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.     

Use of sandwich IgG ELISA for the detection and quantification of adulteration of milk and soft cheese

The aim of this work was to develop an assay capable of detecting adulteration of soft goat, sheep and buffalo milk cheese with bovine milk from cheaper sources. A previously developed indirect competitive ELISA had a lower sensitivity when applied to cheese, compared with milk. A sandwich ELISA was developed utilising the same monoclonal antibody in combination with a polyclonal goat anti-bovine IgG antibody. Once optimised, the ELISA was found to be highly specific. Detection limits in milk were 0.001% cows’ milk adulteration of sheep or buffalo milk, and 0.01% cows’ milk adulteration of goat milk. Detection limits in soft cheese were 0.001% in goat cheese and 0.01% in sheep or buffalo cheese. The assay was highly reproducible with both intra- and inter-assay coefficient of variation <10%. The ELISA performance makes it suitable for development as a kit for use in routine surveillance of milk and soft cheese.     

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.     

Staphylococcus aureus and Listeria monocytogenes in Norwegian raw milk cheese production

The aim of this study was to survey the presence of Staphylococcus aureus and Listeria monocytogenes during the cheese making process in small-scale raw milk cheese production in Norway.The prevalence of S. aureus in bovine and caprine raw milk samples was 47.3% and 98.8%, respectively. An increase in contamination during the first 2-3 h resulted in a 73.6% prevalence of contamination in the bovine curd, and 23 out of 38 S. aureus-negative bovine milk samples gave rise to S. aureus-positive curds. The highest contamination levels of S. aureus were reached in both caprine and bovine cheese after 5-6 h (after the first pressing). There was no contamination of L. monocytogenes in caprine cheeses and only one (1.4%) contaminated bovine cheese.This work has increased our knowledge about S. aureus and L. monocytogenes contamination during the process of raw milk cheese production and gives an account of the hygiene status during the manufacture of Norwegian raw milk cheeses.     

A gas chromatography-mass spectrometry-based metabolomic approach for the characterization of goat milk compared with cow milk

In this work, the polar metabolite pool of commercial caprine milk was studied by gas chromatography-mass spectrometry and multivariate statistical data analysis. Experimental data were compared with those of cow milk and the discriminant analysis correctly classified milk. By the same means, differences due to heat treatments (UHT or pasteurization) on milk samples were also investigated. Results of the 2 discriminant analyses were combined, with the aim of finding the discriminant metabolites unique for each class and shared by 2 classes. Valine and glycine were specific to goat milk, talose and malic acid to cow milk, and hydroxyglutaric acid to pasteurized samples. Glucose and fructose were shared by cow milk and UHT-treated samples, whereas ribose was shared by pasteurized and goat milk. Other discriminant variables were not attributed to specific metabolites. Furthermore, with the aim to reduce food fraud, the issue of adulteration of caprine milk by addition of cheaper bovine milk has been also addressed. To this goal, mixtures of goat and cow milk were prepared by adding the latter in a range from 0 to 100% (vol/vol) and studied by multivariate regression analysis. The error in the level of cow milk detectable was approximately 5%. These overall results demonstrated that, through the combined approach of gas chromatography-mass spectrometry and multivariate statistical data analysis, we were able to discriminate between milk typologies on the basis of their polar metabolite profiles and to propose a new analytical method to easily discover food fraud and to protect goat milk uniqueness. The use of appropriate visualization tools improved the interpretation of multivariate model results.     

Composition and aptitude for cheese-making of milk from cows,buffaloes, goats,sheep, dromedary camels,and donkeys

Bovines produce about 83% of the milk and dairy products consumed by humans worldwide, the rest represented by bubaline, caprine, ovine, camelid, and equine species, which are particularly important in areas of extensive pastoralism. Although milk is increasingly used for cheese production, the cheese-making efficiency of milk from the different species is not well known. This study compares the cheese-making ability of milk sampled from lactating females of the 6 dairy species in terms of milk composition, coagulation properties (using lactodynamography), curd-firming modeling, nutrients recovered in the curd, and cheese yield (through laboratory model-cheese production). Equine (donkey) milk had the lowest fat and protein content and did not coagulate after rennet addition. Buffalo and ewe milk yielded more fresh cheese (25.5 and 22.9%, respectively) than cow, goat, and dromedary milk (15.4, 11.9, and 13.8%, respectively). This was due to the greater fat and protein contents of the former species with respect to the latter, but also to the greater recovery of fat in the curd of bubaline (88.2%) than in the curd of camelid milk (55.0%) and consequent differences in the recoveries of milk total solids and energy in the curd; protein recovery, however, was much more similar across species (from 74.7% in dromedaries to 83.7% in bovine milk). Compared with bovine milk, the milk from the other Artiodactyla species coagulated more rapidly, reached curd firmness more quickly (especially ovine milk), had a more pronounced syneresis (especially caprine milk), had a greater potential asymptotical curd firmness (except dromedary and goat milk), and reached earlier maximum curd firmness (especially caprine and ovine milk). The maximum measured curd firmness was greater for bubaline and ovine milk, intermediate for bovine and caprine milk, and lower for camelid milk. The milk of all ruminant species can be used to make cheese, but, to improve efficiency, cheese-making procedures need to be optimized to take into account the large differences in their coagulation, curd-firming, and syneresis properties.     

