免疫学检测羊乳中掺入牛乳成分

由于营养、价格和原料等原因，市场上在羊乳中掺入牛乳的现象常有发生。针对这种乳源性掺假陆续产生了多种检测方法，本文主要综述了以ELISA为主的免疫学快速检测方法。     

An indirect competitive ELISA for the detection of cows’ milk and caseinate in goats’ and ewes’ milk and cheese using polyclonal antibodies against bovine γ-caseins

 An indirect competitive enzyme-linked immunosorbent assay (ELISA) method was developed for the detection of bovine milk and caseinate in goats’ and ewes’ milk and cheese. Polyclonal antibodies were raised in rabbits and chickens against bovine γ₃-casein. In a first affinity chromatography step, antibodies recognizing caseins were absorbed on bovine casein-Sepharose. From the dialysed eluate, antibodies crossreacting with ewes’ and goats’ milk protein were completely removed by immunoadsorption onto stationary phases containing ovine casein and protein extracted from genuine ewes’ and goats’ milk cheese. The detection limit of the ELISA test was 0.1% and the method was applied successfully during an EU collaborative study of the evaluation of methods for the detection of cows’ milk. Received: 15 February 1996     

Real-time TaqMan PCR for quantitative detection of cows’ milk in ewes’ milk mixtures

A real-time PCR based on the amplification of a fragment of the mitochondrial 12S ribosomal RNA gene was developed and evaluated for the detection and quantification of cows’ milk in raw and heat-treated cow/sheep milk mixtures. The method combines the use of cow-specific primers that amplify a 252 bp fragment from cow DNA, and mammalian-specific primers amplifying a 428 bp fragment from mammalian species DNA, which is used as an endogenous control. The method measures PCR product accumulation through a 6-carboxyfluorescein-labeled fluorogenic probe (TaqMan). A comparison of the cycle number at which mammalian and cow-specific PCR products were first detected, in combination with the use of reference standards of known bovine content, allowed the determination of the percentage of cows’ milk in mixtures. Experimental raw and heat-treated binary mixtures were analyzed, demonstrating the specificity and sensitivity of the assay for detection and quantification of cows’ milk in the range 0.5–10%.     

Opportunities for varying the composition of cows' milk*

At present, only two approaches for modifying the composition of bovine milk can be applied in practice: (a) changing to a breed other than the predominant Friesian and (b) dietary manipulation. Although other breeds may produce milk of high fat and protein content, the high yield of the Friesian, allied to the fact that it is an acceptable meat animal, probably ensures its continued dominance. In terms of diet, the content, yield and type of milk fat are easily susceptible to manipulation. However, the specialized diets required for this type of manipulation would probably incur extra costs, and it is not clear if the improved properties of the butter, eg, a butter having better low-temperature spreadability, could attract a better return in the market. Protein content and yield are fairly insensitive to diet, and the consensus is that protein composition is invariant. Currently, the most sensible strategy for the dairy farmer appears to be to produce milk as cheaply as possible, and to leave it to the technologists to convert the components into a range of attractive, nutritious foodstuffs.     

Rapid detection of cows' milk in sheeps' and goats' milk by a species-specific polymerase chain reaction technique

A polymerase chain reaction (PCR) assay was developed for the specific identification of cows' milk in sheep's and goats' milk by using primers targeting the mitochondrial 12S rRNA gene. The use of a forward primer complementary to a conserved DNA sequence, along with a reverse primer specific for cow, yielded a 223-bp fragment from cows' milk DNA, whereas no amplification signal was obtained in sheep's and goats' milk DNA. The technique was applied to raw, pasteurized, and sterilized milk binary mixtures of cow-sheep and cow-goat, enabling the specific detection of cows' milk with a good sensitivity threshold (0.1%). The proposed PCR assay represents a rapid and straightforward method applicable to the authentication of milk and other dairy products in routine analysis.     

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.     

Spectral surface plasmon resonance biosensor for detection of staphylococcal enterotoxin B in milk

This work evaluates a newly developed wavelength modulation-based SPR biosensor for the detection of staphylococcal enterotoxin B (SEB) in milk. Two modes of operation of the SPR biosensor are described: direct detection of SEB and sandwich assay. In the sandwich assay detection mode, secondary antibodies are bound to the already captured toxin to amplify sensor response. Samples including SEB in buffer and SEB in milk were analyzed in this work. The SPR biosensor has been shown to be capable of directly detecting concentrations of SEB in buffer as low as 5 ng/ml. In sandwich detection mode, the lowest detection limit was determined to be 0.5 ng/ml for both buffer and milk samples. The reported wavelength modulation-based SPR sensor provides a generic platform which can be tailored for detection of various foodborne pathogens and agents for food analysis and testing.     

