Age-specific composition of milk microbiota in Tibetan sheep and goats.

This study investigates the dynamic changes in milk nutritional composition and microbial communities in Tibetan sheep and goats during the first 56 days of lactation. Milk samples were systematically collected at five time points (D0, D7, D14, D28, D56) post-delivery. In Tibetan sheep, milk fat, protein, and casein contents were highest on D0, gradually decreased, and stabilized after D14, while lactose and galactose levels showed the opposite trend. Goat milk exhibited similar initial peaks, with significant changes particularly between D0, D7, D14, and D56. 16S rRNA gene sequencing revealed increasing microbial diversity in both species over the lactation period. Principal coordinates analysis identified distinct microbial clusters corresponding to early (D0-D7), transitional (D14-D28), and mature (D56) stages. Core phyla, including Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria, dominated the milk microbiota, with significant temporal shifts. Core microbes like Lactobacillus, Leuconostoc, and Streptococcus were common in both species, with species-specific taxa observed (e.g., Pediococcus in sheep, Shewanella in goats). Furthermore, we observed a highly shared core microbiota in sheep and goat milk, including Lactobacillus, Leuconostoc, and Streptococcus. Spearman correlation analysis highlighted significant relationships between specific microbial genera and milk nutrients. For instance, Lactobacillus positively correlated with total solids, non-fat milk solids, protein, and casein, while Mannheimia negatively correlated with protein content. This study underscores the complex interplay between milk composition and microbial dynamics in Tibetan sheep and goats, informing strategies for livestock management and nutritional enhancement. KEY POINTS: • The milk can be classified into three types based on the microbiota composition • The changes of milk microbiota are closely related to the variations in nutrition • Filter out microbiota with species specificity and age specificity in the milk.

Mammary fat globules as a source of mRNA to model alterations in the expression of some milk component genes during lactation in bovines.

BACKGROUND: The milk's nutritional value is determined by its constituents, including fat, protein, carbohydrates, and minerals. The mammary gland's ability to produce milk is controlled by a complex network of genes. Thereby, the fat, protein, and lactose synthesis must be boost in milk to increase milk production efficiency. This can be accomplished by fusing genetic advancements with proper management practices. Therefore, this study aimed to investigate the association between the Lipoprotein lipase (LPL), kappa casein CSN3, and Glucose transporter 1 (GLUT1) genes expression levels and such milk components as fat, protein, and lactose in different dairy breeds during different stages of lactation. METHODS: To achieve such a purpose, 94 milk samples were collected (72 samples from 36 multiparous black-white and red-white Holstein-Friesian (HF) cows and 22 milk samples from 11 Egyptian buffaloes) during the early and peak lactation stages. The milk samples were utilized for milk analysis and genes expressions analyses using non- invasive approach in obtaining milk fat globules (MFGs) as a source of Ribonucleic acid (RNA). RESULTS: LPL and CSN3 genes expressions levels were found to be significantly higher in Egyptian buffalo than Holstein-Friesian (HF) cows as well as fat and protein percentages. On the other hand, GLUT1 gene expression level was shown to be significantly higher during peak lactation than early lactation. Moreover, lactose % showed a significant difference in peak lactation phase compared to early lactation phase. Also, fat and protein percentages were significantly higher in early lactation period than peak lactation period but lactose% showed the opposite pattern of Egyptian buffalo. CONCLUSION: Total RNA can be successfully obtained from MFGs. The results suggest that these genes play a role in glucose absorption and lactose synthesis in bovine mammary epithelial cells during lactation. Also, these results provide light on the differential expression of these genes among distinct Holstein-Friesian cow breeds and Egyptian buffalo subspecies throughout various lactation phases.

A retrospective comparison of IgE-mediated cow's milk protein allergy management strategies in pediatric cohorts.

