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Human and animal milk contain a rich variety of milk proteins that meet the needs of their newborns. In total, 1263 skim milk proteins and 1754 MFGM proteins were identified in human milk and six types of animal milk, respectively. Both similarities and differences were observed among the species. Human milk contained more immunoglobulins involved in the adaptive immune response, playing a crucial role in mucosal defense in newborn babies. In contrast, ruminant milk contained higher amounts of antimicrobial proteins, which protect newborns from bacterial infections. The most dominant difference in MFGM proteins between human and animal milk was related to protein processing in the endoplasmic reticulum. Goat milk and camel milk were more similar to human milk in terms of skim milk and MFGM proteins compared to the other five types of animal milk. Moreover, immunoglobulins and complement and coagulation cascade proteins in goat milk were most similar to those in human milk. A higher content of immunoglobulin A was observed in donkey milk, which could be considered as a source of IgA in infant formula. These results provide more comprehensive and novel insights into protein variation among animal milk, which may support improving dairy products such as infant formula.

Cow milk (CM) is an important food source for humans, and food allergy caused by CM has attracted attention worldwide. To our knowledge, systematic studies about the effects of Lactobacillus paracasei, Lactobacillus plantarum, and Pediococcus pentosaceus on the IgE-/IgG-binding ability and nutritional properties of CM are very rare. In this study, L. paracasei, L. plantarum, and P. pentosaceus fermentation on the IgE-/IgG-binding ability was determined by Enzyme-Linked Immunosorbent Assay (ELISA), and the protein quality, amino acid profile, and color were systematically evaluated. The results showed that these LAB strains exhibited higher protein degradation ability, and the IgE reactivity reduction rate was 41.03%-60.00% and the IgG reduction rate was 29.86%-67.20%, respectively. Additionally, the nutritional value was improved obviously, and the color was altered significantly, which was conductive to develop dairy products. These findings provided a theoretical foundation for the development of hypoallergenic dairy products. PRACTICAL APPLICATION: In this study, L. paracasei, L. plantarum and P. pentosaceus could be considered as good potential candidates for solving cow milk allergy owing to their decreased IgE/IgG binding ability andimproved nutritional and sensory properties, which provide a promising strategy to develop hypoallergenic dairy products., (© 2024 Institute of Food Technologists.)

Purpose: Bovine mastitis poses a significant economic burden on the dairy industry worldwide. This pioneering proteomic study conducted a comparative profiling of milk somatic cell (SC) proteins contributing to mammary immune defense during subclinical and clinical mastitis (CM) in Sahiwal (Bos indicus) cows., Experimental Design: Based on California mastitis test (CMT) scores, milk SC counts, differential leukocyte counts (DLCs), and bacteriological culture results, quarter milk SC samples were categorized into healthy (H), subclinical mastitis (SCM), and CM groups. Comparative proteome profiling of milk SCs was done using a label-free liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) proteomic approach., Results: The identified upregulated proteins in mastitis groups such as Vanin 2, Thioredoxin reductase-like selenoprotein T, Ceramidase, Lymphocyte antigen 75, Misshapen-like kinase 1 (MINK1), Thrombospondin 1, Macrophage scavenger receptor 1, Leupaxin, and Lipoamide acyltransferase, involved in immune responses. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed immune functions and pathways like antigen processing, complement cascades, extracellular matrix receptor interaction, efferocytosis, leukocyte migration, chemokine, peroxisome proliferator-activated receptors (PPARs), and transforming growth factor (TGF)-beta signaling., Conclusions and Clinical Relevance: These findings provide essential information on proteomic profiling in milk SCs and contribute valuable insights into immune-related proteins regulated during mastitis in dairy cows. Further, validated proteins (Vanin 2, MINK1, and Thrombospondin 1) offer potential inflammatory biomarkers for early mastitis detection in dairy cows., (© 2024 Wiley‐VCH GmbH.)

