Linking variation in the casein fraction and salt composition to casein micelle size in milk of Dutch dairy goats.

The casein (CN) composition, salt composition and micelle size varies largely between milk samples of individual animals. In goats, the link between those casein characteristics are unknown and could provide useful insights into goat casein micelle structure. In this study, the casein- and salt composition of 42 individual Dutch goats from 17 farms was studied and linked to casein micelle size. Micelle size, proportions of individual caseins, and protein content were associated with each other. Milk with smaller casein micelles was higher in protein content, salt content, and proportion of αs1-CN, but lower in αs2-CN and ß-CN. The higher salt content in milk with small casein micelles was mainly attributed to a higher protein content, but changes in casein composition might additionally contribute to differences in mineralization. The non-sedimentable casein content in goat milk correlated with non-sedimentable fractions of ß-CN and κ-CN and was independent of micelle size. Between large and small casein micelles, goat casein micelles showed more differences in casein and salt composition than bovine micelles, indicating differences in internal structure. Nevertheless, the casein mineralization in goat milk was similar to casein mineralization in bovine milk, indicating that mineralization of casein micelles follows a general principle. These results can help to better understand how composition and micelle structure in goat milk are related to each other, which may be useful to improve processing and product properties of goat milk in the future.

Exploring Human Milk Dynamics: Interindividual Variation in Milk Proteome, Peptidome, and Metabolome.

Human milk is a dynamic biofluid, and its detailed composition receives increasing attention. While most studies focus on changes over time or differences between maternal characteristics, interindividual variation receives little attention. Nevertheless, a comprehensive insight into this can help interpret human milk studies and help human milk banks provide targeted milk for recipients. This study aimed to map interindividual variation in the human milk proteome, peptidome, and metabolome and to investigate possible explanations for this variation. A set of 286 milk samples was collected from 29 mothers in the third month postpartum. Samples were pooled per mother, and proteins, peptides, and metabolites were analyzed. A substantial coefficient of variation (>100%) was observed for 4.6% and 36.2% of the proteins and peptides, respectively. In addition, using weighted correlation network analysis (WGCNA), 5 protein and 11 peptide clusters were obtained, showing distinct characteristics. With this, several associations were found between the different data sets and with specific sample characteristics. This study provides insight into the dynamics of human milk protein, peptide, and metabolite composition. In addition, it will support future studies that evaluate the effect size of a parameter of interest by enabling a comparison with natural variability.

Nutritional content, protein quantity, protein quality and carbon footprint of plant-based drinks and semi-skimmed milk in the Netherlands and Europe.

OBJECTIVE: To compare the nutritional composition of bovine milk and several plant-based drinks with a focus on protein and essential amino acid content and to determine the ratio of essential amino acids to greenhouse gas emission. DESIGN: Nutritional information on the label was extracted for semi-skimmed milk, soy, oat, almond, coconut and rice drink from the Innova database between January 2017 and March 2020 for the Netherlands, Belgium, Germany, Spain, Italy, and Sweden. Protein and amino acids were measured and carbon footprint was calculated for a selection of Dutch products. Protein quality was determined by calculating the contribution to the WHO essential amino acids requirements. SETTING: The bovine milk and plant-based drinks market in Netherlands, Belgium, Germany, Spain, Italy, and Sweden. PARTICIPATING PRODUCTS: Semi-skimmed bovine milk and soy-, oat-, almond-, coconut- and rice drink. RESULTS: Nutritional label information was collected for 399 products. Milk naturally contains many micronutrients, e.g. vitamin B2, B12, and calcium. Approximately 50% of the regular plant-based drinks was fortified with calcium, whereas the organic plant-based drinks were mostly unfortified. Protein quantity and quality were highest in milk. Soy drink had the best protein quality to carbon footprint ratio and milk came second. CONCLUSIONS: The nutrition - climate change balance presented in this study, is in line with previous literature, which shows that semi-skimmed bovine milk and fortified soy drink deserve a place in a sustainable diet.

Feeding hydrogenated palm fatty acids and rumen-protected protein to lactating Holstein-Friesian dairy cows modifies milk fat triacylglycerol composition and structure, and solid fat content.

