Delineating Bovine Milk Derived Microvesicles from Exosomes Using Proteomics.

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

LacdiNAc to LacNAc: remodelling of bovine α-lactalbumin N-glycosylation during the transition from colostrum to mature milk.

α -Lactalbumin, an abundant protein present in the milk of most mammals, is associated with biological, nutritional and technological functionality. Its sequence presents N-glycosylation motifs, the occupancy of which is species-specific, ranging from no to full occupancy. Here, we investigated the N-glycosylation of bovine α-lactalbumin in colostrum and milk sampled from four individual cows, each at 9 time points starting from the day of calving up to 28.0 d post-partum. Using a glycopeptide-centric mass spectrometry-based glycoproteomics approach, we identified N-glycosylation at both Asn residues found in the canonical Asn-Xxx-Ser/Thr motif, i.e. Asn45 and Asn74 of the secreted protein. We found similar glycan profiles in all four cows, with partial site occupancies, averaging at 35% and 4% for Asn45 and Asn74, respectively. No substantial changes in occupancy occurred over lactation at either site. Fucosylation, sialylation, primarily with N-acetylneuraminic acid (Neu5Ac), and a high ratio of N,N'-diacetyllactosamine (LacdiNAc)/N-acetyllactosamine (LacNAc) motifs were characteristic features of the identified N-glycans. While no substantial changes occurred in site occupancy at either site during lactation, the glycoproteoform (i.e. glycosylated form of the protein) profile revealed dynamic changes; the maturation of the α-lactalbumin glycoproteoform repertoire from colostrum to mature milk was marked by substantial increases in neutral glycans and the number of LacNAc motifs per glycan, at the expense of LacdiNAc motifs. While the implications of α-lactalbumin N-glycosylation on functionality are still unclear, we speculate that N-glycosylation at Asn74 results in a structurally and functionally different protein, due to competition with the formation of its two intra-molecular disulphide bridges.

Chemo-Enzymatic Functionalization of Bovine Milk Exosomes with an EGFR Nanobody for Target-specific Drug Delivery.

Bovine milk exosomes (BmExo) have been identified as versatile nanovesicles for anti-cancer drugs delivery due to their natural availability and biocompatibility. However, tumor-specific delivery based on BmExo often requires post-isolation modifications of the membrane surface with active-targeting ligands. In this study, we report an alternative approach to functionalize BmExo with nanobody combining facile chemical modification and Sortase A-mediated site-specific ligation, as demonstrated by the development of an epidermal growth factor receptor (EGFR)-targeted drug delivery system. The BmExo membrane was first coated with a diglycine-containing amphiphile molecule, NH2-GG-PEG2000-DSPE, by hydrophobic insertion. The diglycine as nucleophiles displayed on the membrane enabled the subsequent ligation of the EGFR nanobody (7D12) by Sortase A (SrtA)-mediated site-specific transpeptidation. The successful construction of BmExo-7D12 was confirmed by Western blotting analysis, electron microscopy, and dynamic light scattering (DLS). As a demonstration model, BmExo-7D12 loaded with the chemotherapeutic drug doxorubicin (Dox) was shown to be able to deliver Dox to cancer cells in response to the expression of EGFR as manifested by immunocytochemistry and flow cytometry analysis. Finally, the cytotoxicity assay showed that BmExo-7D12-Dox was more effective in killing tumor cells with high EGFR expression while significantly reduced the non-specific toxicity to EGFR negative cells. In conclusion, these results demonstrate that 7D12-functionalized BmExo can serve as a target-specific delivery system for Dox to selectively kill EGFR-expressing tumor cells. This approach should prove to be versatile and efficient for the generation of protein-ligands modified BmExo.

Coagulase-negative staphylococci from bovine milk: Antibiogram profiles and virulent gene detection.

