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to identify bovine and water buffalo DNA in a single PCR assay in milk and mozzarella cheese (a typical Italian cheese, originally made from pure water buffalo milk).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.     

High resolution melting analysis for quantitative detection of bovine milk in pure water buffalo mozzarella and other buffalo dairy products

Identification of buffalo dairy products has become an important issue to ascertain product quality, consumer rights and absence of food-borne allergic reactions. A polymerase chain reaction (PCR) followed by a high resolution melting (HRM) analysis was developed and applied for species specific detection of bovine milk in nine different commercial buffalo dairy products. A specific buffalo 12S rRNA and a bovine d-loop primer pair, targeting the mitochondrial genome, were employed in a duplex PCR assay. The analysis developed was found capable of identifying the presence of bovine milk down to 1% in commercial buffalo milk products and also of quantifying the ratio of bovine into buffalo milk. HRM was proven to be a fast and accurate technique for a routine authentication testing of mozzarella and other buffalo milk products.     

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.     

Robust PCR‐based method for quantification of bovine milk in cheeses made from caprine and ovine milk

To prevent fraud and enhance quality assurance, credible analysis of dairy products is crucial. A common problem is the addition of cheaper bovine milk to caprine and/or ovine dairy products and when not declared addition of bovine milk constitutes fraud. The aim was to develop a rapid, robust and sensitive method for the identification of adulteration of caprine and/or ovine cheeses with bovine milk. New quantitative real‐time polymerase (qPCR) assays were designed for the specific determination of bovine DNA (Cow1) and bovine, caprine and ovine DNA (BoCaOv). These were applied to 17 samples of caprine cheese and 24 of ovine cheese. Results showed that 17% (7/41) of these cheeses contained >5% bovine milk. As bovine milk was not declared as an ingredient in any of the samples, this represents adulteration. Other cheeses that contained detectable bovine milk at ≤5% (22%; 5/41) might pose a health risk to people allergic to bovine 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%.     

DNA-based qualitative and quantitative identification of bovine whey powder in goat dairy products

As one of the main ingredients in some milk powders, whey powder is sometimes added to pure goat milk products, which can cause health risks, economic fraud, and unfair competition of food industries. This study is the first to explore qualitative and quantitative methods to identify adulteration of bovine whey powder in goat dairy products based on DNA. We extracted DNA from whey powder using a modified DNA extraction method; this exhibited good quality and integrity, with purity of 1.53 to 1.75 and concentration of 122 to 179 ng/μL. Conventional PCR and real-time PCR were compared for qualitative detection of bovine whey powder; real-time PCR demonstrated sensitivity of 0.01 ng/μL, which was higher than the 0.05 ng/μL detected by the conventional PCR method. Furthermore, real-time PCR was conducted for DNA quantitative detection, with good linearity (R² = 0.9858) obtained for bovine whey powder contents from 0.1% to 30%. Relative error decreased with increase of the mixing proportion of whey powder; the coefficient of variation above 0.1% of the mixing ratio was close to or less than 5%; and the relative standard deviation of repeatability results was less than 5%. Considering the economic costs of testing, conventional PCR could be performed first, and samples with obvious intentional adulteration detected can be further accurately quantified by real-time PCR. Overall, this research provides a realistic and effective method for qualitative and quantitative identification of bovine whey powder in goat dairy products, thus laying a good foundation for verification of goat dairy product label claims and industrial control.     

A simultaneous triplex TaqMan real-time PCR approach for authentication of caprine and bovine meat,milk and cheese

Goat foodstuffs are considered as healthy foods with high nutritional value. This study demonstrated the development and validation of a triplex real-time PCR on the basis of species-specific and species-conservative TaqMan probes for the simultaneous identification of caprine and bovine DNA in meats, milk and cheeses with a prerequisite designed endogenous control. In this research, caprine and bovine meat, milk and cheese were specifically identified via developed primers and probes, and the limits of detection of this methodology were 0.005 and 0.01 ng DNA of milk and cheese from goat, and 0.01 and 0.05 ng DNA of milk and cheese from cow. Taken together, this approach was elaborated to address dairy adulteration issues to eliminate the fraud of economically motivated goat milk and cheese adulteration by adding cow milk.     

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.     

