LTQ-Orbitrap 液- 质联用技术对水牛奶酪蛋白的鉴定

建立一种快速、高效的分析酪蛋白组分氨基酸序列的LTQ-Orbitrap质谱方法。为了研究水牛奶酪蛋白、乳牛奶酪蛋白和山羊奶酪蛋白的氨基酸序列组成及氨基酸的替换现象,采用LTQ-Orbitrap液-质联用技术分别对乳源酪蛋白的4种主要组分进行分析,搜索数据库获得4种组分的氨基酸全序列。与水牛奶4种酪蛋白组分进行比对。结果表明,乳牛奶的4种酪蛋白发生氨基酸替换的部位和比率明显小于山羊奶。这意味着与水牛奶酪蛋白相比,乳牛奶酪蛋白氨基酸的稳定性优于山羊奶酪蛋白。     

流式分析技术快速定量检测牛乳中大肠杆菌O157:H7

建立一种基于流式分析技术的快速定量检测牛乳中大肠杆菌O157:H7的方法。用偶联有异硫氰酸荧光素的大肠杆菌O157单克隆抗体对大肠杆菌O157:H7进行特异性标记,通过优化抗体反应条件,建立流式检测方法,然后对磷酸盐缓冲溶液（phosphate buffer saline,PBS）和人工污染牛乳样品中不同浓度的大肠杆菌O157:H7进行定量检测。本研究建立的流式检测方法的在PBS中的检测范围为2.57×103～1.12×108?CFU/mL,灵敏度达到2.57×103?CFU/mL。将所建立的流式检测方法应用于牛乳样品检测,当人工污染牛乳样品中大肠杆菌O157:H7的浓度在2.31×104～1.48×108?CFU/mL之间时,流式检测方法与平板计数方法检测结果基本一致,方法的灵敏度为2.31×104?CFU/mL,检测时间为35?min。该方法能快速、定量地检测出牛乳样品中的大肠杆菌O157:H7,在食源性致病菌的快速筛查和监控中具有重要的应用价值。     

不同泌乳期人乳与牛乳中游离氨基酸的对比

利用同位素标记相对和绝对定量技术结合高效液相色谱-串联质谱技术对不同泌乳期人乳与牛乳中游离氨基酸的种类及含量进行检测及对比分析。结果表明,人乳中游离氨基酸种类较牛乳更为丰富,含量也高于牛乳,且随着泌乳时间的延长其总量呈下降趋势。牛初乳、牛常乳、人初乳和人常乳中游离氨基酸总量分别为0.32、0.16、0.63?g/L和0.37?g/L。实验测定的42?种游离氨基酸中,人常乳中检出35?种,牛常乳中测得31?种,其中人常乳中有25?种游离氨基酸的含量高于牛常乳,人乳中组氨酸、苯丙氨酸、苏氨酸等含量显著高于牛乳（P＜0.05）,游离谷氨酸在人初乳、人常乳、牛常乳中含量均为最高,而牛初乳中游离牛磺酸含量最高。本研究分析了人乳、牛乳中游离氨基酸种类和含量的差异,可为详细的研究母乳氨基酸功能和氨基酸代谢组学提供了一定的理论依据,也可为生产婴幼儿奶粉和功能性乳制品提供参考。     

Meta-analysis of the effect of white clover inclusion in perennial ryegrass swards on milk production

There is increased demand for dairy products worldwide, which is coupled with the realization that consumers want dairy products that are produced in a sustainable and environmentally benign manner. Forage legumes, and white clover (Trifolium repens L.; WC) in particular, have the potential to positively influence the sustainability of pasture-based ruminant production systems. Therefore, there is increased interest in the use of forage legumes because they offer opportunities for sustainable pasture-based production systems. A meta-analysis was undertaken to quantify the milk production response associated with the introduction of WC into perennial ryegrass swards and to investigate the optimal WC content of dairy pastures to increase milk production. Two separate databases were created. In the grass-WC database, papers were selected if they compared milk production of lactating dairy cows grazing perennial ryegrass-WC (GC) swards with that of cows grazing perennial ryegrass-only swards (GO). In the WC-only database, papers were selected if they contained milk production from lactating dairy cows grazing on GC swards with varying levels of WC content. Data from both databases were analyzed using mixed models (PROC MIXED) in SAS (SAS Institute, Cary, NC). Within the grass-WC database, where mean sward WC content was 31.6%, mean daily milk and milk solids yield per cow were increased by 1.4 and 0.12 kg, respectively, whereas milk and milk solids yield per hectare were unaffected when cows grazed GC compared with GO swards. Stocking rate and nitrogen fertilizer application were reduced by 0.25 cows/ha and 81 kg/ha, respectively, on GC swards compared with GO swards. These results highlight the potential of GC production systems to achieve similar levels of production to GO systems but with reduced fertilizer nitrogen inputs, which is beneficial from both an economic and environmental point of view. In the context of increased demand for dairy products, there may be potential to increase the productivity of GC systems by increasing fertilizer nitrogen use to increase stocking rate and carrying capacity while also retaining the benefit of WC inclusion on milk production per cow.     

