Use of sandwich IgG ELISA for the detection and quantification of adulteration of milk and soft cheese

The aim of this work was to develop an assay capable of detecting adulteration of soft goat, sheep and buffalo milk cheese with bovine milk from cheaper sources. A previously developed indirect competitive ELISA had a lower sensitivity when applied to cheese, compared with milk. A sandwich ELISA was developed utilising the same monoclonal antibody in combination with a polyclonal goat anti-bovine IgG antibody. Once optimised, the ELISA was found to be highly specific. Detection limits in milk were 0.001% cows’ milk adulteration of sheep or buffalo milk, and 0.01% cows’ milk adulteration of goat milk. Detection limits in soft cheese were 0.001% in goat cheese and 0.01% in sheep or buffalo cheese. The assay was highly reproducible with both intra- and inter-assay coefficient of variation <10%. The ELISA performance makes it suitable for development as a kit for use in routine surveillance of milk and soft cheese.     

Milk adulteration: Detection of bovine milk in bulk goat milk produced by smallholders in northeastern Brazil by a duplex PCR assay

The aim of this study was to investigate the adulteration of goat milk produced by smallholders in semiarid northeastern Brazil with bovine milk as an adulterant. The study was requested by the association of smallholder producers in the region to investigate and to inhibit adulteration practices as a need to ensure the quality and safety of goat milk. A duplex PCR assay has been developed and standardized. Further validation was performed in 160 fresh bulk goat milk samples. The detection limit of the duplex PCR was 0.5% bovine milk in goat milk and the results indicated that 41.2% of the goat milk presented to market was positive for bovine milk. Making the test available to the association of producers, together with extension activities, have been applied to reduce adulteration in goat milk sold to small-scale dairy plants and to ensure the species origin for goat milk in the state of Paraíba.     

Fourier transform infrared spectroscopy and multivariate analysis for the detection and quantification of different milk species

The authenticity of milk and milk products is important and has extended health, cultural, and financial implications. Current analytical methods for the detection of milk adulteration are slow, laborious, and therefore impractical for use in routine milk screening by the dairy industry. Fourier transform infrared (FT-IR) spectroscopy is a rapid biochemical fingerprinting technique that could be used to reduce this sample analysis period significantly. To test this hypothesis we investigated 3 types of milk: cow, goat, and sheep milk. From these, 4 mixtures were prepared. The first 3 were binary mixtures of sheep and cow milk, goat and cow milk, or sheep and goat milk; in all mixtures the mixtures contained between 0 and 100% of each milk in increments of 5%. The fourth combination was a tertiary mixture containing sheep, cow, and goat milk also in increments of 5%. Analysis by FT-IR spectroscopy in combination with multivariate statistical methods, including partial least squares (PLS) regression and nonlinear kernel partial least squares (KPLS) regression, were used for multivariate calibration to quantify the different levels of adulterated milk. The FT-IR spectra showed a reasonably good predictive value for the binary mixtures, with an error level of 6.5 to 8% when analyzed using PLS. The results improved and excellent predictions were achieved (only 4-6% error) when KPLS was employed. Excellent predictions were achieved by both PLS and KPLS with errors of 3.4 to 4.9% and 3.9 to 6.4%, respectively, when the tertiary mixtures were analyzed. We believe that these results show that FT-IR spectroscopy has excellent potential for use in the dairy industry as a rapid method of detection and quantification in milk adulteration.     

Validation of a rapid lateral flow method for the detection of cows’ milk in water buffalo,sheep or goat milk