The Microstructure and Physicochemical Properties of Probiotic Buffalo Yoghurt During Fermentation and Storage: a Comparison with Bovine Yoghurt

The physicochemical and rheological properties of yoghurt made from unstandardised unhomogenised buffalo milk were investigated during fermentation and 28 days of storage and compared to the properties of yoghurt made from homogenised fortified bovine milk. A number of differences observed in the gel network can be linked to differences in milk composition. The microstructure of buffalo yoghurt, as assessed by confocal laser scanning microscopy (CLSM) and cryo scanning electron microscopy (cryo-SEM), was interrupted by large fat globules and featured more serum pores. These fat globules have a lower surface area and bind less protein than the homogenised fat globules in bovine milk. These microstructural differences likely lead to the higher syneresis observed for buffalo yoghurt with an increase from 17.4 % (w/w) to 19.7 % (w/w) in the weight of whey generated at days 1 and 28 of the storage. The higher concentration of total calcium in buffalo milk resulted in the release of more ionic calcium during fermentation. Gelation was also slower but the strength of the two gels was similar due to similar protein and total solids concentrations. Buffalo yoghurt was more viscous, less able to recover from deformation and less Newtonian than bovine yoghurt with a thixotropy of 3,035 Pa.s^?1 measured for buffalo yoghurt at the end of the storage, at least four times higher than the thixotropy of bovine yoghurt. While the titratable acidity, lactose consumption and changes in organic acid concentrations were similar, differences were recorded in the viability of probiotic bacteria with a lower viability of Lactobacillus acidophilus of 5.17 log (CFU/g) recorded for buffalo yoghurt at day 28 of the storage. Our results show that factors other than the total solids content and protein concentration of milk affect the structural properties of yoghurt. They also illustrate the physicochemical reasons why buffalo and bovine yoghurt are reported to have different sensory properties and provide insight into how compositional changes can be used to alter the microstructure and properties of dairy products.     

Presence of Campylobacter and Arcobacter species in in-line milk filters of farms authorized to produce and sell raw milk and of a water buffalo dairy farm in Italy

The objectives of this study were to investigate the presence of Campylobacter spp. and Arcobacter spp. in dairy herds authorized for the production and sale of raw milk and in a water buffalo dairy farm, and to test the antimicrobial susceptibility of the isolates. A total of 196 in-line milk filters were collected from 14 dairy farms (13 bovine and 1 water buffalo) for detection of Campylobacter spp. and Arcobacter spp. by microbiological culture. For each farm investigated, 1 isolate for each Campylobacter and Arcobacter species isolated was tested using the Etest method (AB Biodisk, Solna, Sweden) to evaluate the susceptibility to ciprofloxacin, tetracycline, chloramphenicol, ampicillin, erythromycin, and gentamicin. A total of 52 isolates were detected in 49 milk filters in 12 farms (85.7%) out of 14 and the isolates were identified as Campylobacter jejuni (6), Campylobacter hyointestinalis ssp. hyointestinalis (8), Campylobacter concisus (1), Campylobacter fetus ssp. fetus (1), Arcobacter butzleri (22), and Arcobacter cryaerophilus (14). The small number of isolates tested for antimicrobial susceptibility precludes any epidemiological consideration but highlights that all Campylobacter isolates were susceptible to macrolides, which are the first-choice drugs for the treatment of campylobacteriosis, and that resistance to fluoroquinolones and tetracycline was detected; for Arcobacter isolates, resistance to ampicillin and chloramphenicol was detected. The sale of raw milk for human consumption by self-service automatic vending machines has been allowed in Italy since 2004 and the presence of C. jejuni in in-line milk filters confirms that raw milk consumption is a significant risk factor for human infection. The high occurrence of emerging Campylobacter spp. and Arcobacter spp. discovered in dairy farms authorized for production and sale of raw milk represents an emerging hazard for human health.     

