Exploration of bovine milk proteome in colostral and mature whey using an ion-exchange approach

In addition to milk's nutritional role, it contains immunoglobulins (antibodies) and immunoregulatory proteins that are active in the digestive tract of newborns. However, knowledge of the repertoire of milk proteins remains meager. In this work, we report an ion-exchange-based protein fractionation method that allows in-depth exploration of the whey proteome in bovine milk; 293 unique gene products were identified, of which 176 were newly identified in whey. This work also demonstrated qualitatively for the first time the consistency, albeit differing in protein levels, in milk proteome between colostrum and mature milk (3 mo. post calving). Semiquantitative analysis showed a number of up-regulated proteins in colostrum that may provide extra natural defenses for the neonate. Increased understanding of the composition and functions of bovine milk proteins and their potential health benefits may, in the future, play an important role in nutritional and biomedical applications as properly processed cow's milk proteins could potentially confer the same bioactivity as their human counterparts.     

Characterisation of host defence proteins in milk using a proteomic approach

Besides providing nutrition to the newborn, milk also protects the neonate and the mammary gland against infection. As well as the six major proteins, bovine milk contains minor proteins, not all of which have been characterized. In this study, we have subjected bovine skim milk, whey, and milk fat globule membrane (MFGM) fractions to both direct liquid chromatography-tandem mass spectrometry (LC-MS/MS), and two-dimensional electrophoresis (2-DE) followed by matrix assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry (MS) of individual protein spots to better characterize the repertoire of minor milk proteins, particularly those involved with host defense. Milk from peak lactation as well as during the period of colostrum formation and during mastitis were analyzed to gain a more complete sampling of the milk proteome. In total, 2903 peptides were detected by LC-MS and 2770 protein spots by 2-DE. From these, 95 distinct gene products were identified, comprising 53 identified through direct LC-MS/MS and 57 through 2-DE-MS. The latter were derived from a total of 363 spots analyzed with 181 being successfully identified. At least 15 proteins were identified that are involved in host defense. These results demonstrate that the proteome of milk is more complex than has previously been reported and a significant fraction of minor milk proteins are involved in protection against infection.     

Changes of insulin concentration in colostrum and milk of women, cows and sows

The method of whey extraction in order to determine insulin concentration in colostrum and milk was worked out, checked and applied. The observation of insulin changes showed high and similar insulin concentration in whey samples of women, cows and sows colostrum on the day before and in the day of parturition. One day after delivery hormone concentration maintained high in case of women and sows but in cow colostrum it decreased to 1/12 comparing to the level on the parturition day. During the postpartum following days it was decreasing gradually in the colostrum of all three examined species; the process was very fast in cows, slower in women and the slowest in sows.     

Proteome mapping of human skim milk proteins in term and preterm milk

The abundant proteins in human milk have been well characterized and are known to provide nutritional, protective, and developmental advantages to both term and preterm infants. However, relatively little is known about the expression of the low abundance proteins that are present in human milk because of the technical difficulties associated with their detection. We used a combination of electrophoretic techniques, ProteoMiner treatment, and two-dimensional liquid chromatography to examine the proteome of human skim milk expressed between 7 and 28 days postpartum by healthy term mothers and identified 415 in a pooled milk sample. Of these, 261 were found in human skim milk for the first time, greatly expanding our understanding of the human skim milk proteome. The majority of the proteins identified were involved in either the immune response (24%) or in cellular (28%) or protein (16%) metabolism. We also used iTRAQ analysis to examine the effects of premature delivery on milk protein composition. Differences in protein expression between pooled milk from mothers delivering at term (38-41 weeks gestation) and preterm (28-32 weeks gestation) were investigated, with 55 proteins found to be differentially expressed with at least 90% confidence. Twenty-eight proteins were present at higher levels in preterm milk, and 27 were present at higher levels in term milk.     

Secretion of whey acidic protein and cystatin is down regulated at mid-lactation in the red kangaroo (Macropus rufus)

Milk collected from the red kangaroo (Macropus rufus) between day 100 and 260 of lactation showed major changes in milk composition at around day 200 of lactation, the time at which the pouch young begins to temporarily exit the pouch and eat herbage. The carbohydrate content of milk declined abruptly at this time and although there was only a small increase in total protein content, SDS PAGE analysis of milk revealed asynchrony in the secretory pattern of individual proteins. The levels of alpha-lactalbumin, beta-lactoglobulin, serum albumin and transferrin remain unchanged during lactation. In contrast, the protease inhibitor cystatin, and the putative protease inhibitor whey acidic protein (WAP) first appeared in milk at elevated concentrations after approximately 150 days of lactation and then ceased to be secreted at approximately 200 days. In addition, a major whey protein, late lactation protein, was first detected in milk around the time whey acidic protein and cystatin cease to be secreted and was present at least until day 260 of lactation. The co-ordinated, but asynchronous secretion of putative protease inhibitors in milk may have several roles during lactation including tissue remodelling in the mammary gland and protecting specific proteins in milk required for physiological development of the dependent young.     