The aim of this study was to analyze the effect of fat and protein supplementation to dairy cattle rations on milk fat triacylglycerol (TAG) composition, fatty acid (FA) positional distribution in the TAG structure, and milk solid fat content (SFC). Fifty-six lactating Holstein-Friesian cows were blocked into 14 groups of 4 cows and randomly assigned 1 of 4 dietary treatments fed for 28 d: (1) low protein, low fat, (2) high protein, low fat, (3) low protein, high fat, and (4) high protein, high fat. The high protein and high fat diets were obtained by isoenergetically supplementing the basal ration (low protein, low fat) with rumen-protected soybean meal and rumen-protected rapeseed meal, and hydrogenated palm FA (mainly C16:0 and C18:0), respectively. Fat supplementation modified milk TAG composition more extensively compared with protein supplementation. Fat supplementation resulted in decreased concentrations of the low molecular weight TAG carbon number (CN) 26 to CN34 and medium molecular weight TAG CN40, CN44, and CN46, and increased concentrations of CN38 and the high molecular weight TAG CN50 and CN52. Increased contents of C16:0, C18:0, and C18:1cis-9 in TAG in response to fat supplementation were related to increases in the relative concentrations of C16:0 and C18:0 at the sn-2 position and C18:0 and C18:1cis-9 at the sn-1(3) positions of the TAG structure. Increased concentrations of high molecular weight TAG species CN50 and CN52 in response to fat supplementation was associated with increased milk SFC at 20, 25, and 30°C. Our study shows that important alterations in milk TAG composition and structure occur when feeding hydrogenated palm FA to lactating dairy cattle, and that these alterations result in an increased SFC of milk fat. These changes in milk SFC and TAG composition and structure may improve absorption of both fat and minerals in milk-based products for infants and may affect processing of milk fat.

Current progress of emerging technologies in human and animals' milk processing: Retention of immune-active components and microbial safety.

Human milk and commercial dairy products play a vital role in humans, as they can provide almost all essential nutrients and immune-active components for the development of children. However, how to retain more native immune-active components of milk during processing remains a big question for the dairy industry. Nonthermal technologies for milk processing are gaining increasing interest in both academic and industrial fields, as it is known that thermal processing may negatively affect the quality of milk products. Thermosensitive components, such as lactoferrin, immunoglobulins (Igs), growth factors, and hormones, are highly important for the healthy development of newborns. In addition to product quality, thermal processing also causes environmental problems, such as high energy consumption and greenhouse gas (GHG) emissions. This review summarizes the recent advances of UV-C, ultrasonication (US), high-pressure processing (HPP), and other emerging technologies for milk processing from the perspective of immune-active components retention and microbial safety, focusing on human, bovine, goat, camel, sheep, and donkey milk. Also, the detailed application, including the instrumental design, technical parameters, and obtained results, are discussed. Finally, future prospects and current limitations of nonthermal techniques as applied in milk processing are discussed. This review thereby describes the current state-of-the-art in nonthermal milk processing techniques and will inspire the development of such techniques for in-practice applications in milk processing.

How processing may affect milk protein digestion and overall physiological outcomes: A systematic review.

Dairy is one of the main sources for high quality protein in the human diet. Processing may, however, cause denaturation, aggregation, and chemical modifications of its amino acids, which may impact protein quality. This systematic review covers the effect of milk protein modifications as a result of heating, on protein digestion and its physiological impact. A total of 5363 records were retrieved through the Scopus database of which a total of 102 were included. Although the degree of modification highly depends on the exact processing conditions, heating of milk proteins can modify several amino acids. In vitro and animal studies demonstrate that glycation decreases protein digestibility, and hinders amino acid availability, especially for lysine. Other chemical modifications, including oxidation, racemization, dephosphorylation and cross-linking, are less well studied, but may also impact protein digestion, which may result in decreased amino acid bioavailability and functionality. On the other hand, protein denaturation does not affect overall digestibility, but can facilitate gastric hydrolysis, especially of ß-lactoglobulin. Protein denaturation can also alter gastric emptying of the protein, consequently affecting digestive kinetics that can eventually result in different post-prandial plasma amino acid appearance. Apart from processing, the kinetics of protein digestion depend on the matrix in which the protein is heated. Altogether, protein modifications may be considered indicative for processing severity. Controlling dairy processing conditions can thus be a powerful way to preserve protein quality or to steer gastrointestinal digestion kinetics and subsequent release of amino acids. Related physiological consequences mainly point towards amino acid bioavailability and immunological consequences.

Stability of fat globules in UHT milk during proteolysis by the AprX protease from Pseudomonas fluorescens and by plasmin.

