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identify bovine and water buffalo DNA in a single PCR assay in milk and mozzarella cheese (a typical Italian cheese, originally made from pure water buffalo milk).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

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.     

Evaluation of oxidative damage in mozzarella cheese produced from bovine or water buffalo milk

Technological processes are the main sources of protein and lipid oxidation in food. The oxidative status was determined in a soft Italian cheese, namely mozzarella, produced from water buffalo or bovine milk. The amount of protein-bound carbonyls, dityrosine and α-lactalbumin aggregates were measured to evaluate the extent of protein oxidation. The α-tocopherylquinone/α-tocopherol ratio and the trolox-equivalent antioxidant capacity were used as redox markers in the fat fraction. The levels of protein-bound carbonyls and α-lactalbumin aggregates were found significantly higher in bovine mozzarella than in buffalo mozzarella. On the other hand, higher amounts of redox markers were found in buffalo mozzarella. The levels of dityrosine aggregates were similar in the two types of cheese. The data suggest that protein and fat are more protected against oxidative structure alterations in buffalo mozzarella than in bovine mozzarella.     

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.     

Low-moisture part-skim mozzarella cheese made from blends of camel and bovine milk: Gross composition,proteolysis, functionality,microstructure, and rheological properties

Camel (CM) milk is used in variety of ways; however, it has inferior gelling properties compared with bovine milk (BM). In this study, we aimed to investigate the physicochemical, functional, microstructural, and rheological properties of low-moisture part-skim (LMPS) mozzarella cheese, made from BM, or BM mixed with 15% CM (CM15%) or 30% CM (CM30%), at various time points (up to 60 d) of storage at 4°C after manufacture. Low-moisture part-skim mozzarella cheeses using CM15% and CM30% had high moisture and total Ca contents, but lower soluble Ca content. Compared with BM cheese, CM15% and CM30% LMPS mozzarella cheese exhibited higher proteolysis rates during storage. Adding CM affected the color properties of LMPS mozzarella cheese manufactured from mixed milk. Scanning electron microscopy images showed that the microstructure of CM15% and CM30% cheeses had smooth surfaces, whereas the BM cheese microstructures were rough with granulated surfaces. Low-moisture part-skim mozzarella cheeses using CM15% and CM30% showed significantly lower hardness and chewiness, but higher stringiness than BM cheese. Compared with BM cheese, CM15% and CM30% cheeses showed lower tan δ levels during temperature surges, suggesting that the addition of CM increased the meltability of LMPS mozzarella cheese during temperature increases. Camel milk addition affected the physicochemical, microstructural, and rheological properties of LMPS mozzarella cheese.     

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.     

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.     

Environmental life cycle assessment of Italian mozzarella cheese: Hotspots and improvement opportunities

The present study investigated a cradle-to-grave life cycle assessment to estimate the environmental impacts associated with Italian mozzarella cheese consumption. The differences between mozzarella produced from raw milk and mozzarella produced from curd were studied, and differences in manufacturing processes have been emphasized in order to provide guidance for targeted improvements at this phase. Specifically, the third-largest Italian mozzarella producer was surveyed to collect site-specific manufacturing data. The Ecoinvent v3.2 database was used for secondary data, whereas SimaPro 8.1 was the modeling software. The inventory included inputs from farm activities to end of life disposal of wasted mozzarella and packaging. Additionally, plant-specific information was used to assign major inputs, such as electricity, natural gas, packaging, and chemicals to specific products; however, where disaggregated information was not provided, milk solids allocation was applied. Notably, loss of milk solids was accounted during the manufacture, moreover mozzarella waste and transport were considered during distribution, retail, and consumption phases. Feed production and animal emissions were the main drivers of raw milk production. Electricity and natural gas usage, packaging (cardboard and plastic), transport, wastewater treatment, and refrigerant loss affected the emissions from a farm gate-to-dairy plant gate perspective. Post-dairy plant gate effects were mainly determined by electricity usage for storage of mozzarella, transport of mozzarella, and waste treatment. The average emissions were 6.66 kg of CO₂ equivalents and 45.1 MJ of cumulative energy demand/kg of consumed mozzarella produced directly from raw milk, whereas mozzarella from purchased curd had larger emissions than mozzarella from raw milk due to added transport of curd from specialty manufacturing plants, as well as electricity usage from additional processes at the mozzarella plant that are required to process the curd into mozzarella. Normalization points to ecotoxicity as the impact category most significantly influenced by mozzarella consumption. From a farm gate-to-grave perspective, ecotoxicity and freshwater and marine eutrophication are the first and second largest contributors of mozzarella consumption to average European effects, respectively. To increase environmental sustainability, an improvement of efficiency for energy and packaging usage and transport activities is recommended in the post-farm gate mozzarella supply chain.     

