东华理工教授发明牛奶检测新技术

“不到3min,我们的技术就可检出牛奶是否合格。”东华理工大学教授陈焕文博士介绍,该校质谱研究空在牛奶现场检测技术研究领域取得重大突破,成功研制出3种新技术,不仅能够检出牛奶中各种添加剂的成分,还能在1s内分析出三聚氰胺是否超标。     

代谢组学方法研究奶牛注射氯霉素后牛奶中的生物标志物

建立了高效液相色谱-质谱(HPLC-MS)技术研究牛奶代谢组学的方法.样品用乙腈提取,经浓缩富集,采用HPLC-MS技术研究了注射氯霉素后牛奶中内源性代谢物随时间的变化情况.结果表明,注射氯霉素后,牛奶的代谢指纹图谱中8个谱峰的变化随给药及休药时间呈现一定升降规律;采用偏最小二乘判别分析法(PLS-DA)可将给药组与空白组样品完全分开;鉴定了对组间差异贡献较大的化合物为单羟基十八碳二烯酸(HODE),HODE可以作为注射氯霉素后奶牛体内代谢应激产生的内源性生物标志物.     

单分散Au纳米粒子液相合成与快速检测三聚氰胺——本科生综合化学实验

利用Au纳米粒子与三聚氰胺相互作用产生的颜色变化来快速评估牛奶中三聚氰胺的含量,采用透射电子显微镜表征Au纳米粒子与三聚氰胺作用前后的形貌变化,通过用紫外-可见吸收光谱数据绘制标准工作曲线来定量测定牛奶中三聚氰胺的含量。该实验贴近生活,学以致用,同时也有助于学生掌握无机化学和分析化学基本操作,对增强学生分析解决问题的能力,提升学生的科研素养也有帮助。     

基于二维相关近红外光谱判定牛奶中掺杂的目标物

分别配制含有不同浓度尿素(1～20g.L-1)、葡萄糖(1～20g.L-1)和三聚氰胺(0.01～3g.L-1)的牛奶溶液共120个,采用二维相关近红外光谱对掺杂牛奶进行了快速判别分析。以牛奶中掺杂目标物浓度为外扰,构建二维相关近红外光谱,研究了各掺杂牛奶的二维相关近红外特性,并进行对比、分析。掺杂尿素牛奶在同步图上出现两个较强的自相关峰,分别在4 552,4 648cm-1处;掺杂葡萄糖牛奶,在同步图中出现3个较强的自相关峰,分别在4 456,4 392,4 360cm-1处;而掺杂三聚氰胺牛奶也有两个较强的自相关峰,出现在4 256,4 328cm-1处。结果表明:所提出的方法能实现对含有上述掺杂物牛奶的正确识别,为快速判别牛奶的安全性提供新方法。     

牛奶样品中新霉素残留量的离子色谱法测定

建立了阴离子交换色谱-脉冲安培检测分析牛奶中新霉素残留量的方法.实验采用三氯乙酸沉淀蛋白,弱阳离子交换固相萃取柱富集净化,处理牛奶样品,并考察了三氯乙酸浓度、样品过柱速度以及洗脱剂浓度等因素对牛奶中新霉素测定结果的影响.牛奶中新霉素的回收率在50.3%～76.9%范围内,基底加标工作曲线的相关系数为0.9949(n=6),线性范围为10.0～160.0ng/mL.所建立的方法可满足牛奶中低新霉素残留量的检测需要.     

复合近红外光谱法在牛奶测定中的应用

研究了用于快速测定牛奶主要成分含量的近红外透射光谱法和漫反射光谱法,分析了两种方法在实际测量中的差异,并提出将两种方法结合形成复合光谱法用于牛奶成分含量的检测。结果表明:复合光谱法的应用有效地提高了多变量回归分析中牛奶成分含量的预测精度。     

限进材料的制备及其应用——介绍一个仪器分析综合实验

介绍一个仪器分析综合实验,利用原子转移自由基聚合技术制备反相限进材料;以该材料为基础,建立牛奶中4种塑化剂的固相萃取-高效液相色谱分析方法。     

EDTA滴定法测定牛奶包装中碳酸钙的迁移

根据GB/T 5009.92-2016,以水、20%乙醇、4%乙酸和正己烷作为食品模拟液,把牛奶塑料包装袋装入食品模拟液,以时间和温度为变量,蒸干浸泡后的模拟液后消解定容,然后以钙红作为指示剂,用EDTA滴定检测迁移出的碳酸钙的浓度。结果表明,牛奶塑料包装袋中的碳酸钙在不同的模拟液体中的迁移量与温度和时间的变化成正比。     