BACKGROUND: Coagulase-negative Staphylococcus species are an emerging cause of intramammary infection, posing a significant economic and public health threat. The aim of this study was to assess the occurrence of coagulase-negative Staphylococcus species in bovine milk and dairy farms in Northwestern Ethiopia and to provide information about their antibiotic susceptibility and virulence gene profiles. METHODS: The cross-sectional study was conducted from February to August 2022. Coagulase-negative Staphylococcus species were isolated from 290 milk samples. Species isolation and identification were performed by plate culturing and biochemical tests and the antimicrobial susceptibility pattern of each isolate was determined by the Kirby-Bauer disc diffusion test. The single-plex PCR was used to detect the presence of virulent genes. The STATA software version 16 was used for data analysis. The prevalence, proportion of antimicrobial resistance and the number of virulent genes detected from coagulase-negative Staphylococcus species were analyzed using descriptive statistics. RESULTS: Coagulase-negative Staphylococcus species were isolated in 28.6%, (95% CI: 23.5-34.2) of the samples. Of these, the S. epidermidis, S. sciuri, S. warneri, S. haemolyticus, S. simulans, S. chromogens, S. cohnii, and S. captis species were isolated at the rates of 11, 5.2, 3.4, 3.1, 3.1, 1, 1, and 0.7% respectively. All the isolates showed a high percentage (100%) of resistance to Amoxicillin, Ampicillin, and Cefotetan and 37.5% of resistance to Oxacillin. The majority (54.2%) of coagulase-negative isolates also showed multidrug resistance. Coagulase-negative Staphylococcus species carried the icaD, pvl, mecA, hlb, sec, and hla virulent genes at the rates of 26.5%, 22.1%, 21.7%, 9.6%, 9.6% and 8.4% respectively. CONCLUSION: The present study revealed that the majority of the isolates (54.2%) were found multidrug-resistant and carriage of one or more virulent and enterotoxin genes responsible for intramammary and food poisoning infections. Thus, urgent disease control and prevention measures are warranted to reduce the deleterious impact of coagulase-negative species. To the best of our knowledge, this is the first study in Ethiopia to detect coagulase-negative Staphylococcus species with their associated virulent and food poisoning genes from bovine milk.

Heat Treatment and Homogenization Influence the Gastric Digestion of Bovine Milk Protein in Growing Pigs as an Adult Human Model.

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.

Analysis of Inflammatory and Regulatory Cytokines in the Milk of Dairy Cows with Mastitis: A Comparative Study with Healthy Animals.

Bovine mastitis remains a major problem in the global dairy cattle industry. The acute invasion of udder by pathogens induces innate immune response as the first defence mechanism in subclinical and clinical mastitis. The aim of the study was to determine inflammatory and regulatory cytokines IL-2, IL-4, TGF-ß1, IL-17A, beta-defensin 3 and IL-10 and their potential changes in milk of dairy cows with subclinical and clinical mastitis, and to compare the findings with healthy animals. Milk samples from 15 holstein Friesian breed cows were used in the study. Cows were divided into three groups based on their health status (5 healthy, 5 subclinical and 5 clinical animals). All samples were tested using immunohistochemistry to evaluate IL-2, IL-4, IL-10, IL17A, TGF-ß1 and ß-Def 3 proteins. Expression of all proteins was detected in all milk samples. High expression of IL-2, IL-4, IL17A, TGF-ß1 was detected in healthy cows' milk and in milk of cows with subclinical and clinical mastitis. However, expression of IL-10 and ß-Def 3 in milk samples of healthy cows was significantly higher compared to the milk of cows with subclinical and clinical mastitis (p < .001). IL-10 and ß-Def 3 can be considered as informative biomarkers in diagnosis of subclinical and clinical mastitis.

Fluorescence Entrenched Probe for Onsite Detection of Amoxicillin Residue in Bovine Milk.