Processes that contribute to radiocesium decontamination of feta cheese

In a series of experiments, the transfer of radiocesium from ovine milk to feta cheese was investigated through modifications of the standard cheese making procedure. All variations explored showed no significant change in the percentage of radiocesium transfer and the milk-to-cheese transfer coefficient was determined as f=.79 plus/minus .04 L.kg-1. It is concluded that cesium, like the rest of the alkali metals, remains in the water phase and thus follows very closely the distribution of moisture into the products of cheese making. The possibility of radiocesium decontamination of mature feta during the customary storage of the product in brine was also explored in a second series of experiments. The theoretical model employed in the analysis of cesium transport from feta to brine is presented in the Appendix to this paper. Predictions of the model were validated by experiments. A procedure is thus proposed for decontaminating mature feta during storage through successive replacements of the storage medium. Nomograms are presented for the determination of the optimum time interval between changes of the brine and the radiocesium concentration remaining in the feta. Changes in the properties of the product induced by the proposed treatment were also investigated with respect to composition, taste, and overall quality.     

Potential growth of Listeria monocytogenes in Italian mozzarella cheese as affected by microbiological and chemical-physical environment

In the present study, 33 brands of mozzarella cheese (pasteurized cow milk mozzarella obtained by direct acidification through the addition of food-grade citric acid or obtained by natural acidification through the addition of thermophilic starter cultures, mozzarella for pizza mainly obtained by addition of citric acid, and pasteurized buffalo milk mozzarella obtained by adding microbial rennet) were characterized for the factors potentially influencing the growth of Listeria monocytogenes (microbial populations, moisture, pH, and organic acids). Then, the growth potential of L. monocytogenes in mozzarella was investigated by challenge tests performed at different temperatures. The presence of heterogeneous microflora (lactobacilli, streptococci, Pseudomonas spp., and, for buffalo mozzarella, yeasts) was evidenced. Almost all the product typologies were classified as high-moisture mozzarella cheese because moisture was >52%. Moreover, pH varied from 5.32 to 6.43 depending on the manufacturing methodology applied. Organic acid concentrations too showed great variability depending on the mozzarella production method, with values ranging from less than limit of detection (LOD; 16 mg/kg) to 14,709 mg/kg, less than LOD (216 mg/kg) to 29,195 mg/kg, and less than LOD (47 mg/kg) to 1,725 mg/kg in the water phase of lactic, citric, and acetic acids, respectively. Despite this presence, the concentration of undissociated acids was lower compared with the minimum inhibitory concentrations estimated for L. monocytogenes by other authors. This was confirmed by the results of the challenge tests conducted inoculating the pathogen in mozzarella produced with the addition of citric acid, as the microorganism grew fast at each temperature considered (4, 9, 15, and 20°C). Good hygiene practices should be strictly applied, especially with the aim of avoiding postproduction contamination of mozzarella, as the presence of organic acids and microflora is insufficient to prevent L. monocytogenes growth.     

Improvement of low fat mozzarella cheese properties using denatured whey protein

Mozzarella cheese was made from buffalo milk (6% fat) or from partially skimmed buffalo milk (2 and 4% fat) with 0.5 and 1% denatured whey protein. Adding whey protein to buffalo milk decreased rennet coagulation time and curd tension whereas increased curd synaeresis. Addition of whey protein to cheese milk increased the acidity, total solids, ash, salt, salt in moisture, also some nitrogen fractions. The meltability and oiling‐off values increased but the calcium values of mozzarella cheese decreased. The sensory properties of low fat mozzarella cheese were improved by addition of whey protein to the cheese milk.     

Detection of nontuberculous mycobacteria from water buffalo raw milk in Brazil

Milk is an important nutritional source to man and water buffalo raw milk is used to produce mozzarella cheese. Products from unpasteurized milk have been associated with certain infectious diseases and can carry pathogenic mycobacteria. Nontuberculous mycobacteria (NTM) are emerging pathogens causing opportunistic infections in humans and animals. The objectives of this study were to demonstrate the presence of mycobacteria in water buffaloes’ milk and to determine their role as possible sources of NTM infections. In this study, raw milk samples from dairy water buffaloes (Bubalus bubalis) (N = 23) were decontaminated by Petroff method and inoculated on to Löwenstein–Jensen and Stonebrink medium. After confirming positive colonies for acid fast bacilli (AFB) by Ziehl-Neelsen technique, the isolated mycobacteria were identified by PCR-Restriction Enzyme Analysis (PRA) and mycolic acids analysis by thin-layer chromatography (TLC). Mycobacterium simiae (2 isolates), Mycobacterium kansasii (2 isolates), Mycobacterium flavescens (2 isolates), Mycobacterium gordonae (3 isolates) and Mycobacterium lentiflavum (1 isolate) were identified by these techniques. The isolation of opportunistic pathogens such as M. kansasii, M. simiae and M. lentiflavum from raw milk represent a risk for the consumers of mozzarella cheese made by this milk.     

