Lateral lipid organization of the bovine milk fat globule membrane is revealed by washing processes

Evidence for the asymmetric distribution of phospholipids in the milk fat globule membrane (MFGM) was obtained by applying 3 washing processes using aqueous solutions with different degrees of stringency (mild, intermediate, and intensive) to milk fat globule (MFG) surfaces in simulated milk ultrafiltrate buffer. We detected no change in the amount of cholesterol after the mild washing process; however, intensive washing yielded a relative enrichment of surface cholesterol with concomitant damage to the outer bilayer of the MFGM. This finding supports the hypothesis of repartitioning of cholesterol on MFG surfaces during mechanical treatments. An updated model system of lipid organization was developed according to the results of relative depletion of individual phospholipids, as analyzed by HPLC.     

Determination of free fatty acids in milk and dairy products by reversed-phase HPLC with fluorescence detection

A highly sensitive HPLC with fluorescence detection was developed for the determination of free fatty acids (FFAs) in milk and dairy products. For this purpose, the FFAs were extracted from small amounts of milk (1.0 mL), cheese (0.5 g) and butter (0.5 g) using Sep-Pak cartridge columns, and then derivatized to 9-anthrylmethyl esters with 9-anthryldiazomethane (ADAM). Good separations of the ADAM derivatives of 16 FFAs (C4-C18) that exist in milk, cheese and butter, which permitted quantitative estimation of their individual FFAs, were achieved by HPLC on a C18 column (Cadenza CD-C18, 150 x 3 mm i.d.) using gradient elution with methanol and water. The acid values calculated from the contents of individual FFAs were in good agreement with those obtained by the conventional titration method. These results demonstrate that the present HPLC method is simple, sensitive and precise, and could be utilized widely for determination of the FFAs in milk and dairy products.     

HPLC测定乳制品中的甲醛含量

针对奶糖及奶制品的甲醛残留问题,建立了一种灵敏、快速和有效的HPLC-UV检测乳制品中甲醛的方法。牛奶和奶粉样品中甲醛经三氯乙酸(TCA)提取,奶糖另添加乙酸锌和亚铁氰化钾进行辅助沉淀,用1mg/mL2,4-二硝基苯肼(DNPH),在60℃条件下衍生30min后,用HPLC-UV分离测定,外标法定量。该方法对3种奶制品平均加标回收率在95%以上,日内精密度和日间精密度在5%以下,甲醛含量在0.03～4.00μg/mL范围内呈良好的线性关系,检出限和定量限分别为0.03μg/mL和0.10μg/mL,对常见醛类和醇类有较好的抗干扰性。该方法具有操作简单,准确性高,稳定性好,抗干扰性强的特点,是一种测定乳制品中甲醛的有效分析技术。     

Technical note: Measurement of total estrone content in foods. Application to dairy products

Estrone is a powerful growth-inducing hormone that is present in milk, mainly in the form of fatty acid esters, at concentrations that promote growth in experimental animals. We present here a method useful for the measurement of this natural hormone in foods and applied it to several common dairy products. Samples were frozen, finely powdered, and lyophilized then extracted with trichloromethane/methanol; the dry extract was saponified with potassium hydroxide. The free estrone evolved was extracted with ethyl acetate and was used for the estimation of total estrone content through radioimmunoassay. Application of the method to dairy products showed high relative levels of total estrone (essentially acyl-estrone) in milk, in the range of 1 microM, which were halved in skimmed milk. Free estrone levels were much lower, in the nanomolar range. A large proportion of estrone esters was present in all other dairy products, fairly correlated with their fat content. The amount of estrone carried by milk is well within the range, where its intake may exert a physiological response in the sucklings for which it is provided. These growth-inducing and energy expenditure-lowering effects may affect humans ingesting significant amounts of dairy products.     