The gelation of milk proteins can be achieved by various means, enabling the development of diverse products. In this study, heat-set milk protein gels (15 % protein) of diverse textures were made by pH modulation and two gels were selected for dynamic in vitro gastric digestion: a spoonable soft gel (SG, pH 6.55' G' of ∼100 Pa) and a sliceable firm gel (FG, pH 5.65; G' of ∼7000 Pa). The two gels displayed markedly different structural changes and digestion kinetics during gastric digestion. The SG underwent substantial structural compaction during the first 120 min of gastric digestion into a denser and firmer gastric chyme (26.3 % crude protein, G* of ∼8500 Pa) than the chyme of the FG (15.7 % crude protein, G* of ∼3000 Pa). These contrasting intragastric structural changes of the gels reversed their original textural differences, which led to slower digestion and gastric emptying of proteins from the SG compared with the FG. The different intragastric pH profiles during the digestion of the two gels likely played a key role by modulating the proteolytic activity and specificity (to κ-casein) of pepsin. Preferential early cleavage of κ-casein in SG stimulated coagulation and compaction of solid chyme, whereas rapid hydrolysis of α S - and β-caseins in the FG weakened coagulation. This study provided new insights into controlling the structural development of dairy-based foods during gastric digestion and modulating digestion kinetics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The main objective of this study was to assess the ability of the NEar Real Digestive Tract (NERDT), a computer-controlled biomimetic in vitro digestion system that considers the biomechanics of the stomach, to reproduce physiologically relevant features of skimmed milk gastric digestion. A second objective was to evaluate the influence of pepsin on the gastric coagulation and emptying of milk proteins from experiments performed with and without pepsin. A mass balance model over the stomach, assuming a perfectly stirred reactor behaviour, has been developed. The results show that the NERDT can adequately reproduce the targeted kinetics of gastric acidification and emptying, with a sieving effect that naturally leads to a delayed emptying of caseins. Milk coagulated earlier and more chyme was emptied towards the end of the experiments in the presence of pepsin than without, hence illustrating the key influence of pepsin on the gastric coagulation of caseins and subsequent hydrolysis and emptying of dairy particles. Overall, this study shows that the NERDT can be adequately controlled to achieve desired gastric digestion conditions, and appears to be a very useful tool to further improve the knowledge of the gastric digestion behaviour of complex foods such as milk., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Milk serves as an important dietary source of bioactive peptides, offering notable benefits to individuals. Among the antioxidant short peptides (di- and tripeptides) generated from gastrointestinal digestion are characterized by enhanced bioavailability and bioaccessibility, while assessing them individually presents a labor-intensive and expensive challenge. Based on 4 distinct types of AA descriptors (physicochemical, 3-dimensional structural, quantum, and topological attributes) and genetic algorithms for feature selection, 1 and 4 machine learning-predicted models separately for di- and tripeptides with 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt radical scavenging capacity exhibited excellent fitting and prediction ability with random forest regression as machine learning algorithm. Intriguingly, the electronic properties of N-terminal AA were considered as only factor affecting the antioxidant capacity of dipeptides containing both tyrosine and tryptophan. Four peptides from the potential di- and tripeptides exhibited highly predicted values by the constructed predicted models. Subsequently, a total of 45 dipeptides and 52 tripeptides were screened by a customized workflow in goat milk during in vitro simulated digestion. In addition to 5 known antioxidant dipeptides, 9 peptides were quantified during digestion, exhibiting concentrations ranging from 0.04 to 1.78 mg L -1 . Particularly noteworthy was the promising in vivo functionality of antioxidant dipeptides with N-terminal tyrosine, supported by in silico assays. Overall, this investigation explored crucial molecular properties influencing antioxidant short peptides and high-throughput screening potential peptides with antioxidant activity from goat milk aided by machine learning, thereby facilitating the discovery of novel functional peptides from milk-derived proteins and paving the way for understanding their metabolites during digestion., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Regarding the limited information on species protein differences between sheep, goat, and cow milk, we analyzed the differentially expressed proteins in sheep, goat, and cow milk and their functional differences using label-free proteomics technology to identify potential biomarkers. In all, 770 proteins and 2,914 peptide segments were identified. Statistical analysis showed significant differences in the relative abundances of the 74 proteins among sheep, goat, and cow milk. CSN3 and LALBA can be used as potential biomarkers for goat milk, XDH can be used as a potential biomarker for cow milk, and CTSB and BPIFB1 can be used as potential biomarkers for sheep milk. Functional analysis using Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes showed that these significantly different proteins were enriched by different pathways, including thyroid hormone synthesis and glycerol phospholipid metabolism. The data revealed differences in the amounts and physiological functions of the milk proteins of different species, which may provide an important basis for research on the nutritional composition of dairy products and adulteration identification technology., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Intracellular AA regulate milk protein synthesis within the mammary gland by modifying mammary plasma flow (MPF) and AA transporter activity. Amino acid transporters catalyze translocation using Na + gradient, substrate gradient (uniporters), and exchange mechanisms; further, they exhibit specificity for individual AA or groups of AA with similar side-chain properties within each transport system. Nonessential AA are actively transported through Na + -dependent transporters and, thus, are often used as intracellular currencies for EAA transport through exchange transporters. Therefore, it was hypothesized that individual EAA supplementation would compete with other EAA for shared transporters, and supplementation with Ala, Gln, and Gly would stimulate EAA transport through exchange transporters. Ten primiparous lactating dairy cows were divided into 2 groups based on milk production and were randomly assigned to treatment sequences within 2 balanced 5 × 5 Latin squares by group. Period length was 14 d. Treatments were 9-d jugular infusions of (1) saline; (2) 34.5 g of Val per day; (3) a ratio of 32.7 g of Ala to 40 g of Gln to 26.7 g of Gly per day (AQG); (4) 43 g of Lys per day; or (5) 33.5 g of Ile per day. All cows were fed a common base diet formulated to contain 15.0% CP. Infusions of Ile, Lys, or AQG did not affect milk protein or milk production; however, Val infusion decreased both. The effects of Val infusion on milk protein production appeared to be partially driven by decreased DMI. The decline in milk protein percentage indicated that milk lactose production was also affected. Additionally, Val infusion increased MPF efficiency (MPF/milk; L/L) by approximately 44%. Infusion of Val tended to decrease or decreased mammary net uptakes of Lys, Leu, Met, and total AA. Infusion of Ile tended to increase its mammary net uptakes but did not affect any other AA. Infusions of Lys and AQG did not affect any mammary net uptakes. Infusion of Val tended to decrease Phe and total NEAA mammary clearance rates. Infusion with AQG stimulated Tyr clearance rates and tended to decline system N mammary clearance rates. Ratios of branched-chain AA mammary uptake to milk protein output (U:O) did not differ from 1 for Val-infused cows, which indicated that little intramammary catabolism was occurring. Additionally, the average NEAA U:O in response to all treatments except Val was 0.70, but Val-infused cows had NEAA U:O that averaged 0.09, indicating increased synthesis within the glands. The effects of Val on mammary net clearance rates of multiple EAA support the incorporation of AA limitations in ration optimizers to prevent AA imbalances. It is possible that oversupplementation of EAA other than Val may also decrease DMI and mammary activity. Identifying efficiency apexes for each of the EAA will allow more precise diet formulation and supplementation, leading to improved production efficiency., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Successful embryo implantation relies on synchronized dialog between the embryo and endometrium, and the role of extracellular vesicles (EVs) in facilitating this cross-talk has been recently established. In our previous study, milk fat globule-EGF factor 8 protein (MFGE8) was identified as increasing in receptive endometrial epithelial cells (EECs) in response to trophoblastic EVs. However, the dynamics of MFGE8 protein in this context are not completely understood. Therefore, we examined its expression and secretion in EECs exposed to estrogen, progesterone, and trophoblastic EVs to gain deeper insights into its potential as an indicator of EV-mediated embryo-maternal dialogue. Our findings revealed that MFGE8 secretion is sensitive to estrogen and progesterone, and that trophoblastic EVs stimulate their release in both receptive and non-receptive EECs. Furthermore, trophoblast EV function was dose and time-dependent. Notably, the secretion of MFGE8 increased within a short timeframe of 30 min after addition of EVs, suggesting the possibility of rapid processes such as binding, fusion or internalization of trophoblastic EVs within EECs. Interestingly, MFGE8 released from EECs was associated with EVs, suggesting increased EV secretion from EECs in response to embryonic signals. In conclusion, increased MFGE8 secretion in this embryo implantation model can serve as an indicator of EV-mediated embryo-maternal dialogue., (© 2024. The Author(s).)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The impact of nutritional modification to increase functional polyunsaturated fatty acids (PUFA), such as n-3 and n-6 fatty acids (FA) or conjugated linoleic acid (CLA), on milk proteome profile during early lactation remains largely unknown. We used an untargeted proteomics approach to investigate the impact of lactation day and PUFA supplementation on the proteome signature in skimmed milk over the course of early lactation. Sixteen Holstein dairy cows received abomasal infusion of saturated FA (CTRL) or a mixture of essential FA and CLA (EFA + CLA group) from - 63 to + 63 days relative to parturition. Using quantitative proteomics, 479 unique proteins were identified in skimmed milk at days 1, 28, and 63 postpartum. The top discriminating proteins between transition milk (day 1) and mature milk (days 28 and 63), including members of complements (i.e. C2 and C5), growth factor (TGFB2), lipoproteins (i.e. APOE and APOD), and chaperones (i.e. ST13 and CLU), are associated with calves' immune system and gut development. The EFA + CLA supplementation moderately affected a few proteins associated with regulating mammary glands' lipogenesis through the (re)assembly of lipoprotein particles, possibly under the PPAR signaling pathway. Collectively, skimmed milk proteome is dynamically regulated initially by cow's metabolic and physiological changes and to a lesser extent by nutritional PUFA modifications., (© 2024. The Author(s).)

Mare milk has a unique protein composition that makes it a preferred option for adult and infant nutrition. Several functional properties have been attributed to this milk, but with little current evidence. In fact, knowledge on mare milk composition is still limited. In particular, studies addressing the performance of mare milk proteins during human gastrointestinal digestion are scarce, which limits the understanding of mare milk nutritional quality and functionality. For this reason, the present study describes the digestibility of mare milk proteins and the release of peptides as affected by management and lactation stage, factors which are known to affect milk composition. Mare milk samples from 3 different farms collected during 6 mo of lactation (n = 54) were subjected to a static in vitro gastrointestinal model to measure peptide release and protein digestibility. In the present study, a detailed description of protein and individual amino acid behavior during the digestion process was given. For the first time, digestion of the 2 equine β-lactoglobulin isoforms (I and II) was described individually. In addition, it was found that lactation stage and management system can significantly affect protein digestibility and peptide release during gastrointestinal digestion of mare milk. Presumably, differences in the composition of mare milk influence the protein structure and enzyme accessibility, which might have an impact on digestion behavior. Although no specific bioactive peptides were identified, several precursors of previously described bioactive peptides were found. These findings could support the idea of mare milk as a food with added value., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Commercial dairy production occurs in a complex management environment, but increasingly, the dairy manager is expected to provide detailed reporting of productivity and environmental outcomes, for which conventional research methods double-blind crossover or case:control trials are inappropriate. This paper demonstrates the development of a milk protein production monitoring tool using a temporal (baseline) control in longitudinal, census-type investigations of modulation of system performance in response to factor change. It utilises farm-derived current and historical data, and contrasts seasonal responses with those achieved on neighbouring farms in a 2 × 2 contingency table. The approach is then shown to be useful in assessing the effect of two approaches to moderating milk urea concentration. Firstly, milk urea content can be monitored as it falls due to reduced feed protein content, and this fall can be arrested when milk protein content starts to decline relative to the value expected for the herd at any lactation stage. Secondly, by providing a dietary intervention aimed at increasing the availability of metabolic energy in the last month before calving, udder development can be augmented, leading to greater protein secretion capacity, meaning greater utilisation of circulating amino acids, and thus more limited substrate for urea synthesis. Thus, the changing impact of differing nutrition practices on dairy herd nitrogen excretion to environment can be followed with daily precision. In principle this approach can provide useful insights into a wide range of practical management interventions., Competing Interests: Graeme Coles is a director of Knewe Biosystems Ltd, the beneficial owner of Rich Technology Solutions Ltd. Jacqueline Rowarth is a farmer-elected director of Dairy NZ and of Ravendown Ltd. None of these entities have had any input into this submission., (©2024 Coles and Rowarth.)

