α_S1-casein in goat milk: identification of genetic variants by Capillary Zone Electrophoresis compared to Isoelectric Focusing

AlphaS1 casein fraction in caprine milk is characterized by an important polymorphism due to substitution, deletion of amino acids and post trascriptional modifications (Grosclaude et al., 1994; Ferranti et al., 1997). This structural polymorphism is associated to a quantitative variability in protein expression related to different milk quality and dairy properties (Pierre et al., 1998; Remeuf, 1993; Vassal et al., 1994). Classical electrophoretic methods were applied to characterize the phenotypic variants at αS1-casein fraction (Addeo et al., 1988; Russo et al., 1986). During the last ten years capillary electrophoresis became an analytical technique for rapid and automated analysis requiring small sample volume and small solvent waste. These characteristics, together with the high resolution and the chance to give quantitative results, made this technique a useful tool in studying milk protein characterization and in detecting adulteration (Cattaneo et al., 1996a; 1996b) in different application fields. CZE was applied to the study of caprine milk proteins to quantify high, medium and low αS1- casein content and to identify genetic variants αS1 A, B and C on the basis of their different migration time (Recio et al., 1997). The aim of this work was to test a CZE procedure able to identify and discriminate the main αS1 caprine variants A, B, E and F through specific and repeatable electromigration patterns. Comparison between CZE and IEF assays is discussed

Polymorphism of α_S1-casein in goat milk: identification of A, B, E and F variants by biochemical and genetic analysis

Many researches, related to genetic polymorphism of αS1-casein in goat milk, showed a marked variability and implications in milk and dairy product traits. The genetic variants (from A to G) were associated with four levels of expression: 3,6 g/L per A, B and C (“strong” alleles); 1,6 g/L per E (“medium” allele); 0,6 g/L per F (“weak” allele) and 0 g/L per O (“null” allele). Differences existing among the A, B, C, D, E, F, G and O have been described (Grosclaude et al., 1994). To analyze genetic polymorphism of caseins from goat milk, distinct electrophoretic (Russo et al., 1986; Addeo et al., 1988) and chromatographic techniques (Jaubert and Martin, 1992; Iametti et al., 1999) were applied. In last years, the advancement in technological field is allowing the research of primary structure of protein variants (Ferranti et al., 1997; Trujillo et al., 2000) and basic sequence of new alleles (Martin et al., 1999; Bevilacqua et al., 2002; Ramunno et al., 2002). In this work, we employed electrophoretic protein separation (IEF and SDS-PAGE), chromatographic analysis (RP-HPLC) and molecular biology techniques based on polymerase chain reaction (real-time PCR) to detect αS1-CN genetic variants from goat milk. The utility of protein and DNA analysis combination was discussed

Effects of lactation stage, parity, beta-lactoglobulin genotype and milk SCC on whey protein composition in Sarda dairy ewes

In 90 Sarda dairy ewes the effects of lactation stage, parity, beta-lactoglobulin genotypes, and somatic cell count (SCC) on the milk content of total protein (TP), casein (CN), whey protein (WP) and its fractions alpha-lactalbumin (ALA), beta-lactoglobulin (BLG), serum albumin (SA), immunoglobulin (IG) and lactoferrin (LF) were analysed using a linear mixed model. Mean values of variables (g/l) were: TP (54.0), CN (43.0), WP (11.0), BLG (4.78), ALA (1.37), SA (0.61), IG (3.83) and LF (0.28). The lactation stage significantly affected all the variables analysed. TP, CN and WP concentrations tended to increase throughout lactation, with the increase of WP being more pronounced than the corresponding variation in CN. There was no definite trend in BLG content, whereas ALA concentration decreased as lactation progressed. The parity affected almost all variables studied. WP concentration differed significantly only between the second and fourth parity (10.45 vs 11.44 g/l). BLG and SA concentrations were significantly lower in the youngest ewes. The BLG genotype affected milk yield, but no effects were observed on the components of the milk. The SCC influenced almost all variables studied. The TP concentration was significantly higher in milk with SCC >1,000,000 (55.0 g/l) than in milk with lower SCC (53.4 g/l). This was mainly due to the increase of WP (12.52 and 10.24 g/l in milk with SCC above and below 1,000,000/ml respectively), especially in those WP fractions originating from blood

