基于旋光法的原料乳中乳糖掺伪鉴别技术研究

旨在建立一种简便、高效的原料乳乳糖掺伪鉴别方法。以三氯乙酸为沉淀剂进行前处理,借助旋光仪,在589.30⁵89.44nm下,测定标准乳糖溶液的旋光度,绘制标准曲线,建立了一种基于标准曲线的原料乳乳糖掺伪定量鉴别法。所得回归方程的线性相关系数R2=0.9993,平均回收率达99.0%,相对标准偏差（RSD）为0.67%。与莱因-埃农氏法进行比对实验,结果无差异,但更快捷,成本更低。研究结果提示,本文所建方法实用、可靠。      

指纹图谱技术在生鲜乳掺假检测中的研究进展

生鲜乳是乳制品行业发展的主要原料,是决定乳制品质量的关键因素。然而近年来国内外在乳制品方面的食品安全事件频发,不法分子通过在生鲜乳中掺入虚假物质以获取经济利益的行为已经成为严重的安全问题,对人们健康以及整个乳制品行业造成不良影响。指纹图谱技术是对通过一定的分析工具产生的图像进行判别的一种检测技术,可以对生鲜乳的掺假进行更灵敏、准确和快速的检测。本文通过对生鲜乳的安全现状进行剖析,总结了电泳法、光谱法、色谱法和电子感官技术法4种指纹图谱技术在牛乳掺假检测中的应用,比较了4种技术的优点和局限性,并对未来的研究方向进行了展望,为提高生鲜乳的品质与安全以及保证消费者健康提供理论依据与参考。     

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,...     

水牛乳及其乳制品中掺入牛属动物乳源的检测方法研究进展

综述了电泳、色谱、免疫化学、DNA技术等在掺假水牛乳检测中的研究进展及各自优劣,旨在为我国开展类似研究提供理论依据。     

综合型传感器阵列对掺杂生乳的识别

以钯、铂、金、钛、钨和银6种自制电极组为工作电极,与Ag/AgCl参比电极和铂对电极组成非选择性传感器组,再结合pH电极、电导率电极和钠度计电极3种选择性电极组成综合型传感器阵列,连接到电化学工作站和安装SPSS统计学软件电脑后,构成生乳掺杂检测系统。对荷斯坦牛生乳及掺杂不同浓度尿素和三聚氰胺,碳酸氢钠的样本乳、陈放乳、巴氏乳、酸败乳采用电化学方法进行检测,获得开路电位、交流阻抗谱、微分脉冲伏安数据,非选择性与选择性传感器组数据分别采用模式识别法进行计算,并获得各被测样本与生乳标准数据库的欧氏距离,再以不同权重得出综合欧氏距离,通过图表得到表达。结果表明,非选择性传感器阵列组对掺杂生乳具有良好的判别能力,在此基础上构建的加装有选择性传感器的综合型传感器阵列对掺杂的甑别效果优于前者。      

Changes during ripening in chemical composition,proteolysis, volatile composition and texture in Kashar cheese made using raw bovine,ovine or caprine milk

Kashar cheeses were manufactured from pure ovine (OV), bovine (BV) and caprine (CP) milk, and the chemical composition, cheese yield, proteolysis, hardness, meltability and volatile composition were studied during 90 days. Gross chemical composition, cheese yield and level of proteolysis were higher in OV cheeses than those of BV or CP cheeses. Glu, Val, Leu, Phe and Lys were the most abundant free amino acids (FAA) in the samples, and the concentrations of individual FAA were at the highest levels in OV cheeses with following BV and CP cheeses. Urea‐PAGE patterns and RP‐HPLC peptide profiles of the BV cheeses were completely different from the small ruminants’ milk cheeses (OV or CP). Higher and lower hardness and meltability values were observed in CP cheeses, respectively. OV cheeses resulted in higher levels of the major volatile compounds. In conclusion, the Kashar cheese made using OV milk can be recommended due to high meltability, proteolysis and volatiles.     

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.     