BACKGROUND: Complete avoidance of milk is the usual management for IgE-mediated cow's milk protein allergy (CMPA). A baked milk ladder is a method of dietary advancement therapy in IgE-mediated CMPA in Ireland, while in Spain, avoidance of milk awaiting natural tolerance acquisition through an oral food challenge (OFC) is employed. The aim of this study was to evaluate the use of dietary advancement therapy using a milk ladder compared with complete avoidance of milk for managing IgE-mediated CMPA. METHODS: This is a retrospective chart review of 371 pediatric patients from the population who have been treated for IgE-mediated CMPA between 2011 and 2020, with the milk ladder (Ireland) or complete avoidance followed by an OFC (Spain). The main outcome was the introduction of cow's milk. RESULTS: Milk ladder patients were 3.67 times more likely to succeed in comparison with milk avoidance (p < .001). Anaphylaxis during the treatment period occurred in 34 patients in the milk avoidance groups, while three patients in the milk ladder group experienced anaphylaxis due to accidental exposure to milk (p < .001). Failure to complete treatment was associated with a higher skin prick test in the milk avoidance group and a raised specific IgE in the milk ladder group. CONCLUSION: This is the first study that compares outcomes of dietary advancement therapy to complete avoidance for CMPA management, demonstrating that cow's milk can be successfully and safely reintroduced using dietary advancement therapy using a milk ladder.

Risks of dairy derived excipients in medications for lactose intolerant and cow milk protein allergic patients.

The use of lactose and cow milk protein (CMP) as potential allergens in pharmaceuticals and their ability to cause allergic reactions remains a significant concern in medicine. Lactose, a common pharmaceutical excipient due to its inert, inexpensive, and stable properties, is found in many prescription-only and over-the-counter medications. However, despite their widespread use, individuals with lactose intolerance (LI) or cow milk protein allergy (CMPA) may experience adverse reactions to these excipients. This study investigated the prevalence of lactose and other dairy-derived ingredients in pharmaceuticals marketed in Portugal. Using the Summary of Product Characteristics (SmPC) from the INFOMED database, various medications, including analgesics, antipyretics, non-steroidal anti-inflammatory drugs (NSAIDs), and antiasthmatics, were analyzed. Results showed a high prevalence of dairy-derived excipients, particularly in antiasthmatic drugs (62.6%) and NSAIDs (39%). Although CMP are not explicitly mentioned in SmPCs, the presence of lactose as an ingredient poses a risk of cross-contamination. The findings emphasize the need for healthcare professionals to be aware of potential allergens in medications and the importance of developing lactose-free alternatives to ensure the safety of patients with LI and CMPA. Further research is required to assess the safety and implications of lactose in medicines for these populations.

Shift from Cow's Milk Food Protein-Induced Enterocolitis Syndrome to IgE-Mediated Allergy: Case Series and Literature Review.

Background: Food protein-induced enterocolitis syndrome (FPIES) is a non-IgE-mediated food allergy characterized by gastrointestinal symptom onset within 1-4 hours from trigger food ingestion. In the literature, some authors have previously described the possibility that a patient with FPIES may develop an IgE-mediated allergy to the same trigger food, especially cow's milk (CM). Case Presentation: We reported five cases of CM-FPIES converting to IgE-mediated CM allergy presented at our tertiary pediatric Allergy Unit and performed a review of the literature, aiming to characterize the clinical features of patients who are at risk of developing such conversion. Conclusions: This phenomenon raises the question of whether IgE-mediated and non-IgE-mediated allergies represent a spectrum of the same disease and highlights the need for further investigation to understand the pathophysiological mechanisms of this process.

Influence of production level, number, and stage of lactation on milk quality in compost barn systems.

This study evaluated the influence of milk production, number of lactations, and days in milk (DIM) on the quality and composition of milk from dairy cows housed in a compost barn (CB) system. The study was carried out using a six-year database, counting 31,268 observations from 2,037 cows of European breeds. Multiparous cows showed higher fat and protein production. Lactose showed high levels for primiparous and the initial stage of lactation (4.65%) and was negatively influenced by somatic cell count (SCC). Milk urea nitrogen was higher (14.01%) from 106 to 205 days in milk, and the other components were higher at >305 days. Therefore, the solids content was higher in the first and second lactations due to the high contents of lactose, fat, and milk protein, but lactose was reduced over lactations. In contrast, high DIM increased SCC and concentrated solids due to lower milk production. The effect of milk production, stage, and lactation order on the composition and milk quality of herds housed in CB showed the same pattern as in other production systems.

