高效液相色谱法测定羊乳中的乳铁蛋白

为开发羊乳中乳铁蛋白的高效液相色谱测定方法及报告羊乳中乳铁蛋白的分布规律。本研究采集哈尔滨地区羊乳样品(n=24)和经过巴氏杀菌的羊乳样品(n=24),采用高效液相色谱测定其乳铁蛋白质量浓度。样品脱脂后,调整pH沉淀酪蛋白,采用C8色谱柱分离,二极管阵列检测器210nm检测,以水、乙腈、三氟乙酸为流动相进行线性梯度洗脱。方法回收率94.2%,线性范围50~300μg/mL,检出限12μg/mL,定量限40μg/mL。原料羊乳中乳铁蛋白的质量浓度的平均值122.1μg/mL,巴氏杀菌后羊乳中乳铁蛋白的平均值103.9μg/mL。巴氏杀菌对羊乳中乳铁蛋白浓度无影响。     

反相高效液相色谱法快速测定乳铁蛋白含量

目的建立一种反相高效液相色谱法(RP-HPLC)快速测定乳铁蛋白在乳原料及乳制品中含量的方法。方法根据酪蛋白在等电点(p H 4.6)凝聚沉淀特点,采用RP-HPLC法,用p H4.6醋酸盐缓冲液溶解分离乳铁蛋白,用C18色谱柱快速分离,用流动相A为0.1%三氟乙酸水溶液,流动相B为含有0.09%(V:V)三氟乙酸的90%(V:V)乙腈水溶液,进行梯度洗脱,检测波长214 nm,流速0.7 m L/min。结果乳铁蛋白在10~500 mg/L之间呈现良好的线性,相关系数为0.9999,平均回收率为98.0%,RSD=0.8%(n=9)。结论该方法推广性高、快速,简便、准确,可用于乳铁蛋白原料、奶粉、牛奶及保健品等中乳铁蛋白的含量检测。     

高效液相色谱法检测乳及乳制品中的呋喃苯烯酸钠残留

建立乳及乳制品中呋喃苯烯酸钠残留检测的高效液相色谱方法。采用乙腈提取,正己烷液液净化,高效液相色谱法测定。结果表明：在10.0～200.0ng/mL 线性范围内,色谱峰面积和呋喃苯烯酸钠浓度的线性关系良好,相关系数大于0.9996,回收率为83.5%～91.9%,检测限为20.0μg/kg。本方法简便、快速,适合大量进出口样品的监控和检测。     

超高效液相色谱法测定乳及乳制品中维生素D的含量

目的建立用正相高效液相色谱硅胶柱净化维生素D,通过反相超高效液相色谱测定乳及乳制品中维生素D的含量的一种快速检测方法。方法样品经氢氧化-–钾皂化后,用石油醚萃取,萃取液经无水硫酸钠脱水、旋蒸,定容,用正向色谱净化,反相超高效液相色谱用以0.1mL/min流速的甲醇/水(97:3V:V)为流动相,采用WatersACQUITY UPLC BEH C18色谱柱分离,紫外检测器检测,内标法定量。结果维生素D3浓度在0.05–1.00μg/mL内标准曲线相关系数(r~2)均大于0.995。在质量分数为0.5、1.0、2.0μg/mL 3个加标水平下,加标回收率为65.0–115.0%,相对标准偏差为0.1%–1.4%。结论该方法简便、快速、实用、准确,各项技术指标满足标准的要求,可用于乳及乳制品中维生素D的定量检测。     

乳与乳制品中三聚氰胺快速检测方法

研究了高效液相色谱法检测乳与乳制品中三聚氰胺的检测方法。采用三氯乙酸和乙腈提取后,用高效液相色谱测定。该方法的检出限为0.5 mg/kg,相对标准偏差为1.09%~1.99%,回收率在95.15%~99.12%之间。与GB/T22388-2008《原料乳与乳制品中三聚氰胺检测方法》第一法高效液相色谱法(HPLC法)相比,处理时间缩短了约3 h。     

高效液相色谱法测定牛乳中α-乳白蛋白

利用常规的C18色谱柱的高效液相色谱建立测定牛乳中主要过敏蛋白α-乳白蛋白含量的方法。色谱条件:色谱柱Alltima-C18(4.6 mm×200 mm,5μm),柱温为45℃,UV检测波长215 nm,流动相A为含0.1%三氟乙酸的超纯水,流动相B为乙腈:超纯水:三氟乙酸=400:100:0.5,采用梯度洗脱方法,流速0.8 mL/min。结果表明:α-乳白蛋白的线性范围分别为50～1 000μg/mL(r=0.990 9);α-乳白蛋白平均回收率为97.27%,RSD=0.76%(n=5);该方法简便、准确,适合于乳制品中α-乳白蛋白含量的测定。     