MicroRNA (miRNA) is a type of endogenous non-coding small RNA, which is abundant in living organisms. miRNAs play an important role in regulating gene expression and myriad cellular processes by binding to target messenger RNAs through complementary base pairing, and cross-species regulation mammalian cells by plant-derived xeno-miRNAs has been described. Here, we examined the miRNA species in two alfalfa (Medicago sativa, lucerne) cultivars commonly grown in Ningxia, China: cv. Zhongmu 1 and cv. Xinyan 52. Both cultivars have good salt and drought resistance. We found that the miRNA profiles were similar between the cultivars, with a slightly higher number of miRNAs present in the newer cv. Xinyan 52, which may contribute to its improved salt and drought tolerance. miRNAs were stable during drying, and some miRNAs were increased in dry versus fresh alfalfa, suggesting some miRNAs may be upregulated during drying. Alfalfa-derived miRNAs could be detected in exosomes from serum and whey collected from dairy cows, confirming the ability of the exogenous miRNAs (xeno-miRNAs) to enter the circulation and reach the mammary epithelium. In vitro studies confirmed that overexpression of mtr-miR156a could downregulate expression of Phosphatase 2 Regulatory Subunit B'gamma ( PPP2R5D) and Phosphoinositide-3-kinase Regulatory Subunit 2 (PIK3R2). Overexpression of mtr-miR156a also modulated PI3K-AKT-mTOR signaling as well as the casein content of milk produced by bovine mammary epithelial cells. Based on the known roles of PPP2R5D and PIK3R2 in regulating the PI3K-AKT-mTOR pathway as well as the effect of PI3K-AKT-mTOR on milk protein content, our findings implicate alfalfa-derived miR156a as a new cross-species regulator of milk quality in dairy cows., (© 2024. The Author(s).)

Background: Goat milk is gaining popularity as a superior alternative to bovine milk due to its closer resemblance to human milk. Understanding the molecular processes underlying lactation is crucial for improving milk quality and production in goats. However, the genetic mechanisms governing lactation in goats, particularly in indigenous breeds like the Jakhrana, remain largely unexplored., Results: In this study, we performed a comprehensive transcriptomic analysis of Jakhrana goat mammary glands during early and late lactation stages. We isolated milk somatic cells and conducted RNA sequencing, followed by transcript quantification and mapping against the ARS1.2 Capra hircus reference assembly. Our analysis identified differentially expressed genes (DEGs) and commonly expressed genes (CEGs) across the lactation phases. Early lactation showed enrichment of genes encoding antimicrobial peptides and lubrication proteins, while late lactation exhibited heightened expression of genes encoding major milk proteins. Additionally, DEG analysis revealed upregulation of pivotal genes, such as the ABC transporter gene MRP4, implicated in modulating milk composition and quality., Conclusion: Our findings provide insights into the genetic mechanisms underlying lactation dynamics in the Jakhrana goat. Understanding these mechanisms could help in improving milk production and quality in goats, benefiting both the dairy industry and consumers., (© 2024. The Author(s).)

Climate change with increasing ambient temperatures negatively influences the biology of dairy cows and their milk production in the mammary gland (MG). This study aimed to elucidate the MG proteome, differences in milk composition, and ruminal short-chain fatty acid concentrations of dairy cows experiencing 7 days of heat stress [HS, 28 °C, temperature humidity index (THI) = 76], pair-feeding (PF), or ad libitum feeding (CON) at thermoneutrality (16 °C, THI = 60). Ruminal acetate, acetate/propionate ratio, and milk urea concentrations were greater, whereas milk protein and lactose were lower in HS than in control cows. Proteome analysis revealed an induced bacterial invasion of epithelial cells, leukocyte transendothelial migration, reduction of the pyruvate and carbon metabolism, and platelet activation in the MG of HS compared to CON or PF cows. These results highlight adaptive metabolic and immune responses to mitigate the negative effects of ambient heat in the MG.

Selenomethionine (SeMet) is an important nutrient, but its role in milk synthesis and the GPCR related to SeMet sensing is still largely unknown. Here, we determined the dose-dependent role of SeMet on milk protein and fat synthesis and proliferation of mammary epithelial cells (MECs), and we also uncovered the GPCR-mediating SeMet function. At 24 h postdelivery, lactating mother mice were fed a maintenance diet supplemented with 0, 5, 10, 20, 40, and 80 mg/kg SeMet, and the feeding process lasted for 18 days. The 10 mg/kg group had the best increase in milk production, weight gain of offspring mice, and mammary gland weight and acinar size, whereas a higher concentration of SeMet gradually decreased the weight gain of the offspring mice and showed toxic effects. Transcriptome sequencing was performed to find the differentially expressed genes (DEGs) between the mammary gland tissues of mother mice in the 10 mg/kg SeMet treatment group and the control group. A total of 258 DEGs were screened out, including 82 highly expressed genes including GPR37 and 176 lowly expressed genes. SeMet increased milk protein and fat synthesis in HC11 cells and cell proliferation, mTOR and S6K1 phosphorylation, and expression of GPR37 in a dose-dependent manner. GPR37 knockdown decreased milk protein and fat synthesis in HC11 cells and cell proliferation and blocked SeMet stimulation on mTOR and S6K1 phosphorylation. Taken together, our data demonstrate that SeMet can promote milk protein and fat synthesis and proliferation of MECs and functions through the GPR37-mTOR-S6K1 signaling pathway.

Liriodendrin is a lignan compound that is involved in a wide variety of physiological functions, however it is unknown whether liriodendrin plays an important role in milk production in the mammary glands. In this study, we explored the role and molecular mechanism of Liriodendrin in milk synthesis of mammary epithelial cells (MECs). Bovine MECs were treated with liriodendrin (0, 0.45, 0.9, 1.35, 1.8, and 2.25 mM) for 24 h. Liriodendrin dose-dependently increased cell number, cell cycle transition, and milk protein synthesis, as well as Cyclin D1 and mTOR phosphorylation, with the maximal effects observed at a dose of 1.35 mM. Liriodendrin increased the expression of DDX18, which mediated liriodendrin stimulation of Cyclin D1 and mTOR mRNA expression. PI3K inhibition and DDX18 knockdown experiments further confirmed that liriodendrin regulates the mRNA expression of Cyclin D1 and mTOR via the PI3K-DDX18 signaling. Mouse feeding experiment showed that liriodendrin dose-dependently promotes β-casein and DDX18 expression in mouse mammary gland. In this study, DDX18 was found to be a novel positive regulator that plays a role in cell proliferation and synthesis of milk protein. These findings reveal that liriodendrin stimulates proliferation and milk protein synthesis of MECs via the PI3K-DDX18 signaling., (© 2024. The Society for In Vitro Biology.)