乳粉中坂崎肠杆菌的快速检验

采用一种选择性显色培养基开展了乳粉中坂崎肠杆菌的检测，该培养基针对坂崎肠杆菌产生α-葡萄糖苷酶活性的特点．在显色培养基中添加了一种底物5-溴-4-氯-吲哚-α-D-吡喃葡萄糖苷（XαGlc），α-葡萄糖苷酶水解XαGlc,使菌落呈现蓝绿色．与传统检测方法相比，该检测方法选择性更强，操作更为简便，检测时间明显缩短，是一种较为理想的检测乳粉中坂崎肠杆菌的方法．     

Chemometric studies of some trace elements in cows' milk

The matrix of the correlation between the concentrations of 11 trace elements determined in 45 samples of cows' milk was used as a starting matrix for principal component analysis. The space dimension was reduced from 11 variables to 5 principal components, accounting for about 80% of the total variance. After an orthogonal rotation, the first factor (F1) was found to be positively correlated with Cr, Mn, and Fe, F2 positively correlated with Ni and negatively with Cu, F3 positively with Cd and Pb, F4 positively with Zn, Sr, and Mo and F5 positively correlated with A1 and Sr. Element clusters appear to be determined by their origin. Some clusters are confirmed by principal factor analysis. A plot of the principal component scores was also applied to the differentiation of Italian Milk from different geographical origins.     

Chemometric studies of some trace elements in cows' milk

Summary The matrix of the correlation between the concentrations of 11 trace elements determined in 45 samples of cows' milk was used as a starting matrix for principal component analysis. The space dimension was reduced from 11 variables to 5 principal components, accounting for about 80% of the total variance. After an orthogonal rotation, the first factor (F1) was found to be positively correlated with Cr, Mn, and Fe, F2 positively correlated with Ni and negatively with Cu, F3 positively with Cd and Pb, F4 positively with Zn, Sr, and Mo and F5 positively correlated with Al and Sr. Element clusters appear to be determined by their origin. Some clusters are confirmed by principal factor analysis. A plot of the principal component scores was also applied to the differentiation of Italian Milk from different geographical origins.

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Chemometrische Untersuchungen über einige Spurenelemente in pasteurisierter Kuhmilch

Zusammenfassung Die Analyse der Hauptkomponenten wurde auf die Korrelationsmatrix zwischen Konzentrationen von 11 Spurenelementen in 45 Kuhmilchproben angewandt. Die Raumdimension von 11 Variablen wurde auf 5 Hauptkomponenten die 80% der totalen Varianz ausmachen, reduziert. Die orthogonal-rotierte Faktorenmatrix zeigt, daß Cr, Mn und Fe an die erste Hauptkomponente gebunden sind, Ni und Cu an die zweite, Cd und Pb an die dritte, Zn, Sr und Mo an die vierte and Al and Sr an die fünfte. Die Herkunft der Gruppen von Spurenelementen werden diskutiert. Einige Gruppen wurden bei der Analyse der Hauptfaktoren bestatigt. Das Diagramm der Hauptkomponentenwerte wurde auf die Unterscheidung von Milch aus zwei geographischen Herkünften angewandt.

     

Application of an indirect ELISA and a PCR technique for detection of cows’ milk in sheep's and goats’ milk cheeses

We have applied a polymerase chain reaction (PCR) procedure and an indirect enzyme-linked immunosorbent assay (ELISA) for detecting the presence of cows’ milk in sheeps’ and goats’ milk cheeses. The PCR used a cattle-specific primer set targeting a 223 bp fragment from the mitochondrial 12S rRNA gene. This technique was applied to experimental cheese mixtures, industrial cheeses produced with known amounts of cow's milk, and several commercial cheeses, enabling the detection of low percentages of cows’ milk (1%). An indirect ELISA using a monoclonal antibody (AH4) against bovine β-casein was also assayed in this study for the specific detection of bovine milk in cheese. Results suggested that both ELISA and PCR may provide specific and reliable tools for detection of low percentages of undeclared cows’ milk in sheeps’ and/or goats’ milk cheeses and other dairy products.     