For many years, the adulteration of milk from sheep, goats or water buffalos with cows’ milk has been a widespread practice due to the higher cost of milk from those other species. Because of this, great concern has been shown by many Protected Designation of Origin councils that have to assure the quality and genuineness of the cheese produced by their associates. Therefore, the whole production chain needs analytical tools that allow the control of potential adulteration. Rapid methods to be used in the field are scarce and have not been validated according to international guidelines. The aim of this work has been to validate a rapid test based on lateral flow immunochromatography to detect cows’ milk in milk from other species, including buffalo’s milk, according to AOAC guidelines. No false-positive result was found after analysing 146 known negative samples from individual animals. The lowest level of adulteration with a Probability of Detection (POD) of 1.00 (confidence interval between 0.94 and 1.00) was found at 0.5% of cows’ milk. This level is below the current EU allowed level of cows’ milk, set at 1%. Variations in the time of assay, volume of the analysis buffer and different batches of the test were evaluated to detect any effect on the false-positive rate or on the limit of detection of the test. The effects of compositional factors (such as high level of fat, protein and somatic cell counts) were also evaluated. The new rapid test to detect cows’ milk in milk from other species is shown to be an adequate tool to control milk quality in routine analysis. This kind of test is very easy to use and it can be performed by untrained staff during milk collection at the farm or upon arrival at dairies.     

Use of MALDI-TOF MS technology to evaluate adulteration of small ruminant milk with raw bovine milk

Detection of adulteration of small ruminant milk is very important for health and commercial reasons. New analytical and cost-effective methods need to be developed to detect new adulteration practices. In this work, we aimed to explore the ability of the MALDI-TOF mass spectrometry to detect bovine milk in caprine and ovine milk using samples from 18 dairy farms. Different levels of adulteration (0.5, 1, 5, 10, 20, 40, 60, and 80%) were analyzed during the lactation period of goat and sheep (in May, from 60 to 90 d in milk, and in August, from 150 to 180 d in milk). Two different ranges of peptide-protein spectra (500–4,000 Da; 4–20 kDa) were used to establish a calibration model for predicting the concentration of adulterant using partial least squares and generalized linear model with lasso regularization. The low molecular weight part of the spectra together with the generalized linear model with lasso regularization regression model appeared to have greater potential for our aim of detection of adulteration of small ruminants' milk. The subsequent prediction model was able to predict the concentration of bovine milk in caprine milk with a root mean square error of 11.4 and 17.0% in ovine milk. The results offer compelling evidence that MALDI-TOF can detect the adulteration of small ruminants' milk. However, the method is severely limited by (1) the complexity of the milk proteome resulting from the adulteration technique, (2) the potential degradation of thermolabile proteins, and (3) the genetic variability of tested samples. Additionally, the root mean square error of prediction based only on one individual sample adulteration series can drop down to 6.34% for quantification of adulterated caprine milk and 6.28% for adulterated ovine milk for the full set of concentrations or down to 2.33 and 4.00%, respectively, if we restrict only to low concentrations of adulteration (0, 0.5, 1, 5, 10%).     

Measurement of bovine IgG by indirect competitive ELISA as a means of detecting milk adulteration

The aim of this work was to develop an assay capable of detecting adulteration of high premium milk with milk from cheaper sources. An indirect, competitive ELISA was developed for the rapid detection of cows' milk in the milk of goat, sheep, and buffalo. The assay uses a monoclonal antibody produced against bovine IgG. This antibody recognizes a species-specific epitope on the heavy chain of both bovine IgG1 and IgG2. A peroxidase-conjugated anti-mouse IgG antibody was used to detect bound monoclonal antibody and subsequent enzymatic conversion of substrate resulted in clear differences in absorbance when assaying different mixtures of milks adulterated with cows' milk. Once optimized, the ELISA was found to be highly specific. Detection limits of the assay are 1.0 microg/mL of bovine IgG, or 0.1% (vol/vol) adulteration with cows' milk. The assay was highly reproducible (CV < 10%) and performed equally well when used to detect bovine IgG in mixtures with the 3 types of milk tested. The ELISA performance makes it suitable for development as a kit, for use in the field as a high throughput screening ELISA.     

