Milk urea nitrogen (MUN) is widely used as an indicator of dietary energy–protein balance and nitrogen utilization efficiency in dairy cows, but the ruminal microbial and metabolic basis of MUN variation remains insufficiently understood. Here, we integrated milk trait measurements with rumen metagenomics and untargeted metabolomics to compare eight high-milk urea nitrogen (HMUN, 16.6 ± 0.48 mg/dL) and seven low-milk urea nitrogen (LMUN, 11.7 ± 0.34 mg/dL) cows. Compared with LMUN cows, HMUN cows exhibited higher MUN yield (P < 0.01) and urinary nitrogen excretion (P < 0.05), with a tendency toward higher ruminal NH₃-N concentration (P = 0.059). In contrast, LMUN cows exhibited higher MCP yield (P < 0.05), a greater MPY/MUNY ratio (P < 0.001), and a tendency toward higher milk protein yield (P = 0.058). Dry matter intake, milk yield, and major ruminal volatile fatty acid concentrations did not differ substantially between groups, suggesting that divergence in MUN phenotype was primarily associated with differences in ruminal nitrogen capture and partitioning rather than overall production performance. Multi-omics analyses revealed distinct and coordinated ruminal microbial, functional, and metabolic profiles between the two phenotypes. HMUN cows were characterized by enrichment of Prevotella, Bacteroides, Hallella, and Treponema and by greater representation of amino acid catabolic pathways, particularly branched-chain amino acid degradation. In contrast, LMUN cows showed enrichment of Butyrivibrio, Pseudobutyrivibrio, Eubacterium, and Clostridium, together with pathways related to pyruvate metabolism, the pentose phosphate pathway, sulfur metabolism, and amino acid biosynthesis. Metabolomic and correlation analyses further supported coordinated microbe–metabolite interactions associated with MUN variation. Variance partitioning analysis showed that rumen microbial composition, microbial functional pathways, and metabolite profiles explained similar proportions of MUN variation (13.59%, 13.42%, and 13.38%, respectively). Collectively, these findings indicate that low-MUN cows possess ruminal microbial and metabolic features consistent with more efficient ammonia incorporation into microbial biomass output, whereas high-MUN cows exhibit features associated with enhanced amino acid degradation and less efficient nitrogen capture. These results provide preliminary evidence that these coordinated differences may underlie the development of distinct MUN phenotypes.

Soybean proteins function as complex food hydrocolloids whose performance is governed by hierarchical structural organization spanning molecular conformation, colloidal assembly, and network formation. Conventional thermal processing often drives uncontrolled aggregation of soy proteins, leading to impaired interfacial activity, brittle gel networks, and reduced digestibility. In contrast, emerging processing technologies offer new opportunities to deliberately engineer soy protein hydrocolloids by modulating structure-function relationships rather than relying on empirical formulation adjustments. This review critically examines how high-pressure processing, ultrasound, pulsed electric fields, microwave and ohmic heating, and enzyme-assisted treatments act as structure-engineering tools that reprogram protein assembly pathways across multiple length scales. Mechanistic links are established between processing-induced molecular unfolding, colloidal disintegration or reassembly, interfacial adsorption kinetics, rheological behavior, and gastrointestinal disintegration. Particular emphasis is placed on pressure-and cavitation-driven modulation of protein-protein and protein-lipid interactions, electroporation-enhanced mass transfer with minimal denaturation, and controlled proteolysis for tuning gel elasticity and interfacial viscoelasticity. Comparative analysis reveals that hybrid and sequential processing strategies can generate synergistic effects, producing soy hydrocolloids with enhanced solubility, improved emulsifying and foaming performance, tailored gelation profiles, and accelerated protein and phytochemical bioaccessibility. Finally, key knowledge gaps are identified, including the need for standardized interfacial and digestion models, multiscale structural characterization, and predictive design frameworks. In conclusion, this review positions innovative processing technologies as rational tools for designing next-generation soy protein hydrocolloids with targeted functional and nutritional performance.