Influence of different milking methods on milk quality based on somatic cell count and basic composition.

INTRODUCTION AND OBJECTIVE: Correlations between the number of milk somatic cells (SCC), the number of microorganisms, and the content of basic components of milk were studied on five farms (F1-F5) with cows of the same breed, but with different milking systems. MATERIAL AND METHODS: From each farm, 50 Holstein Friesien milk samples were collected once a month (250 samples/month; n=3,000) during March 2022 - February 2023. Samples from farms F1 and F5 were tested for fat, protein, lactose, no fat dry matter content (FTIR spectroscopy), for the SCC (Fossomatic 7), and for the differential cells (Vetscan DC-Q). RESULTS: The highest fat content was confirmed on farm F5 (3.85 ± 1.70%) and F4 (3.82 ± 0.21%) with automatic milking system (AMS). However, from the point of view of protein content, these farms showed slightly lower values (<0.05). F1 did not meet the minimum required amount for fat content (2.84 ± 0.81%) set by the legislation of the Slovakia. The comparison shows that there is not much difference in cell size between healthy cells and mastitis cells. The average size of healthy cells was approximately 8.77 ± 0.49 µm. In the monitored period, the average values determined were at the level of 292,000/mL (5.46 ± 0.72 log10 SCC) in cow milk samples, while for the rest of the year, the values remained at 256,000/mL (5.40 ± 0.80 log10 SCC). F1 was categorized as a positive farm with a high TLC (total milk leucocyte count) concentration (5.58 log10 cells/mL, 406.65 ± 53.80 × 103 cells/mL) and a predominant NEU fraction (61%). Farms F2, F4, and F5 were classified as negative farms (TLC was 4.70 ± 0.26 log10 cells/ml). CONCLUSIONS: According to the results, the size of SCCs in healthy milk does not differ from SCCs found in mastitis milk. From the results, it can be concluded that the transition to the latest generation of robotic milking method can positively affect milk production and its quality.

Reference Material Production and Milk Protein Concentration as Elements to Improve Bluetongue Serological Diagnosis in Bulk Tank Milk.

The serological surveillance of bluetongue in bulk tank milk is an efficient and cost-effective method for the early detection of bluetongue virus incursions in unvaccinated free areas of the disease. In addition, the availability of standardized and reliable reagents and refined diagnostic procedures with high sensitivity and specificity are essential for surveillance purposes. However, no available reference materials for bluetongue virus serological surveillance in bulk tank milk exist. This study shows the production and characterization of reference material for the implementation of a commercially available bluetongue milk ELISA test in official laboratories, as well as the evaluation of a procedure to increase the sensitivity in samples with low levels of antibodies. This procedure, based on milk protein concentration, allowed us to notably increase the ELISA test's analytical sensitivity, which is useful for milk samples from farms with low within-herd prevalence or pools of bulk tank milk samples. The standardized milk reference material produced here, together with the evaluated procedure to improve analytical sensitivity, could be applied as tools to ensure an accurate diagnosis by official laboratories in bluetongue unvaccinated free areas.

Science and Faith to Understand Milk Bioactivity for Infants.

