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.     

Integration of high and low field 1H NMR to analyse the effects of bovine dietary regime on milk metabolomics and protein-bound moisture characterisation of the resulting mozzarella cheeses during ripening

The influence of dairy cow feeding regime was investigated using ¹H nuclear magnetic resonance (NMR). Two different NMR analytical systems were deployed: high field ¹H NMR to investigate the influence on milk metabolomics and low field NMR to characterise proton relaxation linked to changes in the state of mozzarella cheese moisture during ripening. The metabolomics results showed that grass-based feeding increased the concentration of a biological marker that signifies near-organic milk production conditions. On the other hand, the investigation of cheese moisture distribution showed that grass-based diets reached final moisture partitioning in a shorter time, which implied the formation of a more compact protein structure in the cheese matrix. These results indicate that pasture-based dairying may be differentiated in terms of the provenance of milk produced along with the accrual of additional benefits during ripening of the resulting mozzarella cheeses.     

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.     

Correlation between multielement stable isotope ratio and geographical origin in Peretta cows' milk cheese

The aim of this study was to characterize the isotopic composition and protect “Peretta” cows’ milk cheese, a typical product of Sardinia, against other cheeses of the same appearance sold under the same name, but made of raw materials from northern Europe. The study was concerned with 3 types of cheese: those produced in local dairies from milk from free-grazing or pasture-grazing cows in Sardinia (local dairy product), cheeses made on an industrial scale from milk produced by intensive farming in Sardinia (factory cheese), and cheeses made with raw materials imported from other countries (imported product). To distinguish the Sardinian cheeses from the imported product, the stable isotope ratios ¹³C/¹²C, ¹⁵N/¹⁴N, D/H, ³⁴S/³²S, and ¹⁸O/¹⁶O were used. Determination of the isotopic data δ¹³C, δ¹⁵N, δ²H, and δ³⁴S was performed in the casein fraction, whereas δ¹⁸O and δ¹³C were determined in the glycerol fraction. Measurements were performed by isotope ratio mass spectrometry. A comparison between mean values of the isotope ratios by statistical analysis (ANOVA and Tukey's test) showed that the greatest difference between the 3 types of cheese (local dairy, factory, and imported products) was in the ¹³C/¹²C, ³⁴S/³²S, and ¹⁸O/¹⁶O isotope ratios. In the other parameters, either no differences (δ¹⁵N) or minimal differences (δ²H) were found. Evaluation of the data by multivariate statistical analysis (principal component analysis and hierarchical cluster analysis) revealed that the isotope characteristics of the factory products were similar to those of the cheeses produced from imported raw materials, whereas a difference was found between the local dairy-produced cheeses and the products in the other 2 categories.     

DETECTION OF COW MILK IN BUFFALO "MOZZARELLA" BY POLYMERASE CHAIN REACTION (PCR) ASSAY

The authors used a polymerase chain reaction (PCR) assay on buffalo mozzarella, a typical Italian dairy product, from the Apulia markets to evaluate the presence of cow milk and verification of the mozzarella label. The results obtained from 30 mozzarella samples demonstrated the presence of the cow genome in 22/30 samples, highlighting contamination as probable fraudulent adding of cow's milk or use of the same equipments in both working cycles.     

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.     

Stable Isotope Ratio Analysis for Assessing the Authenticity of Food of Animal Origin

The main elemental constituents (H, C, N, O, and S) of bio‐organic material have different stable isotopes (²H, ¹H; ¹³C,¹²C; ¹⁵N,¹⁴N; ¹⁸O,¹⁷O,¹⁶O; ³⁶S, ³⁴S, ³³S, and ³²S). Isotopic ratios can be measured precisely and accurately using dedicated analytical techniques such as isotope ratio mass spectrometry (IRMS). Analysis of these ratios shows potential for assessing the authenticity of food of animal origin. In this review, IRMS analysis of food of animal origin and variability factors related to stable isotope ratios in animals are described. The study also lists examples of application of stable isotope ratio analysis to meat, dairy products, fish, and shellfish and emphasizes the strengths and weaknesses of the technique. Geographical, climatic, pedological, geological, botanical, and agricultural factors affect the stable isotope ratios (SIR) of bio‐elements, and SIR variations are ultimately incorporated into animal tissue through eating, drinking, breathing, and exchange with the environment, being recorded in the resulting foods. SIR analysis was capable of determining geographical origin, animal diet, and the production system (such as organic/conventional or wild/farmed) for pork, beef, lamb, poultry, milk, butter, cheese, fish, and shellfish. In the case of the hard PDO (protected designations of origin) cheeses Grana Padano and Parmigiano Reggiano it is also used in real‐life situations to assess the authenticity of grated and shredded cheese on the market.     

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.     