Effects of duodenal infusion of free α-linolenic acid on the plasma and milk proteome of lactating dairy cows

This study is an exploratory analysis for understanding the effect of a duodenal infusion of an α-linolenic acid (LNA) on the plasma and milk proteome of lactating dairy cows. Four primiparous Holstein cows were fitted with duodenal cannulas and received 0, 100, 200, 300 and 400 g/day of LNA in a two-treatment crossover design. Blood and milk were collected for determination of protein composition by two-dimensional gel electrophoresis. Alteration of protein spots was detected and identified using matrix-assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry (MALDI-TOF-TOF MS). Plasma haptoglobin levels, and milk β-casein A2, α_s1-casein variant and albumin, did not differ in cows after infusion of 0, 100, 200 and 300 g/day of LNA, but were increased after the cows received duodenal infusion of 400 g/day of LNA. Western blot analysis of haptoglobin expression in plasma confirmed the alterations in protein expression seen using MS. This study demonstrated that infusion of high doses of LNA by duodenal cannula can result in metabolic stress within the bovine intestine and in changes in milk composition.     

Bovine milk exosome proteome

Exosomes are 40-100 nm membrane vesicles of endocytic origin, secreted by cells and are found in biological fluids including milk. These exosomes are extracellular organelles important in intracellular communication, and immune function. Therefore, the proteome of bovine milk exosomes may provide insight into the complex processes of milk production. Exosomes were isolated from the milk of mid-lactation cows. Purified exosomes were trypsin digested, subjected offline high pH reverse phase chromatography and further fractionated on a nanoLC connected to tandem mass spectrometer. This resulted in identification of 2107 proteins that included all of the major exosome protein markers. The major milk fat globule membrane (MFGM) proteins (Butyrophilin, Xanthine oxidase, Adipophilin and Lactadherin) were the most abundant proteins found in milk exosomes. However, they represented only 0.4-1.2% of the total spectra collected from milk exosomes compared to 15-28% of the total spectra collected in the MFGM proteome. These data show that the milk exosome secretion pathway differs significantly from that of the MFGM in part due to the greatly reduced presence of MFGM proteins. The protein composition of milk exosomes provides new information on milk protein composition and the potential physiological significance of exosomes to mammary physiology.     

Transcriptome profiling of bovine milk oligosaccharide metabolism genes using RNA-sequencing

This study examines the genes coding for enzymes involved in bovine milk oligosaccharide metabolism by comparing the oligosaccharide profiles with the expressions of glycosylation-related genes. Fresh milk samples (n = 32) were collected from four Holstein and Jersey cows at days 1, 15, 90 and 250 of lactation and free milk oligosaccharide profiles were analyzed. RNA was extracted from milk somatic cells at days 15 and 250 of lactation (n = 12) and gene expression analysis was conducted by RNA-Sequencing. A list was created of 121 glycosylation-related genes involved in oligosaccharide metabolism pathways in bovine by analyzing the oligosaccharide profiles and performing an extensive literature search. No significant differences were observed in either oligosaccharide profiles or expressions of glycosylation-related genes between Holstein and Jersey cows. The highest concentrations of free oligosaccharides were observed in the colostrum samples and a sharp decrease was observed in the concentration of free oligosaccharides on day 15, followed by progressive decrease on days 90 and 250. Ninety-two glycosylation-related genes were expressed in milk somatic cells. Most of these genes exhibited higher expression in day 250 samples indicating increases in net glycosylation-related metabolism in spite of decreases in free milk oligosaccharides in late lactation milk. Even though fucosylated free oligosaccharides were not identified, gene expression indicated the likely presence of fucosylated oligosaccharides in bovine milk. Fucosidase genes were expressed in milk and a possible explanation for not detecting fucosylated free oligosaccharides is the degradation of large fucosylated free oligosaccharides by the fucosidases. Detailed characterization of enzymes encoded by the 92 glycosylation-related genes identified in this study will provide the basic knowledge for metabolic network analysis of oligosaccharides in mammalian milk. These candidate genes will guide the design of a targeted breeding strategy to optimize the content of beneficial oligosaccharides in bovine milk.     