Myocardial Infarction (MI) is a leading cause of death worldwide. Metabolic modulation is a promising therapeutic approach to prevent adverse remodeling after MI. However, whether material-derived cues can treat MI through metabolic regulation is mainly unexplored. Herein, a Cu 2+ loaded casein microgel (CuCMG) aiming to rescue the pathological intramyocardial metabolism for MI amelioration is developed. Cu 2+ is an important ion factor involved in metabolic pathways, and intracardiac copper drain is observed after MI. It is thus speculated that intramyocardial supplementation of Cu 2+ can rescue myocardial metabolism. Casein, a milk-derived protein, is screened out as Cu 2+ carrier through molecular-docking based on Cu 2+ loading capacity and accessibility. CuCMGs notably attenuate MI-induced cardiac dysfunction and maladaptive remodeling, accompanied by increased angiogenesis. The results from unbiased transcriptome profiling and oxidative phosphorylation analyses support the hypothesis that CuCMG prominently rescued the metabolic homeostasis of myocardium after MI. These findings enhance the understanding of the design and application of metabolic-modulating biomaterials for ischemic cardiomyopathy therapy., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Information regarding protein expression and phosphorylation modifications in the bovine milk fat globule membrane is scarce, particularly throughout various lactation periods. This study employed a complete proteome and phosphoproteome between bovine colostrum and mature milk. A total of 11 proteins were seen in both protein expression and phosphorylation levels. There were 400 proteins identified in only protein expression, and 104 phosphoproteins identified in only phosphorylation levels. A total of 232 significant protein characteristics were identified within the proteome and significant phosphorylation sites within 86 phosphoproteins of the phosphoproteome. Biological activities and pathways primarily exhibited associations with the immune system. Simultaneously, a comprehensive analysis of proteins and phosphorylation sites using a multi-omics approach. Hence, the data we have obtained has the potential to expand our understanding of how the bovine milk fat globule membrane might be utilized as a beneficial component in dairy products., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The role of the Maillard reaction and the accumulation of non-enzymatic glycation compounds in human milk have been scarcely considered. In this study, we investigated the proteins most susceptible to glycation, the identity of the corresponding modified residues and the quantitative relationship between protein-bound and free glycation compounds in raw human milk and, for comparison, in minimally processed infant formula and pasteurized bovine milk. In human milk, total protein-bound lysine modifications were up to 10% of the counterparts in infant formula, while Nε-carboxymethyllysine reached up to 27% of the concentration in the other two products. We demonstrated that the concentration of free pyrraline and methylglyoxal-hydroimidazolone were of the same order of magnitude in the three milk types. Our results delineate how the occurrence of some glycation compounds in human milk can be an unavoidable part of the breastfeeding and not an exclusive attribute of infant formulas and pasteurized bovine milk., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships, which may be considered as potential competing interests: Joost W. Gouw and Bernd Stahl reports a relationship with Danone Research & Innovation that includes: employment. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Fortification of human milk (HM) is often necessary to meet the nutritional requirements of preterm infants. The present experiment aimed to establish whether the supplementation of HM with either an experimental donkey milk-derived fortifier containing whole donkey milk proteins, or with a commercial bovine milk-derived fortifier containing hydrolyzed bovine whey proteins, affects peptide release differently during digestion. The experiment was conducted using an in vitro dynamic system designed to simulate the preterm infant's digestion followed by digesta analysis by means of LC-MS-MS. The different fortifiers did not appear to influence the cumulative intensity of HM peptides. Fortification had a differential impact on the release of either donkey or bovine bioactive peptides. Donkey milk peptides showed antioxidant/ACE inhibitory activities, while bovine peptides showed opioid, dipeptil- and propyl endo- peptidase inhibitory and antimicrobial activity. A slight delay in peptide release from human lactoferrin and α-lactalbumin was observed when HM was supplemented with donkey milk-derived fortifier., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Laura Cavallarin has patent #Food Composition - EP 3057450 B1 issued to n.a. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

High-pressure processing (HPP) of donor human milk (DM) minimally impacts the concentration and bioactivity of some important bioactive proteins including lactoferrin, and bile salt-stimulated lipase (BSSL) compared to Holder pasteurization (HoP), yet the impact of HPP and subsequent digestion on the full array of proteins detectable by proteomics remains unclear. We investigated how HPP impacts undigested proteins in DM post-processing and across digestion by proteomic analysis. Each pool of milk (n = 3) remained raw, or was treated by HPP (500 MPa, 10 min) or HoP (62.5 °C, 30 min), and underwent dynamic in vitro digestion simulating the preterm infant. In the meal, major proteins were minimally changed post-processing. HPP-treated milk proteins better resisted proximal digestion (except for immunoglobulins, jejunum 180 min) and the extent of undigested proteins after gastric digestion of major proteins in HPP-treated milk was more similar to raw (e.g., BSSL, lactoferrin, macrophage-receptor-1, CD14, complement-c3/c4, xanthine dehydrogenase) than HoP., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Hypoxia stress has been demonstrated to impede animal embryonic development, spermatogenesis, and lactation, leading to decreased animal production performance. However, the impact of hypoxia-induced activation of hypoxia inducible factor-1 (HIF-1) signaling on milk protein and fat synthesis remains unclear. L-leucine, a branched-chain amino acid, is known to modulate milk protein and fat synthesis. Therefore, our study aimed to evaluate the effect of L-leucine on milk protein and fat synthesis under hypoxic conditions and shed light on the molecular mechanism using an in vitro model. The results indicated that hypoxia treatment significantly decreased the synthesis of α-casein and β-casein, as well as inhibited factors related to milk fat synthesis in bovine mammary epithelial cells (MAC-T). Additionally, hypoxia stress suppressed the activities of the mammalian target of rapamycin (mTOR) and protein kinase B (AKT). Interfering with HIF-1α significantly reversed the expression of AKT, mTOR and factors related to milk synthesis. Importantly, supplementation with L-leucine activated AKT/mTOR signaling, thereby enhancing milk protein and fat synthesis in MAC-T cells to some extent. In conclusion, these findings suggest that HIF-1 signaling plays an important role in milk synthesis and that L-leucine may stimulate the synthesis of milk protein and fat by activating the AKT/mTOR signaling pathway under hypoxic conditions, making it a potential additive for promoting milk synthesis inhibited by hypoxia., Competing Interests: Declaration of competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

This study was conducted to investigate whether methionyl-tRNA synthetase (MetRS) is a mediator of methionine (Met)-induced crop milk protein synthesis via the janus kinase 2 (JAK2)/signal transducer and activator of transcription 5 (STAT5) signalling pathway in breeding pigeons. In Experiment 1, a total of 216 pairs of breeding pigeons were divided into three groups (control, Met-deficient, and Met-rescue groups). In Experiments 2 and 3, forty pairs of breeding pigeons from each experiment were allocated into four groups. The second experiment included a control group and three MetRS inhibitor (REP8839) groups. The third experiment included a Met-deficient group, Met-sufficient group, REP8839 + Met-deficient group and REP8839 + Met-sufficient group. Experiment 1 showed that Met supplementation increased crop development, crop milk protein synthesis, the protein expression of MetRS and JAK2/STAT5 signalling pathway, and improved squab growth. Experiment 2 showed that crop development, crop milk protein synthesis and the protein expression of MetRS and the JAK2/STAT5 signalling pathway were decreased, and squab growth was inhibited by the injection of 1·0 mg/kg body weight REP8839, which was the selected dose for the third experiment. Experiment 3 showed that Met supplementation increased crop development, crop milk protein synthesis and the expression of MetRS and JAK2/STAT5 signalling pathway and rescued squab growth after the injection of REP8839. Moreover, the co-immunoprecipitation results showed that there was an interaction between MetRS and JAK2. Taken together, these findings indicate that MetRS mediates Met-induced crop milk protein synthesis via the JAK2/STAT5 signalling pathway, resulting in improved squab growth in breeding pigeons.

