15-fold more alpha-lactalbumin than in milk of control mice. Transgenic and control mice produced milk with the same concentrations of lactose, cream, and total solids, and showed similar mammary gland growth, morphology, and histology. Milk from transgenic mice had 0.6% less protein than milk from control mice (P < 0.05). The in vitro lactose synthase activity in mammary gland homogenates from alpha-lactalbumin transgenic mice was increased (P < 0.05), demonstrating that bovine alpha-lactalbumin could interact with murine beta1,4-galactosyltransferase. Pups reared by lactating transgenic mice showed a 4% increase in growth on d 10 of lactation, suggesting that milk production was increased (P = 0.06). Milk volume, estimated using the weigh-suckle-weigh technique, tended to be higher (although not significantly) in transgenic mice (P = 0.11). These results suggest that augmenting alpha-lactalbumin expression in the dam increases the growth of suckling offspring.     

A comparison of the kinetics of the thermal denaturation of the immunoglobulins in caprine and bovine skim milk samples

The irreversible thermal denaturation of the immunoglobulins in bovine and caprine milk samples was determined over a wide temperature and time range so that kinetic and thermodynamic parameters could be determined. The immunoglobulins in the bovine milk were more heat labile than those in the caprine milk. For both milk samples, the denaturation followed second order reaction kinetics. The activation energies and thermodynamic parameters (ΔH^#, ΔG^# and ΔS^#) were similar for both bovine caprine milk samples, and these parameters were in the range expected for unfolding reactions as the rate determining step for the irreversible denaturation process.     

Variation in milk protein concentrations associated with genetic polymorphism and environmental factors

Concentrations of alpha s-casein, beta-casein, kappa-casein, beta-lactoglobulin, alpha-lactalbumin, serum albumin, and immunoglobulin in milk from 1888 Holstein cows were determined monthly over the lactation period. Cows were phenotyped for genetic variants of alpha s1-casein, beta-casein, kappa-casein, and beta-lactoglobulin. Least squares analyses showed variations in individual proteins due to parity number, month of test, stage of lactation, somatic cell count, fat content, milk yield, and phenotypes of cows for milk proteins. beta-Casein declined and serum proteins increased with advancing age of cows. Concentration of individual proteins decreased during the first 2 to 3 mo in lactation and then increased as lactation progressed. alpha s1-Casein variants significantly affected concentrations of alpha s-casein (BC greater than BB greater than AB) and beta-lactoglobulin (AB greater than BB greater than BC). Variant B for beta-casein is associated with lower alpha s-casein, beta-lactoglobulin, immunoglobulins, and higher beta-casein and alpha-lactalbumin concentrations than variant A1, A2, or A3. Milk from BB kappa-casein, and BB beta-lactoglobulin cows contained more alpha s-casein, kappa-casein, and less beta-lactoglobulin than milk from AA cows for the two proteins. Concentrations of all proteins were negatively correlated with milk production. Increased somatic cell counts were associated with lower beta-casein and higher concentrations of other proteins. Fat content of milk was positively correlated with the three casein fractions and beta-lactoglobulin.     

Phosphorylation of beta-casein and alpha-lactalbumin by casein kinase from lactating bovine mammary gland

Two milk proteins, beta-casein and alpha-lactalbumin, were compared as substrates for casein kinase from bovine mammary gland. beta-Casein could be rephosphorylated after removal of its phosphate groups, whereas alpha-lactalbumin was an effective substrate after the protein had been reduced and carboxymethylated. The native proteins could not be phosphorylated. Magnesium2+, Ca2+, and Mn2+ stimulated phosphorylation of the modified proteins. Calcium2+ was the most effective cation for alpha-lactalbumin and Mn2+ for beta-casein. Michaelis constants were 144 microM for alpha-lactalbumin in the presence of Ca2+ and 142 microM for beta-casein in the presence of Mn2+; however, the maximum velocity for alpha-lactalbumin was three times that of beta-casein. After phosphorylation with [gamma-32P] ATP, partial hydrolysis showed that only serine residues were phosphorylated in both proteins. Chymotryptic peptides of phosphorylated alpha-lactalbumin and tryptic peptides of phosphorylated beta-casein were examined by HPLC and selected peptides were analyzed for amino acid content. Comparison of the analyses with sequence data showed that serine at position 47 in alpha-lactalbumin is the major site of phosphate incorporation. Dephosphorylated beta-casein was only partially rephosphorylated. However, the sites identified correspond to the phosphorylated residues in native beta-casein, namely, serine at position 35 and the cluster of four serines between residues 15 and 20.     