The occurrence of Staphylococcus aureus on a farm with small-scale production of raw milk cheese

In recent years, the small-scale production of raw milk products has increased in Norway, and there is some concern that such foods may pose a risk of staphylococcal food poisoning to consumers. The aim of the study was to evaluate potential sources of contamination of raw milk cheese with Staphylococcus aureus on a bovine dairy farm with small-scale production. Samples for bacteriological analyses (n = 144) were collected from the animals, the environment, processing equipments, from humans, and from cheeses at various stages of production. Staphylococcus aureus was isolated from 10 of 11 cows, the farmer, equipment, the environment, and the cheese. Seventy-five Staph. aureus isolates were genotyped by pulsed-field gel electrophoresis, tested for enterotoxin (SE) production by reversed passive latex agglutination, for SE genes by multiplex polymerase chain reaction, and for penicillin resistance by the cloverleaf method. Five different pulsotypes were identified and SE gene fragments were identified in 11 isolates, but no isolates produced SE or were penicillin resistant. Staphylococcus aureus was found throughout the farm, and appeared to be spread with the milk to the environment, equipment, and to products. One pulsotype dominated and was identified from most sample sites on the farm. Raw milk products are vulnerable to contamination with Staph. aureus. Strategies to reduce the occurrence of Staph. aureus in bulk milk are of particular importance on farms where milk is used for raw milk 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.     

Species origin of milk in Italian mozzarella and Greek feta cheese

Several animal species such as cattle, goats, sheep, and water buffalo provide milk for dairy products. We describe a simple procedure for detecting the species origin of milk used for cheese production. DNA was isolated from Italian mozzarella or Greek feta by sequential organic extractions and resin purification. This DNA was analyzed by polymerase chain reaction-restriction fragment length polymorphism as described previously for meat samples. This procedure differentiated mozzarella made from water buffalo milk and from less expensive bovine milk and also feta cheeses made from bovine, ovine, and caprine milk.     

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.     

A novel method for species identification in milk and milk-based products

This study describes a method for species-specific detection of animal DNA from different species (cattle, sheep, goat, water buffalo) in milk and dairy products. A primer set was designed in conserved region on the basis of the alignment of the sequence codifying the genomic kappa-casein gene in order to amplify all four species with a single primer pair. Polymorphisms were detected via minisequencing with extension primers designed in conserved sequences for haplotype determination that allow unambiguous assignment to each species. The method was successfully applied to the detection of raw and pasteurized milk from the four different species considered as well as to cheese products from the retail trade. Estimation of the limit of detection was carried out using a progression of dilutions of genomic DNA as well as DNA isolated from milk of a known number of somatic cells from different species in order to be able to achieve detection rates as low as 0.1% bovine milk mixed with buffalo milk.     

Prevalence and risk factors for multidrug-resistant Escherichia coli isolated from subclinical mastitis in the western Chitwan region of Nepal

Subclinical mastitis (SCM) represents a significant burden and challenge to modern dairy management. Multidrug-resistant Escherichia coli (MDR E. coli) in milk poses a public health threat to humans especially via the consumption of unpasteurized dairy products. This study aimed to determine the occurrence of MDR E. coli in cows and buffalo in the households of the western part of the Chitwan district of Nepal. A total of 243 lactating cows and buffalo were included in this study. Milk samples (n = 972) were screened using the California Mastitis Test (CMT). The E. coli was isolated from milk samples that were positive for CMT using standard bacteriological protocols. A semi-structured questionnaire was administered to farmers to identify the risk factors associated with the occurrence of SCM in cows and buffalo. Of the 243 dairy animals screened, 42.8% (n = 104/243) showed positive CMT results. However, of the 972 quarters sampled, only 19.3% (n = 188/972) were positive for SCM. The prevalence of E. coli in these animals was found to be 16.5% in animals (n = 40/243). However, E. coli was isolated from only 5% (n = 49/972) of the quarters. Of the 49 E. coli isolated, the resistance to ceftriaxone (38.8%, n = 19/49) and ciprofloxacin (37.7%, n = 17/49) were the most prevalent. Animals with a history of mastitis were 3.57 times more likely to have SCM than other animals. Similarly, lactating animals with previous teat abrasions were 3.22 times more likely to develop SCM than animals without teat injuries. As expected, cleaning the barn once in 2 to 3 d was associated with an increased occurrence of SCM in lactating cows. This study reports the occurrence of MDR E. coli in SCM, which poses a public health threat. Creating awareness of milk pasteurization, and food safety practices are necessary among the farmers.     

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.     