Many improvements have been made to bring infant formula (IF) closer to human milk (HM) regarding its nutritional and biological properties. Nevertheless, the protein components of HM and IF are still different, which may affect their digestibility. This study aimed to evaluate and compare the protein digestibility of HM and IF using the infant INFOGEST digestion method. Pooled HM and a commercial IF were subjected to the infant INFOGEST method, which simulates the physiological digestion conditions of infants, with multiple directions, i.e. the curd state, gel images of SDS-PAGE, molecular weight distribution, free amino acid concentrations and in vitro protein digestion rate. HM underwent proteolysis before digestion and tended to have a higher protein digestion rate with finer curds during gastric digestion, than the IF. However, multifaceted analyses showed that the protein digestibility of HM and IF was not significantly different after gastrointestinal digestion. In conclusion, the infant INFOGEST method showed that the digestibility of HM and IF proteins differed to some extent before digestion and after gastric digestion, but not at the end of gastrointestinal digestion. The findings of this study will contribute to the refinement of IF with better protein digestibility in infant stomach.

Neuronal loss is a hallmark of stroke and other neurodegenerative diseases, and as such, neuronal loss caused by microglia has been thought to be a contributing factor to disease progression. Here, we show that microglia indeed contribute significantly to neuronal loss in a mouse model of stroke, but this microglial-dependent process of neuronal clearance specifically targets stressed and degenerating neurons in the ischemic cortical region and not healthy non-ischemic neurons. Nonspecific stimulation of microglia decreased the density of neurons in the ischemic cortical region, whereas specific inhibition of MFG-E8 signaling, which is required for microglial phagocytosis of neurons, had the opposite effect. In both scenarios, the effects were microglia specific, as the same treatments had no effect in mice whose microglia were depleted prior to stroke. Finally, even though the inhibition of MFG-E8 signaling increased neuronal density in the ischemic brain region, it substantially exacerbated the development of cortical infarction. In conclusion, microglia through MFG-E8 signaling contribute to the loss of ischemic neurons and, in doing so, minimize the development of cortical infarction after stroke., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Wang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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 β-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., Competing Interests: Declaration of competing interest Tanguy Saviard, Linda Le Roux, and Raphaël Chacon are employees of Sill Entreprises. Other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Milk protein production is the largest draw on AA supplies for lactating dairy cattle. Prior NRC predictions of milk protein production have been absorbed protein (MP)-based and used a first-limiting nutrient concept to integrate the effects of energy and protein, which yielded poor accuracy and precision (root mean squared error [RMSE] >21%). Using a meta-data set gathered, various alternative equation forms considering MP, absorbed total EAA, absorbed individual EAA, and digested energy (DE) supplies as additive drivers of production were evaluated, and all were found to be superior in statistical performance to the first limitation approach (RMSE = 14%-15%). Inclusion of DE intake and a quadratic term for MP or absorbed EAA supplies were found to be necessary to achieve intercept estimates (nonproductive protein use) that were similar to the factorial estimates of the National Academies of Sciences, Engineering, and Medicine (2021). The partial linear slope for MP was found to be 0.409, which is consistent with the observed slope bias of -0.34 g/g when a slope of 0.67 was used for MP efficiency in a first-limiting nutrient system. Replacement of MP with the supplies of individual absorbed EAA expressed in grams per day and a common quadratic across the EAA resulted in unbiased predictions with improved statistical performance as compared with MP-based models. Based on Akaike's information criterion and biological consistency, the best equations included absorbed His, Ile, Lys, Met, Thr, the NEAA, and individual DE intakes from fatty acids, NDF, residual OM, and starch. Several also contained a term for absorbed Leu. These equations generally had RMSE of 14.3% and a concordance correlation of 0.76. Based on the common quadratic and individual linear terms, milk protein response plateaus were predicted at approximately 320 g/d of absorbed His, Ile, and Lys; 395 g/d of absorbed Thr; 550 g/d of absorbed Met; and 70 g/d of absorbed Leu. Therefore, responses to each except Leu are almost linear throughout the normal in vivo range. De-aggregation of the quadratic term and parsing to individual absorbed EAA resulted in nonbiological estimates for several EAA indicating over-parameterization. Expression of the EAA as g/100 g total absorbed EAA or as ratios of DE intake and using linear and quadratic terms for each EAA resulted in similar statistical performance, but the solutions had identifiability problems and several nonbiological parameter estimates. The use of ratios also introduced nonlinearity in the independent variables which violates linear regression assumptions. Further screening of the global model using absorbed EAA expressed as grams per day with a common quadratic using an all-models approach, and exhaustive cross-evaluation indicated the parameter estimates for BW, all 4 DE terms, His, Ile, Lys, Met, and the common quadratic term were stable, whereas estimates for Leu and Thr were known with less certainty. Use of independent and additive terms and a quadratic expression in the equation results in variable efficiencies of conversion. The additivity also provides partial substitution among the nutrients. Both of these prevent establishment of fixed nutrient requirements in support of milk protein production., (© 2024, The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

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., (© 2024. The Author(s).)

Human breast milk promotes maturation of the infant gastrointestinal barrier, including the promotion of mucus production. In the quest to produce next generation infant milk formula (IMF), we have produced IMF by membrane filtration (MEM-IMF). With a higher quantity of native whey protein, MEM-IMF more closely mimics human breast milk than IMF produced using conventional heat treatment (HT-IMF). After a 4-week dietary intervention in young pigs, animals fed a MEM-IMF diet had a higher number of goblet cells, acidic mucus and mucin-2 in the jejunum compared to pigs fed HT-IMF (P < 0.05). In the duodenum, MEM-IMF fed pigs had increased trypsin activity in the gut lumen, increased mRNA transcript levels of claudin 1 in the mucosal scrapings and increased lactase activity in brush border membrane vesicles than those pigs fed HT-IMF (P < 0.05). In conclusion, MEM-IMF is superior to HT-IMF in the promotion of mucus production in the young gut., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Aim: Few studies investigate factors that might influence the content of expressed breastmilk. This study aims to investigate the influence of the intervals between breastmilk pumping and the time of the day on protein and fat concentration in breastmilk., Methods: Mothers of very preterm infants in a neonatal ward who expressed more than 400 mL per day were included. Expressed breastmilk was obtained from each mother over 30 h who were pumping at strictly planned and varying intervals: 2, 3, 4 and 6 h. All samples were analysed using infrared transmission spectroscopy., Results: Ten mothers participated at a median of 22 days postpartum. A total of 176 milk samples were analysed, and the average protein and fat concentrations in g/100 mL were 1.1 ± 0.23 and 4.2 ± 1.3, respectively. The time intervals between breast pumping sessions did not impact protein content, but fat content decreased by longer intervals (p < 0.01). The time of the day for milk pumping did not influence the protein or fat content., Conclusion: A single milk sample collected after any 2-6 h interval, at any time during the day, represents the protein content in the breastmilk, but not the fat content which decreased with longer intervals., (© 2024 The Authors. Acta Paediatrica published by John Wiley & Sons Ltd on behalf of Foundation Acta Paediatrica.)