基于特征肽段结合超高效液相色谱-串联质谱法的乳制品真实性鉴别方法研究

目的 基于超高效液相色谱-串联质谱法(ultra performance liquid chromatography-tandem mass spectrometry, UPLC-MS/MS)建立乳制品中乳铁蛋白、乳桥蛋白、奶牛A1 β-酪蛋白和A2 β-酪蛋白、水牛A2 β-酪蛋白的检测方法, 开展市售乳制品中功能性蛋白质检测和乳制品真实性鉴评分析。方法 乳蛋白经酶解后, 应用超高效液相色谱-四极杆-静电场轨道阱高分辨质谱法(ultra performance liquid chromatography-triple quadrupole-Orbitrap high resolution mass spectrometry, UPLC-Q/Orbitrap HRMS)和UPLC-MS/MS筛选获得可用于定性定量分析的特征肽段, 基于UPLC-MS/MS建立同时检测LF、LPN、奶牛A1 β-CN、奶牛A2 β-CN和水牛A2 β-CN的方法并应用于乳制品检测。结果 5种待测蛋白在相应的浓度范围内线性关系良好(r2>0.99), 加标回收率在82.1%~105.5%之间, 相对标准偏差均小于5%, 78份乳制品的检测结果表明同类样品LF含量差异较大,不同类液体乳中LPN均值差异不大。结论 本方法性能良好, 适用于乳制品中乳桥蛋白等功能性蛋白的同时检测, 也可以实现乳制品中乳蛋白物种来源的鉴别。     

肝素亲和柱在乳铁蛋白检测中的应用优化

乳铁蛋白是一种极具营养价值的活性糖蛋白,广泛应用到婴幼儿乳粉中,目前国内外未建立乳铁蛋白测定相关的标准。将肝素亲和柱(HiTrap TM Heparin HP柱)或相当者应用到乳铁蛋白检测前处理的净化过程中,建立一种高效、便捷的食品中乳铁蛋白测定的分析方法。试样中的乳铁蛋白经磷酸氢二钠溶液(0.20 mol/L,p H=8.00±0.5)提取,提取液经肝素亲和柱净化后,用磷酸氢二钠溶液淋洗,磷酸氢二钠-氯化钠溶液洗脱,并定容至3.0 mL。采用反相高效液相色谱柱分离,以紫外或者二极管阵列检测器于280 nm处检测,外标法定量。该方法系首次提出将肝素亲和柱应用到乳铁蛋白检测前处理过程中。该检测方法适合检测机构或乳品企业日常样品中乳铁蛋白的检测。     

乳制品中低聚木糖的测定研究

<正>运用酸水解方法和高效液相色谱-示差折光技术测定木糖,从而建立乳制品食品中低聚木糖的检测方法。乳制品经醇沉淀后,酸水解成木糖,液相色谱示差检测器检测。木糖在0.5~4.0 mg/ml测定线性范围内,呈良好线性关系,相关系数均在0.999以上,加标回收率为90%~105%。本方法快速、灵敏、重现性好,满足测定乳制品中的低聚木糖。低聚木糖是新型低聚糖类产品,低聚木糖是由2-7个木糖分子以β-1,4糖苷键结合而成的功能性聚合糖,以其功效强而受到业内人士的关注。《低聚木糖》QB/T2984-2008标准的实施,     

钙型糖柱—高效液相色谱—蒸发光散射检测器法测定牛乳中的乳糖

目的：实现牛乳中乳糖含量快速检测。方法：建立基于钙型糖柱和高效液相色谱测定牛乳中乳糖的新方法。牛乳通过0.2 g/mL三氯乙酸溶液沉淀蛋白质，所得滤液稀释100倍并过滤膜后进入高效液相色谱系统，经过钙型糖柱分离，蒸发光散射检测器进行检测。结果：乳糖在线性范围（20～100 mg/L）内，色谱峰面积和质量浓度之间具有较好的相关性，R²为0.999 8。乳糖加标水平为15，40，80 mg/g时，回收率为90.96%～98.23%，检出限为3.6 μg/g，定量限为12 μg/g，在6 min内实现乳糖浓度的测定。并且用该方法测定11种市售乳样品，测得结果与国标（GB 5009.8—2016）法基本一致。结论：该方法的精密度、重复性、加标回收率均符合有关规定，检测结果准确且耗时短，适用于快速检测牛乳中的乳糖含量。     