UPLC-Triple-TOF-MS/MS法分析中国东北地区人乳磷脂的组成

借助超高效液相色谱联用质谱(UPLC-Triple-TOF-MS/MS)分析法,对不同泌乳期人乳中的磷脂进行定性和相对定量,为中国人乳磷脂组成的研究提供参考。本研究从哈尔滨(黑龙江省)、齐齐哈尔(黑龙江省)和吉林(吉林省)地区采集到汉族人初乳(1~5 d)、过渡乳(6~20 d)和成熟乳(21 d~)共计73份。LipidView 1.2用于检索磷脂分子式,PeakView 2.2及其插件MasterView 1.1用于磷脂定性分析,MultiQuant用于磷脂相对定量分析(基于色谱峰面积)。结果显示,人乳中检出磷脂共计60种。其中,磷脂酰胆碱(PC)17种,磷脂酰乙醇胺(PE)25种,磷脂酰丝氨酸(PS)4种,磷脂酰肌醇(PI)5种,鞘磷脂(SM)9种。磷脂酰胆碱(PC)、磷脂酰乙醇胺(PE)、鞘磷脂(SM)、磷脂酰丝氨酸(PS)和磷脂酰肌醇(PI)在总磷脂中的相对含量分别为38.12%、26.97%、29.54%、4.43%和0.94%,各种磷脂的相对含量随泌乳期变化表现出不同的变化趋势,但大多数磷脂的相对含量差异并不显著。     

高效液相色谱法测定乳及乳制品中泛酸分析方法的改进

目的改进和优化了高效液相色谱法(high performance liquid chromatography,HPLC)检测乳及乳制品中泛酸的分析方法。方法对测定泛酸的GB 5413.17-2010《食品安全国家标准婴幼儿食品和乳品中泛酸的测定》的HPLC分析方法的流动相进行优化,然后对优化后的方法进行方法学的验证。结果选择0.05%磷酸水溶液+乙腈作为流动相,色谱图上的目标峰得到完全分离,分离效果好。线性方程为Y=1.3735X+23.0911,r~2=0.99996。方法回收率为83.20%~110.75%,相对标准偏差在3.43%~5.96%之间,具有较好的精确度和重现性。结论该方法快速、简便、准确,适用性好,有效地去除了干扰物质,克服了高效液相色谱检测中泛酸背景值高、干扰多的问题。     

A sensitive HPLC method for measuring bacterial proteolysis and proteinase activity in UHT milk

A sensitive quantitative reversed-phase HPLC method is described for measuring bacterial proteolysis and proteinase activity in UHT milk. The analysis is performed on a TCA filtrate of the milk. The optimum concentration of TCA was found to be 4%; at lower concentrations, non-precipitated protein blocked the HPLC while higher concentrations yielded lower amounts of peptides. The method showed greater sensitivity and reproducibility than a fluorescamine-based method. Quantification of the HPLC method was achieved by use of an external dipeptide standard or a standard proteinase.     

基于三重实时荧光聚合酶链反应构建黄牛和牦牛源性成分同步检测方法

目的 建立一种拥有内源质控的三重实时荧光聚合酶链反应(polymerase chain reaction,PCR)技术,用以快速鉴定肉和乳中黄牛和牦牛的源性成分。方法 首先对黄牛肉、牦牛肉、黄牛奶、牦牛奶等7种动物产品进行特异性检测,同时通过稀释梯度方法验证此方法的绝对检出限(limit of detection, LOD),最后通过黄牛和牦牛肉掺假模拟试验确定该方法的相对灵敏度。结果 该方法的特异性强,能特异性地检测到来源于黄牛和牦牛肉和乳的DNA,稳定扩增的内源质控有效地避免了假阴性结果,本方法针对黄牛奶的绝对LOD为2.5×10^-3～5.0×10^-3 ng,针对牦牛奶的绝对LOD为2.5×10^-3～5.0×10^-2 ng,本方法对黄牛肉和牦牛肉混合肉的相对灵敏度可达0.1%牦牛肉。结论 所建立的三重实时荧光PCR方法特异性强、灵敏度高,可实现黄牛和牦牛源性以及内源质控的同步检测,又能通过内源质控排除实验假阴性结果。     