核磁共振在食品掺假检测中的应用

食品掺假作为当今社会最热门的食品问题之一,如何更有效地检测掺假物或掺假比例对解决这一问题至关重要。核磁共振(nuclear magnetic resonance,NMR)技术因能快速无损地得到样品的理化特性及结构等信息,在近年来受到极大的关注。本文从碘值、固体脂肪含量、弛豫特性、扩散率和特定信号等常用检测参数方面来介绍核磁共振在食用油脂、牛奶和蜂蜜等食品掺假检测中的应用。作为一种较新的掺假检测技术,核磁共振技术目前在国内食品掺假方面的应用还不广泛,相较于现有的一些食品掺假检测技术,NMR技术具有快速无损、操作方便等优点,在未来的食品掺假检测方面将会有很大的发展前景。     

乳及乳制品蛋白质掺假检测研究进展

阐述两类方法、一个原则：乳及乳制品蛋白质掺假特异性检测方法和乳及乳制品蛋白质掺假非特异性检测方法;任何一种乳品蛋白质掺假检测方法的建立都要以某一特定蛋白质差异性为依据的原则。     

Estimation of sugars in milk by HPLC and its application in detection of adulteration of milk with soymilk

A high-performance liquid chromatography method is described for the detection of adulteration of milk with soymilk, based on separation of sugars on NH₂ column and their detection by refractive index detector. Sugars were extracted with 20% acetonitrile in the presence of Carrez solutions and quantified. Recovery of added raffinose to soymilk was 96.3%. Owing to relative high concentration of lactose in milk or adulterated milk, lactose peak was very broad and spread to retention time corresponding to sucrose and raffinose. However, stachyose peak remained well separated. Presence of stachyose peak in milk can be used for the detection of adulteration of milk with soymilk and the method can detect upto 5% soymilk in milk.     

Detection of adulteration in milk: A review

Milk is a wholesome nutritious dairy product and is consumed by a majority of the population worldwide for drinking as such, as well as via dairy products. However, the practice of adulteration of milk invariably reduces its quality and may introduce hazardous substances into the dairy supply chain jeopardising consumers’ health. Various instances of adulteration of milk have been reported globally, wherein substances such as extraneous water, foreign proteins, whey proteins, melamine and urea, vegetable or animal fats, plus many minor constituents of milk fat have been added as potential adulterants in milk and milk products. This review focusses on the different methods of detection of these adulterants in milk using techniques such as DSC, RP‐HPLC, LC‐GC, HPTLC, immunoassays: CE, ELISA, FAMPST, FTIR, NIR spectroscopy, PAGE, IEF, DNA‐based methods and MALDI‐MS that have been developed and employed for the last 25 years. The combination of advanced IR spectroscopy and chemometrics provides a powerful tool for quality and authenticity analysis of milk. An electronic tongue is an easy and economic tool for the detection of caprine milk adulterations with bovine milk. Biosensors having the ability to furnish real‐time signals have been developed for the detection of urea in milk. An attempt has been made to give a clear understanding of the most suitable methods for the determination of various sources of adulteration.     

基于电子舌的掺假羊奶快速定量预测模型

为实现对掺假羊奶的快速、客观辨别,模仿人体味觉感知机理研制了一套便携式电子舌检测系统,并建立了一种能够快速鉴别掺假羊奶的新方法。系统检测时,首先对样本溶液进行大幅脉冲扫描,用以获取掺假羊奶的"指纹"信息,然后利用离散小波变换(discrete wavelet transform,DWT)对"指纹"数据中的特征信息进行提取,最后在此基础上,采用主成分分析(principal component analysis,PCA)方法对不同掺假比例的羊奶进行定性辨别。采用粒子群优化极限学习机(Particle swarm optimization extreme learning machine,PSO-ELM)对不同掺假比例的羊奶进行了定量预测。通过试验数据得出,PCA对6种不同掺假比例的羊奶区分达到100%,区分效果好。PSO-ELM羊奶纯度预测模型拟合曲线非常接近实测值曲线,因此采用PSO-ELM方法建立掺假羊奶纯度定量预测模型具有较高的预测精度。     