Milk bioactivity refers to the specific health effects of milk components beyond nutrition. The science of milk bioactivity involves the systematic study of these components and their health effects, as verified by empirical data, controlled experiments, and logical arguments. Conversely, 'faith in milk bioactivity' can be defined as personal opinion, meaning, value, trust, and hope for health effects that are beyond investigation by natural, social, or human sciences. Faith can be strictly secular, but also influenced by spirituality or religion. The aim of this paper is to show that scientific knowledge is frequently supplemented with faith convictions to establish personal and public understanding of milk bioactivity. Mammalian milk is an immensely complex fluid containing myriad proteins, carbohydrates, lipids, and micronutrients with multiple functions across species, genetics, ages, environments, and cultures. Human health includes not only physical health, but also social, mental, and spiritual health, requiring widely different fields of science to prove the relevance, safety, and efficacy of milk interventions. These complex relationships between milk feeding and health outcomes prevent firm conclusions based on science and logic alone. Current beliefs in and understanding of the value of breast milk, colostrum, infant formula, or isolated milk proteins (e.g., immunoglobulins, α-lactalbumin, lactoferrin, and growth factors) show that both science and faith contribute to understand, stimulate, or restrict the use of milk bioactivity. The benefits of breastfeeding for infants are beyond doubt, but the strong beliefs in its health effects rely not only on science, and mechanisms are unclear. Likewise, fear of, or trust in, infant formula may rely on both science and faith. Knowledge from science safeguards individuals and society against 'milk bioactivity superstition'. Conversely, wisdom from faith-based convictions may protect science from unrealistic 'milk bioactivity scientism'. Honesty and transparency about the potentials and limitations of both scientific knowledge and faith convictions are important when informing individuals and society about the nutritious and bioactive qualities of milk.

Human milk proteins differentiate over the sex of newborns and across stages of lactation.

BACKGROUND & AIMS: Human milk (HM) is a complete food that meets the nutritional and energy demands of the newborns. It contains numerous bioactive components, including functional proteins. Variations in HM energy and lipid content have already been reported related to the newborn's sex, but differences between protein profiles are still scarce. This work aimed to identify differences between HM proteins produced by mothers of female and male newborns, in the lactation stages of colostrum and mature milk, and the metabolic pathways involved. METHODS: A total of 98 HM samples were collected from 39 lactating women and classified according to the newborn's sex, stages of lactation, and three mothers' age groups, and evaluated about protein concentration and one-dimensional electrophoretic profile. Next, to assess samples with the greatest differences, the HM proteins regarding the newborn's sex and the stages of lactation were compared using nano-LC-MS/MS, in 24 HM samples randomly rearranged into four groups: female and male infants, and colostrum and mature milk. Functional classification, metabolic pathways, and protein interaction networks were analyzed by Gene Ontology, KEGG, and STRING, respectively. RESULTS: The soluble protein content of HM decreased throughout lactation, with differences regarding isolated factors, such as mothers' age group, child's sex and stages of lactation, and also in terms of their interactions. A total of 146 proteins were identified, 42 of which showed different abundances over the sexes of newborns and 53 between the stages of lactation. In general, proteins related to metabolic processes were up-regulated for mothers of male infants and in the mature stage of lactation, while proteins related to defense were up-regulated in mothers of female infants and in the colostrum phase. CONCLUSION: This study indicated that there are differentiated and specific nutritional and defense needs of newborns, by sex and by lactation phase, which is highly relevant for a more appropriate supply of food to infants receiving HM from donor mothers.

Assessment of protein and phospholipid bioaccessibility in ultrafiltered buttermilk cheese using TIM-1 in vitro gastrointestinal methods.

To meet the high consumer demand, butter production has increased over the last few years. As a result, the buttermilk (BM) co-produced volumes require new ways of adding value, such as in cheese manufacturing. However, BM use in cheese milk negatively influences the cheesemaking process (e.g., altered coagulation properties) and the product's final quality (e.g., high moisture content). The concentration of BM by ultrafiltration (UF) could potentially facilitate its use in cheese manufacturing through an increased protein content while maintaining the milk salt balance. Simultaneously, little is known about the digestion of UF BM cheese. Therefore, this study aimed to characterize the impact of UF BM on cheese manufacture, its structure, and its behavior during in vitro digestion. A 2-fold UF concentrated BM was used for cheese manufacture (skim milk [SM] - control). Compositional, textural, and microstructural analyses of cheeses were first conducted. In a second step, the cheeses were fed into an in vitro TNO gastrointestinal digestion model (TIM-1) of the stomach and small intestine and protein and phospholipid (PL) bioaccessibility was studied. The results showed that UF BM cheese significantly differed from SM cheese regarding its composition, hardness (p < 0.05) and microstructure. However, in TIM-1, UF BM and SM cheeses showed similar digestion behavior as a percentage of protein and PL intake. Despite relatively more non-digested and non-absorbed PL in the ileum efflux of UF BM cheese, the initially higher PL concentration contributes to an enhanced nutritional value compared to SM cheese. To our knowledge, this study is the first to compare the bioaccessibility of proteins and PL from UF BM and SM cheeses.