This study aimed to investigate the therapeutic effects of camel milk (CM) on chronic fatigue syndrome (CFS) and elucidate the mechanisms underlying the microbiota-gut-brain axis. Using a murine model of CFS induced by chronic restraint and forced swimming stress, we administered lyophilized CM (1500 mg/kg/day, equivalent to approximately 121.5 mg/kg/day in humans based on body surface area conversion using the standard allometric scaling formula) orally. CM supplementation was significantly associated with ameliorated fatigue-like behaviors, as evidenced by prolonged swimming endurance and reduced immobility time. Metagenomic analysis revealed that CM was associated with reshaping of the small intestinal microbiota, including enrichment of beneficial Lactococcus lactis and suppression of pathobionts (H. hepaticus and H. typhlonius). These microbial shifts correlated with increased luminal lactic acid, improved intestinal barrier integrity (increased villus height, reduced crypt depth), and attenuated local inflammation (reduced TNF-α and IL-6, elevated IL-10). Consequently, CM was associated with reduced bacterial translocation and systemic inflammation, and normalization of hypothalamic-pituitary-adrenal (HPA) axis hyperactivity. We conclude that CM is associated with prevention of CFS-like symptoms through modulation of the gut ecosystem and strengthening of the intestinal barrier, potentially breaking the vicious cycle of gut inflammation and HPA axis dysfunction, although causality remains to be established through fecal microbiota transplantation or similar mechanistic studies.

Yak and cattle-yak milk are important dairy resources in high-altitude regions, but their lipidomic differences remain poorly characterized. The objective of this study was to compare the milk lipid profiles of 5 Tibetan yak groups and 2 cattle-yak groups produced under plateau conditions. Milk lipids were analyzed by liquid chromatography–tandem mass spectrometry, followed by multivariate analysis, differential lipid screening, and pathway enrichment analysis. A total of 901 lipid species were identified, with glycerophospholipids representing the largest proportion of detected lipids. Multivariate analysis showed distinct lipidomic profiles between yak and cattle-yak milk. Among the 5 yak groups, 28 differential lipids were identified, mainly involving glycerophospholipids, sphingolipids, glycerolipids, and fatty acyl-related molecules. No false discovery rate-confirmed differential lipids were detected between Holstein × yak and Jersey × yak milk, although exploratory analysis suggested group-associated lipid variation. Comparison between yak and cattle-yak milk identified 18 differential lipids after accounting for breed nested within animal type. These lipids were mainly related to membrane-associated polar lipids and glycerolipids. Pathway analysis indicated that glycerophospholipid metabolism was the main pathway distinguishing yak and cattle-yak milk, with additional evidence for fatty acid- and glycerolipid-related differences. A panel of false discovery rate-adjusted lipids showed potential for discriminating among the 7 milk groups, supporting their use as candidate lipid signatures for milk-group characterization. Overall, these findings provide a lipidomic basis for evaluating plateau dairy resources, but broader validation under more controlled production conditions is needed before these lipid signatures can be applied to milk quality assessment or product development.

The study fingerprints the heat-induced metabolic changes in milk across 15 temperature-time treatments (63-136 °C) using an untargeted metabolomics approach. A total of 2277 metabolites were detected, including organic acids, benzene, heterocyclic compounds, aldehydes, ketones, esters, amino acids, carbohydrates, alcohols, amines, fatty acids, lipids, and their derivatives, from thermally processed milk samples across 15 treatment groups. The multivariate analyses (PCA, HCA) and the KEGG pathway enrichment revealed heat-induced metabolic disruptions, particularly in the Maillard reaction products and lipid oxidation pathways. Five thousand three hundred twenty-nine differentially regulated metabolites were observed, with the most pronounced changes observed in the 135 °C vs. raw milk group. Key pathways, including those associated with amino acid and carbohydrate metabolism, were significantly enriched. Amino acid and lipid pathways were most affected by heat processing. Lactulose was consistently downregulated, while L-Proline, Adenosine, and Carnitine were upregulated, suggesting they are candidate thermal biomarkers. The findings fingerprint the thermal-induced biochemical changes in milk, and Lactulose as a potential thermal biomarker for milk quality and authenticity. Further research is required to assess the practical feasibility of the biomarker and the practices needed to optimize dairy processing techniques.