Determination of volatile compounds in cows' milk using headspace GC-MS

The composition of the volatile fraction of milk from cows was investigated in a survey of milk samples using a headspace sampling technique and gas chromatography coupled to mass spectrometry analysis (GC-MS). Milk samples were collected from 12 farms, selected for similar management, breed and level of production. Farms were also grouped according to the type of forage in the ration: (1) hay; (2) hay and maize silage; (3) hay, maize silage and grass silages. Forty-one compounds in milk were isolated and identified from GC-MS headspace analysis. Quantitatively, the most representative chemical class was ketones (eight compounds, 170 microg/kg), followed by aldehydes (nine compounds, 63 microg/kg), alcohols (eight compounds, 36 microg/kg), and lower amounts of hydrocarbons (six compounds), sulphur compounds (three compounds), esters (four compounds) and terpenes (three compounds). The novel headspace sampling technique, and the consequent reduction of sample pre-treatment, allowed the identification of low-molecular weight volatile compounds, and reduced the risk of producing artefacts during analysis. Discriminant analysis was used to identify a classification criterion for milk samples, using type of forage in the ration as a grouping variable. Posterior probability error rate indicated that aldehydes provided one of the best discriminant criteria for grouping milks according to ration composition. When all 41 identified volatile compounds were included, discriminant analysis selected nine compounds (acetone, 2,3-butanedione, 2-butanone, ethanol, acetaldehyde, ethylacetate, ethvlisovalerate, dimethylsulphone) that did not fail the tolerance test and which correctly classified 100% of the original cases.     

Residual concentrations of the flukicidal compound triclabendazole in dairy cows' milk and cheese

Triclabendazole (TCBZ) is a flukicidal halogenated benzimidazole compound extensively used in veterinary medicine. Liver fluke control in lactating dairy cattle is difficult because treatment should be implemented only during the dry period to avoid milk residues. However, control in endemic areas is usually implemented as regular treatments three to four times a year, even during the lactating period. Thus, information on TCBZ milk excretion and the risk of the presence of drug residues in fluid milk and milk-derivate products is essential. The experimental aims were to evaluate the comparative disposition kinetics of TCBZ and its sulpho-metabolites in plasma and milk in lactating dairy cattle after the oral administration (12 mg kg(-1)) of TCBZ and to assess the pattern of residues in cheese made with milk from treated dairy cows. Both TCBZ sulphoxide and sulphone metabolites but not TCBZ were detected in milk (up to 36 and 144 h, respectively) and plasma (up to 144 h) after oral administration of TCBZ. Residual concentrations of TCBZ sulpho-metabolites were found in cheese made with milk from treated animals. The total average residual concentration in fresh cheese was 13.0-fold higher than that obtained in milk used for its elaboration. The high concentrations of TCBZ sulpho-metabolites recovered in fresh cheese should be seriously considered before milk from treated cows is used for making dairy products.     

Alternative strategies for activation of the natural lactoperoxidase system in cows' milk: trials in Tanzania

Thiocyanate content and lactoperoxidase activity of individual cow's milk of different breeds were determined, and the effects of different lactoperoxidase system (LP-s) activation strategies were compared. Lactoperoxidase activity varied significantly between Friesian and both Ayrshire and Tanzania Short Horn Zebu (TSHZ), but differences between Ayrshire and TSHZ were not significant. There was no significant variation in SCN- content between breeds. The LP-s was activated using three strategies based on SCN-: namely; equal concentrations of SCN- and H2O2 (7:7, 10:10, 15:15 mg/l), excess SCN- concentrations (15:10, 20:10, 25:10 mg SCN-:H2O2/l), and excess H2O2 concentrations (10:15, 10:20, 10:25 mg SCN-:H2O2/l), plus a fourth strategy based on I- (15:15 mg I-:H2O2/l). The keeping quality (KQ) was assessed using pH, titratable acidity, clot on boiling and alcohol stability tests. All activation strategies enhanced the shelf life of milk (typically increasing KQ from around 10 to around 20 h), but it was clear that the effectiveness of the LP-s depends on the type and concentrations of the activators of the system. The LP-s activated using I- as an electron donor was more effective than the LP-s activated using SCN- as an electron donor, increasing the KQ by a further 6-8 h compared with SCN-.     

Fast real-time polymerase chain reaction for quantitative detection of Lactobacillus delbrueckii bacteriophages in milk

One of the main microbiological problems of the dairy industry is the susceptibility of starter bacteria to virus infections. Lactobacillus delbrueckii, a component of thermophilic starter cultures used in the manufacture of several fermented dairy products, including yogurt, is also sensitive to bacteriophage attacks. To avoid the problems associated with these viruses, quick and sensitive detection methods are necessary. In the present study, a fast real-time quantitative polymerase chain reaction assay for the direct detection and quantification of L. delbrueckii phages in milk was developed. A set of primers and a TaqMan MGB probe was designed, based on the lysin gene sequence of different L. delbrueckii phages. The results show the proposed method to be a rapid (total processing time 30 min), specific and highly sensitive technique for detecting L. delbrueckii phages in milk.