Fat separation is a limiting factor for the shelf life of UHT milk. It may be promoted by the proteolysis of fat surface-adsorbed proteins (FSAP) by proteases that remain active after UHT treatment. The aim of this research was to explore the relationship between the proteolysis of FSAP and fat destabilization. In this study, we developed a full-fat UHT milk-based model system and added either the major bacterial protease AprX from Pseudomonas fluorescens or the major native milk protease plasmin at high levels to induce fast destabilization of the milk fat globules. We monitored changes in physical properties and FSAP composition, and structural changes in fat globules, over 24 h. Our results showed that AprX-induced sedimentation as a result of the flocculation of fat globules, and plasmin induced cream to float as a result of the coalescence of fat globules. This study confirmed that AprX and plasmin can both lead to fat destabilization in full-fat UHT milk, and it provides insights in the underlying mechanisms.

Binding of CML-Modified as Well as Heat-Glycated ß-lactoglobulin to Receptors for AGEs Is Determined by Charge and Hydrophobicity.

Intake of dietary advanced glycation end products (AGEs) is associated with inflammation-related health problems. Nε-carboxymethyl lysine (CML) is one of the best characterised AGEs in processed food. AGEs have been described as ligands for receptors present on antigen presenting cells. However, changes in protein secondary and tertiary structure also induce binding to AGE receptors. We aimed to discriminate the role of different protein modifications in binding to AGE receptors. Therefore, ß-lactoglobulin was chemically modified with glyoxylic acid to produce CML and compared to ß-lactoglobulin glycated with lactose. Secondary structure was monitored with circular dichroism, while hydrophobicity and formation of ß-sheet structures was measured with ANS-assay and ThT-assay, respectively. Aggregation was monitored using native-PAGE. Binding to sRAGE, CD36, and galectin-3 was measured using inhibition ELISA. Even though no changes in secondary structure were observed in all tested samples, binding to AGE receptors increased with CML concentration of CML-modified ß-lactoglobulin. The negative charge of CML was a crucial determinant for the binding of protein bound CML, while binding of glycated BLG was determined by increasing hydrophobicity. This shows that sRAGE, galectin-3, and CD36 bind to protein bound CML and points out the role of negatively charged AGEs in binding to AGE receptors.

Human Milk Short-Chain Fatty Acid Composition is Associated with Adiposity Outcomes in Infants.

BACKGROUND: Presumed benefits of human milk (HM) in avoiding rapid infancy weight gain and later obesity could relate to its nutrient composition. However, data on breast milk composition and its relation with growth are sparse. OBJECTIVE: We investigated whether short-chain fatty acids (SCFAs), known to be present in HM and linked to energy metabolism, are associated with infancy anthropometrics. METHODS: In a prospective birth cohort, HM hindmilk samples were collected from 619 lactating mothers at 4-8 wk postnatally [median (IQR) age: 33.9 (31.3-36.5) y, body mass index (BMI) (kg/m2): 22.8 (20.9-25.2)]. Their offspring, born at 40.1 (39.1-41.0) wk gestation with weight 3.56 (3.22-3.87) kg and 51% male, were assessed with measurement of weight, length, and skinfold thickness at ages 3, 12, and 24 mo, and transformed to age- and sex-adjusted z scores. HM SCFAs were measured by 1H-nuclear magnetic resonance spectroscopy (NMR) and GC-MS. Multivariable linear regression models were conducted to analyze the relations between NMR HM SCFAs and infancy growth parameters with adjustment for potential confounders. RESULTS: NMR peaks for HM butyrate, acetate, and formic acid, but not propionate, were detected. Butyrate peaks were 17.8% higher in HM from exclusively breastfeeding mothers than mixed-feeding mothers (P = 0.003). HM butyrate peak values were negatively associated with changes in infant weight (standardized B  = -0.10, P = 0.019) and BMI (B = -0.10, P = 0.018) between 3 and 12 mo, and negatively associated with BMI (B = -0.10, P = 0.018) and mean skinfold thickness (B = -0.10, P = 0.049) at age 12 mo. HM formic acid peak values showed a consistent negative association with infant BMI at all time points (B < = -0.10, P < = 0.014), whereas HM acetate was negatively associated with skinfold thickness at 3 mo (B = -0.10, P = 0.028) and 24 mo (B = -0.10, P = 0.036). CONCLUSIONS: These results suggest that HM SCFAs play a beneficial role in weight gain and adiposity during infancy. Further knowledge of HM SCFA function may inform future strategies to support healthy growth.