Short-chain fatty acids (SCFAs) are key microbial metabolites that support intestinal and skeletal development, yet their coordinated effects during early life remain poorly defined. In this study, neonatal mice were administered SCFAs for 28 days to evaluate their impacts on growth, intestinal barrier integrity, immune modulation, bone development, and gut microbiota composition. Valerate supplementation significantly increased body weight and intestinal length. It enhanced the villus structure, crypt depth, and goblet cell number, alongside upregulation of tight junction and mucin genes, indicating improved barrier function. Valerate and propionate also promoted the expression of interleukin-4 (IL-4) and interleukin-10 (IL-10) and reduced pro-inflammatory cytokines, suggesting an immunomodulatory shift. In the skeletal system, valerate improved the microarchitecture, increased bone mineral density (BMD), and upregulated osteogenic genes runt-related transcription factor 2 (Runx2), fibroblast growth factor receptor 1 (FGFR1), and growth hormone receptor (GHR). Microbiota profiling showed enrichment of several genera (e.g., Fructobacillus, Pantoea, and Ralstonia) that correlated with intestinal and bone parameters. Collectively, these data indicate that valerate supplementation is associated with concurrent improvements in neonatal intestinal and skeletal outcomes, accompanied by shifts in microbiota and changes related to the barrier and immune systems; however, causal links among these intermediate steps remain to be established.

Geographical origin may be a key factor contributing to variations in dairy product quality. This study focuses on the fatty acid composition and lipidome of camel milk and bovine milk from different regions, aiming to highlight lipid biomarkers that remain unaffected by regional variations in both camel and bovine milk, as well as region-specific lipid biomarkers present in camel milk. The results showed that camel milk consistently exhibited higher levels of long-chain fatty acids and monounsaturated fatty acids, whereas bovine milk showed a higher proportion of medium-chain and short-chain saturated fatty acids, regardless of the region. Lipidomic analysis revealed that phospholipids were more abundant in camel milk, while triglycerides and diglycerides were characteristic lipids in bovine milk. Certain lipid biomarkers were more abundant in specific types of milk, such as PC (21:4/21:6CHO), FA (29:3), and Hex3Cer (d18:0/24:2) in camel milk, and CerPE (d41:6) in bovine milk, and could be used to distinguish camel milk from bovine milk across all 3 regions. Additionally, fatty acids and lipid molecules distinguishing camel milk from different regions were identified, including C18:3n3 and PE (18:2/22:2CHO) in XJNC, C18:2n6c in XJSC, and C14:0 and PS (16:0/18:1) in IMC. These findings provide new insights into developing species-specific nutritional strategies and regional traceability of dairy products.

Chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) is a complex and debilitating disorder with limited treatment options. Camel milk (CM), known for its rich nutrients and anti-fatigue properties, may offer multi-target benefits for managing this condition. This study utilized an integrated approach combining metabolomics, network pharmacology, and animal experiments. CM metabolites were profiled and screened via ADME. Potential targets were predicted and intersected with CFS/ME-associated genes. Male BALB/c mice were subjected to chronic restraint and forced swimming to evaluate the effects of CM (1000 mg/kg) on behavioral, inflammatory, neuroendocrine, and metabolic parameters. CM administration significantly improved exhaustive swimming time and reduced immobility. It attenuated systemic inflammation (restored IL-10), normalized brain CREB and DRD2/OPRM1 mRNA, and enhanced skeletal muscle AKT/GLUT4 expression and glycogen levels. Camel milk alleviates CFS/ME symptoms through the multi-component, multi-target regulation of neuroendocrine, inflammatory, and energy metabolism pathways. These preclinical findings suggest that CM may have potential as a supportive nutritional intervention for alleviating chronic fatigue, pending validation in human studies.