蛋白质组学技术在不同物种乳掺假检测中的应用

乳品是人类重要的营养源，然而乳品掺假现象时常发生，近年来尤以向乳品中掺假动、植物蛋白，向特色畜乳中掺假牛乳等方式为主，这不仅损害了消费者的利益，甚至会危害消费者的健康。该研究总结了目前常见的掺假行为及相关检测方法，并介绍了蛋白质组学技术——一种通过确立特定生物标记物来区别不同物种乳的技术。作者查询了国内外近十年来牛乳和特色乳掺假方面的研究报道，关键词设置为“蛋白质组学”、“乳品”、“真实性”、“生物标记物”等，按照奶畜乳类别将所得文献进行分类。分别对奶牛乳、羊乳、驼乳、水牛乳、牦牛乳、驴乳的掺假物、潜在标记物和检出限等方面进行了总结和分析，以期为乳品真实性鉴定和保障乳品质量安全提供有效的工作思路。     

核磁共振波谱技术在食品掺假鉴别中的应用研究

核磁共振波谱技术具有快速无损、操作简单及重复性好等优点,近年来被广泛应用于食品掺假鉴别领域。利用核磁共振技术中的低场核磁、定量核磁以及杂核核磁等技术能够对掺假不同成分的牛乳(掺水、食盐、尿素、豆浆及复原乳等)、掺假低价值油(大豆油、玉米油等)的橄榄油、掺假的高价值米、蜂蜜、红酒等进行检测,结合统计学分析方法,包括主成分分析法、偏最小二乘判别法和相似分类法等可以实现对这些食品掺假成分的有效鉴别。本文就近年来核磁共振技术在国内外食品掺假鉴别中的应用研究进行综述,以推动核磁共振技术在食品质量安全检测领域的进一步应用,并为其它领域的掺假鉴别提供新的思路和方法。     

A duplex polymerase chain reaction for the quantitative detection of cows’ milk in goats’ milk cheese

A duplex polymerase chain reaction (PCR) has been applied for the detection of both cows’ and goats’ milk in goats’ milk cheeses using primers targeting the mitochondrial 12S rRNA genes. By means of a linear normalised calibration curve between the log of the ratio of the cows’ band intensity and the sum of cows’ and goats’ intensities vs. the log of cows’ milk percentage, it was possible to quantify cheese adulteration with cows’ milk in the range of 1–60%. The duplex PCR technique allowed the detection of 0.1% of cows’ milk in goats’ milk cheese. The proposed method was successfully applied to cheeses with defined amounts of cows’ milk and to 15 of a total of 17 commercial cheese samples. The results showed the fraudulent addition of cows’ milk in three samples labelled as pure goats’ milk cheeses and the omission of goats’ milk mentioned on the label of two cheeses containing mixed milk.     

实时荧光P C R 在鉴别奶粉中掺入大豆成分的应用研究

首次利用实时荧光PCR 方法,以大豆内源参照基因lectin 为指标,对奶粉中掺入豆粉等植物成分进行定性、定量分析研究。结果表明,通过正己烷抽提奶粉中的油脂,苯酚抽提奶粉中的蛋白质等处理步骤后,应用植物基因组DNA 提取试剂盒能够得到高质量的DNA 模板。实时荧光PCR 反应结果表明,该方法判定奶粉中掺入植物成分的灵敏度大大提高;奶粉中掺入大豆粉或大豆制品的掺入量与实时荧光PCR扩增曲线及循环数呈良好的对应关系。实时荧光PCR 法能够对奶粉中掺入的植物成分进行快速准确地定性、定量分析,可以作为市场监督和检验鉴定的可行性方法。     

一对同时鉴定8种动物源性成分的通用引物的制备及应用

肉类真假鉴定是食品检测工作的内容之一,目前已有多种基于PCR的肉类鉴定方法,但是鉴定种类和效率受限。本研究设计了一对基于普通PCR技术可同时鉴定8种动物源性成分的通用引物并建立了鉴定方法。该引物以线粒体DNA为靶标,利用扩增产物中不同物种间的插入缺失多态性片段大小即可鉴定山羊、绵羊、鹿、水牛、牛、牦牛、猪和骆驼8个物种,扩增后分别得到728 bp、704 bp、504 bp、453 bp、448 bp、431 bp、396 bp和326 bp的片段,每种PCR产物经SspI酶切后产生数量和大小不同的片段,可以进一步清晰鉴别8个物种。引物特异性测试表明和其他常见肉类动物DNA无交叉反应,DNA检测最低限度在0.01~0.05 ng。应用本方法对40份市场肉类及产品的检测表明,羊肉串、羊肉卷以及特色畜产品如驼肉、鹿肉和驴肉存在较多的掺假行为。与其他现有PCR检测方法相比,该方法具有简便易行和高通量的优点,可以作为肉类掺假筛选检测的常规方法。     

