Detection of Adulteration in Italian Mozzarella Cheese Using Mitochondrial DNA Templates as Biomarkers

Considering the importance of monitoring adulterations of genuine cheeses in the dairy industry, a polymerase chain reaction–based method was developed to detect bovine- specific mitochondrial DNA sequence in Italian water buffalo Mozzarella cheese. DNA was isolated from cheese matrix and governing liquid by organic extractions and kit purifications. Amplifications of a 134-bp fragment were performed with a bovine–specific set of primers designed on the sequence alignment of bovine and buffalo mitochondrial cytochrome oxidase subunit I. The specificity of the primers was tested using DNA from the blood of two species (water buffalo and bovine), which are present together in adulterated Italian Mozzarella cheese. This method reliably detected a content of 0.5 % of bovin milk, making it suitable for routine fraud monitoring

Polimorfizam κ-kazeina talijanskih pasmina koza: novi ACRS-PCR test za razlikovanje A i B alela

The objective of this study was to develop a DNA test for rapid characterisation of goat κ-casein (κ-CN) A and B variants and to study this polymorphism in Italian goat breeds. Genetic polymorphism of κ-CN gene was, in addition to isoelectric focusing, analysed according to a new technique designated as amplification created restriction site. Two alleles that differ in one nucleotide mutation (G Ø A) in exon 4 were characterised. The 167-bp PCR product surrounding the nucleotide mutation was amplified from genomic DNA and the PCR product was digested with MaeIII. After digestion the A allele gives three fragments of 77, 65 and 25 bp in comparison with the B allele which gives two fragments of 90 and 77 bp. The analysis of allele frequency distribution at κ-CN locus, based on 401 individual samples, revealed significant differences among three goat breeds from the north of Italy (Nera di Verzasca, Frontalasca and Alpine) with frequency of κ-CN B allele around 0.3, versus two goat breeds from the south of Italy (Maltese and Sarda) with frequency of κ-CN B allele around 0.5. While two goat breeds (Maltese and Nera di Verzasca) did not show significant deviations from the Hardy-Weinberg equilibrium, a highly significant excess of heterozygote genotype (AB) was observed in Alpine, Frontalasca and Sarda goats. Here the developed DNA method and observed relatively high frequency of κ-CN B allele give a prerequisite for the assessment of research related to the simultaneous estimation of the effects of composite αs1/κ-CN genotypes on milk production and cheese-making properties.Svrha je bila razviti DNA test za brzo određivanje genetičkih varijanti κ-kazeina u talijanskih pasmina koza. Genetički je polimorfizam istraživan izoelektričnim fokusiranjem (IEF) i amplifikacijom nastalim restrikcijskim mjestom na genomu (ACRS). PCR produkt na ekzonu 4, dug 167-bp koji okružuje nukleotidnu mutaciju (G Ø A), amplificiran s genomske DNA i razgrađen je s MaeIII enzimom. Identificirana su dva alela koja se razlikuju u jednoj nukleotidnoj mutaciji (G Ø A). Dobiveni su fragmenti 77 i 65 bp za alel A te 90 i 77 bp za alel B. Signifikantna razlika za frekvenciju alela B ustanovljena je za tri pasmine koza sa sjevera Italije (Nera di Verzasca, Frontalasca i Alpine) s frekvencijom κ-kazeina B oko 0,3, u usporedbi s dvjema pasminama s juga Italije (Maltese i Sarda), s frekvencijom κ-kazeina B otprilike 0,5. Pasmine Maltese i Nera di Verzasca nisu pokazale signifikantno odstupanje od Hardy-Weinbergove ravnoteže, za razliku od pasmina Alpine, Frontalasca i Sarda u kojima je uočeno veliko signifikantno odstupanje. Prikazana DNA metoda i promatrana relativno velika frekvencija κ-kazeina B alela omogućavaju daljnja istraživanja zajedničkog utjecaja αs1-kazeina i κ-kazeina na proizvodnju mlijeka i sira

Caprine αs1-Casein Polymorphism: Characterisation of A, B, E and F Variants by Means of Various Biochemical and Molecular Techniques

Considering a wide interest for the characterisation of caprine αs1-casein variants and a large number of differently equipped laboratories, the objective of this study was to analyse and compare characteristics of caprine αs1-casein variants by means of various biochemical and molecular techniques. The most frequent caprine αs1-casein variants (A, B, E and F) were characterized by employing electrophoretic protein separation analyses (capillary electrophoresis, isoelectric focusing, and sodium dodecylsulfate polyacrylamide gel electrophoresis), chromatographic analysis (reversed phase-high performance liquid chromatography) as well as DNA analyses (ASA and real-time polymerase chain reaction approach). Further, we stressed weak and strong points for each method applied and provided information for the optimal and complementary use of those methods with respect to time, resolution and costs

Caprine αs1-Casein Polymorphism: Characterisation of A, B, E and F Variants by Means of Various Biochemical and Molecular Techniques

Considering a wide interest for the characterisation of caprine αs1-casein variants and a large number of differently equipped laboratories, the objective of this study was to analyse and compare characteristics of caprine αs1-casein variants by means of various biochemical and molecular techniques. The most frequent caprine αs1-casein variants (A, B, E and F) were characterized by employing electrophoretic protein separation analyses (capillary electrophoresis, isoelectric focusing, and sodium dodecylsulfate polyacrylamide gel electrophoresis), chromatographic analysis (reversed phase-high performance liquid chromatography) as well as DNA analyses (ASA and real-time polymerase chain reaction approach). Further, we stressed weak and strong points for each method applied and provided information for the optimal and complementary use of those methods with respect to time, resolution and costs

