Short communication: Detection of undeclared presence of bovine milk in buffalo yogurt

The authenticity of buffalo (Bubalus bubalis) dairy products is a focal issue, considering the increasing demand for buffalo milk products. Therefore, the aim of this study was to investigate the undeclared presence of bovine (Bos taurus) milk in buffalo yogurt, to understand which risk factors might make the product vulnerable to fraud. Real-time PCR assay showed the undeclared presence of bovine DNA in addition to buffalo DNA in 18 of 72 samples. Given the widespread lack of data on the presence of undeclared milk species in buffalo dairy products, the study provides a significant insight into the incidence of fraud in the buffalo dairy field. The data from this study could help improve the analysis of food safety risks along the buffalo milk supply chain and in the dairy processing industry, perceived as being highly vulnerable to food fraud, and prioritize target areas for food policy making to steer and enforce European food fraud regulations.     

Identification of cow's milk in "buffalo" cheese by duplex polymerase chain reaction

A duplex polymerase chain reaction (PCR) was developed to identify the milk of bovine and buffalo species in cheese products, particularly in mozzarella cheese, a typical Italian cheese made from buffalo's milk. Two sets of primers were designed on the basis of the alignment of the sequence codifying mitochondrial cyt b available in the GenBank database. The primers proved to be species-specific, giving rise to 279-bp (bovine) and 192-bp (buffalo) amplified fragments. Since the amplification conditions for bovine and buffalo primers were identical, a duplex PCR was successfully applied to identify the two species in a single reaction step. This technique, when used to test cheese products from the retail trade, allowed the detection of partial or even total substitution of cow's milk for buffalo's milk, in some cases in samples of cheese misleadingly labeled "pure buffalo" mozzarella.     

A fast and accurate method for controlling the correct labeling of products containing buffalo meat using High Resolution Melting (HRM) analysis

The substitution of high priced meat with low cost ones and the fraudulent labeling of meat products make the identification and traceability of meat species and their processed products in the food chain important. A polymerase chain reaction followed by a High Resolution Melting (HRM) analysis was developed for species specific detection of buffalo; it was applied in six commercial meat products. A pair of specific 12S and universal 18S rRNA primers were employed and yielded DNA fragments of 220 bp and 77 bp, respectively. All tested products were found to contain buffalo meat and presented melting curves with at least two visible inflection points derived from the amplicons of the 12S specific and 18S universal primers. The presence of buffalo meat in meat products and the adulteration of buffalo products with unknown species were established down to a level of 0.1%. HRM was proven to be a fast and accurate technique for authentication testing of meat products.     

Use of duplex polymerase chain reaction (duplex-PCR) technique to identify bovine and water buffalo milk used in making mozzarella cheese

Molecular biology techniques have been used for species identification in food of animal origin in relatively recent years. A polymerase chain reaction (PCR) based method, the multiplex PCR, was recently applied to species identification in meat and meat products. It allows co-amplification of separate regions of a single gene or specific fragments, each typical of a different animal species in a single PCR reaction, using different pairs of primers in the same reaction mix. In the present paper, the duplex-PCR technique is proposed to identify bovine and water buffalo DNA in a single PCR assay in milk and mozzarella cheese (a typical Italian cheese, originally made from pure water buffalo milk). Because of its lower cost, undeclared bovine milk is added to water buffalo milk for making different kinds of mozzarella cheese. The results of this experiment indicate the applicability of this method, which showed an absolute specificity for the two species and a high sensitivity even down to low DNA concentrations (1 pg). In bovine and water buffalo mixtures of both milk and mozzarella cheese, the minimum concentration tested was 1% of bovine in water buffalo milk and water buffalo in bovine milk. The importance of the somatic cell content in raw milk is also discussed with special reference to the evaluation of mixtures (milk or cheese) of the two species.     