Symposium review: The relevance of bovine milk phospholipids in human nutrition—Evidence of the effect on infant gut and brain development

This paper reflects the concepts reviewed during the presentation in the Joint MILK/Lactation Biology Symposium at the ADSA 2018 Annual Meeting. Our intention is to update the concepts and advances in the area of research regarding milk phospholipids or polar lipid fraction as part of a dairy ingredient used today in nutritional studies that focus on gut health as well as brain development of infants. Although processing advances have allowed the production of novel ingredients rich in milk fat globule membrane (MFGM) components, mostly monitored by phospholipid concentration and presence of membrane proteins, there is wide variability in their composition and structure. Furthermore, we aimed to include in the phospholipid fraction of milk nanovesicles designated as milk exosomes, which are secreted into milk by different secretion mechanisms than those of the fat globules but are also made up of a unique mixture of polar lipids. We consider imperative the study of polar lipid-derived structures from milk regarding composition and structure to gain insights into their biological effect in human health. Nevertheless, and tolerating the differences in composition and concentration of their components, studies supplementing the diet of infants with polar lipids (i.e., MFGM components) have shown significant advances in several areas of human health and well-being. Here we present a summary of the important components of MFGM and milk exosomes as well as an overview of the effects on gut health and brain and cognitive development when added to the diet of infants.     

Enzyme-linked immunosorbent assays for bovine alpha-lactalbumin and beta-lactoglobulin in serum and tissue culture media.

Enzyme-linked immunosorbent assays for bovine alpha-lactalbumin and beta-lactoglobulin have been developed for measurements of serum and tissue culture samples. Either alpha-lactalbumin or beta-lactoglobulin antiserum was coated on ELISA plates. Biotinylated proteins were used in competition with unknown amount of proteins in samples. After unbound proteins were washed off, ExtrAvidin-peroxidase and tetramethylbenzidine were then used as a detection system. Crossreactivity of caseins or bovine serum albumin was less than .0001% in either alpha-lactalbumin or beta-lactoglobulin ELISA. Parallel curves from serial dilutions were obtained in serum and media samples. The additivity of alpha-lactalbumin and beta-lactoglobulin ELISA was validated in either serum or medium samples. The intraassay and interassay coefficients of variation for alpha-lactalbumin and beta-lactoglobulin ELISA were below 10% over 51 and 47 assays. The ELISA are useful in mammary gland biology studies for measuring milk whey protein in serum or culture media.     

Proteomics and microstructure profiling of goat milk protein after homogenization

This study investigated the protein changes in goat milk during the homogenization process using label-free quantification. We quantified 310 and 315 proteins in the control group (CG) and homogenized group (HG), respectively, and 16 proteins were significantly different between the 2 groups. For HG, the goat milk protein particle sizes were smaller and more evenly distributed and exhibited an increase in the regular arrangement of the secondary structures. Proteomics analysis verified that xanthine dehydrogenase and asparaginase-like 1 expression in CG were higher than in HG, whereas the opposite was observed for fructose-bisphosphate aldolase, κ-casein, and β-casein. Significant changes were found in the homogenization-treated goat milk proteome that were related to goat milk glycolysis/gluconeogenesis metabolism. This work provides updated information on the current proteome characteristics of homogenized goat milk, which may be important for applying the protein component of goat milk to human nutrition and health.     

Serum concentrations of the milk proteins alpha-lactalbumin and beta-lactoglobulin in pregnancy and lactation: correlations with milk and fat yields in dairy cattle.

Serum concentrations of alpha-lactalbumin and beta-lactoglobulin in first pregnancy, parturition, lactation, involution, and second parturition in 37 Holstein cattle were determined and used as an index of mammary status and in predicting milk yield. During first pregnancy, serum alpha-lactalbumin increased in the last 3 mo and reached a peak at parturition (approximately 1100 ng/ml). Changes in alpha-lactalbumin could not be described by a simple exponential equation, whereas changes in serum beta-lactoglobulin were described by a single exponential from second trimester until 4 wk prepartum and reached a peak at parturition (approximately 460 ng/ml). By 2 wk after parturition, alpha-lactalbumin had dropped to approximately 140 ng/ml, and beta-lactoglobulin dropped to approximately 25 ng/ml. In late lactation, alpha-lactalbumin was approximately 70 ng/ml and beta-lactoglobulin approximately 20 ng/ml. Short-term elevations were found after cessation of milking in both alpha-lactalbumin and beta-lactoglobulin in serum. The concentrations of alpha-lactalbumin and beta-lactoglobulin at second parturition were similar to those at first parturition with no differences found between parity. Both alpha-lactalbumin and beta-lactoglobulin in serum were functionally associated with mammary growth and development. In heifers late in pregnancy, both serum concentrations of alpha-lactalbumin and beta-lactoglobulin were positively correlated with mature equivalent milk and fat yields in the subsequent lactation. Serum beta-lactoglobulin concentrations at 16 wk prepartum in heifers were highly correlated with the sum of first and second lactation milk (r = .60) and fat (r = .60) yields. The potential value of using serum beta-lactoglobulin as an index for prescreening of heifers for lactation potential is discussed.     