Varying the ratio of Lys:Met while maintaining the ratios of Thr:Phe,Lys:Thr,Lys:His,and Lys:Val alters mammary cellular metabolites,mammalian target of rapamycin signaling,and gene transcription

Amino acids are not only precursors for but also signaling molecules regulating protein synthesis. Regulation of protein synthesis via AA occurs at least in part by alterations in the phosphorylation status of mammalian target of rapamycin (mTOR) pathway proteins. Although the ideal profile of Lys:Met to promote milk protein synthesis during established lactation in dairy cows has been proposed to be 3:1, aside from being the most-limiting AA for milk protein synthesis, the role of Met in other key biologic pathways such as methylation is not well characterized in the bovine. The objective of this study was to determine the influence of increasing supplemental Met, based on the ideal 3:1 ratio of Lys to Met, on intracellular metabolism related to protein synthesis and mTOR pathway phosphorylation status. MAC-T cells, an immortalized bovine mammary epithelial cell line, were incubated (n = 5 replicates/treatment) for 12 h with 3 incremental doses of Met while holding Lys concentration constant to achieve the following: Lys:Met 2.9:1 (ideal AA ratio; IPAA), Lys:Met 2.5:1 (LM2.5), and Lys:Met 2.0:1 (LM2.0). The ratios of Thr:Phe (1.05:1), Lys:Thr (1.8:1), Lys:His (2.38:1), and Lys:Val (1.23:1) were the same across the 3 treatments. Applying gas chromatography–mass spectrometry metabolomics revealed distinct clusters of differentially concentrated metabolites in response to Lys:Met. Lower Phe, branched-chain AA, and putrescine concentrations were observed with LM2.5 compared with IPAA. Apart from greater intracellular Met concentrations, further elevations in Met level (LM2.0) led to greater intracellular concentrations of nonessential AA (Pro, Glu, Gln, and Gly) compared with IPAA and greater essential AA (EAA; Met, Ile, and Leu) and nonessential AA (Pro, Gly, Ala, Gln, and Glu) compared with LM2.5. However, compared with IPAA, mRNA expression of β-casein and AA transporters (SLC7A5, SLC36A1, SLC38A2, SLC38A9, and SLC43A1) and mTOR phosphorylation were lower in response to LM2.5 and LM2.0. Overall, the results of this study provide evidence that increasing Met while Lys and the ratios of Phe, Thr, His, and Val relative to Lys were held constant could increase the concentration and utilization of intracellular EAA, in particular branched-chain AA, potentially through improving the activity of AA transporters partly controlled by mTOR signaling. Because EAA likely are metabolized by other tissues upon absorption, a question for future in vivo studies is whether formulating diets for optimal ratios of EAA in the metabolizable protein is sufficient to provide the desired levels of these AA to the mammary cells.     

Short communication: Variability in milk urea nitrogen and dairy total mixed ration composition in the northeastern United States

The main objective of this survey was to examine variability in milk urea nitrogen (MUN) for Dairy Herd Improvement Association (DHIA) herds in the northeastern United States (the Northeast), examine trends in dairy cow diet composition, and determine potential relationships for MUN and diet composition. Trends in milk fat and protein concentrations, milk yield, days in milk on test day, and lactation number of the cows were also evaluated. The data set for the survey included 10,839,461 DHIA dairy cow records from 2004 to 2015 for 13 states (CT, DE, MA, MD, ME, NH, NJ, NY, PA, RI, VA, VT, and WV) and was retrieved from Dairy Records Management Systems (Raleigh, NC). Average (across states and years) milk yield, milk fat, and milk protein were 31.6 ± 0.24 kg/d, 3.85 ± 0.021%, and 3.13 ± 0.013%, respectively. No obvious trends were observed for milk fat or protein content, but milk yield steadily increased during the survey period. Milk urea N concentration averaged 13.3 ± 0.13 mg/dL, with no obvious or consistent trends. Examination of variability in dairy feed cost and all milk price for the Northeast indicated that high MUN generally coincided with high feed cost and high milk price. For the diet composition survey, 9,707 records of total mixed ration (TMR) analyses, unrelated to the milk composition data set, from the Cumberland Valley Analytical Service (Maugansville, MD) database were examined. Concentration of TMR crude protein (CP) decreased from 17.1% in 2007 to 16.4% in 2015, but there was not an obvious trend in soluble protein concentration. Concentration of TMR neutral detergent fiber (NDF) and 24-h in vitro NDF degradability declined steadily during the survey period and was accompanied by a steady increase in TMR starch concentration. Examination of these unrelated data sets revealed lack of correlation between MUN and diet chemical composition. Thus, we conclude that individual cow MUN in Northeast dairy herds fluctuated between 2004 and 2015. It appeared that MUN followed variability in feed cost; however, ration feed ingredient data were not available to better define the reasons for the variations in MUN.     

