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Red clover (Trifolium pratense) isoflavone was supplemented to dairy cows, and antioxidant capacity of milk was assessed. Treated cows increased the activities of antioxidant enzymes, reduced production of oxidation products, and enhanced the concentrations of vitamin E and vitamin C. Moreover, milk fatty acid profile was positive influenced by 8 g/kg red clover isoflavone, with changes in the lower saturated and higher unsaturated fatty acids. We further demonstrated the efficacy of antioxidant capacity of milk in mice, found that milk from cows feeding red clover isoflavone increased the expressions of antioxidant enzymes, and alleviated lipopolysaccharide (LPS)-stimulated tissue damage of duodenum and jejunum, which was related to upregulated metabolism pathways of carbohydrate, lipid, and amino acid, as well as downregulated inflammatory related pathways. Together, dietary supplementation of red clover isoflavone is an effective way to improve milk antioxidant capacity, providing a natural strategy for developing functional foods., 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.)

Pseudomonas spp. are common microorganisms from cold-storage raw milk, and protease secreted by Pseudomonas spp. can cause the deterioration of stored milk. However, analyses of Pseudomonas spp. diversity and proteolytic activity in raw milk from different regions of China have not been extensively examined. With this aim, the diversity and proteolytic activity of Pseudomonas isolated from 25 raw cow milk samples from Inner Mongolia, Heilongjiang, Gansu, Henan, Anhui, Jiangsu, Chongqing and Hunan of China in different seasons were evaluated by PCR targeting 16S rDNA and rpoD, as well as TNBS method, respectively. A total of 116 Pseudomonas isolates from 25 raw cow milk samples were identified at the species level, including P. fluorescens, P. veronii, P. psychrophila, P. lundensis, P. lactis, P. azotoformans, P. granadensis, P. lurida, P. rhizosphaerae, P. rhodesiae and P. extremorientalis. P. fluorescens accounted for 75.8% of the total. Of all 116 Pseudomonas isolates, 68.9% of them displayed proteolytic activity at 4 °C, 81.9% at 10 °C and 85.3% at 25 °C, respectively. The aprX gene encoded a secreted and heat-resistant metalloprotease that was present in 60.3% of the Pseudomonas isolates tested. The proteases showed residual activity ranged from 73 ± 4% to 84 ± 7% residual activity after the heat treatment at 72 °C for 15 s and 62 ± 3% to 74 ± 2% after the heat treatment at 132 °C for 4 s. This is the first report to compare Pseudomonas spp. diversity and proteolytic activity at species levels in raw milk from different regions of China. The results of this study provide valuable data about the diversity and spoilage potential of Pseudomonas species in raw milk and the thermal resistance of the proteases. Therefore, these findings provide a reference for the importance to prevent Pseudomonas spp. contamination of raw cow milk to ensure the quality and safety of milk and dairy products., Competing Interests: Declaration of competing interest All authors have approved the submission and none declare any conflict of interest in the work performed or in the submission of the manuscript., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The milk fat globule membrane (MFGM) plays an important role in stabilizing fat in the aqueous phase, and the components of this membrane are involved in multiple biological functions. Here, we investigated changes in the protein composition of the MFGM fraction between raw and heated whole milk using a label free proteomic approach. In total, 612 MFGM-related proteins were identified in all groups. Compared with raw milk, the number of proteins that were not identified in the MFGM fraction was increased from pasteurized milk to ultra-high-temperature milk, whereas the number of milk proteins incorporated into the MFGM fraction was similar among heated groups. The abundances of several milk proteins (β-lactoglobulin and β-casein) were increased in the heated milk groups in a temperature-dependent manner. From our functional analysis, proteins that were not identified in the MFGM fraction of heated milk were mainly