In vitro gastrointestinal digestion of cow's and sheep's dairy products: Impact of species and structure.

Sheep's milk (SM) is known to differ from cow's milk (CM) in nutritional composition and physicochemical properties, which may lead to different digestion behaviours. This work aimed to investigate the impact of the species (cow vs sheep) and the structure (milk vs yogurt) on the digestion of dairy products. Using an in vitro static gastrointestinal digestion model, CM, SM, cow's milk yogurt (CY) and sheep's milk yogurt (SY) were compared on particle size evolution, microscopic observations, degree of lipolysis, degree of proteolysis, specific protein degradation and calcium bioaccessibility. Species and structure affected particle size evolution during the gastric phase resulting in smaller particles for yogurts compared to milks as well as for CM products compared to SM products. Species impacted lipid composition and lipolysis, with SM products presenting higher short/medium-chain fatty acids content and higher intestinal degree of lipolysis. Proteolysis was influenced by structure, with milks showing higher intestinal degree of proteolysis compared to yogurts. Caseins were digested faster in CM, ⍺-lactalbumin was digested faster in SM despite its higher concentration, and during gastric digestion ß-lactoglobulin was more degraded in CM products compared to SM products and more in yogurts compared to milks. Lastly, SM products released more bioaccessible calcium than CM products. In conclusion, species (cow vs sheep) impacted more the digestion compared to the structure (milk vs yogurt). In fact, SM was different from CM mainly due to a denser protein network that might slow down the accessibility of the enzyme to its substrate which induce a delay of gastric disaggregation and thus lead to slower the digestion of the nutrients.

Peptidome profiling of human, bovine, and donkey colostrum through label-free quantitative analysis reveals proteolysis of milk proteins.

Proteins and peptides play vital roles in different biological processes in vivo. As a dynamic hydrolysis system, milk is rich in proteins and proteases and provides a constant supply of endogenous bioactive peptides to newborn mammals. Previous studies have primarily focused on researching bioactive peptides by adding exogenous enzymes to milk samples. However, such an approach overlooks the significance of endogenous peptides and parent proteins that naturally exist in milk. Herein, we analyzed and compared parent proteins and their releasing peptides in human colostrum (HC), bovine colostrum (BC), and donkey colostrum (DC). The predominant proteins and hydrolyzed peptides in the three types of milk were identified. Among them, peptides were found to possess common bioactivities, including ACE inhibitory, antioxidant, antibacterial and immunomodulatory properties in HC, BC, and DC. Furthermore, the biological functions of these parent proteins were clarified using bioinformatics. These insights offer a novel perspective on natural bioactive peptides and the potential utilization of specific parent proteins and peptides to develop infant formulae derived from diverse milk sources.

Quantitation of milk proteins in thermally treated milk samples and commercial food products by ELISA test kits.

This study evaluated four ELISA kits for quantitation of milk proteins in thermally treated milk samples and food products. How reference materials may be used for comparison of kit performance was examined. Protein contents determined by Veratox Total Milk generally reflected those determined by the 660 nm total protein assay. BioKits BLG Kit was less affected by thermal treatment but resulted in overestimation of protein contents in samples that were boiled, autoclaved or dry-heated at ≤149 °C, while ELISA Systems Casein (ES Casein) and Beta-Lactoglobulin (ES BLG) assays underestimated protein levels in these samples. The four kits gave similar results for ice cream. Veratox registered higher concentrations in all products tested but its sensitivity was greatly lowered in retorted products. ES Casein underperformed Veratox for baked and retorted products. BioKits BLG maintained a better sensitivity towards fried, baked and retorted products while ES BLG exhibited reduced sensitivity for these products.