Cyclopropyl and ω-cyclohexyl fatty acids as quality markers of cow milk and cheese

Content of cyclopropyl and ω-cyclohexyl fatty acids of microbial origin, respectively, from silage and rumen, was determined by GC–MS and confirmed by ¹H NMR in more than 200 milk samples from dairy cows fed with different forages. Cyclopropyl fatty acids (about 0.1% of milk fat) were detected for the first time in milk and they were present only in milk samples from cows fed with forages containing maize silage, which is not allowed to produce milk for some PDO cheeses as Parmigiano-Reggiano. Their determination can be proposed as a quality parameter of milk or feed and it can be useful especially to distinguish cheeses sold as Parmigiano Reggiano from others cheeses. The content of ω-cyclohexyl tridecanoic acid varied from 0.0% to 0.15% of milk fat, and it was higher in milk samples from cows fed with diets richer in cereal meals.     

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.     

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%.     

Occurrence of β-casomorphins 5 and 7 in commercial dairy products and in their digests following in vitro simulated gastro-intestinal digestion

The occurrence of β-casomorphin-5 (BCM5) and β-casomorphin-7 (BCM7) was investigated in commercial dairy products and in their digests, following in vitro simulated gastro-intestinal digestion (SGID), by means of HPLC–MS. The analysed dairy products were as follows: 10 cheeses (Gorgonzola, Caprino, Brie, Taleggio, Gouda, Fontina, Cheddar and Grana Padano 10-, 15- or 25-m ripened); 4 samples of drinking milk (unprocessed, pasteurised, UHT and in bottle-sterilised); 2 yoghurts and 4 fermented milks containing probiotics; 7 infant formulas; and 4 dried milk-derivatives (skim milk powder, calcium caseinate and milk protein concentrates). β-Casomorphin-5 was not detected in dairy products, either prior to or after SGID. β-Casomorphin-7 was detected only in cheeses with the exception of Taleggio, Caprino and Grana Padano samples. Peptide amount was in the range 0.01–0.15 mg kg⁻¹ the highest level being recovered in Brie sample. Following SGID, BCM7 formed in all dairy samples or increased up to 21.77 mg kg⁻¹ in digests of cheeses. The peptide level ranged from 0.29 to 1.23 mg kg⁻¹in fermented milks and from 3.46 to 22.18 mg kg⁻¹ in dried milk-derivatives. Digests of commercial infant formulas contained BCM7 at concentrations of 0.04–0.21 mg l⁻¹. For the first time, this work reports quantitative values for BCM5 and BCM7 in a range of dairy products providing evidence that, during processing, only proteolytic systems involved in manufacturing and ripening of cheese can potentially hydrolyse β-CN to BCM7. Nevertheless, formation or further release of BCM7 is mainly promoted by the action of gastrointestinal proteinases during in vitro digestion irrespective of the type of dairy product.     

Quality evaluation of mozzarella cheese made from buffalo milk by using paneer booti (Withania coagulans) and calf rennet

This systematic research was conducted to assess the suitability of Withania coagulans for the preparation of mozzarella cheese from buffalo milk. The extract of dried berries of paneer booti (W. coagulans) was prepared in three buffers separately viz. Tris‐HCl, phosphate, NaCl and appropriate concentration of crude extracts were tested for their milk coagulating activity. NaCl solution with 0.85% strength was found to be suitable for milk coagulation without any objectionable taste and flavour. The results revealed that 15 μL crude enzyme extract per mL of milk was the optimum concentration for coagulation while 4.25 and 37 °C were the best levels of pH and incubation temperature for coagulation respectively. Mozzarella cheese prepared from crude enzyme extract was compared with the cheese prepared by calf rennet using acidification process. There was a nonsignificant difference among all tested parameters of both the coagulants. These findings support the suitability of an aqueous extract of W. coagulans for the commercial preparation of different types of mozzarella cheeses.     

Determining the geographic origin of milk in Australasia using multi-element stable isotope ratio analysis

A pilot study was conducted to evaluate the suitability of multi-element isotope ratio analysis for determining the origin of cows’ milk produced within Australasia. A milk sample from pasture-fed cows was obtained from seven dairying regions in Australia and New Zealand and analysed for the ratios of ¹³C/¹²C, ¹⁵N/¹⁴N, ¹⁸O/¹⁶O, ³⁴S/³²S, and ⁸⁷Sr/⁸⁶Sr. Each milk sample displayed a distinct isotopic fingerprint. Isotope ratios for ¹⁸O/¹⁶O and ¹³C/¹²C, in particular, conformed to predicted isotope fractionation patterns based on the latitude and climate of each region. Relative to skim milk, casein was enriched in ¹³C and ¹⁵N, and depleted of ³⁴S, whereas, predictably, ⁸⁷Sr/⁸⁶Sr ratios remained equivalent in both skim milk and casein. The milk samples from Australasia were considerably enriched in ¹⁸O and ³⁴S compared with reported values for most European dairy products. Overall, multi-element isotopic analysis has good potential for determining the geographic origin of dairy products produced within Australasia.     