二维相关红外光谱与支持向量机和灰度共生矩阵统计法相结合判别掺杂牛奶

应用二维相关红外光谱与支持向量机(SVM)和灰度共生矩阵统计方法相结合对分别掺杂有尿素、三聚氰胺和葡萄糖的牛奶进行判别。以质量浓度为外扰,建立掺杂牛奶的二维相关红外光谱图,选择角二阶矩、主对角线惯性矩、相关系数、熵的均值和标准差作为图像纹理特征并分别建立3种掺杂牛奶的SVM判别模型。结果表明:在对同步谱的分析中,上述3种掺杂牛奶样品中,掺杂尿素的牛奶样品训练集分类准确率为91.7%,预测集的准确率为85.0%;掺杂三聚氰胺和葡萄糖的牛奶的训练集分类准确率和预测集分类准确率分别为96.7%,90.0%和91.7%,100%。用同步谱和异步谱相结合的方法对准确率较低的掺杂尿素的牛奶做进一步试验,两种准确率分别提升为98.1%和92.3%;这一数据的提升是由于两者相结合提供了更大的信息量,有利于掺杂牛奶的判别。据此认为,此方法对掺杂牛奶的判别是可行的。     

应用双向电泳和质谱联用技术研究乳源蛋白的差异性

应用双向电泳和质谱联用技术,对不同乳源蛋白的差异性进行了研究。根据ImageMaster 2DPlatinum图像分析软件对不同乳源酪蛋白和乳清蛋白的双向电泳(2-DE)图谱进行蛋白斑点的匹配分析,获得21个存在于水牛奶中主要分布在低丰度蛋白区的酪蛋白差异蛋白点和24个存在于水牛奶中乳清蛋白差异蛋白点。这些差异蛋白点经质谱鉴定分析,得到4个属于水牛奶酪蛋白的主要组分和2个与水牛奶中酪蛋白有较高同源性的新组分,同时获得4个属于水牛奶乳清蛋白的主要组分和3个与水牛奶中乳清蛋白有较高同源性的组分。     

Determination of Penicillins in Milk by a Dual-Optrode Biosensor

Penicillins are the most frequently found antibiotic residues in milk, as they are commonly used for the treatment of bacterial infections in cows. In the present study, we introduce a method for the rapid detection of penicillin residues in raw milk based on the determination of glucose concentration in milk with a dual flow-through biosensor. The molar concentration of glucose in milk is typically over 500 times lower than the concentration of lactose and is highly dependent on the rate of lactose hydrolysis, which is catalyzed by β-galactosidase. Glucose concentrations in milk change with variation in the β-galactosidase activity. β-Galactosidase is an enzyme produced in the microbiota in milk and its activity is inhibited by benzylpenicillin. Spiking milk with benzylpenicillin lowers glucose concentrations in comparison to high-quality milk after short storage intervals. The presence of penicillin in the milk of treated animals resulted in decreased glucose concentrations in comparison with high-quality milk that contained no antibiotics. The glucose concentration in milk samples was followed by the system enabling the elimination of the effects of bacterial respiration in the output with reliable results in less than 1?min.     

Application of transmission diffusing wave spectroscopy to the study of gelation of milk by acidification and rennet

Transmission diffusing wave spectroscopy has been used to study and compare three milk gelling systems (acid gelation of heated and unheated milks and rennet coagulation of unheated milk). In all cases, DWS was able to demonstrate the point of gelation as indicated by a rapid increase in particle size, as well as the small decreases in casein micelle radius attributed to the collapse or removal of the hairy kappa-casein layer. More importantly, the photon transport mean free path (l(*)) was measured. This parameter is unique to transmission DWS and can potentially give information about developing microstructures and the mechanical properties between different types of gels. The values of l(*) changed during the gelation processes, and these changes were manifested earlier than any change in particle aggregation or rheology of the systems. All three different gelling systems showed different changes in l(*) with time, showing the development of different interactions as the acidification or renneting reactions proceeded. Although a full analysis of the l(*) parameter and its changes cannot be made, it is concluded that they can provide important information on the pre-gelation states of aggregating systems.     