PCR detection of cows’ milk in water buffalo milk and mozzarella cheese

Polymerase chain reaction (PCR) has been applied for the specific detection of cows’ DNA in water buffalo milk and mozzarella cheese by using primers targeting the mitochondrial 12S ribosomal RNA gene. The use of specific primers for cow yielded a 346 bp fragment from cows’ milk DNA, whereas no amplification signal was obtained in sheep's, goats’ and water buffalo's milk DNA. Analysis of both buffalo milk and buffalo mozzarella cheese mixtures containing different percentages of cows’ milk or bovine mozzarella cheese, enabled the specific detection of cow's DNA with a sensitivity threshold of 0.1%. The proposed PCR assay represents a rapid and straightforward method for the detection of adulterations in water buffalo milk and mozzarella cheese.     

水牛奶乳制品深加工的研究进展

简单介绍了国内外奶水牛业的发展,详细展望了水牛奶深加工的产品,对发展奶水牛业和提升水牛奶的附加值具有一定的指导作用。     

Simultaneous detection of cow and buffalo species in milk from China, India, and Pakistan using multiplex real-time PCR

Asian countries are major producers of cow and buffalo milk. For quality and authenticity purposes, a multiplex real-time PCR assay was developed to specifically and simultaneously detect DNA from these 2 bovine species. Targeting the cytochrome b gene of mitochondrial DNA, common PCR primers amplified a 105-bp fragment, and 2 fluorescent probes specific to either cow or buffalo were designed for their identification. Specificity was successfully tested on 6 other species, including sheep and goat, and sensitivity reached 1% of cow DNA in buffalo DNA and vice versa. As an evaluation, the method was tested using 119 freeze-dried Asian milk samples from regional industrial milk facilities. Although these samples did not cover the entire Asian zone, the multiplex assay indicated that approximately 20% of the samples (mainly from India) showed high levels of cross-contamination of cow milk by buffalo milk, and vice versa. Fast, sensitive, and straightforward, this method is fit-for-purpose for the authenticity control of Asian milk.     

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%.     

Simultaneous detection of cow and buffalo milk in mozzarella cheese by Real-Time PCR assay

A Real-Time PCR Allelic Discrimination TaqMan assay based on the analysis of one diagnosis position enabling the identification of cows’ and buffalo milk in dairy products was developed. Specific primers and probes were designed on the mitochondrial cytochrome b gene. In particular, primers were designed upstream and downstream the chosen diagnosis site in a well conserved region for both Bos taurus and Bubalus bubalis. Two probes were designed to specifically hybridise to B. taurus and B. bubalis sequences.     

Survey on mycoflora of cow and buffalo dairy products from Southern Italy

Economic losses of dairy products due to spoilage by yeasts have been increasing in European companies because of the reduced use of preservatives, packaging in modified atmospheres, or new formulations that do not strictly control the growth of these organisms. This study reports the results of a survey of yeast species and populations in 145 samples of cow and buffalo dairy products collected in some regions of Southern Italy. Yeasts were isolated from 74% and 57% of cow and buffalo products, respectively. Candida inconspicua was the predominant species in unripened products from cow's milk, while C. famata was detected in medium and long-term ripened dairy products, mostly in association with other yeasts and with moulds belonging to the genus Penicillium. For dairy products produced from buffalo milk, C. inconspicua was the most important yeast frequently isolated from dairy products. Total yeast populations ranged from 5 x 10(2) to 5 x 10(5) cfu/g, indicating a good hygienic quality of the products. The isolation of C. albicans from one stracciatella sample is noteworthy, as this yeast represents a potential contamination by human. Even though yeasts are considered as environmental contaminants, the occurrence of some of them in dairy products at high levels could represent a risk for human health, in particular for immunocompromised patients.     

Comparison of the principal proteins in bovine, caprine, buffalo, equine and camel milk

Proteomic analysis of bovine, caprine, buffalo, equine and camel milk highlighted significant interspecies differences. Camel milk was found to be devoid of β-lactoglobulin, whereas β-lactoglobulin was the major whey protein in bovine, buffalo, caprine, and equine milk. Five different isoforms of κ-casein were found in camel milk, analogous to the micro-heterogeneity observed for bovine κ-casein. Several spots observed in 2D-electrophoretograms of milk of all species could tentatively be identified as polypeptides arising from the enzymatic hydrolysis of caseins. The understanding gained from the proteomic comparison of these milks may be of relevance both in terms of identifying sources of hypoallergenic alternatives to bovine milk and detection of adulteration of milk samples and products.     