EFFECTS of STERILIZING DOSES of GAMMA IRRADIATION ON LEVELS of VITAMIN A, AMINO ACIDS, and FATTY ACIDS IN SELECTED DAIRY PRODUCTS

Frozen dairy products intended for low microbial diets of immunosuppressed patients were sterilized by exposure to 40 kGy (4 Mrad) of cobalt-60 irradiation. Levels of vitamin A, amino acids, and selected fatty acids were measured in yogurt bars, ice cream, Cheddar cheese, mozzarella cheese, and nonfat dry milk before and after irradiation. Addition of antioxidants to the frozen dairy desserts prior to irradiation at - 78C was not effective in preventing degradation of vitamin A. There were significant decreases of approximately 90% in levels of vitamin A in Cheddar cheese and mozzarella cheese irradiated at -78C and 0–5C compared to the corresponding nonirradiated products. Levels of vitamin A in nonfat dry milk irradiated at three different temperatures (22C, 0–5C, or - 78C) were variable compared to levels in the nonirradiated product. Levels of palmitic, stearic, oleic, linoleic, and linolenic acids were retained in all irradiated dairy products analyzed. Irradiation at the specified temperatures did not affect levels of individual amino acids in nonfat dry milk or mozzarella cheese.     

H HRMAS-NMR metabolomic to assess quality and traceability of mozzarella cheese from Campania buffalo milk

The production of Mozzarella di Bufala Campana (MBC) is relevant for the agro-food economy of the Campania Region of Italy and the mark of Protected Designation of Origin (PDO) has been assigned to MBC in relation to its geographical origin. Advanced analytical methods must be then employed to assess authenticity, traceability, and quality of MBC. ¹H HRMAS-NMR (High Resolution Magic Angle Spinning Nuclear Magnetic Resonance) spectroscopy was applied here to directly identify specific metabolites in MBC intact samples without time-consuming sample pre-treatments. Overcrowded conventional ¹H HRMAS-NMR spectra were selectively simplified with two NMR pulse sequences: eCPMG and eDiff, by modulating spin-spin relaxation times and diffusion of MBC molecular components, respectively. Signal elaboration of edited spectra was combined with multivariate analyses to enable significant metabolic differentiation between MBC samples from two different production sites in Campania. Principal Components Analysis (PCA) for eCPMG spectra explained 97.54% of total variance between the two MBC groups for four metabolites (β-galactose, β-lactose, acetic acid, and glycerol). Less efficient was groups distinction by PCA for eDiff spectra, although differences in polyunsaturated acids, such as linoleic and linolenic acids, were highlighted. Similarly, Discriminant Analysis (DA) provided MBC group classification with 100% success in validation tests for eCPMG spectra, while DA prediction ability was reduced to 94.12% for eDiff spectra. Hierarchical Cluster Analysis (HCA) gave a totally correct classification between the two MBC groups only for eCPMG spectra. eCPMG spectra were also used to identify metabolites during MBC aging. As compared to fresh samples, 2 days old MBC samples showed increasing signals for isobutylic alcohol, lactic acid, and acetic acid. This work shows that ¹H HRMAS-NMR spectroscopy can rapidly characterise the metabolic profile of intact MBC samples and statistically distinguish the geographical origin of buffalo milk mozzarella and its freshness.     

Effects of Gamma Irradiation at -78°C on Microbial Populations in Dairy Products

The effect of low temperature (-78°C) gamma irradiation was investigated on microbial populations in selected dairy products to determine the irradiation dosage needed to produce commercially sterile dairy products for immunosuppressed patients. 40 kGy irradiation was sufficient to sterilize ice cream and frozen yogurt, but not mozzarella or Cheddar cheeses. Up to 8 wk continued incubation of the 40 kGy irradiated products at 7°C or 35°C resulted in no resuscitative growth in ice cream or yogurt, but identifiable growth in the cheeses. The 12D for B. cereus preinoculated into cheese and ice cream was 43-50 kGy.     

The effect of varying casein/fat ratio on physicochemical and sensory qualities of Feta‐type cheese made using buffalo milk

The Feta‐type cheese was prepared with different casein/fat (C/F) ratios of buffalo milk using microbial rennet. The manufactured Feta cheeses were subjected to physicochemical and sensory quality at 15‐day interval up to 60 days of ripening. Sensory analysis discriminated the different level of C/F ratio of buffalo milk cheeses predominantly by age. There was no significant difference (P < 0.01) observed in cheese made from C/F ratio of 0.6–0.7 in terms of flavour. The titratable acidity (TA), soluble protein and free fatty acid appear to be age‐dependent and increased throughout the ripening in all experimental cheeses.