Determination of minor proteins of bovine milk and colostrum by optical biosensor analysis

Automated, rapid, sensitive, and label-free biosensor-based immunoassays for immunoglobulin G (IgG), folate binding protein, lactoferrin, and lactoperoxidase in bovine milk using surface plasmon resonance optical detection with direct binding assay format are described. Samples are prepared for analysis by direct dilution into buffer. Analysis conditions, including ligand immobilization, flow rate, contact time, and regeneration are defined and nonspecific binding considerations evaluated. The technique has been applied to the measurement of these proteins in consumer milks, colostrum, milk products, and infant formulas, and their temporal change during early bovine lactation followed.     

The Influence of Bacteria Causing Subclinical Mastitis on the Structure of the Cow’s Milk Microbiome

Mastitis is the most expensive disease of dairy cattle across the world and is the main reason for the use of antibiotics in animal husbandry. The aim of this study was to analyze the microbiome of raw milk obtained from a semi-subsistence farm located in the Kuyavian–Pomeranian Voivodeship in Poland. Milk from healthy cows and from cows with subclinical mastitis was analyzed. The following pathogenic bacteria were found in milk from individuals with subclinical mastitis: Escherichia coli or Streptococcus agalactiae. The composition of drinking milk was assessed on the basis of 16S rRNA gene sequencing using the Ion Torrent platform. Based on the conducted research, significant changes in the composition of the milk microbiome were found depending on the physiological state of the cows. The microbiome of milk from healthy cows differed significantly from the milk from cows with subclinical mastitis. Two phyla dominated in the milk from healthy cows: Firmicutes and Proteobacteria, in equal amounts. On the contrary, in the milk from cows with diagnosed subclinical mastitis, one of the types dominated: either Firmicutes or Proteobacteria, and was largely predominant. Moreover, the milk microflora from the ill animals were characterized by lower values of the determined biodiversity indicators than the milk from healthy cows. The presence of pathogenic bacteria in the milk resulted in a significant reduction in the share of lactic acid bacteria in the structure of the population of microorganisms, which are of great importance in the production technology of regional products.     

Charakterisierung und Quantifizierung von Mineralstoffbindungen in Lebensmitteln

Summary Ion-exchange on Chelex-100, donnan dialysis and the method of ion-retardation are applied to characterize the bonding strengths of the minerals Ca, Mg and Zn in milk and extracts of meat and flour. Another ion-exchange procedure and direkt potentiometry with an Ca-selective electrode are applicate for the quantification of weak bindings. By use of the above mentioned methods it is possible to calculate element species stabilities, which represent characteristic values. Both methods give comparable results for milk, extracts of meat and spinach, grape-juice, red and white wine. Detailed examinations of binding conditions in milk proved citrate to the strength binding compound for Ca and Mg. Model-examinations with citrate, caseine and phosphate agree well with calculations and measurements in milk. Furthermore differences in the binding of Ca and Mg in raw and cream spinach are examined. The above mentioned methods are suitable to characterize and quantify the binding of elements in foodstuffs. The results give informations about chemical changes due to technological processes e.g., in regard to storage life.     

On-Site Measurement of Fat and Protein Contents in Milk Using Mobile NMR Technology

Robust and easy-to-use NMR sensor technology is proposed for accurate, on-site determination of fat and protein contents in milk. The two parameters are determined using fast consecutive ¹H and ³⁵Cl low-field NMR experiments on milk samples upon the 1:1 addition of a low-cost contrast solution. Reliable and accurate measurements are obtained without tedious calibrations and the need for extensive database information and may readily be conducted by non-experts in production site environments. This enables on-site application at farms or dairies, or use in laboratories harvesting significant reductions in costs and time per analysis as compared to wet-chemistry analysis. The performance is demonstrated for calibration samples, various supermarket milk products, and raw milk samples, of which some were analyzed directly in the milking room. To illustrate the wide application range, the supermarket milk products included both conventionally/organically produced, lactose-free milk, cow’s, sheep’s and goat’s milk, homogenized and unhomogenized milk, and a broad nutrient range (0.1–9% fat, 1–6% protein). Excellent agreement between NMR measurements and reference values, without corrections or changes in calibration for various products and during extensive periods of experiment conduction (4 months) demonstrates the robustness of the procedure and instrumentation. For the raw milk samples, correlations between NMR and IR, NMR and wet-chemistry, as well as IR and wet-chemistry results, show that NMR, in terms of accuracy, compares favorably with the other methods.     