Detection of bovine milk adulteration in caprine milk with N-acetyl carbohydrate biomarkers by using 1H nuclear magnetic resonance spectroscopy

In a return to tradition, the popularity of caprine milk is on the rise. However, particularly in countries with developed dairy industries based on bovine milk, there is the risk of adulteration with bovine milk, which is a cheaper alternative. Thus, a rapid, robust, and simple method for the detection of bovine milk added to caprine milk is necessary, and ¹H nuclear magnetic resonance spectroscopy appears to provide a solution. A matrix of 115 pure and artificially adulterated pasteurized milk samples was prepared and used to discover biomarkers of bovine milk that are independent of chemical and biological variation caused by factors such as genetics, diet, or seasonality. Principal component analysis and orthogonal projections to latent structures discriminant analysis of pure bovine milk and pure caprine milk revealed spectral features that were assigned to the resonances of 4 molecules. Of these, the peaks corresponding to protons in the N-acetylglucosamine and N-acetylgalactosamine acetyl moieties showed significant applicability for our method. Receiver operating characteristic curve analysis was used to evaluate the performance of the peak integrals as biomarkers of adulteration. This approach was able to distinguish caprine milk adulterated with 5% of bovine milk with 84.78% accuracy and with 10% of bovine milk an excellent 95.65% accuracy. This study demonstrates that N-acetyl carbohydrates could be used as biomarkers for the detection of bovine milk in caprine milk and could help in protecting caprine milk authenticity.     

Direct sandwich ELISA to detect the adulteration of human breast milk by cow milk

The demand for commercially available human breast milk has significantly increased in recent years. For various reasons, a significant amount of commercially available human breast milk is being adulterated with other types of milk. This fraudulent practice poses a threat to consumers' health due to potential adulterants such as cow milk, which may put the infant at risk due to intolerance or allergy. A direct sandwich anti-bovine IgG ELISA has been developed for the sensitive and specific detection of cow milk in adulterated human breast milk. This assay uses polyclonal anti-bovine IgG antibody as a capture antibody and monoclonal anti-bovine IgG-alkaline phosphatase antibody as a detection antibody. Once optimized, the assay was found to be highly sensitive, and specific to bovine IgG. The assay had no significant cross-reaction with human breast milk, indicating that it was highly specific. The anti-bovine IgG ELISA was able to detect the presence of cow milk in adulterated human breast milk with a detection limit of 0.001% cow milk. The developed assay was highly reproducible (coefficient of variation <10%). The developed direct sandwich anti-bovine IgG ELISA is simple, reliable, and reproducible, making it an ideal test for this purpose.     

Peptidome comparison following gastrointestinal digesta of bovine versus caprine milk serum

Infant formula is used as a supplement for newborns. Although bovine milk-based infant formulas dominate the market, caprine milk-based infant formula has attracted increasing attention because of its lower allergenicity. This study compared the digestive peptidome of bovine and caprine milk serum proteins by using in vitro infant simulating conditions. The result showed that the degradation pattern of milk proteins was similar, whereas the digestive rates of milk proteins differed between bovine and caprine milks. Several proteins, such as α-lactalbumin (LALBA), β-lactoglobulin (LGB), serum amyloid A protein (SAA1), glycosylation-dependent cell adhesion molecule 1 (GLYCAM1), and lactotransferrin (LTF), released more peptides during digestion of caprine milk serum than during digestion of bovine milk serum; however, more peptides derived from α_S1-casein (CSN1S1) were found in bovine digesta. In addition, antimicrobial-related peptides were mostly only found in caprine intestinal digesta. The results of this study may be useful in understanding the digestion characteristics of milk serum proteins and providing guidance on the improvement of infant formula.     

Qualitative and quantitative identification of adulteration of milk powder using DNA extracted with a novel method

The extraction of high-quality DNA from processed dairy products is often the crucial step in an authentication process by PCR-based methods. In this study, we optimized a novel DNA extraction method for milk powder and used the extracted DNA for identification of milk powder based on PCR analysis. The DNA quality was assessed by amplifying target sequences from mitochondrial genes, as well as by monitoring the yield, purity, and integrity of the extracted DNA. In addition, a laboratory adulteration model of milk powder was detected by PCR-based methods (PCR and real-time PCR) using primers targeting the mitochondrial 12S rRNA gene. Results showed that a sufficient amount and quality of DNA could be isolated from milk powder with this method. Both PCR and real-time PCR detection of cow milk compositions in goat milk powder further confirmed the DNA extracted with this extraction method could be widely used in addressing milk powder adulterant by a PCR-based method.     