Detection of Adulteration in Italian Mozzarella Cheese Using Mitochondrial DNA Templates as Biomarkers

Considering the importance of monitoring adulterations of genuine cheeses in the dairy industry, a polymerase chain reaction–based method was developed to detect bovine- specific mitochondrial DNA sequence in Italian water buffalo Mozzarella cheese. DNA was isolated from cheese matrix and governing liquid by organic extractions and kit purifications. Amplifications of a 134-bp fragment were performed with a bovine–specific set of primers designed on the sequence alignment of bovine and buffalo mitochondrial cytochrome oxidase subunit I. The specificity of the primers was tested using DNA from the blood of two species (water buffalo and bovine), which are present together in adulterated Italian Mozzarella cheese. This method reliably detected a content of 0.5 % of bovin milk, making it suitable for routine fraud monitoring

Polimorfizam κ-kazeina talijanskih pasmina koza: novi ACRS-PCR test za razlikovanje A i B alela

The objective of this study was to develop a DNA test for rapid characterisation of goat κ-casein (κ-CN) A and B variants and to study this polymorphism in Italian goat breeds. Genetic polymorphism of κ-CN gene was, in addition to isoelectric focusing, analysed according to a new technique designated as amplification created restriction site. Two alleles that differ in one nucleotide mutation (G Ø A) in exon 4 were characterised. The 167-bp PCR product surrounding the nucleotide mutation was amplified from genomic DNA and the PCR product was digested with MaeIII. After digestion the A allele gives three fragments of 77, 65 and 25 bp in comparison with the B allele which gives two fragments of 90 and 77 bp. The analysis of allele frequency distribution at κ-CN locus, based on 401 individual samples, revealed significant differences among three goat breeds from the north of Italy (Nera di Verzasca, Frontalasca and Alpine) with frequency of κ-CN B allele around 0.3, versus two goat breeds from the south of Italy (Maltese and Sarda) with frequency of κ-CN B allele around 0.5. While two goat breeds (Maltese and Nera di Verzasca) did not show significant deviations from the Hardy-Weinberg equilibrium, a highly significant excess of heterozygote genotype (AB) was observed in Alpine, Frontalasca and Sarda goats. Here the developed DNA method and observed relatively high frequency of κ-CN B allele give a prerequisite for the assessment of research related to the simultaneous estimation of the effects of composite αs1/κ-CN genotypes on milk production and cheese-making properties.Svrha je bila razviti DNA test za brzo određivanje genetičkih varijanti κ-kazeina u talijanskih pasmina koza. Genetički je polimorfizam istraživan izoelektričnim fokusiranjem (IEF) i amplifikacijom nastalim restrikcijskim mjestom na genomu (ACRS). PCR produkt na ekzonu 4, dug 167-bp koji okružuje nukleotidnu mutaciju (G Ø A), amplificiran s genomske DNA i razgrađen je s MaeIII enzimom. Identificirana su dva alela koja se razlikuju u jednoj nukleotidnoj mutaciji (G Ø A). Dobiveni su fragmenti 77 i 65 bp za alel A te 90 i 77 bp za alel B. Signifikantna razlika za frekvenciju alela B ustanovljena je za tri pasmine koza sa sjevera Italije (Nera di Verzasca, Frontalasca i Alpine) s frekvencijom κ-kazeina B oko 0,3, u usporedbi s dvjema pasminama s juga Italije (Maltese i Sarda), s frekvencijom κ-kazeina B otprilike 0,5. Pasmine Maltese i Nera di Verzasca nisu pokazale signifikantno odstupanje od Hardy-Weinbergove ravnoteže, za razliku od pasmina Alpine, Frontalasca i Sarda u kojima je uočeno veliko signifikantno odstupanje. Prikazana DNA metoda i promatrana relativno velika frekvencija κ-kazeina B alela omogućavaju daljnja istraživanja zajedničkog utjecaja αs1-kazeina i κ-kazeina na proizvodnju mlijeka i sira

Polymorphism of κ-Casein in Italian Goat Breeds: A New ACRS-PCR Designed DNA Test for Discrimination of A and B Alleles

The objective of this study was to develop a DNA test for rapid characterisation of goat κ-casein (κ-CN) A and B variants and to study this polymorphism in Italian goat breeds. Genetic polymorphism of κ-CN gene was, in addition to isoelectric focusing, analysed according to a new technique designated as amplification created restriction site. Two alleles that differ in one nucleotide mutation (G Ø A) in exon 4 were characterised. The 167-bp PCR product surrounding the nucleotide mutation was amplified from genomic DNA and the PCR product was digested with MaeIII. After digestion the A allele gives three fragments of 77, 65 and 25 bp in comparison with the B allele which gives two fragments of 90 and 77 bp. The analysis of allele frequency distribution at κ-CN locus, based on 401 individual samples, revealed significant differences among three goat breeds from the north of Italy (Nera di Verzasca, Frontalasca and Alpine) with frequency of κ-CN B allele around 0.3, versus two goat breeds from the south of Italy (Maltese and Sarda) with frequency of κ-CN B allele around 0.5. While two goat breeds (Maltese and Nera di Verzasca) did not show significant deviations from the Hardy-Weinberg equilibrium, a highly significant excess of heterozygote genotype (AB) was observed in Alpine, Frontalasca and Sarda goats. Here the developed DNA method and observed relatively high frequency of κ-CN B allele give a prerequisite for the assessment of research related to the simultaneous estimation of the effects of composite αs1/κ-CN genotypes on milk production and cheese-making properties