A novel fluorescent probe (E)-3-(4-hydroxyphenyl)-2-((pyrene-1-ylmethylene) amino)propanoic acid (PyT) was developed for the 'turn-on' detection of amoxicillin(AM), residues. The PyT molecule was developed by a simple condensation reaction between a biologically important tyrosine amino acid and pyrene carboxaldehyde. The small fluorophore molecule has spectacular photoluminescence properties such as large stock shift, high photostability, selectivity and sensitivity toward the analytes. The PyT upon dispersion in the liquid phase becomes highly luminescent possessing the restricted intramolecular rotation (RIR) and excited stated intramolecular proton transfer (ESIPT) properties which are the major criteria for aggregation induced emission enhancement (AIEE) mechanism prevailing the aggregation caused quenching (ACQ). PyT molecule shows a binding constant of 3.285 × 104 L mol-1 for amoxicillin (AM). The limit of detection (LOD) values are found to be 1.67µM. Consuming bovine milk with antibiotic residues exceeding the maximum residue limit (MRL) can lead to food toxicity and life threatening diseases in humans. The milk sample with AM antibiotic residue in presence of PyT probe shows a distinct blue colour which infers the selectivity and sensitivity of the probe towards the analyte. The fluorescence probe adheres with merits like on site and visual examination by naked eye without aid of any instruments.

An interactive R-based custom quantification program for semi-quantitative analysis of triacylglycerols in bovine milk.

The R programming language, RStudio, and open-source software solutions for analysis of liquid chromatography-mass spectrometry (LC-MS) data have been used with user-written R-based custom quantification programs (CQP) for semi-quantification of triacylglycerols (TAGs) in bovine milk lipid extracts. Using the peak-finding capabilities of the package "xcms" in RStudio, peaks were integrated, and retention times aligned, normalized, and then used for semi-quantitative analysis of a custom set of four extraction internal standards (EISs) and 29 TAG regioisomers using the choice of four analytical internal standards (AISs). Alternating stereospecific numbering (sn) 1,3 TAG regioisomers (standards 1, 3, and 5 of six calibration standards) and sn-1,2 TAG regioisomers (standards 2, 4, and 6 of six standards) were used to make a set of six calibration standards, which were used for quantification using a linear fit model, polynomial fit model, power fit model, level-bracketed linear fit, replicate-bracketed polynomial fit, replicate-bracketed power fit, and replicate- and level-bracketed linear fit and response factors. For example, the linear fit for EIS1 gave an unacceptable coefficient of determination (CoD), r2 = 0.9616, whereas the polynomial fit gave r2 = 0.9908 and the power fit gave r2 = 0.9928, while the double-bracketed linear fit gave CoDs of r2 = 0.9960, 0.9848, and 0.9781 for the three brackets, yet gave the least % difference to known calibration concentrations. For unparalleled transparency, the CQP produced webpages that allowed every step in the data processing and quantification sequence to be verified and reproduced, and contained interactive figures. The data are publicly available using a digital object identifier (DOI). The R code can be downloaded and used with the downloadable data to reproduce the results, to modify the code and further customize the results, or to copy and paste and adapt the code to other quantification applications.

Bovine milk osteopontin improved intestinal health of pregnant rats fed a high-fat diet through improving bile acid metabolism.

The main purpose of the current study was to investigate the ameliorative effects of bovine milk osteopontin (bmOPN) on the gut dysfunction of pregnant rats fed a high-fat diet (HFD). Bovine milk osteopontin was supplemented at a dose of 6 mg/kg body weight. Bovine milk osteopontin supplementation during pregnancy reduced colonic inflammation of HFD dams, and it also increased the colonic expression of ZO-1 and claudin-4 of HFD dams. Bovine milk osteopontin significantly enriched the relative abundance of Bacteroidetes, whereas it decreased Proteobacteria, Helicobacteraceae, and Desulfovibrionaceae in feces of HFD dams. The levels of isobutyric acid and pentanoic acid in the HFD + bmOPN group were higher than that of the HFD group. Functional predication analysis of microbial genomes revealed that bmOPN supplementation to HFD pregnancies changed 4 Kyoto Encyclopedia of Genes and Genomes pathways including bile acid biosynthesis. Further, bmOPN enriched hepatic taurochenodeoxycholic acid and tauroursodeoxycholic acid plus taurohyodeoxycholic acid in the gut of HFD maternal rats. Our findings suggested that bmOPN improved the gut health of HFD pregnant rats partially through modulating bile acid biosynthesis.