The Extracellular Protease AprX from Pseudomonas and its Spoilage Potential for UHT Milk: A Review.

The negative effects of proteases produced by psychrotrophic bacteria on dairy products, especially ultra-high-temperature (UHT) milk, are drawing increasing attention worldwide. These proteases are especially problematic, because it is difficult to control psychrotrophic bacteria during cold storage and to inactivate their heat-resistant proteases during dairy processing. The predominant psychrotrophic species with spoilage potential in raw milk, Pseudomonas, can produce a thermostable extracellular protease, AprX. A comprehensive understanding of AprX on the aspects of its biological properties, regulation, proteolytic potential, and its impact on UHT milk can contribute to finding effective approaches to minimize, detect, and inactivate AprX. AprX also deserves attention as a representative of all extracellular metalloproteases produced by psychrotrophic bacteria in milk. The progress of current research on AprX is summarized in this review, including a view on the gap in current understanding of this enzyme. Reducing the production and activity of AprX has considerable potential for alleviating the problems that arise from the instability of UHT milk during shelf-life.

Recent Advances in Phospholipids from Colostrum, Milk and Dairy By-Products.

Milk is one of the most important foods for mammals, because it is the first form of feed providing energy, nutrients and immunological factors. In the last few years, milk lipids have attracted the attention of researchers due to the presence of several bioactive components in the lipid fraction. The lipid fraction of milk and dairy products contains several components of nutritional significance, such as ω-3 and ω-6 polyunsaturated fatty acids, CLA, short chain fatty acids, gangliosides and phospholipids. Prospective cohort evidence has shown that phospholipids play an important role in the human diet and reinforce the possible relationship between their consumption and prevention of several chronic diseases. Because of these potential benefits of phospholipids in the human diet, this review is focused on the recent advances in phospholipids from colostrum, milk and dairy by-products. Phospholipid composition, its main determination methods and the health activities of these compounds will be addressed.

An interactomics overview of the human and bovine milk proteome over lactation.

BACKGROUND: Milk is the most important food for growth and development of the neonate, because of its nutrient composition and presence of many bioactive proteins. Differences between human and bovine milk in low abundant proteins have not been extensively studied. To better understand the differences between human and bovine milk, the qualitative and quantitative differences in the milk proteome as well as their changes over lactation were compared using both label-free and labelled proteomics techniques. These datasets were analysed and compared, to better understand the role of milk proteins in development of the newborn. METHODS: Human and bovine milk samples were prepared by using filter-aided sample preparation (FASP) combined with dimethyl labelling and analysed by nano LC LTQ-Orbitrap XL mass spectrometry. RESULTS: The human and bovine milk proteome show similarities with regard to the distribution over biological functions, especially the dominant presence of enzymes, transport and immune-related proteins. At a quantitative level, the human and bovine milk proteome differed not only between species but also over lactation within species. Dominant enzymes that differed between species were those assisting in nutrient digestion, with bile salt-activated lipase being abundant in human milk and pancreatic ribonuclease being abundant in bovine milk. As lactation advances, immune-related proteins decreased slower in human milk compared to bovine milk. Notwithstanding these quantitative differences, analysis of human and bovine co-expression networks and protein-protein interaction networks indicated that a subset of milk proteins displayed highly similar interactions in each of the different networks, which may be related to the general importance of milk in nutrition and healthy development of the newborn. CONCLUSIONS: Our findings promote a better understanding of the differences and similarities in dynamics of human and bovine milk proteins, thereby also providing guidance for further improvement of infant formula.

Short communication: Influence of shortening the dry period of Swedish dairy cows on plasmin activity in milk.

The aim of this study was to evaluate the influence of shortening the dry period of Swedish dairy cows on plasmin activity and casein composition in milk. Swedish Holstein and Swedish Red cows, 45 in total, were assigned to a dry period of either 4 or 8wk. Milk samples were taken 10 and 5wk prepartum, and 6 and 12wk postpartum. Plasmin activity and plasminogen activity were measured with a spectrophotometric assay. Casein composition was measured by capillary zone electrophoresis. Prepartum plasminogen activity increased by 22% between 10 and 5wk prepartum, whereas no change in plasmin activity was observed during the same period. Cows with a 4-wk dry period had 61% higher plasmin activity in postpartum milk than cows with an 8-wk dry period. Cows of third or greater parity tended to have a stronger increase in plasmin activity as a result of applying a short dry period than cows of second parity. Although the αS1- and ß-casein fractions declined with increasing plasmin activity, no dry period effects were found. Based on postpartum differences in plasmin activity, it was concluded that particularly multiparous cows require more than 4wk between lactations for recovery of the mammary epithelium. Changes in casein composition as an effect of plasmin activity are not expected to have a great effect on processing quality of milk, although future work is needed to verify this.