associated with multiple biological functions. These findings provided novel insights into the effects of heat procedures on MFGM protein components and their potential physiological functions, thereby yielding data on the appropriate heating procedures to use for raw milk., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Commercial milk products worldwide come not only from cows, but also from goats, buffaloes, camels, and yaks. Milk from non-bovine animals is important culturally and economically. Pseudomonas spp. are frequently linked to milk spoilage under storage temperatures. The objectives of this study were to identify Pseudomonas spp. isolated from goat, buffalo, camel, and yak milks, and to measure proteolytic activity of Pseudomonas spp. under different storage temperatures. Raw milk samples of goat (n = 50), buffalo (n = 25), camel (n = 25), and yak (n = 25) were collected from 5 provinces in China. Pseudomonas spp. were analyzed by Pseudomonas-specific 16S, universal 16S rRNA, and rpoB gene sequence analyses. Proteolytic activity on milk agar, quantification via the trinitrobenzenesulfonic acid assay at 2°C, 4°C, 7°C, 10°C and 25°C, as well as alkaline peptidase gene (aprX) identification were performed to ascertain the proteolytic activity of these isolates. Pseudomonas spp. were found in 46 samples out of total 125 samples. A total of 67 Pseudomonas spp. were identified. Of Pseudomonas isolates, we obtained extracellular peptidase activity in 7 (10.4%) at 2°C, 17 (25.4%) at 4°C, 24 (35.8%) at 7°C, 39 (58.2%) at 10°C, and 41 (61.2%) at 25°C. The results revealed that a wide diversity of Pseudomonas spp. were present in different non-bovine raw milks, with the ability to produce peptidases at storage temperatures. However, proteolytic activity varied widely among the peptidase-positive isolates. A majority of isolates from yak milk had high proteolytic activity., (Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Glycosylated proteins in milk have been implicated in multiple biological roles. However, the N-glycoprotein components and their complexity in milk whey from dairy animals are not well characterized. Here, a modified proteomics approach consisting of N-glycopeptide enrichment and identification was used to map the N-glycoproteome profile of milk whey from Holstein and Jersey cows, buffaloes, yaks, goats, camels, and horses. A total of 233 N-glycosylation sites, corresponding to 147 N-glycoproteins, were detected in the studied animals. Most of the identified N-glycosylation sites were not characterized in the database and were considered as unknown. Functional analysis of the identified glycoproteins demonstrated that response to stimulus was the most abundant GO category shared in the studied animals according to their annotation. Lysosome, glycosaminoglycan degradation, and extracellular matrix-receptor interaction pathways were shared between Holstein and Jersey cows, and yaks. N-glycoprotein components of milk whey from Holstein and Jersey cows, buffaloes, yaks, and goats were more similar to each other than to those of camels and horses. These results significantly extend the number of known N-glycosylation sites and reveal in-depth composition and potential functions of N-glycoproteins in milk whey, which in turn provides insights to further explore N-glycoprotein biosynthesis in the studied animals., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Milk contains free amino acids (AAs) that play essential roles in maintaining the growth and health of infants, and D-AA isomers are increasingly being recognized as important signalling molecules. However, there are no studies of the different characteristics of chiral AA (C-AA) from different milk origins. Here, UPLC coupled to ion-mobility high-resolution MS (IM-HRMS) was employed to characterize 18 pairs of C-AAs in human, cow, yak, buffalo, goat, and camel milk. The results proved that milk origins can be differentiated based on the D- to L- AA ratio-based projection scores by principal component analysis. The present study gives a deeper understanding of the D- to L- AA ratio underlying the biological functions of different animal milks, and provide a new strategy for the study of AA metabolic pathways.