Analysis of milk adulteration by means of a potentiometric electronic tongue.

The adulteration of milk presents significant challenges in the food industry, promoting the need for efficient detection methods. This study introduces a potentiometric electronic tongue for rapid and accurate milk adulteration detection. Utilizing polymeric membranes integrated with various additives, the electronic tongue distinguishes between different milk types and detects common adulterants. Experimental results demonstrate its effectiveness in discriminating raw, pasteurized, and medicated cow milk, as well as goat milk. Moreover, it successfully identifies adulterants like water and bovine milk in goat milk samples. Chemometric analyses, including Principal Component Analysis and Partial Least Squares regression, correlate sensor responses with traditional milk parameters such as fat, protein, and lactose content with up to a 0.97 R2 on the validation step. Strong correlations validate the electronic tongue's potential for rapid milk quality assessment. This innovative approach offers a cost-effective, reliable solution for detecting milk adulteration in contrast with current techniques that require numerous, time consuming experiments.

Microwave Dielectric Response of Bovine Milk as Pregnancy Detection Tool in Dairy Cows.

The most reliable methods for pregnancy diagnosis in dairy herds include rectal palpation, ultrasound examination, and evaluation of plasma progesterone concentrations. However, these methods are expensive, labor-intensive, and invasive. Thus, there is a need to develop a practical, non-invasive, cost-effective method that can be implemented on the farm to detect pregnancy. This study suggests employing microwave dielectric spectroscopy (MDS, 0.5-40 GHz) as a method to evaluate reproduction events in dairy cows. The approach involves the integration of MDS data with information on milk solids to detect pregnancy and identify early embryonic loss in dairy cows. To test the ability to predict pregnancy according to these measurements, milk samples were collected from (i) pregnant and non-pregnant randomly selected cows, (ii) weekly from selected cows (n = 12) before insemination until a positive pregnancy test, and (iii) daily from selected cows (n = 10) prior to insemination until a positive pregnancy test. The results indicated that the dielectric strength of Δε and the relaxation time, τ, exhibited reduced variability in the case of a positive pregnancy diagnosis. Using principal component analysis (PCA), a clear distinction between pregnancy and nonpregnancy status was observed, with improved differentiation upon a higher sampling frequency. Additionally, a neural network machine learning technique was employed to develop a prediction algorithm with an accuracy of 73%. These findings demonstrate that MDS can be used to detect changes in milk upon pregnancy. The developed machine learning provides a broad classification that could be further enhanced with additional data.

Bovine milk ß-casein: Structure, properties, isolation, and targeted application of isolated products.

ß-Casein, an important protein found in bovine milk, has significant potential for application in the food, pharmaceutical, and other related industries. This review first introduces the composition, structure, and functional properties of ß-casein. It then reviews the techniques for isolating ß-casein. Chemical and enzymatic isolation methods result in inactivity of ß-casein and other components in the milk, and it is difficult to control the production conditions, limiting the utilization range of products. Physical technology not only achieves high product purity and activity but also effectively preserves the biological activity of the components. The isolated ß-casein needs to be utilized effectively and efficiently for various purity products in order to achieve optimal targeted application. Bovine ß-casein, which has a purity higher than or close to that of breast ß-casein, can be used in infant formulas. This is achieved by modifying its structure through dephosphorylation, resulting in a formula that closely mimics the composition of breast milk. Bovine ß-casein, which is lower in purity than breast ß-casein, can be maximized for the preparation of functional peptides and for use as natural carriers. The remaining byproducts can be utilized as food ingredients, emulsifiers, and carriers for encapsulating and delivering active substances. Thus, realizing the intensive processing and utilization of bovine ß-casein isolation. This review can promote the industrial production process of ß-casein, which is beneficial for the sustainable development of ß-casein as a food and material. It also provides valuable insights for the development of other active substances in milk.

Bovine Milk Derived Exosomes Affect Gut Microbiota of DSS-Induced Colitis Mice.