Short communication: Prediction of milk coagulation and acidity traits in Mediterranean buffalo milk using Fourier-transform mid-infrared spectroscopy

Milk coagulation and acidity traits are important factors to inform the cheesemaking process. Those traits have been deeply studied in bovine milk, whereas scarce information is available for buffalo milk. However, the dairy industry is interested in a method to determine milk coagulation and acidity features quickly and in a cost-effective manner, which could be provided by Fourier-transform mid-infrared (FT-MIR) spectroscopy. The aim of this study was to evaluate the potential of FT-MIR to predict coagulation and acidity traits of Mediterranean buffalo milk. A total of 654 records from 36 herds located in central Italy with information on milk yield, somatic cell score, milk chemical composition, milk acidity [pH, titratable acidity (TA)], and milk coagulation properties (rennet coagulation time, curd firming time, and curd firmness) were available for statistical analysis. Reference measures of milk acidity and coagulation properties were matched with milk spectral information, and FT-MIR prediction models were built using partial least squares regression. The data set was divided into a calibration set (75%) and a validation set (25%). The capacity of FT-MIR spectroscopy to correctly classify milk samples based on their renneting ability was evaluated by a canonical discriminant analysis. Average values for milk coagulation traits were 13.32 min, 3.24 min, and 39.27 mm for rennet coagulation time, curd firming time, and curd firmness, respectively. Milk acidity traits averaged 6.66 (pH) and 7.22 Soxhlet-Henkel degrees/100 mL (TA). All milk coagulation and acidity traits, except for pH, had high variability (17 to 46%). Prediction models of coagulation traits were moderately to scarcely accurate, whereas the coefficients of determination of external validation were 0.76 and 0.66 for pH and TA, respectively. Canonical discriminant analysis indicated that information on milk coagulating ability is present in the MIR spectra, and the model correctly classified as noncoagulating the 91.57 and 67.86% of milk samples in the calibration and validation sets, respectively. In conclusion, our results can be relevant to the dairy industry to classify buffalo milk samples before processing.     

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.     

Evaluation of the peptide profile with a view to authenticating buffalo mozzarella cheese

Electrophoretic and chromatographic techniques were used to determine water‐soluble peptide profiles aiming to identify the adulteration of buffalo milk mozzarella cheese by the addition of cow's milk. Thus, cheeses were produced with contents of cow's milk varying from 0% to 100%, and the peptides extracted after production and after 20 days of refrigeration. Polyacrylamide gel electrophoresis under denaturing conditions in the presence of sodium dodecyl sulphate (SDS‐PAGE) identified a potential peptide marker of exclusively bovine origin with a size of about 21 kDa for the addition of cow's milk above 30%. Reverse‐phase high‐performance liquid chromatography (RP‐HPLC) indicated the existence of two potential peptides present in higher concentrations in buffalo milk and one exclusive for cow's milk, the latter making it possible to estimate the addition of cow's milk to buffalo milk. Six commercial brands of buffalo mozzarella cheese were evaluated, and indications of adulteration found in four of them.     

Characterisation of the geographical origin of buffalo milk and mozzarella cheese by means of analytical and spectroscopic determinations

Appreciation of qualified national products, together with a guaranteed reference for consumers, has become necessary in the field of dairy products. Indeed, the Protected Designation of Origin (PDO) trademark has been assigned to numerous cheeses, such as buffalo milk mozzarella. In order to receive this designation, the raw materials have to be produced and processed in the specified region from which the product gets its name. Therefore, in order to determine the authenticity of typical dairy products it is necessary to determine the geographical origin of the milk and of the finished product obtained from it. Classical techniques, high performance ion chromatography (HPIC), inductively coupled plasma emission spectroscopy (ICP-AES), nuclear magnetic resonance (NMR) and isotope ratio mass spectrometry (IRMS) were used for determining different compounds in combination with chemometric methods for the geographical characterization of buffalo milk mozzarella cheeses originating from two areas of Southern Italy. Isotopic ratios (¹³C/¹²C and ¹⁵N/¹⁴N) and other variables were affected by the specific area of origin of milk samples, while NMR data, together with isotopic ratios, were useful for the discrimination of mozzarella samples.     