Infradian rhythmicity in milk leukocyte activity together with plasma cortisol and prolactin levels throughout the lactation period in high-yielding crossbred cows

Infradian rhythmicity in milk leukocyte activity together with plasma cortisol and prolactin levels throughout the lactation period in high-yielding crossbred cows has been studied in 10 high-yielding (milk production: 5000 l per lactation) Karan Fries crossbred (Holstein Friesian × Tharparkar) cows. Milk and blood samples were collected from all the experimental animals. Isolation of milk phagocytes (neutrophils and macrophages) and lymphocytes were done by density gradient centrifugation. In vitro phagocytic index of milk neutrophils and macrophages was performed by colorimetric NBT reductive assay. Mitogen-induced milk lymphocyte blastogenic response was estimated by colorimetric MTT (tetrazolium) assay. Total plasma cortisol and prolactin were estimated by enzyme immune assay. Highest value of plasma cortisol and prolactin was observed at calving which decreased significantly (p < 0.01) on 15th day postpartum for both prolactin and cortisol. Immune activity of milk leukocytes was highest on day 0 colostrum and decreased significantly (p < 0.01) on 7th day postpartum. A significant (p < 0.01) rise of plasma prolactin was observed around 135th and 225th days postpartum, whereas a peak level of plasma cortisol was observed at 105th, 180^th, and 270th days postpartum. Phagocytic index of milk neutrophils and macrophages remains almost in a steady state during mid-lactation period (between 100 and 200 days postpartum). A decline in increasing trend of milk phagocytic activity was observed during late lactation. Mitogen-induced milk lymphocyte blastogenic response was highest on day 0 colostrum which decreased significantly (p < 0.01) on 15th day postpartum. Con A-induced milk lymphocyte blastogenic response showed an increasing trend from 120th to 210th days postpartum. Upon correlation study, it showed that the plasma cortisol has a negative effect on milk leukocyte activity, while prolactin has a positive effect, though the effect is lactation stage specific.     

Alteration of the N-glycome of bovine milk glycoproteins during early lactation

Milk provides nutritional, immunological and developmental components for newborns. Whereas identification of such components has been performed by targeting proteins and free oligosaccharides, structural and functional analyses of the N-glycome of milk glycoproteins are scarce. In this study, we investigated, for the first time, the alterations of the bovine milk N-glycome during early lactation (1 day, 1, 2, 3 and 4 weeks postpartum), characterizing more than 80 N-glycans. The glycomic profile of colostrum on day 1 after calving differed substantially from that in other periods during early lactation. The proteins in colostrum obtained 1 day postpartum were more highly sialylated than milk samples obtained at other time points, and the N-glycolylneuraminic acid (Neu5Gc)/N-acetylneuraminic acid (Neu5Ac) ratio was significantly higher on day 1, showing a gradual decline with time. In order to dissect the N-glycome of colostrum, alterations of the N-glycosylation profile of major bovine milk proteins during the early lactation stage were elucidated, revealing that the alteration is largely attributable to qualitative and quantitative N-glycosylation changes of IgG, the major glycoprotein in colostrum. Furthermore, by preparing and analyzing IgGs in which the N-glycan structure and subtypes were well characterized, we found that the interaction between IgG and FcRn was not affected by the structure of the N-glycans attached to IgG. We also found that bovine FcRn binds IgG(2) better than IgG(1) , strongly suggesting that the role of FcRn in the bovine mammary gland is to recycle IgG(2) from the udder to blood, rather than to secrete IgG(1) into colostrum.     

Concentration of lactoferrin and transferrin throughout lactation in cow's colostrum and milk

The evolution of the concentration of lactoferrin and transferrin was studied in cow's colostrum and milk throughout lactation. The highest concentrations of both proteins were found in the first milking (0.83 mg/ml for lactoferrin and 1.07 mg/ml for transferrin), decreasing sharply during the first days of lactation (colostral period). Thereafter, the concentrations of these proteins decreased slowly, reaching their definitive values during the third week post-partum (0.09 mg/ml for lactoferrin and 0.02 mg/ml for transferrin). The ratio transferrin/albumin in colostrum (first milking), mature milk, milk from mastitic cows and serum was determined, and found to be four times greater in colostrum than in mature or mastitic milk, suggesting a specific transport of transferrin from blood to milk.     