酶联免疫吸附法快速检测婴儿配方奶粉中乳铁蛋白含量

目的采用乳铁蛋白快速检测试剂盒,通过酶联免疫吸附法定量测定婴儿配方奶粉中的乳铁蛋白含量,并验证该方法的准确性、稳定性和可靠性。方法按照试剂盒操作说明制作标准曲线,选取乳铁蛋白阴性样品进行添加回收实验,验证方法的准确性;选取一个乳铁蛋白阳性样品,重复检测6次,计算结果偏差,验证方法的稳定性;选取12个乳铁蛋白阳性样品,分别采用试剂盒方法和高效液相色谱法进行检测,对比2种方法的检测结果,验证试剂盒方法的可靠性。结果试剂盒检出限为2 mg/100 g;试剂盒检测方法回收率在101.6%~109%;相对标准偏差(relative standard deviation,RSD)为5.34%;试剂盒方法检测结果与标签值的偏差为-6.90%~6.45%,和高效液相色谱法检测结果的偏差为-6.45%~6.9%,说明试剂盒方法具有很好的可靠性。结论乳铁蛋白试剂盒检测方法灵敏度高、操作简单,能快速准确地测定婴儿配方奶粉中的乳铁蛋白含量。     

牛初乳中乳铁蛋白的分离纯化与免疫功能检测

牛初乳经离心、盐析后获得乳铁蛋白粗提物，通过CM-Sephadex C50离子交换色谱和Heparin-Sepharose CL-6B亲和色谱纯化获得纯度分别为76．98％和92．75％的乳铁蛋白，经SDS-PAGE检测乳铁蛋白存在分子质量分别为76000Da和78000Da的两种异构体，免疫电泳说明提纯样品与乳铁蛋白标准品具有相同的免疫原性。小白鼠喂养和免疫实验说明乳铁蛋白可提高机体免疫功能。     

Potent bactericidal activity of bovine lactoferrin hydrolysate produced by heat treatment at acidic pH.

A hydrolysate of bovine lactoferrin produced by heat treatment under acidic conditions had antibacterial activity at concentrations of 10 micrograms/ml in the culture medium. The optimal degree of hydrolysis for this activity was about 10%. Heat-treated lactoferrin, treated at pH 2.0 and 120 degrees C for 15 min and degree of hydrolysis of about 10%, had no Fe-binding capacity (0%) and less antigenicity (about 10(-6) than untreated lactoferrin. Heat-treated lactoferrin increased in antibacterial activity, and the activity was maintained in an Fe-rich medium. After fractionation of heat-treated lactoferrin by reverse-phase HPLC, several peptide fractions were found that had strong antibacterial activity. It was suggested that lactoferrin latently contains at least one bactericidal domain that is activated upon release by limited acid hydrolysis of the protein. The bactericidal activity of the peptide fragments of lactoferrin was shown to have no relation to Fe chelation, in contrast with the antibacterial mechanism of native lactoferrin.     

乳铁蛋白测定方法的比较

建立了3种乳铁蛋白的测定方法并进行了比较。免疫酶联吸附法灵敏度高，最小检测限低，适合于乳铁蛋白分离纯化的全程分析；高效液相色谱法快速准确，操作简便，可实现乳铁蛋白初步分离时的同步分析；分光光度法快速简便，准确性稍差，适合于纯化后具有较高浓度的乳铁蛋白的在线分析。     

Effect of iron saturation on the recovery of lactoferrin in rennet whey coming from heat-treated skim milk

This study aimed to determine the effect of thermal treatments on the recovery of lactoferrin in whey coming from rennet-coagulated skim milk. The impact of lactoferrin iron saturation was also assessed using skim milk spiked with different lactoferrin iron forms. The recovery of lactoferrin in the rennet whey fraction was determined by reverse-phase HPLC. One- and 2-dimensional sodium dodecyl sulfate PAGE analyses were performed on rennet curds to characterize the protein interactions involving lactoferrin in heated milk. The extent of lactoferrin recovered in the whey fraction was found to reduce as the heating temperature increased. The binding of iron by lactoferrin improved its thermal stability and its recovery in the whey fraction. Poly-acrylamide gel electrophoresis results showed that the association of lactoferrin in the unheated milk rennet curd involved noncovalent interactions, whereas upon heating, lactoferrin also interacted via an intermolecular disulfide link. Depending on the severity of the heat treatment, lactoferrin aggregates with Cys-containing proteins (β-lactoglobulin, α-lactalbumin, α_s2-casein, and κ-casein) occurred by intermolecular thiol/disulfide exchange reactions. These noncovalent and covalent interactions explained the lower recovery of lactoferrin in heated milk.     