Detection of buffalo milk adulteration with cow milk by capillary electrophoresis analysis

The addition of cow milk during the production of buffalo mozzarella is a common fraud in dairy industries because of the lower price and greater availability of cow milk throughout the year. The aim of this study was to develop a new, rapid, and robust capillary electrophoresis method for detecting and quantifying cow milk in buffalo milk by exploiting cow α-lactalbumin as a marker of adulteration. In particular, a linear calibration curve was generated, using a training set of calibrators consisting of 7 series of 17 buffalo/bovine whey mixtures, obtained after casein precipitation, with increasing percentages of cow whey. The capillary electrophoresis method showed high linearity (R² = 0.968), repeatability [relative standard deviation (RSD) = 2.11, 3.02, 4.38, and 1.18%, respectively for 5, 10, 20, and 50% of buffalo/bovine whey mixtures], and intermediate precision (RSD = 2.18, 2.49, 5.09, and 3.19%, respectively, for 5, 10, 20, and 50% buffalo/bovine whey mixtures). Moreover, the minimum amount of detectable fraudulent cow milk was 1%, and the limit of quantification was 3.1%.     

基质辅助激光解析/电离飞行时间质谱结合化学计量学技术筛查原奶掺假研究

目的 利用基质辅助激光解析/电离飞行时间质谱 (Matrix-assisted laser desorption/ionization time of flight mass spectrometry, MALDI-TOF) 结合化学计量学方法快速筛查鲜奶掺假。方法 样品经0.1%三氟乙酸水溶液稀释50倍,与10 mg/mL芥子酸溶液等体积混合,取2 μL混合液点靶板,待样品干燥后载入MALDI-TOF,MALDI-TOF在线性模式下采集数据。通过主成分分析 (Principal Component Analysis, PCA)分析不同来源的数据,建立鲜奶蛋白质信息数据库。通过检测掺假样品并与数据库进行比较,评估检测方法性能。结果 确定了MALDI-TOF蛋白质图谱可做为鲜奶的指纹图谱。建立了一个涉及2个产地和季节的鲜奶蛋白质信息数据库。方法能够检测含有0.3% (w/w) 猪皮明胶、大豆分离蛋白或乳清蛋白的掺假样品。方法操做简单快速,从进样到获得结果,一个样品仅需几分钟。结论 MALDI-TOF结合化学计量学方法能够快速检测在0.3% (w/w) 浓度下含有猪皮明胶、大豆分离蛋白或乳清蛋白的鲜奶掺假。     

Detection of cow milk adulteration in yak milk by ELISA

In the current study, a simple, sensitive, and specific ELISA assay using a high-affinity anti-bovine β-casein monoclonal antibody was developed for the rapid detection of cow milk in adulterated yak milk. The developed ELISA was highly specific and could be applied to detect bovine β-casein (10–8,000 μg/mL) and cow milk (1:1,300 to 1:2 dilution) in yak milk. Cross-reactivity was <1% when tested against yak milk. The linear range of adulterant concentration was 1 to 80% (vol/vol) and the minimum detection limit was 1% (vol/vol) cow milk in yak milk. Different treatments, including heating, acidification, and rennet addition, did not interfere with the assay. Moreover, the results were highly reproducible (coefficient of variation <10%) and we detected no significant differences between known and estimated values. Therefore, this assay is appropriate for the routine analysis of yak milk adulterated with cow milk.     