Simultaneous identification of bovine and equine DNA in milks and dairy products inferred from triplex TaqMan real-time PCR technique

Koumiss is a popular dairy product in many lands, traditionally prepared from mare milk with spontaneous fermentation. Mare milk and its fermented derivates are more expensive than cow milk and its fermented derivates, and the possibility exists for producers and dealers to adulterate equine products with bovine items. In this work, we described the development of a triplex real-time PCR based on species-specific TaqMan probes for identification of bovine and equine DNA in milks and dairy products. In addition, a novel designed endogenous control was simultaneously amplified to eliminate possible false negatives. With this methodology, bovine and equine DNA were specifically identified by employing developed primers and probes. The limits of detection of this method were 0.001 ng for cow milk, yogurt, and mare milk, and 0.005 ng for sour soup and koumiss, respectively. In addition, the triplex real-time PCR assay for authentication of animal-derived products was effectively validated using binary DNA and milk mixtures, exhibiting well in terms of specificity, sensitivity, and reproducibility. In short, the triplex PCR assay was verified to be a time-saving and money-saving technique for the identification of bovine and equine DNA in milks and dairy products.     

Aflatoxin M1 in buffalo and cow milk in Afyonkarahisar,Turkey

Potential hazardous human exposure to aflatoxin M₁ (AFM₁) via consumption of milk and milk products has been demonstrated by many researchers. The aim of this study was to investigate the presence of this mycotoxin in buffalo and cow milk samples in the city of Afyonkarahisar, Turkey. For this purpose, 126 buffalo and 124 cow milk samples were collected from dairy farms in Afyonkarahisar province. AFM₁ levels were determined by high-performance liquid chromatography with tandem mass spectrometric detection. Although AFM₁ was not detected in cow milk samples, AFM₁ was found above the limit of detection (<0.008–0.032 µg/L) in 27% (34 out of 126) of the buffalo milk samples. The results of this study indicated the importance of continuous surveillance of commonly consumed milk or milk product samples for AFM₁ contamination in Turkey.     

Confirmation of buffalo tallow in anhydrous cow milk fat using gas liquid chromatography in tandem with species‐specific polymerase chain reaction

Confirmation of buffalo tallow in anhydrous cow milk fat (cow ghee) is a major problem to date. In the present investigation, a novel strategy has been developed to detect and confirm the buffalo tallow in cow ghee. It involved coupling the gas liquid chromatography (GLC) of triglycerides with the rapid species‐specific polymerase chain reaction (PCR) to confirm the presence of buffalo tallow in anhydrous cow milk fat. Adulteration of cow ghee with buffalo tallow at 10% level could be confirmed using the standardised protocol. The standardised protocol can help in countering the cases of cow ghee adulteration with buffalo tallow.     

Application of immunological methods for the detection of species adulteration in dairy products

A number of enzyme‐linked immunosorbent assays (ELISAs) have been developed for the detection of milk adulteration in dairy products. Target antigens have been caseins, lactoglobulins, immunoglobulins and other whey proteins. Polyclonal and monoclonal antibodies have been used in a variety of formats including direct, indirect, competitive and sandwich ELISAs. ELISAs have been successfully applied to the detection of cows' milk adulteration of sheep, goat and buffalo milk. Goat milk adulteration of sheep milk has also been detected. A number of ELISAs have also been applied to cheese. It is recommended that ELISA should be used in combination with PCR to ensure compliance with current legislation.