Optical biosensor analysis of the heat denaturation of bovine lactoferrin

There is growing interest in bovine lactoferrin as a nutritional ingredient, and since thermal exposure may compromise protein functionality, the heat-induced denaturation of native bovine lactoferrin was monitored by a recently developed optical biosensor-based immunoassay. Kinetic parameters were determined between 70 and 90 °C, which confirmed that thermal exposures typical of pasteurisation were relatively benign with respect to retained conformational integrity, while higher temperature–time combinations resulted in significant loss of conformationally intact lactoferrin. The influence of solution pH was also investigated and the biosensor technique compared with an HPLC procedure. The described biosensor immunoassay provides a useful complement to current analytical methods for investigation of heat-induced protein denaturation.     

Short communication: Determination of lactoferrin in Feta cheese whey with reversed-phase high-performance liquid chromatography

In the current paper, a method is introduced to determine lactoferrin in sweet whey using reversed-phase HPLC without any pretreatment of the samples or use of a separation technique. As a starting point, the most common HPLC protocols for acid whey, which included pretreatment of the whey along with a sodium dodecyl sulfate-PAGE step, were tested. By skipping the pretreatment and the separation steps while altering the gradient profile, different chromatographs were obtained that proved to be equally efficient to determine lactoferrin. For this novel 1-step reversed-phase HPLC method, repeatability was very high over a wide range of concentrations (1.88% intraday to 5.89% interday). The limit of detection was 35.46 μg/mL [signal:noise ratio (S/N) = 3], whereas the limit of quantification was 50.86 μg/mL (S/N = 10). Omitting the pretreatment step caused a degradation of the column’s lifetime (to approximately 2,000 samples). As a result, the lactoferrin elution time changed, but neither the accuracy nor the separation ability of the method was significantly influenced. We observed that this degradation could be easily avoided or detained by centrifuging the samples to remove fat or by extensive cleaning of the column after every 5 samples.     

HPLC测定食品中苯甲酸、山梨酸、糖精钠的样品前处理

采用20%CuSO4沉淀蛋白,增大5倍NaOH的用量处理样品,按国标法通过HPLC测定了食品中的苯甲酸、山梨酸和糖精钠含量。结果表明,改进的样品前处理方法使蛋白沉淀完全、溶液易过滤、过滤液清澈透明,可直接进样测定,对苯甲酸、山梨酸、糖精钠的测定准确可靠。     

Roles of charge interactions on astringency of whey proteins at low pH

Whey proteins are a major ingredient in sports drink and functional beverages. At low pH, whey proteins are astringent, which may be undesirable in some applications. Understanding the astringency mechanism of whey proteins at low pH could lead to developing ways to minimize the astringency. This study compared the astringency of β-lactoglobulin (β-LG) at low pH with phosphate buffer controls having the same amount of phosphate and at similar pH. Results showed that β-LG samples were more astringent than phosphate buffers, indicating that astringency was not caused by acid alone and that proteins contribute to astringency. When comparing among various whey protein isolates (WPI) and lactoferrin at pH 3.5, 4.5, and 7.0, lactoferrin was astringent at pH 7.0 where no acid was added. In contrast, astringency of all WPI decreased at pH 7.0. This can be explained by lactoferrin remaining positively charged at pH 7.0 and able to interact with negatively charged saliva proteins, whereas the negatively charged WPI would not interact. Charge interactions were further supported by β-LG or lactoferrin and salivary proteins precipitating when mixed at conditions where β-LG, lactoferrin, or saliva themselves did not precipitate. It can be concluded that interactions between positively charged whey proteins and salivary proteins play a role in astringency of proteins at low pH.     

酒糟与分离的沉淀产物中的氨基酸分析

在酒糟中含有相当数量的残留蛋白质与氨基酸,采用高效液相色谱柱前衍生法分别对酒糟与其分离产物沉淀中的氨基酸进行检测比较分析,发现酒糟中氨基酸含量高达13.45％,而沉淀产物中的氨基酸更是高达22.72％,其含量相比酒糟中提高了近一倍.通过离心分离后的酒糟的营养价值和经济价值都得到大大地提高.另外,酒糟及其沉淀产物中氨基酸种类十分全面,比例适中,是一种非常优秀的动物饲料.