Seasonal changes in the mineral composition of colostrum and milk whey of cows

It is shown that in summer the level of sodium, potassium and calcium in the colostrum and milk serum of cows for the first ten days of lactation is 1.5-2.0 times higher and the content of inorganic phosphorus lower than the analogous indices for cows in winter-spring. The concentration of iron, copper, zinc and chlorides in the colostrum and milk serum of cows in different seasons is unchanged.     

In-depth analysis of low abundant proteins in bovine colostrum using different fractionation techniques

Bovine colostrum is well known for its large content of bioactive components and its importance for neonatal survival. Unfortunately, the colostrum proteome is complicated by a wide dynamic range, because of a few dominating proteins that hamper sensitivity and proteome coverage achieved on low abundant proteins. Moreover, the composition of colostrum is complex and the proteins are located within different physical fractions that make up the colostrum. To gain a more exhaustive picture of the bovine colostrum proteome and gather information on protein location, we performed an extensive pre-analysis fractionation of colostrum prior to 2D-LC-MS/MS analysis. Physical and chemical properties of the proteins and colostrum were used alone or in combination for the separation of proteins. ELISA was used to quantify and verify the presence of proteins in colostrum. In total, 403 proteins were identified in the nonfractionated colostrum (NF) and seven fractions (F1-F7) using six different fractionation techniques. Fractionation contributed with 69 additional proteins in the fluid phase compared with NF. Different fractionation techniques each resulted in detection of unique subsets of proteins. Whey production by high-speed centrifugation contributed most to detection of low abundant proteins. Hence, prefractionation of colostrum prior to 2D-LC-MS/MS analysis expanded our knowledge on the presence and location of low abundant proteins in bovine colostrum.     

Leptin in bovine colostrum and milk.

We studied leptin content in bovine colostrum, milk and plasma during the first month of lactation, and investigated relationships between selected milk components and milk leptin in five multiparous dairy cows. Colostrum/milk yield and composition were measured on days 0, 10, 20, and 30 of lactation. Leptin was assayed using a multi-species leptin RIA kit. Leptin concentration was 56 % lower in mature milk (day 10) than colostrum (13.90 vs. 6.14 microg/l; p < 0.001), but remained steady over the twenty days afterwards. Daily secretion of leptin into mature milk was 28 % lower than into colostrum (173.2 microg/d vs. 220.0 microg/d; p = 0.09) notwithstanding an 80 % increase in production. Colostrum and milk leptin levels correlated with fat (0.90; p < 0.001) and choline phospholipid (0.76; p < 0.05). Plasma and milk leptin decreased during the first month, but remained higher in milk, and highest in colostrum. Thus, leptin is present in large quantities in colostrum, less so and more variably in untreated milk, and is likely to be decreased in skimmed milk. These findings have implications for the use of untreated milk and colostrum-based (functional) food products.     

Alterations in the milk yield and composition during different stages of lactation cycle in elite and non-elite Karan-Fries cross-bred cows (Holstein Fresian x Tharparkar)

Alterations in the milk constituents throughout the lactation cycle have been studied in 20 Karan-Fries cross-bred cows (Holstein Fresian x Tharparkar) divided into two groups, viz. elite (n = 10) and non-elite (n = 10). About 100 ml of composite milk samples (from all the quarters) were collected through hand milking in sterile tubes from the day of calving at 15-day interval till 300 days of lactation cycle. Different milk constituents (viz. fat, protein, lactose, Solid not fat) were estimated by automatic milk analyzer. The daily milk yield varied significantly (p < 0.01) between group and different days of lactation cycle. Milk fat percentage of milk did not differ significantly between groups and different days of lactation cycle. Milk protein and lactose percentage did not differ significantly between groups but differed significantly (p < 0.001) between days. Solid not fat (SNF) percentage of milk was significantly (p < 0.01) higher in elite cows compared to non-elite cows and also varied significantly (p < 0.05) during different days of lactation cycle. In both the group of cows, milk protein, fat, and SNF percentage was highest and lactose percentage was lowest up to 1st week of lactation cycle and rapidly declined thereafter due to the transition of the colostrum into milk. After 2nd week of lactation, all the milk constituents under study were almost remained unaltered till the end of the cycle. The above stated investigation not only substantiates the already existing information of lactation stage-specific alteration in milk constituents but also depict the exact point of transition of these constituents during the lactation cycle so the nutritional and managemental interventions could be carried out in proper time.     