The objectives of this study were to measure the concentrations of elements in raw milk by inductively coupled plasma-mass spectrometry (ICP-MS) and evaluate differences in element concentrations among animal species and regions of China. Furthermore, drinking water and feed samples were analyzed to investigate whether the element concentrations in raw milk are correlated with those in water and feed. All samples were analyzed by ICP-MS following microwave-assisted acid digestion. The mean recovery of the elements was 98.7 % from milk, 103.7 % from water, and 93.3 % from a certified reference material (cabbage). Principal component analysis results revealed that element concentrations differed among animal species and regions. Correlation analysis showed that trace elements Mn, Fe, Ni, Ga, Se, Sr, Cs, U in water and Co, Ni, Cu, Se, U in feed were significantly correlated with those in milk (p < 0.05). Toxic and potential toxic elements Cr, As, Cd, Tl, Pb in water and Al, Cr, As, Hg, Tl in feed were significantly correlated with those in milk (p < 0.05). Results of correlation analysis revealed that elements in water and feed might contribute to the elements in milk.

Glycosylation of proteins has been implicated in various biological functions and has received much attention; however, glycoprotein components and inter-species complexity have not yet been elucidated fully in milk proteins. N-linked glycosylation sites and glycoproteins in milk fat globule membrane (MFGM) fractions were investigated by combining N-glycosylated peptides enrichment and high-accuracy Q Exactive identification, to map the N-glycoproteome profiles in Holstein and Jersey cows, buffaloes, yaks, goats, camels, horses, and humans. A total of 399 N-glycoproteins with 677 glycosylation sites were identified in the MFGM fractions of the studied mammals. Most glycosylation sites in humans were classified as known and those in the other studied mammals as unknown, according to Swiss-Prot annotations. Functionally, most of the identified glycoproteins were associated with the 'response to stimulus' GO category. N-glycosylated protein components of MFGM fractions from Holstein and Jersey cows, buffaloes, yaks, and goats were more similar to each other compared with those of camels, horses and human. The findings increased the number of known N-glycosylation sites in the milk from dairy animal species, revealed the complexity of the MFGM glycoproteome, and provided useful information to further explore the mechanism of MFGM glycoproteins biosynthesis among the studied mammals., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Several milk metabolites are associated with breeds or species of dairy animals. A better understanding of milk metabolites from different dairy animals would advance their use in evaluating milk traits and detecting milk adulteration. The objective of this study was to characterize the milk metabolite profiles of Chinese Holstein, Jersey, yak, buffalo, goat, camel, and horse and identify any differences using non-targeted metabolomic approaches. Milk samples were tested using nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography-tandem mass spectrometry (LC-MS). Data were analyzed using a multivariate analysis of variance and differences in milk metabolites between Holstein and the other dairy animals were assessed using orthogonal partial least-squares discriminant analysis. Differential metabolites were identified and some metabolites, such as choline and succinic acid, were used to distinguish Holstein milk from that of the other studied animals. Metabolic pathway analysis of different metabolites revealed that glycerophospholipid metabolism as well as valine, leucine, and isoleucine biosynthesis were shared in the other ruminant animals (Jersey, buffalo, yak, and goat), and biosynthesis of unsaturated fatty acids was shared in the non-ruminant animals (camel and horse). These results can be useful for gaining a better understanding of the differences in milk synthesis between Holstein and the other dairy animals., (Copyright © 2016. Published by Elsevier B.V.)

Concentrations of IgA and IgM in milk from normal lactating cows were measured to determine whether they were affected by lactation number, lactation stage, daily milk production, milk composition and somatic cell score (SCS). A total of 284 Chinese Holstein cows showing no signs of mastitis were selected from more than 1600 cows from one farm. Concentrations of IgA and IgM in normal milk were determined by ELISA. Results revealed that average milk IgA and IgM concentrations were 235±11 and 32±3 μg/ml, respectively. IgA and IgM concentrations in milk were found to have an association with lactation number, stage of lactation, daily milk production and SCS. Both SCS and daily milk production were found to be strong significant (P<0·01) direct factors correlating to IgA concentration. Stage of lactation and SCS were found to be strong significant (P<0·01) direct factors correlating to IgM concentration.

BACKGROUND Milk microRNA (miRNA) with bioactivity is beneficial for human health. However, the effect of heat treatment on miRNA in milk is still not clear. In this study, the miRNAs in raw (RM), pasteurized (PM) and ultra‐high‐temperature (UHT) milk (UM) from the same batch were extracted, sequenced and analyzed. RESULTS: The results showed that there was a significant difference in miRNAs between RM and UM, but not between RM and PM. The total read counts of milk miRNAs were significantly decreased by heat treatment, with the least counts in UM (P < 0.05). The average length and GC percentage of miRNAs were significantly reduced by heat treatment (P < 0.05), while there was no significant difference in these terms between RM and PM. The content of miRNAs was verified by qPCR, finding that miR‐17‐5p, miR‐25, miR‐27b and miR‐9‐5p were significantly reduced in UM (P < 0.05) but not significantly affected in PM (except miR‐27b). In addition, the targeting gene ontology enrichment functions of the different presented miRNAs were mostly enriched in biological process, cellular component and molecular function. The top 20 enriched miRNAs with different levels in heat‐treated milk were identified by the Kyoto Encyclopedia of Genes and Genomes enrichment analysis. Interestingly, most of the functions of these miRNA targeting genes are involved in cancer or inflammation activity. CONCLUSION: This study revealed that the bioactive miRNA in RM was lost after UHT treatment but not in pasteurized treatment. © 2021 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