Milk titratable acidity is a key indicator of raw milk freshness and quality, but its variation across different dairy animal species remains incompletely characterized. Based on 16,984 raw milk samples from eight dairy animal species (Holstein cow, goat, buffalo, camel, sheep, yak, donkey, and horse) collected within a retrospective raw milk quality monitoring framework in China from 2016 to 2024, this study provides a large-scale descriptive comparison of milk titratable acidity across species. Distinct titratable acidity profiles were observed among species, with camel and yak milk showing relatively high values, sheep, Holstein, and buffalo milk exhibiting intermediate values, and donkey and horse milk presenting markedly low values. Calendar-season-associated patterns also differed among species. Correlations between titratable acidity and milk components varied by species, with relatively stronger positive associations with protein and solids-not-fat (SNF) in several ruminant milks, suggesting that milk composition may contribute to differences in titratable acidity. However, because this study was based on an unbalanced observational dataset with limited animal-level, farm-level, feeding, management, physiological, and environmental metadata, these observations should be interpreted as descriptive and exploratory patterns rather than causal biological mechanisms. This dataset provides preliminary reference information for future studies on species-associated variation in raw milk titratable acidity and for discussions on species-specific raw milk quality evaluation.

ABSTRACT Sustainable dairy development is crucial for global food security, as it converts feed resources into nutrient‐dense human food while supporting soil fertility through manure recycling. Milk provides high‐quality protein and essential micronutrients, yet rising production demands have intensified challenges related to animal welfare and environmental impacts. Addressing these issues requires integrated strategies, including precision monitoring technologies, enteric methane inhibitors, and improved manure management. A balanced approach aligning productivity with animal health and ecological integrity is essential for future dairy systems.

Milk urea nitrogen, the primary form of nonprotein nitrogen (N) in milk, is an indirect indicator of N metabolism in dairy cows. The MUN concentrations are modulated by various factors, including dietary composition, physiological status, and environmental conditions. However, the potential roles of host gut microbiota and metabolome in the development of distinct MUN phenotypes remain insufficiently elucidated. Here, we compared fecal microbiota and fecal/serum metabolomes of high-MUN (HMUN) and low-MUN (LMUN) cows (n = 7 per group) under uniform feeding management using 16S rRNA gene sequencing and liquid chromatography-MS-based metabolomics. Compared with that of LMUN cows, the feces of HMUN cows exhibited relatively higher abundances of UCG-009, UCG-002, and Christensenellaceae_R-7_group and lower abundances of Succinivibrio, Lachnospiraceae_NK3A20_group, Acetitomaculum, Prevotellaceae_UCG-001, and norank_f__Bifidobacteriaceae. Metabolomic analysis revealed that HMUN cows had relatively lower levels of hydroxypropionic acid, N-myristoyl arginine, and N-eicosapentaenoyl tryptophan in feces, and reduced amounts of l-serine, linoleic acid, and butyrate in serum. Kyoto Encyclopedia of Genes and Genomes pathway mapping revealed that the metabolites relatively elevated in LMUN cows were primarily involved in the β-alanine metabolism pathway. The UCG-009 and Christensenellaceae_R-7_group were positively correlated with MUN, whereas Succinivibrio, Lachnospiraceae_NK3A20_group, Anaeroplasma, and Acetitomaculum were negatively correlated. These microbial taxa were also significantly associated with several fatty and AA metabolites, including adipic acid, undecenoic acid, dodecanedioic acid, dl-tryptophan, and l-leucine. Collectively, this study revealed certain differences in the gut microbiota and metabolome between cows with high and low MUN levels. These findings suggest that alterations in gut microbial composition and metabolic profiles may contribute to variations in MUN phenotypes and provide new insights into host factors influencing MUN metabolism in dairy cows.

Milk and dairy products are essential food globally, due to their rich nutritional content. The nutritional quality of milk provides an excellent medium for microbial growth. While beneficial microbes can enhance nutritional quality, spoilage and pathogenic bacteria pose significant risks. Extended shelf life (ESL), pasteurization, and ultra-high temperature (UHT) processing reduce microbial load but variably impact nutritional quality and the milk microbiome. This review critically compares the microbiome dynamics across these processing types with a specific emphasis on ESL milk, an area where comprehensive metagenomics data is notably lacking. A deeper understanding of raw milk contamination routes and the potential for outgrowth or survival of biofilm forming or spore forming bacteria during processing is critical. This review summarizes processing driven microbiome shifts, safety implications, and nutritional consequences in milk. A key focus is the limitations of current detection strategies, particularly the challenge of distinguishing viable from non-viable cells in processed milk. By synthesizing existing research and highlighting critical knowledge gaps, this review establishes a framework to guide future dairy microbiome research, focusing on the development of safer, more nutritious milk products and practical industry applications.