Milk fat globule membrane proteins (MFGMP) in human milks have positive effects on infant's health. As gestational diabetes mellitus (GDM) causes variations in MFGMP, it is essential to understand the effects of GDMon MFGMP. This study aims to investigate and compare the MFGMP (>3 months postpartum) of GDM and non-GDM (NGDM) women using four-dimensional-data-independent-acquisition proteomics technology. Principal component analysis shows significant differences in the MFGMP of GDM and NGDM women. A total of 4747 MFGMP were identified in maturehuman milk of GDM and NGDM women. Among these proteins, 174 differentially expressed proteins (DEPs) were identified in MFGM of GDM and NGDM women. Albumin (FC = 7.96) and transthyretin (FC = 2.57) which are related to insulin resistance and involved in thyroid hormone synthesis, are significantly up-regulated in MFGMP of GDM mothers indicating insulin resistance, imbalance of glucose homeostasis and poor glucose metabolism might persist in postpartum period., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Bovine milk processing influences the structure of the curd formed during gastric digestion, which may alter gastric protein hydrolysis and impact amino acid (AA) release into the small intestine., Objectives: This study aimed to determine the influence of heat treatment and homogenization on the gastric protein digestion and AA emptying of bovine milk., Methods: Nine-wk-old pigs (n = 144) consumed either raw, pasteurized nonhomogenized (PNH), pasteurized homogenized (PH), or ultra-high-temperature homogenized (UHT) bovine milk for 10 d. On day 11, fasted pigs received the milk treatment (500 mL) before gastric contents were collected at 0, 20, 60, 120, 180, and 300 min postprandially. The apparent degree of gastric protein hydrolysis (based on the release of free amino groups), apparent gastric disappearance of individual proteins [based on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel band intensity], and the gastric emptying of digested protein and AA were determined., Results: During the first 60 min, the rate of apparent gastric protein hydrolysis was fastest in pigs fed UHT milk (0.29%/min compared with on average 0.07%/min in pigs fed raw, PNH, and PH milk). Differences in the apparent degree of gastric protein hydrolysis and emptying were reflected in the rate of digested protein entering the small intestine. The AA gastric emptying half-time was generally shorter in pigs fed PH and UHT milk than in pigs fed raw and PNH milk. For example, the gastric release of total essential AA was >2-fold faster (P < 0.01) in pigs fed PH or UHT milk than that in pigs fed raw or PNH milk (i.e., homogenized compared with nonhomogenized milk)., Conclusions: Heat treatment and homogenization increased the apparent gastric degree of protein hydrolysis and the release of digested protein into the small intestine. However, the rate of AA entering the small intestine was mainly increased by homogenization., (Copyright © 2024 American Society for Nutrition. Published by Elsevier Inc. All rights reserved.)

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.

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.

Objective: Intestinal fibrosis is considered an inevitable consequence of chronic IBD, leading to stricture formation and need for surgery. During the process of fibrogenesis, extracellular matrix (ECM) components critically regulate the function of mesenchymal cells. We characterised the composition and function of ECM in fibrostenosing Crohn's disease (CD) and control tissues., Design: Decellularised full-thickness intestinal tissue platforms were tested using three different protocols, and ECM composition in different tissue phenotypes was explored by proteomics and validated by quantitative PCR (qPCR) and immunohistochemistry. Primary human intestinal myofibroblasts (HIMFs) treated with milk fat globule-epidermal growth factor 8 (MFGE8) were evaluated regarding the mechanism of their antifibrotic response, and the action of MFGE8 was tested in two experimental intestinal fibrosis models., Results: We established and validated an optimal decellularisation protocol for intestinal IBD tissues. Matrisome analysis revealed elevated MFGE8 expression in CD strictured (CDs) tissue, which was confirmed at the mRNA and protein levels. Treatment with MFGE8 inhibited ECM production in normal control HIMF but not CDs HIMF. Next-generation sequencing uncovered functionally relevant integrin-mediated signalling pathways, and blockade of integrin αvβ5 and focal adhesion kinase rendered HIMF non-responsive to MFGE8. MFGE8 prevented and reversed experimental intestinal fibrosis in vitro and in vivo., Conclusion: MFGE8 displays antifibrotic effects, and its administration may represent a future approach for prevention of IBD-induced intestinal strictures., Competing Interests: Competing interests: The Cleveland Clinic receives funds on her behalf from Celgene, Morphic, Pfizer, UCB, GB004 and Helmsley. SDH was consultant to Shionogi, Takeda and Guidepoint and receives research funding from the Crohn’s and Colitis Foundation and the American Society of Colon and Rectal Surgeons. CF receives speaker fees from UCB, Genentech, Sandoz and Janssen, and he is the consultant of Athos Therapeutics, Inc. FR is the consultant of Agomab, Allergan, AbbVie, Boehringer-Ingelheim, Celgene, Cowen, Genentech, Gilead, Gossamer, Guidepoint, Helmsley, Index Pharma, Jansen, Koutif, Metacrine, Morphic, Pfizer, Pliant, Prometheus Biosciences, Receptos, RedX, Roche, Samsung, Takeda, Techlab, Thetis, UCB and 89Bio., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Animal-based agriculture and the production of protein-rich foods from animals, particularly from ruminants, are not sustainable and have serious climate effects. A new type of alternative proteins is now on the menu, namely animal proteins produced recombinantly by microbial fermentation. This new technology, precision fermentation, is projected to completely disrupt traditional animal-based agriculture. Certain milk and egg proteins along with specific meat substitute analog components produced by precision fermentation are already entering the market. This first wave of precision fermentation products targets the use of these proteins as protein additives, and several commercial players are already active in the field. The cost-efficiency requirements involve production titers above 50 g/L which are several orders of magnitude higher than those for pharmaceutical protein manufacture, making strain engineering, process optimization, and scale-up critical success factors. This new development within alternative proteins defines a new research direction integrating biotechnology, process engineering, and sustainable food protein production.