Outdoor grazing of dairy cows on pasture versus indoor feeding on total mixed ration: Effects on gross composition and mineral content of milk during lactation

The influence of feeding system and lactation period on the gross composition, macroelements (Ca, P, Mg, and Na), and trace elements (Zn, Fe, Cu, Mo, Mn, Se, and Co) of bovine milk was investigated. The feeding systems included outdoor grazing on perennial ryegrass pasture (GRO), outdoor grazing on perennial ryegrass and white clover pasture (GRC), and indoors offered total mixed ration (TMR). Sixty spring-calving Holstein Friesian dairy cows were assigned to 3 herds, each consisting of 20 cows, and balanced with respect to parity, calving date, and pre-experimental milk yield and milk solids yield. The herds were allocated to 1 of the 3 feeding systems from February to November. Milk samples were collected on 10 occasions over the period June 17 to November 26, at 2 or 3 weekly intervals, when cows were on average 119 to 281 d in lactation (DIL). The total lactation period was arbitrarily sub-divided into 2 lactation periods based on DIL, namely mid lactation, June 17 to September 9 when cows were 119 to 203 DIL; and late lactation, September 22 to November 26 when cows were 216 to 281 DIL. With the exception of Mg, Na, Fe, Mo, and Co, all other variables were affected by feeding system. The GRO milk had the highest mean concentrations of total solids, total protein, casein, Ca, and P. The TMR milk had the highest concentrations of lactose, Cu, and Se, and lowest level of total protein. The GRC milk had levels of lactose, Zn, and Cu similar to those of GRO milk, and concentrations of TS, Ca, and P similar to those of TMR milk. Lactation period affected all variables, apart from the concentrations of Fe, Cu, Mn, and Se. On average, the proportion (%) of total Ca, P, Zn, Mn, or Se that sedimented with the casein on high-speed ultracentrifugation at 100,000 × g was ≥60%, whereas that of Na, Mg, or Mo was ≤45% total. The results demonstrate how the gross composition and elemental composition of milk can be affected by different feeding systems.     

Short communication: Relationships among plasma and milk vitamin B12, plasma free fatty acids,and blood β-hydroxybutyrate concentrations in early lactation dairy cows

This study was undertaken to evaluate the relationship between plasma and milk concentrations of vitamin B₁₂ as well as the relationship between plasma or milk concentrations of vitamin B₁₂ and plasma concentration of free fatty acids (FFA) or blood concentration of β-hydroxybutyrate (BHB) of early lactating Ayrshire (AY) and Holstein (HO) cows. A total of 44 dairy herds (7 AY and 37 HO herds) and 62 AY (21 in first, 19 in second, and 22 in third and more lactations) and 228 HO (51 in first, 74 in second, and 103 in third and more lactation) cows between 3 and 40 d in milk were involved in the study. Hand-stripped milk samples and blood samples were taken 6 h after the morning milking. Milk and plasma samples were analyzed for vitamin B₁₂ concentration and plasma samples were analyzed for FFA concentration. A handheld device was used for blood BHB concentration determination. Thresholds for elevated plasma FFA concentration and hyperketonemia were set at ≥0.70 and ≥1.2 mmol/L, respectively. Vitamin B₁₂ concentration in milk of AY primiparous cows [2,557 (1,995–3,276) pg/mL] was lower than in milk from HO primiparous cows [3,876 (3,356–4,478) pg/mL], whereas no difference was observed among other parities and breeds. Regardless of breeds, plasma concentration of vitamin B₁₂ of first and second parities was lower than plasma concentration of third and more lactation cows. Milk vitamin B₁₂ concentration was positively correlated with plasma vitamin B₁₂ concentration, but the relationship was stronger for AY (ρ averaging 0.63) than for HO cows (ρ averaging 0.36). For AY and HO breeds, a significant relationship between milk or plasma vitamin B₁₂ concentrations and plasma FFA concentration (ρ between 0.29 and 0.59) was observed. Moreover, cows with elevated plasma FFA concentration had greater milk and plasma vitamin B₁₂ concentrations than cows with normal plasma FFA concentration. No relationship between vitamin B₁₂ concentration in milk or plasma and blood BHB concentration and hyperketonemia was noted. In summary, milk is not well correlated with plasma vitamin B₁₂ concentration for HO. It could be hypothesized that elevated plasma concentration of FFA could have a negative effect on the use of vitamin B₁₂ by cow cells, which increases the concentration of the vitamin in plasma and its secretion in milk.