Volatile organic compounds in milk and mozzarella: Comparison between two different farming systems

The volatile organic compounds (VOC) and sensory characteristics of milk and mozzarella from farms with different rearing systems were compared. Milk samples were taken from 9 intensive (INT) and 13 semi-extensive farms (EXT) in the grazing period. VOC were analysed by SPME-GC-MS, and sensory evaluation was carried out by paired comparison. The volatile profiles of milks were significantly different: acetone and isopropyl alcohol characterised INT milk, pentanoic and decanoic acids, hexanal, ethylacetate, toluene, dimethyl sulphide characterised milk from EXT farms. Also mozzarella was discriminated by VOC, but only a few compounds derived from milk. Based on sensory evaluation, milk from the two sources can be distinguished by odour and colour, mozzarella only by colour. It was hypothesised that heating during the stretching phase caused volatilisation of many milk volatile compounds and formation new aroma active molecules that overcame the ‘primary odours’ of milk.     

Detection of the adulteration of water buffalo milk and mozzarella with cow’s milk by liquid chromatography–mass spectrometry analysis of β-lactoglobulin variants

A liquid chromatography–mass spectrometry method for detecting a fraudulent addition of cow’s milk to water buffalo milk and mozzarella is described. The presented approach utilises the whey protein β-lactoglobulin as marker for an adulteration. It offers a rapid determination combined with unequivocal identification of the marker protein in every run. An in-depth discussion of the subsequent data analysis highlights the potential problems of obtaining quantitative information on the level of adulteration. In an examination of 18 commercial buffalo mozzarella samples three products were found to be adulterated with high levels of cow’s milk.     

The effects of κ-casein polymorphism on the texture and functional properties of mozzarella cheese

The present study compared the texture and functional properties of mozzarella cheese made with milk containing different of genetic polymorphisms κ-casein (AA, AB, AE or BE). The genotype of κ-casein in the milk from individual Holstein cow was determined by pyrosequencing method. Full-fat Mozzarella cheese was made from pooled milk from 3 cows with the same κ-casein genotype and analysed 7 d after manufacture. The cheese made from type AB contained the highest level of fat and Ca/protein, and the lowest moisture content. The cheese made from type AB milk was harder and chewier than cheese made from type AE and BE milk. The cheese made from type AB and AA milk had higher stretchability but lower meltability and flowability than type AE and BE. In summary, the cheese made from type AB milk had different texture and functionality quality than that made from type AE or BE.     

Diversity of Lactic Acid Bacteria Isolated from Brazilian Water Buffalo Mozzarella Cheese

The water buffalo mozzarella cheese is a typical Italian cheese which has been introduced in the thriving Brazilian market in the last 10 y, with good acceptance by its consumers. Lactic acid bacteria (LAB) play an important role in the technological and sensory quality of mozzarella cheese. In this study, the aim was to evaluate the diversity of the autochthones viable LAB isolated from water buffalo mozzarella cheese under storage. Samples were collected in 3 independent trials in a dairy industry located in the southeast region of Brazil, on the 28th day of storage, at 4 ºC. The LAB were characterized by Gram staining, catalase test, capacity to assimilate citrate, and production of CO₂ from glucose. The diversity of LAB was evaluated by RAPD‐PCR (randomly amplified polymorphic DNA‐polymerase chain reaction), 16S rRNA gene sequencing, and by Vitek 2 system. Twenty LAB strains were isolated and clustered into 12 different clusters, and identified as Streptococcus thermophilus, Enterococcus faecium, Enterococcus durans, Leuconostoc mesenteroides subsp. mesenteroides, Lactobacillus fermentum, Lactobacillus casei, Lactobacillus delbrueckii subsp. bulgaricus, and Lactobacillus helveticus. Enterococcus species were dominant and citrate‐positive. Only the strains of L. mesenteroides subsp. mesenteroides and L. fermentum produced CO₂ from glucose and were citrate‐positive, while L. casei was only citrate positive. This is the first report which elucidates the LAB diversity involved in Brazilian water buffalo mozzarella cheese. Furthermore, the results show that despite the absence of natural whey cultures as starters in production, the LAB species identified are the ones typically found in 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%.     

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.     

Methicillin-resistant Staphylococcus aureus (MRSA) in foods of animal origin product in Italy

Methicillin-resistant Staphylococcus aureus (MRSA) strains are a global health concern. The present study regarded 160 S. aureus strains that had been isolated from 1634 foodstuff samples of animal origin in a previous survey conducted in Italy during 2003-2005. The strains were characterized by detecting the mecA gene, the production of type A to D staphylococcal enterotoxins (SEs), and studying their resistance properties against several antibiotics; their ecological origin was determined by biotyping. Of the 160 analyzed S. aureus strains six (3.75%) were mecA positive and derived from six different samples; four isolates were from bovine milk and two from dairy products (pecorino cheese and mozzarella cheese). Two strains isolated from milk belonged to the non-host-specific biovar while the others to the ovine biovar. The strain isolated from mozzarella cheese belonged to the non-host-specific biovar and the strain isolated from pecorino cheese to the ovine biovar. All the MRSA strains isolated were enterotoxigenic; two strains synthesized SEA/SED two SED and one SEC. All the strains showed resistance to at least one of the antibiotics tested but none was resistant to glycopeptides.