A method for separation and quantification of phospholipid classes in human milk

A simple, isocratic method for separating the major phospholipid classes of human milk by HPLC is described. Resolution of phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, lysophosphatidylethanolamine, phosphatidylcholine, and spingomyelin from human milk phospholipids was achieved in 30 min on a silica column. Total phospholipids were injected in 50 microliter of chloroform:diethyl ether (1:2, vol/vol) and eluted with a solvent mixture of acetonitrile:methanol:sulfuric acid (100:3:.05, vol/vol/vol) at a flow rate of 2.5 ml/min. Fractions were collected and each phospholipid class quantified by analysis of inorganic phosphorus after sulfuric acid digestion. A repeatability study with 19 samples had a coefficient of variation of 5.3%. The analytical recoveries of phospholipid standards averaged 98%. Recoveries varied with phospholipid class; variation was greatest with spingomyelin.     

Comparison of ion-specific electrode and high performance liquid chromatography methods for the determination of iodide in milk

Two methods for the determination of I⁻ in raw and processed milk were examined. A simple ion-specific electrode (ISE) method was compared against a more complex HPLC reference technique. Accuracy and precision were evaluated both within and between the 2 methods. Both methods yielded good recoveries for Ion spiked samples, ranging from 87 to 114% for ISE and 91 to 100% for HPLC. Within-run repeatability and between-run reproducibility were superior with the HPLC method, but were still more than acceptable with the ISE technique. Overall agreement of paired results between ISE and HPLC methods was good (r² = 0.85 on raw herd milk; r² = 0.84 on processed milk). The ISE method had a significant positive bias relative to the HPLC reference method. Both methods lend themselves well to the measurement of I⁻ in raw or processed milk. Given its relatively low cost and ease of use, the ISE method is well suited as a screening method. The impressive accuracy, precision, selectivity, and limit of detection of the HPLC technique make it an ideal confirmation method.     

Protein profile of dairy products: Simultaneous quantification of twenty bovine milk proteins

While proteomic techniques allow the identification and relative quantification of thousands of proteins in a single run, methods for absolute quantification remain laborious. In this study, a newly developed multiple reaction monitoring (MRM) method using liquid chromatography mass spectrometry (LC-MS) that enables the simultaneous quantification of twenty key milk proteins is presented. The selected proteins comprise all individual caseins, the major whey proteins and most well-known milk fat globule membrane (MFGM) proteins. For validation, the twenty milk proteins in raw milk, raw cream, raw milk Emmental cheese and whey, were quantified as well as in eighteen commercial heat-treated dairy products. The method presented is ideally suited for various applications, for example, the comparison of the protein patterns in raw milk of cows at different stages of lactation or of different breeds.     

Analysis of phospho- and sphingolipids in dairy products by a new HPLC method

Dairy phospho- and sphingolipids are gaining interest due to their nutritional and technological properties. A new HPLC method, using an evaporative laser light-scattering detector, was developed, which enabled excellent separation of glucosylceramide, lactosylceramide, phosphatidic acid, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylcholine, sphingomyelin, and lysophosphatidylcholine in less than 21 min, including the regeneration of the column. No loss of column performance was observed after 1500 runs because an acid buffer was used. The output signal of the evaporative laser light scattering detector was highly dependent of the flow of the carrier gas and the temperature of the nebulizer, and was maximized by means of a response surface experimental design. Finally, raw milk, cream, butter, buttermilk, Cheddar whey, quarg, and Cheddar cheese were analyzed for their polar lipid content. The absolute values varied substantially (0.018 to 0.181 g/100 g of product). Significant differences were found in the relative content of each polar lipid class among the analyzed products.     