High nitrogen utilization efficiency (NUE) is important for increasing milk protein production and decreasing the feed nitrogen cost and nitrogen emission to the environment. Currently, there is a limited whole picture of the relationship between ruminal bacteriome and the NUE of dairy cows, even though some information has been revealed about the bacteriome and milk or milk protein production of dairy cows. The purpose of this study was to compare the rumen bacterial community in dairy cows with different nitrogen utilization efficiency under the same diet. The natural abundance of 15N between the animal proteins and diet (Δ15N) was used as a simple, non-invasive, and accurate biomarker for NUE in ruminants to mark the individual variation. Dairy cows with high NUE (HE_HP, n = 7), medium NUE (ME_MP, n = 7), and low NUE (LE_LP, n = 7) were selected from 284 Holstein dairy cows with the same diet. Measurement of the rumen fermentation indices showed that the proportion of propionate was higher in HE_HP cows and ME_MP cows than in LE_LP cows (P < 0.05). The diversity of rumen bacterial community was higher in LE_LP cows than in ME_MP cows and HE_HP cows by 16S rRNA sequencing analysis (P < 0.05). Moreover, at the genus level, the relative abundances of Succinivibrionaceae _UCG_001, uncultured_ Selenomonadaceae , and Acidaminococcus were higher in HE_HP cows than in LE_LP cows (P < 0.05). Interestingly, we found that these bacteria were positively correlated with milk protein yield and negatively correlated with Δ15N (P < 0.05). However, Clostridia _UCG_014, Saccharofermentans , Bacilli _RF39, and Desulfovibrio were lower in HE_HP cows and ME_MP cows than in LE_LP cows (P < 0.05), which were negatively correlated with milk protein yield and positively correlated with Δ15N (P < 0.05). In conclusion, the study showed that the diversity and relative abundances of rumen bacteria differed among different NUE cows, indicating that rumen bacteriome contributes to nitrogen metabolism in dairy cows. [ABSTRACT FROM AUTHOR]

In this study, we investigated the species composition and diversity of psychrotrophic bacteria in raw milk from Heilongjiang, Inner Mongolia, Gansu, Henan, Anhui, Jiangsu, Chongqing, and Hunan provinces in China using traditional cultivation and PacBio Single Molecule Real-Time sequencing methods. The isolated psychrotrophic bacteria were highly diverse, which composed of 21 genera and 59 species. Pseudomonas accounted for 58.9% of the total genera while Stenotrophomonas and Enterococcus were also highly represented (above 5.0%). In particular, P. azotoformans occurred at a level of 16.9% and P. paralactis, P. lactis, E. faecalis, and P. marginalis were present in relatively high proportions (above 4.0%). Regional differences were found significantly among the test regions except samples from Heilongjiang and Inner Mongolia were similar. Additionally, differences were observed between days in Henan, Anhui, and Jiangsu samples. Therefore, control strategies must be implemented on regional and season basis. [ABSTRACT FROM AUTHOR]

Psychrotrophic bacteria in raw milk are most well known for their spoilage potential and the economic losses they cause to the dairy industry. Food-related psychrotrophic bacteria are increasingly reported to have antibiotic resistance features. The aim of this study was to evaluate the resistance patterns of Pseudomonas spp. isolated from bulk-tank milk. In total, we investigated the antibiotic susceptibility profiles of 86 Pseudomonas spp. isolates from raw milk. All strains were tested against 15 antimicrobial agents. Pseudomonas isolates were most highly resistant to imipenem (95.3%), followed by trimethoprim-sulfamethoxazole (69.8%), aztreonam (60.5%), chloramphenicol (45.3%), and meropenem (27.9%). Their multiple antibiotic resistance (MAR) index values ranged from 0.0 to 0.8. Whole-genome sequencing revealed the presence of intrinsic resistance determinants, such as BcI, amp C-09, bla CTX-M, opr D, sul 1, dfr E, cat A1, cat B3, cat I, flo R, and cml V. Moreover, resistance-nodulation-cell division (RND) and ATP-binding cassette (ABC) antibiotic efflux pumps were also found. This study provides further knowledge of the antibiotic resistance patterns of Pseudomonas spp. in milk, which may advance our understanding of resistance in Pseudomonas and suggests that antibiotic resistance of Pseudomonas spp. in raw milk should be a concern. [ABSTRACT FROM AUTHOR]

The presence of mycotoxins in feed has the potential to cause significant detriment to animal and human health, and even severe economic implications. Previous studies on the effects of mycotoxins mainly focused on the addition of commercially available mycotoxins into feeds in animals. In the present study, corn meal and cottonseed were kept in warm and humid conditions to allow for mycotoxins produced and then used to substitute 50% and 100% of normal corn meal and cottonseed in diets for lactating cows for 14 days. The results showed that aflatoxin M1, deoxynivalenol, aflatoxin B1, and zearalenone were primary mycotoxins in milk from cows fed the diets. Compared with the control group, feeding the diets containing mildewy corn meal and cottonseed reduced feed intake, milk yield, and milk fat, protein and lactose productions (p > 0.05). No significant difference was observed in the acetate and valerate concentrations, acetate to propionate ratio, and the calculated CH4 production in rumen fluid (p > 0.05), whereas, the propionate, butyrate, isovalerate concentrations were affected (p < 0.05) depending on the content and type of natural mycotoxins. Serum creatinine and total glyceride concentrations were influenced with corn meal and cottonseed fully replaced with the mildewy feeds. Metabolic pathways for small molecule metabolites in milk were altered by dietary mycotoxin exposures, and the changes were mainly associated with amino acid metabolism, glucose metabolism, and energy metabolism. However, cows exposed to natural mycotoxins in the diets were still in healthy conditions and had low somatic cell count in milk. [ABSTRACT FROM AUTHOR]