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.     

Detection of cows' milk in goats' cheeses inferred from mitochondrial DNA polymorphism

A new polymerase chain reaction (PCR)-based method was developed to detect cows milk in goat cheese. This method is based on mitochondrial DNA (mtDNA) control region sequence variations. DNA extractions from 150 mg of cheese were carried out using a spin column-based method. Subsequent PCR amplifications of DNA were performed with cow specific primers, demonstrating the ability to detect cows' milk in a variety of cheeses. This simple approach provides high quality DNA, and is shown to be very sensitive, with a detection limit of less than 0.1% of cows' milk. Analysis of an agarose gel digital image allows a rough estimation of the percentage of cows' milk used in adulteration.     

Detection of milk mixtures in Halloumi cheese

A capillary electrophoresis method has been applied to the detection of illegal addition of milk from goat and/ or cow in Halloumi cheese, traditionally made with sheep milk. The electrophoretic profiles of the casein from Halloumi cheeses have revealed that caprine para-kappa-casein and bovine alphas1-casein peaks point to the presence of low percentages of goat's and/or cow's milk added to Halloumi cheese. Stepwise multiple linear regression has been used to predict these percentages with a standard error of the estimation of 2.14%. The analytical method combined with the statistical application is valid for the prediction of percentages higher than 2% of goat's and percentages of 5% of cow's milk added to the cheese either in fresh or ripened cheese. The standard error of estimation was higher for the prediction of cow's milk than for goat's 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.     

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.     

Functionality and physico-chemical characteristics of bovine and caprine mozzarella cheeses during refrigerated storage

Low moisture part-skim Mozzarella cheeses (MC) were manufactured using fresh bovine and caprine milk to study melting, physico-chemical, textural, and microstructural properties of the cheeses during 8 wk of refrigerated storage. Structural changes in cheese matrix were evaluated by scanning electron microscopy and by proteolytic patterns using nitrogen solubility, SDS-PAGE, and Gel-pro analyzer. Meltability of ripened cow and goat MC were not different when fat content of both milks were standardized, whereas bovine MC formed a significantly larger amount of free oil throughout the experiment. The results of the proteolytic patterns, texture attribute (cohesiveness), and microstructure revealed that bovine MC had a greater structural degradation of cheese matrix than caprine MC during the storage. Elevated protein degradation in bovine MC led to more intense brown color formation than the goat counterpart when the cheeses were baked. The melting characteristics showed high positive correlation (r = 0.51 to 0.80) with proteolysis, whereas it was negatively correlated with textural characteristics. Among textural attributes, cohesiveness was highly inversely correlated with melting characteristics (r = -0.69 to -0.88). High negative correlations were also observed between proteolytic parameters and textural attributes (r = -0.48 to -0.81).     

Effect of partial or total substitution of bovine for caprine milk on the compositional,volatile, non-volatile and sensory characteristics of semi-hard cheeses

Semi-hard cheeses were manufactured from caprine milk and bovine milk mixed in ratios of 100:0; 75:25; 50:50; 25:75; 0:100 (caprine:bovine) and ripened at 12 °C for 150 d. Substitution of up to 75% bovine for caprine milks had no significant effect on levels of cheese protein, moisture-in-non-fat substances, salt, pH, whiteness, total or individual free amino acids and substitution of up to 50% had no significant effect on levels of primary proteolysis and free fatty acid and fruity flavours. Increased bovine milk content resulted in decreased levels of butanoic to octanoic acids, ethyl and methyl esters of acetic to octanoic acids and acyclic, mono and bicyclic terpenes and waxy/goaty flavours to a degree dependent on the proportion of each milk type. Partial substitution of caprine with bovine milks resulted in cheeses with chemical and sensorial characteristics that may provide an available alternative to consumers desiring caprine cheeses.     

Changes in texture,composition and sensory characteristics of Camembert cheese made from a mixture of goat milk and cow milk during ripening

The study was conducted to evaluate the nutritional, textural and sensory characteristics of Camembert cheese made from goat milk, cow milk and their blends. The type of milk used affected yield, physicochemical properties and fatty acid profile of the cheeses. The percentages of C6:0, C8:0 and C10:0 acids were higher in the cheeses containing ≥ 75% goat milk. The sensory evaluation results showed differences in texture, colour, aroma and flavour among the cheeses. Cheeses made with the mixture of cow and goat milk particularly 50% goat milk mixed with 50% cow milk (50G:50C) maintained particular positive nutritional characteristics especially with respect to the mineral contents, fatty acids and consumer acceptability.     