原料乳掺假现场检测箱的设计与检测项目组合

提出了原料乳掺假现场检测箱的设计思路,利用若干化学反应的基本原理和现象,可检测出原料乳中21种掺假物质。通过系统的评价各种检测方法的检测限,给出了优化后试剂组合。该检测箱适用于现场检测,便于携带。     

Sensory analysis and species-specific PCR detect bovine milk adulteration of frescal (fresh) goat cheese

The Brazilian market for dairy products made from goat milk is increasing despite the seasonality of production and naturally small milk production per animal, factors that result in high-priced products and encourage fraud. In Brazil, no official analytical method exists for detecting adulteration of goat dairy products with cow milk. The aim of this study was to design a strategy to investigate the adulteration of frescal (fresh) goat cheeses available in the Rio de Janeiro retail market, combining analysis of cheese composition and the perception of adulteration by consumers. Commercial goat cheeses were tested by using a duplex PCR assay previously designed to authenticate cheeses, by targeting the mitochondrial 12S ribosomal RNA genes of both species simultaneously. The PCR test was able to detect 0.5% (vol/vol) cow milk added during goat cheese formulation. The analysis of 20 locally produced goat cheeses (20 lots of 4 brands) showed that all were adulterated with cow milk, even though the labels did not indicate the addition of cow milk. To estimate the ability of consumers to perceive the fraudulent addition of cow milk, a triangle test was performed, in which cheeses formulated with several different proportions of goat and cow milk were offered to 102 regular consumers of cheese. Detection threshold analysis indicated that almost half of the consumers were able to perceive adulteration at 10% (vol/vol) cow milk. Effective actions must be implemented to regulate the market for goat dairy products in Brazil, considering the rights and choices of consumers with respect to their particular requirements for diet and health, preference, and cost.     

酪蛋白质量分数作为乳品掺假检验指标的探讨

论述了我国乳品掺假的原因、方式和危害,以及掺假鉴定的必要性和重要性,分析了酪蛋白是牛乳中的特征性成分,根据乳品中酪蛋白占乳蛋白的质量比例（即酪蛋白质量分数）一般会稳定在77%～78%左右,提出了酪蛋白质量分数≥73%,可以作为乳品掺假检验的定量指标,探讨了国内外酪蛋白定量检测方法的研究现状。结果表明,等电点沉淀法无疑是一种适合我国国情的、操作简单的酪蛋白检验方法,对防治乳品掺假具有重要的现实意义,并通过分析提出了该检验方法所需研究的内容。     

Short communication: Persistent contamination by Listeria monocytogenes of bovine raw milk investigated by whole-genome sequencing

Following the persistent detection of Listeria monocytogenes in raw bovine milk sold through a vending machine, the 120 lactating cows of the herd producing the milk were subjected to bacteriological investigation. A single cow with subclinical mastitis (1.2–1.3 × 10⁵ somatic cells/mL) and persistent L. monocytogenes excretion was detected. The cow was subjected to antimicrobial therapy, but L. monocytogenes excretion remained high (>3.0 × 10² cfu/mL). Following culling of the infected cow, L. monocytogenes disappeared from the tank milk, and further isolates were recovered from the mammary parenchyma and lymph nodes of the infected cow. To investigate the clonal nature of the contamination, all isolates recovered in the study (n = 13) were analyzed by serogroup PCR, pulsed-field gel electrophoresis, and whole-genome sequencing. Our results demonstrated the clonal nature of the contamination. All isolates belonged to lineage II, serogroup IIa, sequence type 37, clonal complex 37 and harbored some virulence determinants. This case showed that, although relatively rare, prolonged milk contamination by L. monocytogenes can originate from subclinical and persistently infected cows, posing a health risk to consumers.     

近红外光谱法检测奶粉掺假

目的建立近红外光谱法结合Adulterant Screen算法快速鉴别奶粉中大豆蛋白和尿素掺假的方法。方法采用近红外光谱仪获得奶粉未知样的光谱曲线,再用Adulterant Screen算法以及全数据库奶粉分类模型和既定类型的掺假物模型对曲线主要成分和掺假成分进行分析。结果该方法对一定浓度大豆蛋白和尿素掺假奶粉样可以实现掺假鉴别,大豆蛋白和尿素掺假奶粉样的掺假判别限分别为0.3 g/100g和0.2 g/100g,掺假物正确识别限分别为0.5 g/100g和0.8 g/100g。结论利用近红外光谱法结合Adulterant Screen算法可以快速鉴别奶粉中大豆蛋白和尿素的掺假。