The objective of this study was to investigate the effect of bovine milk derived exosomes (MDEs) on the gut microbiota of Dextran sodium sulfate (DSS)-induced colitis mice. Total of 42 specific pathogen free (SPF) male BALB/c mice (3 weeks old) were randomly assigned to three groups including control group, DSS group (DSS) and bovine milk derived exosome group (Exo), with 7 replicates/cages per treatment and two mice in one cage. 16S rRNA gene sequencing of cecal digesta samples was conducted. DSS significantly decreased the average daily feed intake of mice in DSS and Exo groups (P = 0.03). Shannon index of the DSS group was significantly lower than the control group (P < 0.05) whereas no difference between the control group and Exo group was observed. Administration of MDEs tended to increase the relative abundance of Campylobaterota. Compared to the control group, the relative abundance of Roseburia was significantly decreased in the DSS group (P < 0.05) whereas no difference between the Exo group and control group was observed. MDEs also tended to increase the relative abundance of Lachnospiraceae_UCG_006. In conclusion, oral administration of 10 µL MDEs (1 mg/mL) positively affected gut microbiota of DSS-induced colitis mice. The results of this study provided valuable reference for MDEs application in the prevention and treatment of colitis.

Bovine milk-derived extracellular vesicles enhance doxorubicin sensitivity in triple negative breast cancer cells by targeting metabolism and STAT signalling.

Metastatic triple-negative breast cancer (TNBC) has a low 5-year survival rate of below 30% with systemic chemotherapy being the most widely used treatment. Bovine milk-derived extracellular vesicles (MEVs) have been previously demonstrated to have anti-cancer attributes. In this study, we isolated bovine MEVs from commercial milk and characterised them according to MISEV guidelines. Bovine MEVs sensitised TNBC cells to doxorubicin, resulting in reduced metabolic potential and cell-viability. Label-free quantitative proteomics of cells treated with MEVs and/or doxorubicin suggested that combinatorial treatment depleted various pro-tumorigenic interferon-inducible gene products and proteins with metabolic function, previously identified as therapeutic targets in TNBC. Combinatorial treatment also led to reduced abundance of various STAT proteins and their downstream oncogenic targets with roles in cell-cycle and apoptosis. Taken together, this study highlights the ability of bovine MEVs to sensitise TNBC cells to standard-of-care therapeutic drug doxorubicin, paving the way for novel treatment regimens.

Key changes in bovine milk immunoglobulin G during lactation: NeuAc sialylation is a hallmark of colostrum immunoglobulin G N-glycosylation.

We monitored longitudinal changes in bovine milk IgG in samples from four cows at 9 time points in between 0.5 and 28 days following calving. We used peptide-centric LC-MS/MS on proteolytic digests of whole bovine milk, resulting in the combined identification of 212 individual bovine milk protein sequences, with IgG making up >50 percent of the protein content of every 0.5 d colostrum sample, which reduced to ≤3 percent in mature milk. In parallel, we analyzed IgG captured from the bovine milk samples to characterize its N-glycosylation, using dedicated methods for bottom-up glycoproteomics employing product ion-triggered hybrid fragmentation; data are available via ProteomeXchange with identifier PXD037755. The bovine milk IgG N-glycosylation profile was revealed to be very heterogeneous, consisting of >40 glycoforms. Furthermore, these N-glycosylation profiles changed substantially over the period of lactation, but consistently across the four individual cows. We identified NeuAc sialylation as the key abundant characteristic of bovine colostrum IgG, significantly decreasing in the first days of lactation, and barely detectable in mature bovine milk IgG. We also report, for the first time to our knowledge, the identification of subtype IgG3 in bovine milk, alongside the better-documented IgG1 and IgG2. The detailed molecular characteristics we describe of the bovine milk IgG, and their dynamic changes during lactation, are important not only for the fundamental understanding of the calf's immune development, but also for understanding bovine milk and its bioactive components in the context of human nutrition.

Functional attributes of bioactive peptides of bovine milk origin and application of in silico approaches for peptide prediction and functional annotations.