HPLC quantification of biogenic amines in cheeses: correlation with PCR-detection of tyramine-producing microorganisms

The consumption of food and beverages containing high amounts of biogenic amines (BA) can have toxicological effects. BA found in foods and beverages are synthesized by the microbial decarboxylation of certain amino acids. This paper reports the concentrations of BAs in a number of commercial cheeses, as determined by HPLC. The cheeses studied were made from raw and pasteurized milk of different origin, and were subjected to different ripening periods. BA concentrations were lower in short ripening period than in long ripening period cheeses, and higher in cheeses made from raw milk than in those made from pasteurized milk. The highest BA concentrations were recorded in blue cheeses made from raw milk. Tyramine was the most commonly recorded and abundant BA. The presence of tyramine-producing bacteria was determined by PCR, and a good correlation obtained between the results of this method and tyramine detection by HPLC. These methods could be used to complement one another in the detection and quantification of tyramine in cheese prevention of tyramine accumulation in cheese.     

应用傅立叶变换近红外光谱检测婴幼儿配方奶粉中的乳糖含量

利用傅立叶变换近红外光谱（FT-NIR）技术结合偏最小二乘法（PLS）对婴幼儿配方奶粉中的乳糖进行快速检测分析。搜集 94个不同产地、不同品牌的婴幼儿配方奶粉中乳糖的实验室数据,并采集婴幼儿配方奶粉的近红外光谱图,选择最优的光谱预处理方法,优化、验证和建立模型,并预测6个未知品牌的婴幼儿配方奶粉样品的乳糖含量。结果表明,不同阶段的婴幼儿配方奶粉有非常相似 的近红外特征图谱,图谱处理后的方差为95.423 5%,该模型测定的乳糖值与高效液相色谱（HPLC）法测定的乳糖值之间的平均相对误 差均≤0.67%,相对标准偏差（RSD）均≤0.88%,均符合误差范围。该方法可以无损、快速、高效地测定婴幼儿配方奶粉中的乳糖含量。     

液相色谱法与氨基酸分析法测定食品中甘氨酸的比较研究

建立对食品中游离甘氨酸经提取、净化等处理后,邻苯二甲醛衍生,经高效液相色谱测定分析,并将该方法与氨基酸分析仪测定甘氨酸的方法进行比较研究。通过对市售的液体乳、含乳饮料(品)类和植物蛋白饮料(品)类中游离甘氨酸分别应用两种方法进行测定,结果表明,氨基酸分析仪法测定甘氨酸具有测定结果稳定准确、检出限低和操作简单的特点,而液相色谱法(荧光检测器)测定甘氨酸检测时间快,检出限也能满足常规检测的需要,因此,在实际应用中,两种方法均可采用,可作为互相验证的方法。     

Cholesterol content and lipid composition of low fat dairy products

The lipid composition of different products obtained from the same raw milk was investigated. The cholesterol content of skim milk (0.05% total fat) and butter milk (0.57% total fat) lipids was 4.20% and 1.31%, respectively, while raw milk lipids and butter fat contained 0.34% or 0.30%, respectively. As the cholesterol content of these low fat milk products increases with decreasing fat content, equations were derived to estimate the fat-related cholesterol content from the value analysed in butter fat. In contrast to the fat-related cholesterol content, the product-related content is still relatively small in skim and butter milk. Their fat-related cholesterol content was found to be influenced by drying. The high content of phospholipids in skim and butter milk led to considerable changes in the fatty acid composition and affected the detection of foreign fat by butyric acid as well as by triglycerides. Skim and butter milk lipids contained only 2.41% or 2.33% butyric acid, respectively, instead of 3.36% found in raw milk lipids. Further, the overlap of phospholipids as well as cholesterol with the gas chromatographic triglyceride pattern resulted in calculated foreign fat contents of more than 20%.     

Deterioration of protein fraction by Maillard reaction in dietetic milks.

The development of the Maillard reaction in pasteurized, UHT and in-bottle sterilized dietetic milks was studied. In these products damage caused by heat treatments could increase as a result either of the addition of various ingredients or of manufacturing processes that alter their content of reducing carbohydrates. Protein damage was evaluated by measuring furosine by reversed-phase ion-paired HPLC. The levels of furosine detected made it possible to assess the amounts of biologically unavailable lysine. In all milks analysed blocked lysine values were < 340-350 mg/g total lysine, the level at which lysine becomes the limiting amino acid in milk. Pasteurized dietetic milks had levels of blocked lysine similar to that in ordinary pasteurized cows' milk. In some UHT and in-bottle sterilized dietetic milks their different composition resulted in an increase in the blocked lysine content. In some in-bottle sterilized milks, protein damage greatly reduces the beneficial effects of milk as a dietary supplement. Lactose-free milks, which are more susceptible to protein deterioration because of their higher content of reducing carbohydrates, were also analysed after storage at 20 degrees C and at < or = 4 degrees C. At the end of their recommended storage times, they contained limited amounts of blocked lysine only if they had been stored at < or = 4 degrees C.     