Buffalo milk fat globules and their biological membrane: in situ structural investigations

Milk fat globules and their surrounding biological membrane (the MFGM) are not well understood despite the importance of these milk components in human nutrition and the role of fat globules in determining the properties of dairy products. The objectives of this study were to investigate these unique colloidal assemblies and the microstructure of the MFGM in buffalo milk, which is the second largest global source of dairy products. In-situ structural investigations were performed at room temperature using confocal microscopy with multiple fluorescent probes (Nile Red, Rh-DOPE, the lectin WGA-488). Microscopic observations showed cytoplasmic crescents around fat globules and the heterogeneous distribution of glycosylated molecules and polar lipids with the occurrence of lipid domains. The lipid domains in the buffalo MFGM appear to form by the segregation of lipids with a high phase transition temperature (e.g. sphingomyelin and saturated phosphatidylcholine molecular species) and cholesterol resulting in a gel phase or a Lo phase forming circular domains. The structure of the buffalo MFGM results from a non-random mixing of components, consistent with observations for other species. Structural heterogeneities of the MFGM could affect the processability of buffalo fat globules and the bioavailability of milk lipids.     

The prevalence and characterization of Bacillus cereus isolated from raw and pasteurized buffalo milk in southwestern China

Bacillus cereus is an important food-borne pathogenic bacteria and a putrid microorganism in the dairy industry. Raw and pasteurized buffalo milk play important roles in the dairy market in southwestern China. However, the reports on the prevalence and characterization of B. cereus strains isolated from the above sources are lacking. In this study, 150 raw buffalo milk samples and 300 pasteurized buffalo milk samples were collected from 3 provinces in southwestern China. The genotype, virulence gene distribution, antibiotic resistance, and biofilm-forming ability of isolates were analyzed. Ninety-six B. cereus strains were isolated and identified: 50 isolates (33.3%) from buffalo raw milk and 46 isolates (15.3%) from pasteurized buffalo milk. These strains were classified into 41 sequence types (ST) and 5 groups, of which ST857 was the predominant ST. The detection rates of virulence genes nheABC cluster, hblACD cluster, cytK, bceT, entFM, hlyII, and cesB were 89.6%, 13.5%, 64.6%, 71.9%, 84.4%, 62.5%, and 6.25%, respectively. The antimicrobial susceptibility testing showed that more than 90% of the isolates were susceptible to gentamicin, chloramphenicol, ciprofloxacin, erythromycin, vancomycin, and tetracycline, as well as resistant to ampicillin, cefepime, oxacillin, and rifampin. The results of biomass biofilm evaluation of the isolates on the stainless-steel tube showed that the optical density values at a wavelength of 595 nm of all strains in group I were greater than 1, with the strongest overall biofilm-forming ability among 5 groups, and the overall biofilm-forming ability of group III was the weakest. There was a relationship between the biofilm-forming ability and phylogenetic relationship of B. cereus strains. Taken together, our findings are the first to report the contamination situation and characterization of B. cereus isolated from raw and pasteurized buffalo milk in southwestern China as well as indicate the potential risk posed by this pathogen to dairy industry and public health.     

一种适用于乳制品基因组DNA快速提取方法的研究

目的对比NaOH裂解法、PBS裂解法以及直接煮沸法3种方法提取乳制品中核酸的提取效果,优化提取条件,确定一种更适用于现场检测、简便快速的的乳制品DNA快速提取技术。方法以牛奶、水牛奶、牦牛奶、羊奶、骆驼奶、以及驴奶6种常见的乳制品为材料,分别用NaOH裂解法、PBS裂解法以及直接煮沸法3种提取方法提取乳制品中的DNA,并根据裂解液用量和裂解时间进行优化,通过PCR扩增和琼脂糖凝胶电泳分析,检测DNA提取的质量和灵敏度。结果 NaOH裂解法能够提取所有物种的乳制品DNA,而且可以在最佳裂解条件下提取模拟掺假混合乳的DNA进行检测,发现其检测限能达到1%的牛奶含量。结论该方法取样量小,成本低,在15 min内即可完成快速提取,为实验室乳制品DNA定性或定量鉴别,以及乳制品的现场掺假鉴别提供了一种快速灵敏低成本的样品前处理技术。