Genetski polimorfizam β-laktoglobulina u paške ovce

Milk samples from 248 Pag ewes, belonging to 14 different flocks and located through the Pag Island (Croatia), were analyzed by isoelectrofocusing and PCR-RFLP. Two genetic variants (A and B) and three genotypes (AA, AB and BB) of β-lactoglobulin have been identified. According to the allele frequency (A=0.48, B=0.52) and occurrence of genetic variants, the Pag breed is similar to other Mediterranean dairy sheep breeds. The observed genotype frequencies at the β-lactoglobulin locus (AA=0.185, AB=0.589 and BB=0.226) were significantly different from those expected from Hardy-Weinberg equilibrium. In comparison with 12 different studies related to Mediterranean dairy sheep populations, the significant departure from Hardy-Weinberg was also obtained in Valle del Belice sheep. However, the sample size required for detection of the same amount of departure from the Hardy-Weinberg as it was observed in Pag sheep was only sufficient in four studies (including Pag sheep). The superiority of the AB β-lactoglobulin genotype for milk production could be one of possible reasons for the observed excess of heterozygotes and allele frequencies at intermediate level.Uzorci mlijeka od 248 paških ovaca iz 14 različitih stada lociranih po cijelom otoku Pagu analizirani su tehnikama izoelektričnog fokusiranja i PCR-RFLP. Identificirane su dvije genetske varijante (A i B) i tri genotipa (AA, AB i BB) β-laktoglobulina. Po frekvenciji alela (A=0.48, B=0.52) i prisutnosti genetskih varijanti paška je ovca slična ostalim mediteranskim mliječnim pasminama ovaca. Dobivene genotipske frekvencije β-laktoglobulinskog lokusa (AA=0.185, AB=0.588 i BB=0.226) pokazale su signifikantno odstupanje od očekivanog Hardy-Weinbergove ravnoteže. U 12 različitih istraživanja koja se odnose na mediteranske mliječne pasmine ovaca, signifikantno odstupanje primijećeno je još samo u pasmine Valle del Belice. Međutim, samo u četiri istraživanja (obuhvaćajući i istraživanje autora) veličina uzorka bila je dovoljna za detekciju istog odstupanja od Hardy-Weinbergove ravnoteže. Superiornost AB genotipa β-laktoglobulina za proizvodnju mlijeka mogući je uzrok veće prisutnosti heterozigote te intermedijarne frekvencije alela

Effects of lactation stage, parity, β-lactoglobulin genotype and milk SCC on whey protein composition in Sarda dairy ewes

In 90 Sarda dairy ewes the effects of lactation stage, parity, β-lactoglobulin genotypes, and somatic cell count (SCC) onthe milk content of total protein (TP), casein (CN), whey protein (WP) and its fractions α-lactalbumin (ALA), β-lactoglobulin(BLG), serum albumin (SA), immunoglobulin (IG) and lactoferrin (LF) were analysed using a linear mixed model.Mean values of variables (g/l) were: TP (54.0), CN (43.0), WP (11.0), BLG (4.78), ALA (1.37), SA (0.61), IG (3.83) andLF (0.28). The lactation stage significantly affected all the variables analysed. TP, CN and WP concentrations tended toincrease throughout lactation, with the increase of WP being more pronounced than the corresponding variation in CN.There was no definite trend in BLG content, whereas ALA concentration decreased as lactation progressed. The parityaffected almost all variables studied. WP concentration differed significantly only between the second and fourth parity(10.45 vs 11.44 g/l). BLG and SA concentrations were significantly lower in the youngest ewes. The BLG genotype affectedmilk yield, but no effects were observed on the components of the milk. The SCC influenced almost all variables studied.The TP concentration was significantly higher in milk with SCC >1,000,000 (55.0 g/l) than in milk with lower SCC(53.4 g/l). This was mainly due to the increase of WP (12.52 and 10.24 g/l in milk with SCC above and below1,000,000/ml respectively), especially in those WP fractions originating from blood

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

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

Italian Mediterranean river buffalo CSN2 gene structure and promoter analysis

The nucleotide sequence of the whole buffalo β-casein encoding gene (CSN2) plus 1,476 nt at the 5' flanking region and 51 nt at the 3' flanking region was determined. The gene is spread over 10.2 kb and consists of 9 exons varying in length from 24 (exon 5) to 498 bp (exon 7) and 8 introns from 92 bp (intron 5) to 22 59 bp (intron 1). Furthermore, highly conserved sequences, mainly located in the 5' flanking region, were found between this gene and the β-casein encoding genes of other species. The comparison between the obtained promoter and exonic regions and buffalo sequences present in EMBL evidenced different polymorphic sites. Finally, 5 interspersed repeated elements (4 in the bovine CSN2 gene) were also identified at 3 different locations of the sequenced region: 5' untranscribed region, intron 1, and intron 4

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