Metabolomic Profile of Personalized Donor Human Milk

Human milk could be considered an active and complex mixture of beneficial bacteria and bioactive compounds. Since pasteurization drastically reduces the microbial content, we recently demonstrated that pasteurized donor human milk (DHM) could be inoculated with different percentages (10% and 30%) of mother’s own milk (MOM) to restore the unique live microbiota, resulting in personalized milk (RM10 and RM30, respectively). Pasteurization affects not only the survival of the microbiota but also the concentration of proteins and metabolites, in this study, we performed a comparative metabolomic analysis of the RM10, RM30, MOM and DHM samples to evaluate the impact of microbial restoration on metabolite profiles, where metabolite profiles clustered into four well-defined groups. Comparative analyses of DHM and MOM metabolomes determined that over one thousand features were significantly different. In addition, significant changes in the metabolite concentrations were observed in MOM and RM30 samples after four hours of incubation, while the concentration of metabolites in DHM remained constant, indicating that these changes are related to the microbial expansion. In summary, our analyses indicate that the metabolite profiles of DHM are significantly different from that of MOM, and the profile of MOM may be partially restored in DHM through microbial expansion.     

Effects of Melatonin on Dairy Herd Improvement (DHI) of Holstein Cow with High SCS

Mastitis is a common disease in cows breeding. The milk quality will be significantly reduced with increased milk somatic cells, which often occurs in cows with mastitis. In this study, the influence of seasonal changes, age and lactation stages in the Dairy Herd Improvement (DHI) of cows was investigated. Then, the Dairy Herd Improvement (DHI) of cows with high somatic cell score (SCS) after melatonin treatment was systemically investigated. The results showed that melatonin significantly suppressed the milk somatic cell score under all of the tested conditions. The melatonin treatment also improved the milk nutritional value by reducing its fat but increasing its lactose and protein contents. The application of melatonin significantly improved the DHI. The beneficial effects of melatonin on DHI are likely attributed to the antioxidant and anti-inflammatory activities of melatonin.     

Visual detection of melamine in milk products by label-free gold nanoparticles

A simple, rapid, field-portable colorimetric method for the detection of melamine based on melamine-induced color change of label-free gold nanoparticles (Au NPs) was developed in this study. Melamine can induced the aggregation of Au NPs and results in the color change from wine-red to purple, which provided a platform for rapid and field-portable colorimetric detection of melamine. The proposed method can be used to detect melamine in liquid milk and infant formula with a detection limit of 1.0 and 4.2 ppm, respectively, within 30 min by naked eyes observation without the aid of any advanced instrument and the need of any complex pretreatment, and detect as low as 0.15 ppm of melamine in liquid milk and 2.5 ppm of melamine in infant formula with UV-vis-spectroscopy. The proposed method is promising for on-site screening of melamine adulterant in milk products.     

D-optimal designs and N-way techniques to determine sulfathiazole in milk by molecular fluorescence spectroscopy

The present work proposes an analytical procedure to determine sulfathiazole in milk by using molecular fluorescence spectroscopy. For this sulfonamide the European Union in Regulation 37/2010 has established a maximum residue limit in milk of 100 μg kg(-1). The study includes the effect of six factors on the recovery of sulfathiazole. The factors are: (i) The one related to the matrix depending on the heat treatment of the milk (UHT, pasteurized); (ii) Those related to the protein precipitation step, namely the ratio between the volume of trichloroacetic acid (TCA) and milk, centrifugation speed and temperature; (iii) Those affecting the derivatization reaction: derivatization time and volume of fluorescamine. To do this, two chemometric tools are used together: a D-optimal design for studying the effect of the factors on the recovery of sulfathiazole, considerably reducing the number of needed experiments; and the second-order property of the PARAFAC (Parallel Factor Analysis) decomposition that avoids the need of fitting a new calibration model each time that the experimental conditions change. It has been found that the type of milk, the TCA:milk ratio and the volume of fluorescamine have significant effect on the response. The rest of factors and interactions are not significant. The best recovery is obtained with UHT milk, 4:6 rate for TCA:milk volumes and 40 μL of fluorescamine. In UHT milk, the mean recovery (n=5) in the optimal conditions is 88.7% (RSD=12.4%). As some non-linear behaviour may occur when using fluorescence spectroscopy, the calibration model that relates the fluorescence spectra with the concentration is computed by a partial least squares regression and a multi-layer feed-forward neural network. In both cases, the proposed procedures have been validated according to Decision 2002/657/EC, concluding that the two are accurate although the calibration model built with the neural network has better figures of merit, the decision limit (CCα) for x(0)=100 μg L(-1) is 103.3 μg L(-1) and the detection capability (CCβ) is 106.5 μg L(-1), with the probabilities of false noncompliance (α) and false compliance (β) equal to 5%.