Dopamine D2 receptors (D2Rs) play crucial roles in regulating diverse physiological functions of the central nervous system and peripheral organs. D2Rs are also expressed in mammary glands. However, which cell types express D2Rs and whether they are involved in milk production remains unclear. The present findings revealed that D2Rs are expressed in the apical regions of the lateral membranes of mammary epithelial cells (MECs) in lactating mice. We also investigated the effects of the D2R agonist bromocriptine and/or antagonist domperidone on intracellular cAMP levels, milk protein production, and apoptosis in a lactation culture model of MECs that produce major milk components like lactating MECs in vivo. We found that bromocriptine decreased intracellular cAMP levels, whereas domperidone dose-dependently neutralized this effect. Bromocriptine also inhibited casein and lactoferrin production and suppressed activities of STAT5 and glucocorticoid receptors (GRs). Domperidone neutralized the inhibition of casein production as well as STAT5 and GR inactivation induced by bromocriptine. Furthermore, D2R activation by bromocriptine induced apoptosis and inactivated ERK, a signaling molecule responsible for promoting cell proliferation and survival. Domperidone attenuated ERK inactivation and apoptosis induced by bromocriptine. These findings suggest that D2Rs play regulatory roles in milk protein production and apoptosis in MECs., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Although the association of maternal milk production with developmental programming of offspring has been investigated, there is limited information available on the relationship of maternal milk components with productive and reproductive performance of the offspring. Therefore, the present study was conducted to analyze the association of maternal milk fat and protein percentage and milk fat to protein ratio with birth weight, survival, productive and reproductive performance and AMH concentration in the offspring. In study I, data of birth weight, milk yield and reproductive variables of offspring born to lactating dams (n = 14,582) and data associated with average maternal milk fat percentage (MFP), protein percentage (MPP) and fat to protein ratio (MFPR) during 305-day lactation were retrieved. Afterwards, offspring were classified in various categories of MFP, MPP and MFPR. In study II, blood samples (n = 339) were collected from offspring in various categories of MFP, MPP and MFPR for measurement of serum AMH. Maternal milk fat percentage was positively associated with birth weight and average percentage of milk fat (APMF) and protein (APMP) and milk fat to protein ratio (FPR) during the first lactation, but negatively associated with culling rate during nulliparity in the offspring (P < 0.05). Maternal milk protein percentage was positively associated with birth weight, APMF, APMP, FPR and culling rate, but negatively associated with milk yield and fertility in the offspring (P < 0.05). Maternal FPR was positively associated with APMF and FPR, but negatively associated with culling rate, APMP and fertility in the offspring (P < 0.05). However, concentration of AMH in the offspring was not associated with MFP, MPP and MFPR (P > 0.05). In conclusion, the present study revealed that maternal milk fat and protein percentage and their ratio were associated with birth weight, survival, production and reproduction of the offspring. Yet it was a preliminary research and further studies are required to elucidate the mechanisms underlying these associations., Competing Interests: Declaration of competing interest The authors have no conflict of interest to disclose., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Net energy for lactation (NE L ) and metabolizable protein (MP) are the 2 main nutritional forces that drive synthesis of milk components. This study investigated mammary-gland metabolism in dairy cows in response to variations in the supply of NE L and MP. Four Holstein dairy cows were randomly assigned to a 4 × 4 Latin square design, in which each experimental period consisted of 14 d of dietary treatment. The diets provided 2 levels of NE L (low energy, 25.0 Mcal/d vs. high energy, 32.5 Mcal/d) and 2 levels of MP (low protein, 1,266 g/d vs. high protein, 2,254 g/d of protein digestible in the intestine) in a 2 × 2 factorial arrangement. Performance and dry matter intake (DMI) were measured during the last 5 d of each period, and the mammary net balance was measured on d 13 by collecting 6 sets of blood samples from the left carotid artery and left mammary vein. Mammary plasma flow was measured according to the Fick principle for Phe and Tyr. The mammary net balance of carbon equaled the uptake of nutrients expressed as carbon minus the output of lactose, fatty acids (FA) synthesized in the mammary gland, AA of milk protein, and glycerol-3P from triglyceride on d 13. Milk, lactose, fat, and protein yields increased when NE L and MP supplies increased. However, increasing the NE L supply increased FA synthesis more than increasing the protein supply did. In addition, FA secretion increased more than lactose secretion when the NE L supply increased. Increasing the NE L supply increased the left half-udder uptake of all major energy-yielding nutrients by increasing mammary plasma flow. However, nutrient uptake increased more than milk output did, which in turn increased carbon dioxide output. This increase in nutrient oxidation by the mammary gland decreased the mammary efficiency of nutrients utilization when the NE L supply increased. Increasing MP supply tended to increase glucose uptake through mammary clearance and increased mammary AA uptake with no change in mammary plasma flow. In addition, the protein supply did not change the mammary uptake of acetate or β-hydroxybutyrate. The increase in milk-component secretions in response to either NE L or MP supplies occurred through different metabolic adaptations (increase in mammary plasma flow vs. clearances, respectively). These results suggest that the nutrient use by the mammary gland is highly flexible, which helps in maintaining milk and milk-component yields even with limiting nutrient supplies., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Bovine milk and meat factors (BMMFs) are plasmid-like DNA molecules isolated from bovine milk and serum, as well as the peritumor of colorectal cancer (CRC) patients. BMMFs have been proposed as zoonotic infectious agents and drivers of indirect carcinogenesis of CRC, inducing chronic tissue inflammation, radical formation and increased levels of DNA damage. Data on expression of BMMFs in large clinical cohorts to test an association with co-markers and clinical parameters were not previously available and were therefore assessed in this study. Tissue sections with paired tumor-adjacent mucosa and tumor tissues of CRC patients [individual cohorts and tissue microarrays (TMAs) (n = 246)], low-/high-grade dysplasia (LGD/HGD) and mucosa of healthy donors were used for immunohistochemical quantification of the expression of BMMF replication protein (Rep) and CD68/CD163 (macrophages) by co-immunofluorescence microscopy and immunohistochemical scoring (TMA). Rep was expressed in the tumor-adjacent mucosa of 99% of CRC patients (TMA), was histologically associated with CD68 + /CD163 + macrophages and was increased in CRC patients when compared to healthy controls. Tumor tissues showed only low stromal Rep expression. Rep was expressed in LGD and less in HGD but was strongly expressed in LGD/HGD-adjacent tissues. Albeit not reaching statistical significance, incidence curves for CRC-specific death were increased for higher Rep expression (TMA), with high tumor-adjacent Rep expression being linked to the highest incidence of death. BMMF Rep expression might represent a marker and early risk factor for CRC. The correlation between Rep and CD68 expression supports a previous hypothesis that BMMF-specific inflammatory regulations, including macrophages, are involved in the pathogenesis of CRC., (© 2023 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Milk protein content is an important index to evaluate the quality and nutrition of milk. Accumulating evidence suggests that microRNAs (miRNAs) play important roles in bovine lactation, but little is known regarding the cross-kingdom regulatory roles of plant-derived exogenous miRNAs (xeno-miRNAs) in milk protein synthesis, particularly the underlying molecular mechanisms. The purpose of this study was to explore the regulatory mechanism of alfalfa-derived xeno-miRNAs on proliferation and milk protein synthesis in bovine mammary epithelial cells (BMECs). Our previous study showed that alfalfa miR159a (mtr-miR159a, xeno-miR159a) was highly expressed in alfalfa, and the abundance of mtr-miR159a was significantly lower in serum and whey from high-protein-milk dairy cows compared with low-protein-milk dairy cows. In this study, mRNA expression was detected by real-time quantitative PCR (qRT-PCR), and casein content was evaluated by enzyme-linked immunosorbent assay (ELISA). Cell proliferation and apoptosis were detected using the cell counting kit 8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridine (EdU) staining, western blot, and flow cytometry. A dual-luciferase reporter assay was used to determine the regulation of Protein Tyrosine Phosphatase Receptor Type F (PTPRF) by xeno-miR159a. We found that xeno-miR159a overexpression inhibited proliferation of BMEC and promoted cell apoptosis. Besides, xeno-miR159a overexpression decreased β-casein abundance, and increased α-casein and κ-casein abundance in BMECs. Dual-luciferase reporter assay result confirmed that PTPRF is a target gene of xeno-miR159a. These results provide new insights into the mechanism by which alfalfa-derived miRNAs regulate BMECs proliferation and milk protein synthesis., (© 2024. The Author(s).)

Metabolic syndrome (MS) is defined by the outcome of interconnected metabolic factors that directly increase the prevalence of obesity and other metabolic diseases. Currently, obesity is considered one of the most relevant topics of discussion because an epidemic heave of the incidence of obesity in both developing and underdeveloped countries has been reached. According to the World Obesity Atlas 2023 report, 38% of the world population are presently either obese or overweight. One of the causes of obesity is an imbalance of energy intake and energy expenditure, where nutritional imbalance due to consumption of high-calorie fast foods play a pivotal role. The dynamic interactions among different risk factors of obesity are highly complex; however, the underpinnings of hyperglycemia and dyslipidemia for obesity incidence are recognized. Fast foods, primarily composed of soluble carbohydrates, non-nutritive artificial sweeteners, saturated fats, and complexes of macronutrients (protein-carbohydrate, starch-lipid, starch-lipid-protein) provide high metabolic calories. Several experimental studies have pointed out that dairy proteins and peptides may modulate the activities of risk factors of obesity. To justify the results precisely, peptides from dairy milk proteins were synthesized under in vitro conditions and their contributions to biomarkers of obesity were assessed. Comprehensive information about the impact of proteins and peptides from dairy milks on fast food-induced obesity is presented in this narrative review article.