Abstract: Beta-2-microglobulin (β2m) is an integral component of the Fc receptor (FcRn) heterodimer which is involved in IgG transfer across mammary epithelial cells. The aim of this study was to estimate the frequency of alleles of β2m and the association of SNPs genotypes as well as haplotypes with milk IgG1 concentration and mass. Blood DNA from 189 Chinese Holstein dairy cows was extracted, sequenced and genotyping was analyzed. Milk IgG1 concentrations from these cows were determined by ELISA and evaluated with respect to their β2m genotypes. The results showed that two single nucleotide polymorphisms (SNPs) and one insertion/deletion (indel) of two base pairs, assorted into four haplotypes, were identified. The genotypes and haplotypes of β2m were found to be correlated (P <0.05) with milk IgG1 concentration and mass. Dairy cows with homozygous deletion of β2m had the lowest milk IgG1 concentration and mass. These results indicate that β2m genotypes could serve as a marker to identify dairy cows with high IgG1 milk levels. [Copyright &y& Elsevier]

Simple Summary: Microbiota can enter and persist in raw milk at several stages of the farming chain. The development of culture-independent methods and high-throughput DNA sequencing techniques have improved the approach to study microbiota communities in milk and milk products. This work aimed to determine the effects of farming environments on milk microbiota using the high-throughput DNA sequencing technique, and to elucidate the relationship among the microbiota. The effects of the farming environment on the microbiota in milk can guide farm and management practices to ensure that the milk is not contaminated with bacteria during milking and processing, thereby improving the quality of the milk. The aim of the present study was to determine the effects of farming environments on microbiota in raw milk and to assess the relationship among microbes by 16S rRNA sequencing methods. Samples of raw milk, cow trough water, teat dip cup, teat, teat liner, dairy hall air, cowshed air, feces, feed, and bedding from two farms were collected. The two highest abundant bacterial groups of Moraxellaceae and Staphylococcaceae were found in milk and teat liner samples, respectively, at Zhengzhou farm, Henan Province. Moreover, the two highest abundant bacterial groups of Enterobacteriaceae and Moraxellaceae were found in milk and teat dip cup samples, respectively, at Qiqihar farm, Heilongjiang Province. Source Tracker analysis revealed that the teat liner and teat dip cup were the most important contributors of microbes in milk samples at Zhengzhou farm and Qiqihar farm, respectively, which could be attributed to the management level of the farm. Therefore, disinfection and cleaning procedures should be developed to improve the quality of raw milk. [ABSTRACT FROM AUTHOR]

Antibiotics are increasingly found in dairy products. To ensure consumer safety, rapid screening methods which are accurate and cost-effective are highly in demand. A multiplexed detection technology was tested for quantification of these four kinds of antibiotics simultaneously in milk. The visual microarray technology based on enzyme-linked immunosorbent assays was utilized in this method, which can detect multiple target molecules simultaneously. This colorimetric technology provides direct results that can be detected using common commercial microplate scanners. The limit of detection (LOD) were calculated as 3.30, 3.39, 2.42 and 4.88 μg kg−1 for quinolones, tetracycline, lincomycin and streptomycin, respectively. The limits of quantity (LOQ) were defined as 8.97, 8.43, 5.36 and 10.97 μg kg−1 for quinolones, tetracycline, lincomycin and streptomycin, respectively. The accuracy of method was also adequate for these four substances with the recovery rates ranging from 77.6% to 116.4% were obtained for the detection of practical samples. According to our evaluation, this methodology can be used for rapid screening of antibiotics in internal quality control programs in dairy industry. • The multiplexed detection method for testing veterinary antibiotics in milk were evaluation. • The ELISA-based visualization microarray chip technique using 96-well microplate. • The limit of detection are less than the MRL established in China, the CAC and the EU. • The accuracy of method was adequate for these four veterinary antibiotics. [ABSTRACT FROM AUTHOR]