Electrophoretic Distinction of the Origin in Different Dairy Products and Milk Samples

Caseins, lacto-albumin, and lacto-globulin are major milk proteins. These globular proteins could be significant indicators of the milk and dairy products origin. Knowing that caseins, lacto-albumins and lacto-globulins vary in molecular weight and concentration in different types of milk, this fluctuation can be used for determination of milk origin. The aim of this study was to develop an appropriate method for distinction of milk proteins from different origin. Twelve samples of milk, white cheese, yellow cheese and whey cheese from cow, sheep and goat were obtained and studied. The protein separation was made using SDS-PAGE. SDS is an anionic detergent that breaks all inter and intramolecular bonds and leaves the polypeptide subunits of proteins in forms that can be separated on the basis of their molecular weight. Polyacrylamide gels, used as support medium, restrain larger molecules from migrating as fast as smaller molecules. In order to optimize the conditions of the experiment, some of the parameters were modified (polyacrilamide concentration from 10-15% according to the molecules size, duration of electrophoresis, quantity of applied material, sonification treatment of the different samples). Bovine milk proteins standards were used for the determination of the proteins. The results have shown differences, as well as other fractions that can be used for identification of the origin. In yellow and white cheese the differences among the samples from different origins appear in lacto-albumin fractions and some digested fractions below the caseins. The main differences in whey cheese samples were identified in casein fractions. The milk samples showed differences in upper fractions, probably serum albumins that remained in the milk samples

USEFULNESS OF A LOCUS LEEF1Aa IN THE GENETIC DIFFERENTIATION OF TOMATO VARIETIES

The molecular techniques provide new possibilities to characterize advanced genetic materials for registration purposes and for the protection of breeders’ rights. The microsatellites appear as suitable molecular markers due to their highly polymorphic character. Such microsatellites may generate polymorphism useful for the analysis of genetic diversity and relationships within the genus Lycopersicon. The focus of the following study was usefulness of the locus LEEF1Aa in the genetic differentiation among six morphologically different tomato varieties of Lycopersicon esculentum Mill. The fragment analyses were done using Applied Biosystems DNA analyzer (ABI 3130) and GeneMapper®Software program. The obtained data were analyzed using the specific program Power Marker Software. The number of detected alleles for the microsatellites locus LEEF1Aa was six in estimated tomato varieties (219-221-223-225-227-229bp). The allele with the length of 229 bp was noticed only in Lycopersicon esculentum subsp. cultum var. grandifolium, while the alleles (221, 225 and 227 bp) in 4 varieties, the allele of 219 bp in 3 varieties and the allele of 223 bp in 2 varieties. The average PIC value for the locus LEEF1Aa was 0.7552 and it belongs to the group of high informative markers. Obtained results showed that the locus LEEF1Aais good choice for genetic differentiation of tomato varieties in combination with other polymorphic microsatellite loci

DIFFERENT APPROACHES IN THE IDENTIFICATION OF MEAT ORIGIN BASED ON PROTEIN PROFILING AND SIMPLE PCR

The meat origin, as a fundamental factor for the quality and the usability of its products, can be determined using DNA or protein analysis. In this study, different techniques are applied to determine the origin of different raw meat samples. The DNA analysis of meat is based on the Polymerase Chain Reaction whereas the techniques involving protein analysis are carried out using electrophoresis. Using the SDS-PAGE technique optimized by changing the running conditions, the amount of materials and the gel concentration, it is possible to differentiate different types of meat. We determine the differences in protein profile of poultry meat compared to the other samples in which are identified two specific fractions between 116 and 200 kD in the zone of myosin heavy chains and one bellow 45 kD in the zone of actin. In the beef samples there is a specific fraction in the zone of tropomyosin, while in pork and beef samples appeared a fraction in the zone of myosin light chain. This technique is suitable and can be only used for internal control in production and processing environments because of its low sensitivity. In practice, there are commercialized kits for the identification of meat and meat products based on DNA analysis. All those kits are dealing with specific primers for different type of meat (beef, pork, poultry, goat, horse, etc). In this case we used the primers for ryanodine receptor gene 1 (RYR1) which protein is involved in calcium pathways of the skeletal muscle cells. We amplify DNA isolated from beef, pork and poultry and DNA analysis based on partial amplification of the RYR1 gene showed the difference between mammalian and poultry meat because there is no amplification on the DNA sample isolated from chicken meat

DIFFERENT APPROACHES IN ANALYZING CHYMOSIN PURITY

Chymosin is a specific proteolytic enzyme found in rennet, and is the key enzyme in cheese production classified in the aspartic endopeptidases (EC 3.4.23.4). The aim of this study was to determine the purity of different commercially available chymosins and its equivalents using electrophoretic and chromatographic techniques. Chymosins produced by the company Chr. Hansen, CHY-MAX 200 and CHY-MAX Plus, CHY-MAX PowderExtra NB, as well as Maxiren 1800 Granulate from the company DSM, Sirnik from SZR – Travnik, Kraljevo and Planika from Mikroprocessing, Bileca were used as materials for this study. The purity level of the commercially available enzymes was analyzed using electrophoretic (sodium dodecyl sulfate polyacrylamide gel electrophoresis or SDS-PAGE) and chromatographic (Rapid Resolution Liquid Chromatography or RRLC) techniques. Results showed no presence of undeclared protein fractions due to inappropriate purification process in the samples except for CHY-MAX М 200 which had two protein fractions, most likely as a result of a polymorphism. All the CHY-MAX and Maxiren samples have chymosin as the active component (36 kDa), except for Planika and Sirnik which have a natural protease from R. miehei. Chromatographic analysis showed that beside the active component (chymosin), the preservative sodium benzoate was present in varying concentrations in all but CHY-MAX PowderExtra NB