The specific recognition of peripheral membrane-binding proteins for their target membranes is mediated by a complex constellation of various lipid contacts. Despite the inherent complexities of the heterogeneous protein-membrane interface, the binding dependence of such proteins is, surprisingly, often reliably described by simple models such as the Langmuir Adsorption Isotherm or the Hill equation. However, these models were not developed to describe associations with two-dimensional, highly concentrated heterogeneous ligands such as lipid membranes. In particular, these models fail to capture the dependence on the lipid composition, a significant determinant of binding that distinguishes target from non-target membranes. In this work, we present a model that describes the dependence of peripheral proteins on lipid composition through an analytic expression for their association. The resulting membrane-binding equation retains the features of these simple models but completely describes the binding dependence on multiple relevant variables in addition to the lipid composition, such as protein and vesicle concentration. Implicit in this lipid composition dependence is a new form of membrane-based cooperativity that significantly differs from traditional solution-based cooperativity. We introduce the Membrane-Hill number as a measure of this cooperativity and describe its unique properties. We illustrate the utility and interpretational power of our model by analyzing previously published data on two peripheral proteins that associate with phosphatidylserine-containing membranes: The transmembrane immunoglobulin and mucin domain-containing protein 3 (TIM3) that employs calcium in its association, and milk fat globulin epidermal growth factor VIII (MFG-E8) which is completely insensitive to calcium. We also provide binding equations for systems that exhibit more complexity in their membrane-binding., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Milk proteins produced by lactating cells isolated from bovine mammary tissue can offer a sustainable solution to the high protein demand of a global growing population. Serum is commonly added to culture systems to provide compounds necessary for optimal growth and function of the cells. However, in a cellular agricultural context, its usage is desired to be decreased. This study aims at examining the minimum level of fetal bovine serum (FBS) required for the growth and functionality of bovine mammary epithelial cells (MECs). The cells were isolated from dairy cows in early and mid-lactation and cultured in reduced concentrations of FBS (10%, 5%, 1.25%, and 0%). Real-time cell analysis showed a significant effect of lactation stage on growth rate and 5% FBS resulted in similar growth rate as 10% while 0% resulted in the lowest. The effect of reducing FBS on cell functionality was examined by studying the expressions of selected marker genes involved in milk protein and fat synthesis, following differentiation. The gene expressions were not affected by the level of FBS. A reduction of FBS in the culture system of MEC, at least down to 5%, does not assert any negative effect on the growth and expression levels of studied genes. As the first attempt in developing an in-vitro model for milk component production using MEC, our results demonstrate the potential of MEC to endure FBS-reduced conditions., (© 2024 International Federation of Cell Biology.)

The direction and magnitude of immune responses are critically affected when dead cells are disposed of. Milk fat globule-epidermal growth factor-factor 8 (MFG-E8) promotes the engulfment of apoptotic normal and cancerous cells without inducing inflammation. We have previously reported that a certain proportion of the cancer cells express abundant MFG-E8, and that such expression is associated with the shorter survival of patients with esophageal cancer who had received chemotherapy before surgery. However, the influence of tumor-derived and systemically existing MFG-E8 on antitumor immune responses has not yet been fully investigated. Herein, we showed that CTL-dependent antitumor immune responses were observed in mice with no or decreased levels of systemic MFG-E8, and that such responses were enhanced further with the administration of anti-PD-1 antibody. In mice with decreased levels of systemic MFG-E8, the dominance of regulatory T cells in tumor-infiltrating lymphocytes was inverted to CD8 + T cell dominance. MFG-E8 expression by tumor cells appears to affect antitumor immune responses only when the level of systemic MFG-E8 is lower than the physiological status. We have also demonstrated in the clinical setting that lower levels of plasma MFG-E8, but not MFG-E8 expression in tumor cells, before the treatment was associated with objective responses to anti-PD-1 therapy in patients with non-small cell lung cancer. These results suggest that systemic MFG-E8 plays a critical role during the immunological initiation process of antigen-presenting cells to increase tumor-specific CTLs. Regulation of the systemic level of MFG-E8 might induce efficient antitumor immune responses and enhance the potency of anti-PD-1 therapy., (© 2024 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Low crude protein (CP) diets might be fed to dairy cows without affecting productivity if the balance of absorbed AA were improved, which would decrease the environmental effect of dairy farms. The aim of this study was to investigate the effects of supplementing ruminally protected Lys (RPL) and Met (RPM) at 2 levels of dietary CP on nutrient intake, milk production, milk composition, milk N efficiency (MNE), and plasma concentrations of AA in lactating Holstein cows and to evaluate these effects against the predictions of the new NASEM (2021) model. Fifteen multiparous cows were used in a replicated 3 × 3 Latin square design with 21-d periods. The 3 treatments were (1) a high-protein (HP) basal diet containing 16.4% CP (metabolizable protein [MP] balance of -130 g/d; 95% of target values), (2) a medium-protein diet containing 15% CP plus RPL (60 g/cow per day) and RPM (25 g/cow per day; MPLM; MP balance of -314 g/d; 87% of target values), and (3) a low-protein diet containing 13.6% CP plus RPL (60 g/cow per day) and RPM (25 g/cow per day; LPLM; MP balance of -479 g/d; 80% of target values). Dry matter intake was less for cows fed MPLM and LPLM diets compared with those fed the HP diet. Compared with the HP diet, the intake of CP, neutral detergent fiber, acid detergent fiber, and organic matter, but not starch, was lower for cows fed MPLM and LPLM diets. Milk production and composition were not affected by MPLM or LPLM diets relative to the HP diet. Milk urea N concentrations were reduced for the MPLM and LPLM diets compared with the HP diet, indicating that providing a low-protein diet supplemented with rumen-protected AA led to greater N efficiency. There was no significant effect of treatment on plasma AA concentrations except for proline, which significantly increased for the MPLM treatment compared with the other 2 treatments. Overall, the results supported the concept that milk performance might be maintained when feeding lactating dairy cows with low CP diets if the absorbed AA balance is maintained through RPL and RPM feeding. Further investigations are needed to evaluate responses over a longer time period with consideration of all AA rather than on the more aggregated MP and the ratio between Lys and Met., (The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Supplementing a diet with rumen-protected amino acids (AAs) is a common feeding strategy for efficient production. For a cost-effective use of rumen-protected AA, the accurate bioavailability of rumen-protected amino acids should be known and their metabolism after absorption needs to be well understood. The current study determined the bioavailability, absorption, utilization, and excretion of rumen-protected Lys (RP-Lys). Four ruminally cannulated cows in a 4 × 4 Latin square design (12 d for diet adaptation; 5 or 6 d for total collections) received the following treatments: L0, a basal diet; L25, the basal diet and L-Lys infused into the abomasum to provide 25.9 g/d L-Lys; L50, the basal diet and L-Lys infused into the abomasum to provide 51.8 g/d L-Lys; and RPL, the basal diet supplemented with 105 g/d (as-is) of RP-Lys to provide 26.7 g of digestible Lys. During the last 5 or 6 d in each period, 15 N-Lys (0.38 g/d) was infused into the abomasum for all cows to label the pool of AA, and the total collection of milk, urine, and feces were conducted. 15 N enrichment of samples on d 4 and 5 were used to calculate the bioavailability and Lys metabolism. We used a model containing a fast AA turnover (≤ 5 d) and slow AA turnover pool (> 5 d) to calculate fluxes of Lys. The Lys flux to the fast AA turnover pool (absorbed Lys + Lys from the slow AA turnover pool to fast AA turnover pool) was calculated using 15 N enrichment of milk Lys. The flux of Lys from the fast AA turnover pool to milk and urine was calculated using 15 N transfer into milk and urine. Then, absorbed Lys was estimated by the sum of Lys flux to milk and urine assuming no net utilization of Lys by body tissues. Duodenal Lys flow was estimated by 15 N enrichment of fecal Lys. The bioavailability of RP-Lys was calculated from duodenal Lys flows and Lys absorption for RPL. Increasing Lys supply from L25 to L50 increased Lys utilization for milk by 9 g/d but also increased urinary excretion by 10 g/d. For RPL, absorbed Lys was estimated to be 136 g/d where 28 g of absorbed Lys originated from RP-Lys. In conclusion, 68% of bioavailability was obtained for RP-Lys. The Lys provided from RP-Lys was not only utilized for milk protein (48%) but also excreted in urine (20%) after oxidation., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)