摄食不同来源磷脂对大鼠脂质代谢及脑内磷脂脂肪酸组成的影响

研究了摄食不同来源磷脂对大鼠脂质代谢及其脑内磷脂脂肪酸组成的影响。雄性SD大鼠按体重随机分为大豆油对照组(添加9%)、牛乳磷脂组(添加5%)、大豆磷脂组(添加5%)、蛋黄磷脂组(添加5%),喂食3周。检测了血清总胆固醇(TC)、甘油三酯(TG)、高密度脂蛋白胆固醇(HDL-C)、游离脂肪酸(FFA)及肝脏TC、TG、磷脂(PL)的含量,并用气相色谱法测定了脑内磷脂脂肪酸的组成变化。结果显示:与大豆油对照组相比,3种磷脂均不同程度提高了大鼠体重、脏器指数,蛋黄磷脂效果显著;3种磷脂不同程度降低了血清TC、TG和FFA含量,牛乳磷脂降低血清FFA显著,大豆磷脂降低血清TC、TG显著,蛋黄磷脂降低FFA显著,大豆磷脂显著提升了血清HDL-C含量;3种磷脂不同程度降低了肝脏TC、TG、PL含量,牛乳磷脂与大豆磷脂降低肝脏TG、TC显著,而蛋黄磷脂降低肝脏TG显著;3种磷脂对脑内磷脂脂肪酸组成的影响各不相同,牛乳磷脂显著提高了脑内磷脂饱和脂肪酸含量,而大豆磷脂和蛋黄磷脂提高了DHA等多不饱和脂肪酸含量。研究表明,3种磷脂均有降血脂、肝脂作用,以大豆磷脂作用尤为明显,大豆磷脂和蛋黄磷脂的益智作用可能优于牛乳磷脂。     

Comparison of High Performance Liquid Chromatography and Enzymatic Method for the Measurement of Lactose in Milk

A high performance liquid chromatographic (HPLC) procedure was compared with an enzymatic method for the measurement of lactose in milk. A new rapid HPLC sample preparation technique was used for precipitating milk proteins with 2-propanol. Results indicated that lactose detected in milk (expressed as percentage) was consistently lower with the enzymatic method than the HPLC procedure. Mean difference between the two methods was 0.15% with a range of 0.04 to 0.51. Coefficient of variability for the HPLC and enzymatic methods were 1.02 and 2.75, respectively. Recovery of added lactose in milk by HPLC was 100.3% for whole milk, 97.1% for low fat milk, and 99.5% for skim milk.     

实时荧光定量PCR法鉴定发酵乳中嗜热链球菌

该研究通过系统发育树分析确定目标基因,根据目的基因设计特异性引物和探针,建立一种能够快速准确鉴定发酵乳中嗜热链球菌的实时荧光定量聚合酶链式反应(RT-fqPCR)法,通过特异性、灵敏性和抗干扰实验对所建立方法进行验证,并使用该方法对市售的60份标识含有嗜热链球菌的发酵乳样品进行检测。结果表明,rec A基因具有种间特异性,种间差异率>10%,以其为目的基因建立的RT-fqPCR方法能够特异性的检测嗜热链球菌;绝对灵敏度达1 pg/μL,相对灵敏度达10³CFU/m L;在培养物水平和基因组水平抗干扰能力良好。采用该方法从60份标识含有嗜热链球菌的发酵乳样品中均能检测出嗜热链球菌,说明实时荧光定量PCR方法能够快速、准确的对发酵乳中嗜热链球菌进行检测。