Detection of milk mixtures in Halloumi cheese

A capillary electrophoresis method has been applied to the detection of illegal addition of milk from goat and/ or cow in Halloumi cheese, traditionally made with sheep milk. The electrophoretic profiles of the casein from Halloumi cheeses have revealed that caprine para-kappa-casein and bovine alphas1-casein peaks point to the presence of low percentages of goat's and/or cow's milk added to Halloumi cheese. Stepwise multiple linear regression has been used to predict these percentages with a standard error of the estimation of 2.14%. The analytical method combined with the statistical application is valid for the prediction of percentages higher than 2% of goat's and percentages of 5% of cow's milk added to the cheese either in fresh or ripened cheese. The standard error of estimation was higher for the prediction of cow's milk than for goat's milk.     

基于乳清蛋白的骆驼乳中掺假牛乳的检测及热处理对方法的影响

利用超高效液相色谱（ultra-high performance liquid chromatography,UPLC）技术,建立一种以牛β-乳球蛋白为掺假标识物的检测方法,用于定性定量检测骆驼乳中掺假的牛乳,并且探讨不同热处理方式对掺假标识物的影响,以期满足不同商品化驼乳制品的检测需求。结果表明：该方法能有效地检测鲜驼乳、巴氏杀菌驼乳以及驼乳粉中掺假的牛乳,3 种类型掺假乳样本的定量检测回归方程线性良好,线性相关系数（R2）分别为0.997 9、0.996 9和0.997 8;鲜驼乳、巴氏杀菌驼乳和驼乳粉中掺假牛乳的检出限分别为2%、3%和5%,可满足检测需求;利用该方法在10 种不同品牌市售纯驼乳粉中检测出4 种掺假驼乳粉产品。UPLC法可以有效地检测骆驼乳及其制品中掺假的牛乳,为骆驼乳行业的掺假检测提供一定的技术方法支持。     

掺假羊乳及其制品中牛乳的检测技术研究进展

羊乳具有营养价值高、蛋白质组成更接近人乳、脂肪球直径小及致敏性低等优点, 更利于人体消化吸收, 受到消费者和乳品企业的青睐。近年来我国羊乳产业发展迅速且潜力巨大, 但由于受羊乳产量和养殖规模的限制, 羊乳价格昂贵, 市场中存在羊乳及其制品掺假牛乳的现象, 且掺假手段多样, 难以辨别。为了保证消费者的健康和权益, 保障羊乳市场良性发展, 羊乳及其制品的纯正性、真实性检测已经成为热点研究方向。本文通过分析基于乳中蛋白质、脂肪和核酸差异的羊乳中牛乳掺假检测技术的研究现状, 介绍和探讨了各检测技术的基本原理及其在应用中的优缺点, 同时展望羊乳掺假检测技术的发展方向, 旨在为牛羊乳混合掺假检测技术的进一步发展提供资料参考和思路。     

近红外光谱技术在食品掺伪检测应用中的研究进展

近年来,我国食品掺伪现象严重。目前一些常用的食品掺伪检测方法费时费力,需要消耗大量的化学试剂。近红外光谱技术能够实现快速、无损、在线的食品掺伪检测。本文综述近红外光谱技术在食用油、牛奶、蜂蜜、饮料等液态食品和肉制品、奶粉、茶叶、小麦粉等固态食品掺伪检测应用中的研究进展,同时分析近红外光谱技术在掺伪检测中的局限性以及存在问题,并对今后的进一步研究提出展望。