Update on optimized purification and characterization of natural milk allergens

Highly purified allergens namely cow's milk alpha-lactalbumin (ALA). (Bos d 4), beta-lactoglobulin (BLG) (Bos d 5) and casein (Bos d 8) and goat's milk casein were prepared from the raw milk from a single animal with a known genetic background. Consequently the natural isoforms are limited, constant and characterized. Purification included selective precipitations and chromatographical steps. Characterization of structure and allergenic activity assessment of milk allergens were carried out using physicochemical and immunochemical methods. Taken together data demonstrated the absence of impurities and of contamination by other milk allergens in each preparation. NMR and circular dichroism analyses confirmed the native conformation and proper folding of ALA and BLG and the expected absence of folding of bovine and caprine casein. Enzyme immuno assays confirmed the native conformation of BLG and the purity and immunoreactivity of all the proteins. The allergenic activity, e. g. the IgE binding capacity, of purified proteins was identical as that of those proteins when present in milk. The purified proteins also demonstrated the ability to provoke the degranulation of humanized rat basophilic leukaemia cells. All the data thus confirm the purity, identity, structural conformation and functionality of the prepared milk allergens.     

Quantitative determination of complex carbohydrates in bovine milk and in milk-based infant formulas.

Quantitative determination of all structural families of complex carbohydrate micronutrients was performed on bovine milk samples, milk-based infant formulas, and whey-based manufacturing raw materials. Differences found between formulas depended mainly on their whey: casein ratios. A solvent separation procedure was required for quantitative estimation of the gangliosides and neutral glycolipids within the fat fraction. All infant formulas except one contained slightly more gangliosides than bovine milk. Complex carbohydrates were consistently higher in the nonfat fraction. By gel permeation chromatography, an oligosaccharide subfraction was separated from a glycopeptide one. Oligosaccharide content of infant formulas increased as a function of the whey:casein ratio, and glycopeptides were found only in formulas made with whey components. Neuraminic acids from infant formulas were associated primarily with the glycoprotein fraction, except in hydrolysate-based preparations in which "precipitable" glycoproteins were converted into "soluble" glycopeptides by trypsin treatment. Because whey-based raw materials are very rich in all bovine milk glycoconjugates and oligosaccharides their increased use will result in high contents of these micronutrients in modern formulas.     

Milk biologically active components as nutraceuticals: review

Milk contains components that provide critical nutritive elements, immunological protection, and biologically active substances to both neonates and adults. Milk proteins are currently the main source of a range of biologically active peptides. Concentrates of these peptides are potential health-enhancing nutraceuticals for food and pharmaceutical applications. Several bioactive peptides may be used as nutraceuticals, for example, in the treatment of diarrhea, hypertension, thrombosis, dental diseases, as well as mineral malabsorption, and immunodeficiency. Minor whey proteins, such as lactoferrin, lactoperoxidase, lysozyme, and immunoglobulins, are considered antimicrobial proteins. Milk also contains some natural bioactive substances. These include oligosaccharides, fucosylated oligosaccharides, hormones, growth factors, mucin, gangliosides, and endogenous peptides, which are present in milk at secretion. Most of the claimed physiological properties of milk bioactive components have been carried out in vitro or in animal model systems, and these hypothesized properties remain to be proven in humans. Whether these milk bioactive components will replace drugs entirely in the immediate future is still unclear, but the increasing appreciation of "drug foods" or nutraceuticals plays a complementary rather than a substitutional role to the synthetic pharmacological drugs.     

Effect of dose of bovine growth hormone on milk composition: alpha-lactalbumin, fatty acids, and mineral elements

Tissue-specific effects of bovine growth hormone on lactating dairy cows were examined by analysis of milk composition. Milk samples were from 6 cows that received subcutaneous injections of 0, 5, 10, 25, 50, and 100 IU/d of growth hormone in a Latin-square design. Samples from the last 5 d of each 10-d treatment period were pooled for analyses of milk components. Concentration of alpha-lactalbumin in milk increased progressively across the treatment range up to 32% above controls (1.30 mg/nl) at the 100 IU dose. Specific alpha-lactalbumin synthesis (expressed as a percent of total milk protein) was also increased. Secretion of de novo synthesized fatty acids (short and medium chain length) in milk was increased, but response plateaued between the 50 and 100 IU/d. Secretion of preformed (long-chain) fatty acids progressively increased across the entire dose range. Thus, the percentage of long-chain fatty acids in milk increased at the highest doses of hormone. Changes in fatty acid composition of milk were apparently related to energy status; the milk response to 50 and 100 IU/d of growth hormone caused cows to be in or near negative energy balance. Exogenous growth hormone did not affect the concentration of calcium, phosphorus, sodium, iron, copper, and manganese in milk. Results are consistent with growth hormone functioning in homeorhesis to coordinate the partitioning of all nutrients to support the increased secretion of milk and milk components.     

Heat-induced and other chemical changes in commercial UHT milks

The properties of commercial directly and indirectly heated UHT milks, both after heating and during storage at room temperature for 24 weeks, were studied. Thermally induced changes were examined by changes in lactulose, furosine and acid-soluble whey proteins. The results confirmed previous reports that directly heated UHT milks suffer less heat damage than indirectly heated milk. During storage, furosine increased and bovine serum albumin in directly heat-treated milks decreased significantly. The changes in lactulose, alpha-lactalbumin and beta-lactoglobulin were not statistically significant. The data suggest that heat treatment indicators should be measured as soon as possible after processing to avoid any misinterpretations of the intensity of the heat treatment.