The protein and lipid composition of the membrane of milk fat globules depends on their size.

In bovine milk, fat globules (MFG) have a heterogeneous size distribution with diameters ranging from 0.1 to 15 µm. Although efforts have been made to explain differences in lipid composition, little is known about the protein composition of MFG membranes (MFGM) in different sizes of MFG. In this study, protein and lipid analyses were combined to study MFG formation and secretion. Two different sized MFG fractions (7.6±0.9 µm and 3.3±1.2 µm) were obtained by centrifugation. The protein composition of MFGM in the large and small MFG fractions was compared using mass-spectrometry-based proteomics techniques. The lipid composition and fatty acid composition of MFG was determined using HPLC-evaporative light-scattering detector and gas chromatography, respectively. Two frequently studied proteins in lipid droplet biogenesis, perilipin-2 and TIP47, were increased in the large and small MFG fractions, respectively. In the large MFG fraction, besides perilipin-2, cytoplasmic vesicle proteins (heat shock proteins, 14-3-3 proteins, and Rabs), microfilaments and intermediate filament-related proteins (actin and vimentin), host defense proteins (cathelicidins), and phosphatidylinositol were higher in concentration. On the other hand, cholesterol synthesis enzymes [lanosterol synthase and sterol-4-α-carboxylate 3-dehydrogenase (decarboxylating)], cholesterol, unsaturated fatty acids, and phosphatidylethanolamine were, besides TIP47, higher in concentration in the small MFG fraction. These results suggest that vesicle proteins, microfilaments and intermediate filaments, cholesterol, and specific phospholipids play an important role in lipid droplet growth, secretion, or both. The observations from this study clearly demonstrated the difference in protein and lipid composition between small and large MFG fractions. Studying the role of these components in more detail in future experiments may lead to a better understanding of fat globule formation and secretion.

Endogenous human milk peptide release is greater after preterm birth than term birth.

BACKGROUND: Hundreds of naturally occurring milk peptides are present in term human milk. Preterm milk is produced before complete maturation of the mammary gland, which could change milk synthesis and secretion processes within the mammary gland, leading to differences in protein expression and enzymatic activity, thereby resulting in an altered peptide profile. OBJECTIVE: This study examined differences in peptides present between milk from women delivering at term and women delivering prematurely. METHODS: Nano-LC tandem mass spectrometry was employed to identify naturally occurring peptides and compare their abundances between term and preterm human milk samples at multiple time points over lactation. Term milk samples were collected from 8 mothers and preterm milk was collected from 14 mothers. The 28 preterm and 32 term human milk samples were divided into 4 groups based on day of collection (<14, 14-28, 29-41, and 42-58 d). RESULTS: Preterm milk peptide counts, ion abundance, and concentration were significantly higher in preterm milk than term milk. Bioinformatic analysis of the cleavage sites for peptides identified suggested that plasmin was more active in preterm milk than term milk and that cytosol aminopeptidase and carboxypeptidase B2 likely contribute to extensive milk protein breakdown. Many identified milk peptides in both term and preterm milk overlapped with known functional peptides, including antihypertensive, antimicrobial, and immunomodulatory peptides. CONCLUSION: The high protein degradation by endogenous proteases in preterm milk might attenuate problems because of the preterm infant's immature digestive system. This trial was registered at clinicaltrials.gov as NCT01817127.

Peptidomic analysis of healthy and subclinically mastitic bovine milk.

A variety of proteases release hundreds of endogenous peptide fragments from intact bovine milk proteins. Mass spectrometry-based peptidomics allows for high throughput sequence assignment of a large number of these peptides. Mastitis is known to result in increased protease activity in the mammary gland. Therefore, we hypothesized that subclinically mastitic milks would contain higher concentrations of released peptides. In this work, milks were sampled from three cows and, for each, one healthy and one subclinically mastitic teat were sampled for milk. Peptides were analyzed by nano-liquid chromatography quadrupole time of flight tandem mass spectrometry and identified with database searching. In total, 682 peptides were identified. The total number of released peptides increased 146% from healthy to subclinically mastitic milks (p < 0.05), and the total abundance of released peptides also increased significantly (p < 0.05). Bioinformatic analysis of enzyme cleavage revealed increases in activity of cathepsin D and elastase (p < 0.05) with subclinical mastitis.