Bovine milk peptides are the protein fragments with diverse bioactive properties having antioxidant, anticarcinogenic, other therapeutic and nutraceutical potentials. These peptides are formed in milk by enzymatic hydrolysis, gastrointestinal digestion and fermentation processes. They have significant health impact with high potency and low toxicity making them a suitable natural alternative for preventing and managing diseases. Antibiotic resistance has increased the quest for better peptide candidates with antimicrobial effects. This article presents a comprehensive review on well documented antimicrobial, immunological, opioid, and anti-hypertensive activities of bovine milk peptides. It also covers the usage of computational biology tools and databases for prediction and analysis of the food-derived bioactive peptides. In silico analysis of amino acid sequences of Bos taurus milk proteins have been predicted to generate peptides with dipeptidyl peptidase IV inhibitory and ACE inhibitory properties, making them favorable candidates for developing blood sugar lowering drugs and anti-hypertensives. In addition to the prediction of new bioactive peptides, application of bioinformatics tools to predict novel functions of already known peptides is also discussed. Overall, this review focuses on the reported as well as predicted biologically active peptide of casein and whey proteins of bovine milk that can be utilized to develop therapeutic agents.

Whey protein hydrolysates improve high-fat-diet-induced obesity by modulating the brain-peripheral axis of GLP-1 through inhibition of DPP-4 function in mice.

PURPOSE: Obesity is a growing global health concern. Recent literature indicates a prominent role of glucagon-like peptide-1 (GLP-1) in glucose metabolism and food intake. The synergistic action of GLP-1 in the gut and brain is responsible for its satiety-inducing effect, suggesting that upregulation of active GLP-1 levels could be an alternative strategy to combat obesity. Dipeptidyl peptidase-4 (DPP-4) is an exopeptidase known to inactivate GLP-1, suggesting that its inhibition could be a crucial strategy for effectively extending the half-life of endogenous GLP-1. Peptides derived from partial hydrolysis of dietary proteins are gaining traction due to their inhibitory activity on DPP-4. METHODS: Whey protein hydrolysate from bovine milk (bmWPH) was produced using simulated in situ digestion, purified using RP-HPLC, and characterized for DPP-4 inhibition. The antiadipogenic and antiobesity activity of bmWPH was then studied in 3T3-L1 preadipocytes and high-fat diet-induced obesity (HFD) mice model, respectively. RESULTS: The dose-dependent inhibitory effect of bmWPH on the catalytic activity of DPP-4 was observed. Additionally, bmWPH suppressed adipogenic transcription factors and DPP-4 protein levels, leading to a negative effect on preadipocyte differentiation. In an HFD mice model, co-administration of WPH for 20 weeks downregulated adipogenic transcription factors, resulting in a concomitant reduction in whole body weight and adipose tissues. Mice fed with bmWPH also showed a marked reduction in DPP-4 levels in WAT, liver, and serum. Furthermore, HFD mice fed with bmWPH exhibited increased serum and brain GLP levels, which led to a significant decrease in food intake. CONCLUSION: In conclusion, bmWPH reduces body weight in HFD mice by suppressing appetite through GLP-1, a satiety-inducing hormone, in both the brain and peripheral circulation. This effect is achieved through modulation of both the catalytic and non-catalytic activity of DPP-4.

Regulation of iron metabolism is critical for the survival of Salmonella Typhimurium in pasteurized milk.