The nutritional value of milk fat depends on lipid and fatty acid composition. This study characterized the lipidomics and fatty acid positional distribution of milk from nine mammalian species: cow, buffalo, yak, goat, sheep, camel, mare, donkey, and human. Lipid molecular species were profiled by liquid chromatography-tandem mass spectrometry. Fatty acid positional distribution was determined using NH₂ and Si solid-phase extraction, selective sn-1/3 hydrolysis by Candida antarctica lipase B, and sn-2 monoacylglycerol quantification by gas chromatography-mass spectrometry. Ruminant milks showed high compositional similarity and differed from camel and monogastric milks. Compared to human milk, animal milks had higher triacylglycerols, phosphatidylcholines, phosphatidylethanolamines, and sphingomyelins, but lower free fatty acids and diacylglycerols. Short-chain fatty acids consistently occupied sn-1/3 positions, whereas medium-chain, long-chain, odd-chain, and branched-chain fatty acids favored sn-2 position. Monogastric milks exhibited sn-2 preference for palmitic acid and other saturated fatty acids and sn-1/3 preference for unsaturated fatty acids; ruminant and camel milks showed the reverse. Bray-Curtis analysis identified donkey milk as the closest lipidomic analog to human milk, followed by mare milk. These findings provide a systematic reference for evaluating mammalian milks and data for humanized infant formula lipid design.

Red clover extract (RCE) is rich in isoflavones, which have been reported to improve nitrogen utilization in dairy diets. Therefore, the objective of this study was to evaluate the effects of partially replacing soybean meal (SBM) with RCE plus feed-grade urea on lactation performance and nitrogen metabolism in lactating Holstein cows, relative to a slow-release urea (SRU) nitrogen source formulated to provide a similar amount of dietary nitrogen. In a randomized complete block design, 600 cows received one of 3 diets for 35 d (n = 200 per diet): SBM (10.81% SBM), SRU (0.65% SRU replacing 37.74% of SBM), or RCE (0.20% RCE + 0.57% urea replacing 37.74% of SBM). Diets were formulated to be isoenergetic and isonitrogenous. Data were analyzed using a mixed model with diet as a fixed effect and block and pen nested within diet as random effects; the pen was considered the experimental unit. Milk yield and milk composition did not differ between RCE and SBM. The DMI and feed efficiency were similar between these treatments, whereas SRU was associated with greater DMI and lower feed efficiency relative to SBM. Compared with SBM, RCE increased urinary purine derivatives and estimated microbial N, whereas concentrations of urea N in milk, plasma, and urine did not differ. Apparent NDF and ADF digestibility were similar between RCE and SBM but were lower with SRU. Differences among diets were also observed in selected blood biochemical and endocrine variables. These findings indicate that, during the 35-d experimental period, partial replacement of SBM with RCE plus urea maintained lactation performance relative to SBM and was associated with higher estimated microbial N compared with SBM, whereas responses to SRU differed in several nitrogen-related measures.

Escherichia coli (E. coli) is a major pathogen responsible for mastitis and calf diarrhea in dairy cows and has developed heteroresistance (HR) to a wide range of antibiotics. An increasing number of animal studies have shown that the presence of heteroresistance leads to failure of antibiotic therapy. The aims of this study were to investigate whether heteroresistance exists in E. coli and to investigate the biological characteristics of heteroresistant E. coli and its resistant subpopulations in order to investigate possible heteroresistance mechanisms. In this study, we screened heteroresistant E. coli by the Minimum Inhibitory Concentration (MIC) test, Kirby-Bauer (K-B) test, and population analysis profile (PAP), and analyzed the heteroresistance bacteria by a combination of resistance stability test, growth curve test, biofilm formation ability test, transcriptomics and qRT-PCR characterization and mechanism. The results of the K-B test, MIC test and PAP experiments showed that the strains D2, D8, D14, D15, and D19 were heteroresistant to amoxicillin/clavulanic acid (AMC), and the frequency of heteroresistant subpopulations ranged from 3.95 × 10-6 to 8.11 × 10-5. The resistant subpopulation resistance of strain D2 was stable and there was no growth lag. The results of transcriptomics testing confirmed that the heteroresistance mechanism involves ompF, reduced or absent ompC pore protein expression, and increased biofilm formation. This finding provides new insights into the molecular regulatory pathways involved in the development of heteroresistant strains of E. coli.