Application of pressure homogenization on whole human milk pasteurized by high hydrostatic pressure: Effect on protein aggregates in milk fat globule membrane and skim milk phases.

This study explored the impact of homogenization (at pressures of 16, 30, and 45 MPa) on both raw and high hydrostatic pressure (HHP)-treated human milk (HM). It focused on protein compositions and binding forces of soluble and insoluble fractions for both milk fat globule membrane (MFGM) and skim milk. Mild homogenization of HHP-treated milk increased lactoferrin (LF) levels in the insoluble fractions of both MFGM and skim milk, due to insoluble aggregation through hydrophobic interactions. Intense homogenization of HHP-treated milk decreased the LF level in the MFGM fractions due to the LF desorption from the MFGM, which increased LF level in the insoluble skim milk fraction. Homogenized-HHP treated milk showed noticeably higher casein (CN) level at the MFGM compared to homogenized-raw milk, attributed to HHP effect on CN micelles. Overall, the combined use of HHP and shear-homogenization should be avoided as it increased the biological proteins in insoluble fractions.

Differences in proteomic profiles and immunomodulatory activity of goat and cow milk fat globule membrane.

This study aimed to clarify the composition and bioactivity differences between goat and cow milk fat globule membrane (MFGM) protein by proteomic, and the immunomodulatory activity of MFGM proteins was further evaluated by using mouse splenic lymphocytes in vitro. A total of 257 MFGM proteins showed significant differences between goat and cow milk. The upregulated and unique MFGM proteins in goat milk were significantly enriched in the positive regulation of immune response, negative regulation of Interleukin-5 (IL-5) secretion, and involved in nucleotide-binding oligomerization domain (NOD)-like receptor signaling. The contents of IL-2 and Interferon-γ in the supernatant of spleen lymphocytes treated with goat MFGM proteins were much higher than those of IL-4 and IL-5, suggesting a Th1-skewed immune response. These results revealed that goat MFGM proteins could possess better immunomodulatory effects as compared to cow milk. Our findings may provide new insights to elucidate the physiological functions and nutritional of goat milk.

Improved breast milk proteome coverage by DIA based LC-MS/MS method.

The breast milk composition includes a multitude of bioactive factors such as viable cells, lipids and proteins. Measuring the levels of specific proteins in breast milk plasma can be challenging because of the large dynamic range of protein concentrations and the presence of interfering substances. Therefore, most proteomic studies of breast milk have been able to identify under 1000 proteins. Optimised procedures and the latest separation technologies used in milk proteome research could lead to more precise knowledge of breast milk proteome. This study (n = 53) utilizes three different protein quantification methods, including direct DIA, library-based DIA method and a hybrid method combining direct DIA and library-based DIA. On average we identified 2400 proteins by hybrid method. By applying these methods, we quantified body mass index (BMI) associated variation in breast milk proteomes. There were 210 significantly different proteins when comparing the breast milk proteome of obese and overweight mothers. In addition, we analysed a small cohort (n = 5, randomly selected from 53 samples) by high field asymmetric waveform ion mobility spectrometry (FAIMS). FAIMS coupled with the Orbitrap Fusion Lumos mass spectrometer, which led to 41.7% higher number of protein identifications compared to Q Exactive HF mass spectrometer.

Food intake of breastfeeding mothers versus protein content of breastmilk in the coastal area of Bengkulu, Indonesia.