Temporal changes in milk proteomes reveal developing milk functions

Human milk proteins provide essential nutrition for growth and development, and support a number of vital developmental processes in the neonate. A complete understanding of the possible functions of human milk proteins has been limited by incomplete knowledge of the human milk proteome. In this report, we have analyzed the proteomes of whey from human transitional and mature milk using ion-exchange and SDS-PAGE based protein fractionation methods. With a larger-than-normal sample loading approach, we are able to largely extend human milk proteome to 976 proteins. Among them, 152 proteins are found to render significant regulatory changes between transitional milk and mature milk. We further found that immunoglobulins sIgA and IgM are more abundant in transitional milk, whereas IgG is more abundant in mature milk, suggesting a transformation in defense mechanism from newborns to young infants. Additionally, we report a more comprehensive view of a complement system and associated regulatory apparatus in human milk, demonstrating the presence and function of a system similar to that found in the circulation but prevailed by alternative pathway in complement activation. Proteins involved in various aspects of carbohydrate metabolism are also described, revealing either a transition in milk functionality to accommodate carbohydrate-rich secretions as lactation progresses, or a potentially novel way of looking at the metabolic state of the mammary tissue. Lately, a number of extracellular matrix (ECM) proteins are found to be in higher abundance in transitional milk and may be relevant to the development of infants' gastrointestinal tract in early life. In contrast, the ECM protein fibronectin and several of the actin cytoskeleton proteins that it regulates are more abundant in mature milk, which may indicate the important functional role for milk in regulating reactive oxygen species.     

Quantitative milk proteomics – Host responses to lipopolysaccharide‐mediated inflammation of bovine mammary gland

Intramammary infusion of lipopolysaccharide (LPS) in cows induces udder inflammation that partly simulates mastitis caused by infection with Gram‐negative bacteria. We have used this animal model to characterize the quantitiative response in the milk proteome during the time course before and immediately after the LPS challenge. Milk samples from three healthy cows collected 3 h before the LPS challenge were compared with milk samples collected 4 and 7 h after the LPS challenge, making it possible to describe the inflammatory response of individual cows. Quantitative protein profiles were obtained for 80 milk proteins, of which 49 profiles changed significantly for the three cows during LPS challenge. New information obtained in this study includes the quantified increase of apolipoproteins and other anti‐inflammatory proteins in milk, which are important for the cow's ability to balance the immune response, and the upregulation of both complement C3 and C4 indicates that more than one complement pathway could be activated during LPS‐induced mastitis. In the future, this analytical approach may provide valuable information about the differences in the ability of individual cows to resist and recover from mastitis.     

Proteomic characterization of human milk whey proteins during a twelve-month lactation period

Human milk is a rich source of bioactive proteins that support the early growth and development of the newborn. Although the major components of the protein fraction in human milk have been studied, the expression and relative abundance of minor components have received limited attention. We examined the expression of low-abundance proteins in the whey fraction of human milk and their dynamic changes over a twelve-month lactation period. The low-abundance proteins were enriched by ProteoMiner beads, and protein identification was performed by liquid chromatography tandem mass spectrometry. One hundred and fifteen proteins were identified, thirty-eight of which have not been previously reported in human colostrum or milk. We also for the first time described differences in protein patterns among the low-abundance proteins during lactation. These results enhance our knowledge about the complexity of the human milk proteome, which constitutes part of the advantages to the breast-fed infant.     

Soluble form of ICAM-1, VCAM-1, E- and L-selectin in human milk

In breast milk and paired serum from 70 lactating women and 40 of their term, infection-free neonates, on the 2nd and 5th day postpartum slCAM-1, sVCAM-1, sE- and sL-selectin were measured by ELISA and compared with those in 26 healthy adults (controls). Seven infant formulas and fresh milk from five cows were also analyzed. Human colostrum values of slCAM-1, sVCAM-1 (similar to those in maternal and control serum), sE-selectin and sL-selectin (-10 and -100 times lower than in maternal and control serum) were significantly higher than those in milk, while they varied widely. None of the adhesion molecules was detected in fresh cow's milk or infant formulas. Exclusively breast-fed infants showed significantly higher values of slCAM-1 and sL-selectin on the 2nd day of life than those supplemented also with formula. Only slCAM-1 values correlated positively between colostrum and time-matched maternal serum. These findings show in human milk important amounts of slCAM-1 and sVCAM-1 but minimal amounts of sE- and sL-selectin, which could affect the immune system of the neonate.