Equol, a bioactive metabolite of isoflavones, is associated with diverse health benefits, yet its enrichment in bovine milk has not been fully clarified. In this study, we established a liquid chromatography-mass spectrometry method for detecting equol in milk, featured by an acid hydrolysis pretreatment. Equol was found predominantly in the milk fat fraction. A random forest regression model was developed to predict milk equol levels based on its significant correlations with various lactation parameters. Dietary supplementation with red clover isoflavones markedly increased equol concentration in milk by up to 15.6 fold. The milk thermol processing and microbial fermentation showed negligible effects on equol level, indicating its strong stability. Collectively, these findings elucidate the distribution patterns and key determinants of equol in milk. Importantly, a strategy of red clover isoflavones supplementation was developed to produce higher equol-enriched milk as a novel functional food for human health.

The cellulolytic bacteria are crucial for applications in animal feeds, food processing, and bioenergy production. However, isolation of these bacteria from the microbial community has been challenging due to limitations in current methodologies. This study introduces a novel cellulose-functionalized magnetic nanoparticle-mediated isolation technique designed to enhance the recovery of cellulolytic bacteria. We hypothesized that bacteria could adhere to magnetic nanoparticles coated with cellulose, allowing selective isolation of cellulolytic strains through their cellulase activity-induced magnetic loss. The magnetic nanocomposite was synthesized and functionalized with cellulose (magnetic nanoparticle-cellulose composite [MNPC]). The anaerobic Streptococcus bovis was used to evaluate the bacteria capture efficiency of MNPCs. A mixture of Cellulomonas flavigena (a cellulolytic bacterium) and S. bovis (a non-cellulolytic bacterium) was used to evaluate the cellulolytic bacteria isolation specificity of the method. This method was also applied to isolate the novel ruminal cellulolytic bacteria. The results showed that the magnetic nanoparticle had an average size of 20.00 nm and superparamagnetism. MNPC achieved a bacterial capture efficiency of 99% and a high specificity. The method successfully captured the known cellulolytic bacteria from the rumen of cattle, including Fibrobacter, Ruminococcus flavefaciens, and Butyrivibrio, indicating its reliability. Furthermore, a novel cellulolytic species from the family Lachnospiraceae was isolated from the rumen, with genome analysis revealing cellulolytic pathway genes encoding endoglucanase, β-glucosidase, and cellobiose phosphorylase. In conclusion, the cellulose-functionalized magnetic nanoparticle-mediated isolation method presents a promising approach for the efficient enrichment and isolation of active cellulolytic bacteria. This technique and the isolated strains have potential applications in enhancing animal productivity, bioenergy production, and the food industry.IMPORTANCELignocellulosic biomass, as the primary component of cell walls in gramineous plants, is widely recognized as an ideal biofuel feedstock due to its sustainability and renewability. It can be digested and degraded by cellulolytic microorganisms, converting it into absorbable proteins and volatile fatty acids that provide energy for the organism. However, isolation of these bacteria from the microbial community has been challenging due to the limitations in current methodologies. In this work, we developed a new method, the cellulose-functionalized magnetic nanoparticle-mediated isolation technology, which can contribute to capture highly active cellulolytic bacteria. We isolated a novel unclassified cellulolytic strain of the Lachnospiraceae from the rumen of cattle. This methodology presents a promising approach for the efficient enrichment and isolation of active cellulolytic bacteria from the community.