Maternal intake has a role in the composition of nutritional content, especially breast milk protein, but is rarely studied in fish producing areas (coastal). This study aimed to assess protein concentrations in human milk and evaluate their changes according to diet. The type of study was analytical observation, cross-sectional research design with a total sample of 50 from 313 breastfeeding mothers of infants 0-6 months domiciled at the Padang Serai Health Center. Dietary intake is collected using the food recall form 2 x 24 hours. Breast milk is collected in the morning as much as 30 ml using an electric breast pump. T-Test Independent analyzed the data. The results showed that as many as 10% of mothers had breast milk protein content above 0.9 g/100mL, and there was a relationship between maternal age, energy, and protein with breast milk protein content. Maternal diet, especially energy and protein, affects the protein content of breast milk. It is recommended that mothers improve their daily intake through nutritionally balanced and varied foods.

L'apport maternel a un rôle dans la composition du contenu nutritionnel, notamment en protéines du lait maternel, mais est rarement étudié dans les zones piscicoles (littorales). Cette étude visait à évaluer les concentrations de protéines dans le lait maternel et à évaluer leurs évolutions en fonction du régime alimentaire. Le type d'étude était une observation analytique, une conception de recherche transversale avec un échantillon total de 50 personnes provenant de 313 mères allaitantes de nourrissons de 0 à 6 mois domiciliées au centre de santé Padang Serai. L'apport alimentaire est collecté à l'aide du formulaire de rappel d'aliments 2 x 24 heures. Le lait maternel est collecté le matin jusqu'à 30 ml à l'aide d'un tire-lait électrique. T-Test Independent a analysé les données. Les résultats ont montré que jusqu'à 10 % des mères avaient une teneur en protéines du lait maternel supérieure à 0,9 g/100 ml, et qu'il existait une relation entre l'âge de la mère, son énergie et ses protéines et la teneur en protéines du lait maternel. L'alimentation maternelle, notamment énergétique et protéique, affecte la teneur en protéines du lait maternel. Il est recommandé aux mamans d'améliorer leurs apports quotidiens grâce à une alimentation nutritionnellement équilibrée et variée.

Delineating Bovine Milk Derived Microvesicles from Exosomes Using Proteomics.

In the work presented herein, a simple serial-pelleting purification strategy combined with a mass spectrometry-based proteomics analysis was developed as a means of discerning differences in extracellular vesicle (EV) populations found in bovine milk samples. A sequence of ultracentrifugation speeds was used to generate changes in the abundances of EV populations, allowing for the identification of associated proteins. A metric was developed to determine the relative abundances of proteins in large EVs (>200 nm) and small EVs (<200 nm). Of the 476 proteins consistently found in this study, 340 are associated with vesicular components. Of these, 156 were heavily enriched in large EVs, 155 shared between large and small EVs, and 29 heavily enriched in small EVs. Additionally, out of 68 proteins annotated as exosome proteins, 32 were enriched in large EVs, 27 shared between large and small EVs, 5 enriched in small EVs, and 7 were found to be nonvesicular contaminant proteins. The top correlated proteins in the small EV group were predominantly membrane-bound proteins, whereas the top correlated proteins in the large EV group were mostly cytosolic enzymes for molecular processing. This method provides a means of assessing the origins of vesicle components and provides new potential marker proteins within discrete vesicle populations.

Anxiolytic-like effects of milk proteins.

Milk varieties and specific proteins exhibit anxiolytic-like actions in mice and rats exposed to several tests, the most prominent being the elevated plus-maze. Administrations of αs1-casein, its 91-100 (α-casozepine), 91-97, 91-93, and 91-92 fragments, the 60-69 fragment of ß-casein, lactoferrin, ß-lactotensin, wheylin-1, wheylin-2, and α-lactalbumin have been reported to increase open arm exploration relative to enclosed arm exploration. Anxiolytic-like actions have also been described for 91-93 and 91-92 fragments of αs1-casein, wheylin-1, α-lactalbumin, and lactoferrin in the open-field. Some effects appear to be mediated by the GABAA receptor complex, since antagonists mitigated the anxiolytic-like actions of αs1-casein, the 91-92 fragment of αs1-casein, and wheylin-1. Other neurotransmitters purported to affect such behaviors include 5HT, dopamine, and neurotensin. Further research is needed to identify their neuropharmacological actions.