Short communication: Practical issues in implementing volatile metabolite analysis for identifying mastitis pathogens.

Several parameters for improving volatile metabolite analysis using headspace gas chromatography-mass spectrometry (GC-MS) analysis of volatile metabolites were evaluated in the framework of identification of mastitis-causing pathogens. Previous research showed that the results of such volatile metabolites analysis were comparable with those based on bacteriological culturing. The aim of this study was to evaluate the effect of several method changes on the applicability and potential implementation of this method in practice. The use of a relatively polar column is advantageous, resulting in a faster and less complex chromatographic setup with a higher resolving power yielding higher-quality data. Before volatile metabolite analysis is applied, a minimum incubation of 8h is advised, as reducing incubation time leads to less reliable pathogen identification. Application of GC-MS remained favorable compared with regular gas chromatography. The complexity and cost of a GC-MS system are such that this limits the application of the method in practice for identification of mastitis-causing pathogens.

Effect of the DGAT1 K232A genotype of dairy cows on the milk metabolome and proteome.

Diglyceride O-acyltransferase 1 (DGAT1) is the enzyme that catalyzes the synthesis of triglycerides from diglycerides and acyl-coenzyme A. The DGAT1 K232A polymorphism was previously shown to have a significant influence on bovine milk production characteristics (milk yield, protein content, fat content, and fatty acid composition). The mechanism of this influence has, however, not been elucidated. In this study, metabolomics ((1)H-nuclear magnetic resonance) and proteomics (laser chromatography-tandem mass spectrometry) were applied to determine the serum and lipid metabolite composition and milk fat globule membrane proteome of milk samples from cows with the DGAT1 KK and AA genotypes. The milk samples from cows with the DGAT1 KK genotype contained more stomatin, sphingomyelin, choline, and carnitine, and less citrate, creatine or phosphocreatine, glycerol-phosphocholine, mannose-like sugar, acetyl sugar phosphate, uridine diphosphate (UDP)-related sugar, and orotic acid compared with milk samples from cows with the DGAT1 AA genotype. Based on these results, we propose that the differences between the DGAT1 genotypes may be related to stomatin-sphingomyelin lipid rafts as well as structural (cell membrane) differences in epithelial cells of the mammary gland. In conclusion, our study shows that, in addition to previously described changes in triglyceride composition, cows differing in DGAT1 polymorphism differ in their milk proteome and metabolome, which may help in further understanding the effect of the DGAT1 K232A polymorphism on milk production characteristics.

Perspective on calf and mammary gland development through changes in the bovine milk proteome over a complete lactation.

Milk contains all the nutrients for the growth and development of the neonate. However, milk composition is not constant during lactation. To study the changes of the milk proteome over lactation, filter-aided sample preparation combined with dimethyl labeling followed by liquid chromatography tandem mass spectrometry was used to identify and quantify milk proteins from 4 cows. A total of 229 proteins were identified, of which 219 were quantified. An 80% overlap was found in identified and quantified proteins between the 4 individual cows during lactation. Over lactation, the number of quantified proteins changed slightly (less than 10%), whereas the concentration of proteins changed considerably. Transport proteins involved in lipid synthesis (fatty acid-binding protein, perilipin-2, butyrophilin) increased, whereas proteins related to cholesterol transport (apolipoprotein E) decreased. The changes of lipid synthesis proteins are in accordance with the increased milk fat yield over lactation, indicating the increase of de novo mammary fatty acid synthesis as lactation advances. The high abundance of immune-related proteins in early lactation indicates the important role of these proteins for immune system development of calves. The increase in immune-related proteins (immunoglobulins, osteopontin, lactoferrin) and the decrease of proteins related to milk component synthesis (α-lactalbumin, ß-lactoglobulin, fatty acid-binding protein, perilipin-2, butyrophilin) in late lactation can be associated with the protection of the mammary gland. In conclusion, the changes of proteins with different biological functions reflect not only the changing needs of calves but also the development and protection of the mammary gland over lactation.