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.     

Use of MALDI-TOF MS technology to evaluate adulteration of small ruminant milk with raw bovine milk

Detection of adulteration of small ruminant milk is very important for health and commercial reasons. New analytical and cost-effective methods need to be developed to detect new adulteration practices. In this work, we aimed to explore the ability of the MALDI-TOF mass spectrometry to detect bovine milk in caprine and ovine milk using samples from 18 dairy farms. Different levels of adulteration (0.5, 1, 5, 10, 20, 40, 60, and 80%) were analyzed during the lactation period of goat and sheep (in May, from 60 to 90 d in milk, and in August, from 150 to 180 d in milk). Two different ranges of peptide-protein spectra (500–4,000 Da; 4–20 kDa) were used to establish a calibration model for predicting the concentration of adulterant using partial least squares and generalized linear model with lasso regularization. The low molecular weight part of the spectra together with the generalized linear model with lasso regularization regression model appeared to have greater potential for our aim of detection of adulteration of small ruminants' milk. The subsequent prediction model was able to predict the concentration of bovine milk in caprine milk with a root mean square error of 11.4 and 17.0% in ovine milk. The results offer compelling evidence that MALDI-TOF can detect the adulteration of small ruminants' milk. However, the method is severely limited by (1) the complexity of the milk proteome resulting from the adulteration technique, (2) the potential degradation of thermolabile proteins, and (3) the genetic variability of tested samples. Additionally, the root mean square error of prediction based only on one individual sample adulteration series can drop down to 6.34% for quantification of adulterated caprine milk and 6.28% for adulterated ovine milk for the full set of concentrations or down to 2.33 and 4.00%, respectively, if we restrict only to low concentrations of adulteration (0, 0.5, 1, 5, 10%).     

Milk species identification in cheese varieties using electrophoretic,chromatographic and PCR techniques

Different methods were applied to standard mixtures of milk from different species and to model cheeses of different ages to study their applicability for the detection of cows’ milk and determination of the percentage of cows’ milk in mixed milk cheeses. Urea–polyacrylamide gel electrophoresis (urea–PAGE) and anion-exchange high performance liquid chromatography (HPLC) of caseins was restricted to the control of the adulteration of milk. Isoelectric focusing (IEF) of γ-caseins according the EU reference method was well suited for detection of cows’ milk even in matured cheese, but could not discriminate ewes’ milk from goats’ milk. IEF and cation-exchange HPLC of para-κ-caseins were appropriate for differentiation of bovine, ovine and caprine milk. Urea–PAGE and IEF of whey proteins was useful for the species authentication of whey cheeses. Polymerase chain reaction using species–specific primers was a highly sensitive technique for the qualitative detection of cows’ milk even in overripe mixed cheese.     

Indirect competitive enzyme-linked immunosorbent assay for the detection of native and heat-denatured bovine β-lactoglobulin in ewes' and goats' milk cheese

An indirect competitive enzyme-linked immunosorbent assay (ELISA) has been developed for the detection of native and heat-denatured bovineβ-lactoglobulin (hd-β-lg) in goats' and ewes' milk cheese. Polyclonal antibodies raised in chicken against hd-β-lg were purified by immunadsorption chromatography on native bovine, ovine and caprineβ-lactoglobulins conjugated to CNBr-activated Sepharose. Although lactoglobulins from ewe, goat and cow have very similar amino acid sequences, crossreactivity was eliminated completely by this procedure. A commercially available goat-anti-chicken alkaline phosphatase conjugate was used as a secondary antibody to detect the anti-hd-β-lg antibodies bound to immobilized hd-β-lg. The detection limit of the assay was 100 ng/ml bovine native lg or hd-β-lg or 0.1–0.2% of cows' milk equivalent in cheese. Within a method validation study of the European Union, the ELISA test was successfully applied for the detection of native and heat-denatured bovine whey proteins in ewes' and goats' milk cheeses.     

Nutritional and sensory characteristics of Minas fresh cheese made with goat milk,cow milk,or a mixture of both

This study aimed to assess and compare the nutritional, technological, and sensory characteristics of Minas fresh cheese made with goat milk, cow milk, or a mixture of the two stored in cold conditions for 21 d. The yield and centesimal composition of the cheeses were not affected by the type of milk used in their preparation. Reductions were observed in the moisture content, pH, proteolysis index, and instrumental hardness; moreover, increases were observed in the syneresis, acidity index, and depth of proteolysis index in all cheeses. The percentages of caprylic, capric, oleic, and linoleic fatty acids were higher in goat milk cheese and cheese made with a mixture of goat and cow milk compared with cow milk cheese, and a sensory evaluation revealed differences in color, flavor, and aroma between the cheeses. The preparation of Minas fresh cheese with a mixture of goat and cow milk can be a viable alternative for dairy products in the market that can be characterized as high-quality products that meet consumer demands.     

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.