Salmonella is known to survive in raw/pasteurized milk and cause foodborne outbreaks. Lactoferrin, present in milk from all animal sources, is an iron-binding glycoprotein that limits the availability of iron to pathogenic bacteria. Despite the presence of lactoferrins, Salmonella can grow in milk obtained from different animal sources. However, the mechanism by which Salmonella overcomes iron scarcity induced by lactoferrin in milk is not evaluated yet. Salmonella employs the DNA binding transcriptional regulator Fur (ferric update regulator) to mediate iron uptake during survival in iron deplete conditions. To understand the importance of Fur in Salmonella milk growth, we profiled the growth of Salmonella Typhimurium Δfur (ST4/74Δfur) in both bovine and camel milk. ST4/74Δfur was highly inhibited in milk compared to wild-type ST4/74, confirming the importance of Fur mediated regulation of iron metabolism in Salmonella milk growth. We further studied the biology of ST4/74Δfur to understand the importance of iron metabolism in Salmonella milk survival. Using increasing concentrations of FeCl3, and the antibiotic streptonigrin we show that iron accumulates in the cytoplasm of ST4/74Δfur. We hypothesized that the accumulated iron could activate oxidative stress via Fenton's reaction leading to growth inhibition. However, the inhibition of ST4/74Δfur in milk was not due to Fenton's reaction, but due to the 'iron scarce' conditions of milk and microaerophilic incubation conditions which made the presence of the fur gene indispensable for Salmonella milk growth. Subsequently, survival studies of 14 other transcriptional mutants of ST4/74 in milk confirmed that RpoE-mediated response to extracytoplasmic stress is also important for the survival of Salmonella in milk. Though we have data only for fur and rpoE, many other Salmonella transcriptional factors could play important roles in the growth of Salmonella in milk, a theme for future research on Salmonella milk biology. Nevertheless, our data provide early insights into the biology of milk-associated Salmonella.

Continuous enrichment and trace analysis of tetracyclines in bovine milk using dual-functionalized aqueous biphasic system combined with high-performance liquid chromatography.

Two PPG1000 based temperature-sensitive magnetic ionic liquid were synthesized and characterized. The temperature-sensitive magnetic ionic liquid aqueous biphasic system combined with HPLC was applied for the continuous enrichment and trace analysis of tetracycline antibiotics (TC) in bovine milk for the first time. High enrichment factors were achieved and the detection was highly sensitive. The trace analysis of TC was rapid, free of organic solvent, recyclable and magnetically assisted for phase separation. Under optimum conditions, wide linear ranges of 0.25-300 ng/mL for all TC, high enrichment factors of 217.7-231.4, good precisions with relative standard deviation in the range of 0.74-3.97%, very low limits of detection of 0.031-0.067 ng/mL, limits of quantification of 0.103-0.223 ng/mL, and good recoveries of 94.28-99.76% were acquired for the proposed analytical method. Real milk analysis was satisfactory. This developed analytical method is showing great potential for trace analysis of targeted analytes in foods and drinks.

Effect of dietary seaweed (Ascophyllum nodosum) supplementation on milk mineral concentrations, transfer efficiency, and hematological parameters in lactating Holstein cows.

This study investigated the effect of feeding seaweed (Ascophyllum nodosum) to dairy cows on milk mineral concentrations, feed-to-milk mineral transfer efficiencies, and hematological parameters. Lactating Holstein cows (n = 46) were allocated to 1 of 2 diets (n = 23 each): (1) control (CON; without seaweed) and (2) seaweed (SWD; replacing 330 g/d of dried corn meal in CON with 330 g/d dried A. nodosum). All cows were fed the CON diet for 4 wk before the experiment (adaptation period), and animals were then fed the experimental diets for 9 wk. Samples included sequential 3-wk composite feed samples, a composite milk sample on the last day of each week, and a blood sample at the end of the study. Data were statistically analyzed using a linear mixed effects model with diet, week, and their interaction as fixed factors; cow (nested within diet) as a random factor; and data collected on the last day of the adaptation period as covariates. Feeding SWD increased milk concentrations of Mg (+6.6 mg/kg), P (+56 mg/kg), and I (+1,720 µg/kg). It also reduced transfer efficiency of Ca, Mg, P, K, Mn, and Zn, and increased transfer efficiency of Mo. Feeding SWD marginally reduced milk protein concentrations, whereas there was no effect of SWD feeding on cows' hematological parameters. Feeding A. nodosum increased milk I concentrations, which can be beneficial when feed I concentration is limited or in demographics or populations with increased risk of I deficiency (e.g., female adolescents, pregnant women, nursing mothers). However, care should also be taken when feeding SWD to dairy cows because, in the present study, milk I concentrations were particularly high and could result in I intakes that pose a health risk for children consuming milk.