APPLICATION OF ANALYTICAL METHODS FOR FOOD AUTHENTICATION

Autentifikacija proizvoda služi za dokazivanje sastava i podrijetla poljoprivrednih i prehrambenih proizvoda. Republika Hrvatska ima veliki broj poljoprivrednih i prehrambenih proizvoda koji su zbog specifičnih organoleptičkih, tehnoloških i prehrambenih specifičnosti deklarirani kao proizvodi visoke vrijednosti te zaštićeni oznakom izvornosti, zemljopisnog podrijetla ili zajamčeno tradicionalnog specijaliteta. Provođenje postupka autentifikacije predstavlja multidisciplinarni postupak koji uključuje analitičke metode kombinirane s informatikom, matematikom i statistikom, a sve s ciljem dobivanja što točnijih i pouzdanijih podataka kako bi se mogao donijeti zaključak o podrijetlu i sastavu proizvoda. Zanimanje javnosti za konzumiranjem kvalitetne i sigurne hrane dovelo je do brzog razvoja metoda za autentifikaciju proizvoda. U analitičke metode analize ubrajaju se molekularne metode analize, kromatografske metode, spektrometrijske metode i imunološke metode. U radu su opisane analitičke metode koje se primjenjuju u postupcima dokazivanja izvornosti, zemljopisnog podrijetla ili tradicijskog specijaliteta proizvoda u svijetu.Food Authentication is primarily used for testing the composition and origin of agricultural and food products. Croatia has a large number of agricultural and food products, which are due to the specific organoleptic, technological and nutritional specifics declared as high-value and they are protected by designation of origin, geographical indications and traditional specialties guaranteed. The implementation of the authentication procedure is multidisciplinary process which includes analytical methods combined with computer science, mathematics and statistics, all with the goal of achieving a more accurate and reliable information in order to make a conclusion about the origin and composition of the product. Public interest in the consumption of high-quality and safe food has led to a rapid development of methods for products authentication. The analytical approach to the analysis is divided in several categories related to the molecular analysis methods, chromatographic methods, immunological methods and spectroscopic methods. This paper describes the analytical methods used in the procedures of proving the origin, geographical indications or traditional specialties product

PRELIMINARNO ISTRAŽIVANJE ODNOSA POLIMORFIZAMA NA CAST GENU I KVALITETE SVINJSKOG MESA

The present study was performed in order to investigate a relationship between polymorphisms on the calpastatin gene (CAST) and pig meat quality traits. The investigation was carried out on 29 gilts and barrows, crosses of Large White x German Landrace randomly selected at slaughter line. Pigs were slaughtered at 130-150 kg of live weight and blood samples were taken for genomic DNA analysis. The following indicators of meat quality and meat chemical composition were evaluated: pH and electric conductivity measured 45 minutes post mortem in m. Longissimus dorsi (LD muscle) and in M. Semimembranosus (SM muscle); electric conductivity measured at the same locations after 24h of cooling; drip loss (determined by “bag method”); colour (measured with Minolta chromameter and expressed as Hunter L, a, b values); instrumental tenderness assessed as Warner-Bratzler (WB) shear force and moisture, fat, protein and collagen content (%) determined on cooked LD muscle after 24h of thawing. The amplification products of the CAST gene were digested with HinfI restriction endonuclease and three genotypes (AA, BB and AB) were revealed. Statistical analysis showed that meat originated from pigs of AB genotype had the lowest WB shear force and the highest protein content of cooked LD muscle. As for the indicators of technological meat quality, statistically significant differences (p<0.05) were found between genotype AA and both BB and AB genotype for electric conductivity measured in LD muscle after 24h of cooling, as well as between BB and both AA and AB genotypes for drip loss.Istraživanje je provedeno na 29 križanaca velikog jorkšira i njemačkog landrasa nasumično odabranih na liniji klanja u cilju utvrđivanja veze između polimorfizama na kalpastatin genu (CAST) i svojstava kvalitete mesa. Svinje su zaklane pri 130-150 kg tjelesne mase, kada su izuzeti uzorci krvi za izolaciju genomske DNK. Na liniji klanja izmjerena su slijedeća svojstva kvalitete mesa: početna pH vrijednost i početna vrijednost električne provodljivosti (mjereni na musculus Longissimus dorsi i musculus semimembranosus 45 minuta nakon klanja); završne vrijednosti električne provodljivosti (izmjerene na istim mjestima 24 h nakon hlađenja polovica); otpuštanje mesnog soka (“metodom vrećice”); boja (izmjerena Minolta kolorimetrom i izražena kao Hunter L, a i b vrijednosti); tvrdoća/nježnost mesa te udio vlage, masti, proteina i kolagena (na skuhanom odsječku MLD). Umnoženi produkti CAST gena podvrgnuti su restrikciji HinfI endonukleazom te su utvrđena tri genotipa (AA, AB i BB). Statističkom analizom utvrđeno je da su svinje AB genotipa imale najpoželjnije vrijednosti tvrdoće/nježnosti mesa te najviši udio proteina u MLD-u, dok su za tehnološka svojstva kvalitete mesa statistički značajne razlike utvrđene između genotipa AA i BB te AB za završne vrijednosti električne provodljivosti u MLD-u, kao i između BB te AA i AB za vrijednosti otpuštanja mesnog soka

PRELIMINARNO ISTRAŽIVANJE ODNOSA POLIMORFIZAMA NA CAST GENU I KVALITETE SVINJSKOG MESA

The present study was performed in order to investigate a relationship between polymorphisms on the calpastatin gene (CAST) and pig meat quality traits. The investigation was carried out on 29 gilts and barrows, crosses of Large White x German Landrace randomly selected at slaughter line. Pigs were slaughtered at 130-150 kg of live weight and blood samples were taken for genomic DNA analysis. The following indicators of meat quality and meat chemical composition were evaluated: pH and electric conductivity measured 45 minutes post mortem in m. Longissimus dorsi (LD muscle) and in M. Semimembranosus (SM muscle); electric conductivity measured at the same locations after 24h of cooling; drip loss (determined by “bag method”); colour (measured with Minolta chromameter and expressed as Hunter L, a, b values); instrumental tenderness assessed as Warner-Bratzler (WB) shear force and moisture, fat, protein and collagen content (%) determined on cooked LD muscle after 24h of thawing. The amplification products of the CAST gene were digested with HinfI restriction endonuclease and three genotypes (AA, BB and AB) were revealed. Statistical analysis showed that meat originated from pigs of AB genotype had the lowest WB shear force and the highest protein content of cooked LD muscle. As for the indicators of technological meat quality, statistically significant differences (p<0.05) were found between genotype AA and both BB and AB genotype for electric conductivity measured in LD muscle after 24h of cooling, as well as between BB and both AA and AB genotypes for drip loss.Istraživanje je provedeno na 29 križanaca velikog jorkšira i njemačkog landrasa nasumično odabranih na liniji klanja u cilju utvrđivanja veze između polimorfizama na kalpastatin genu (CAST) i svojstava kvalitete mesa. Svinje su zaklane pri 130-150 kg tjelesne mase, kada su izuzeti uzorci krvi za izolaciju genomske DNK. Na liniji klanja izmjerena su slijedeća svojstva kvalitete mesa: početna pH vrijednost i početna vrijednost električne provodljivosti (mjereni na musculus Longissimus dorsi i musculus semimembranosus 45 minuta nakon klanja); završne vrijednosti električne provodljivosti (izmjerene na istim mjestima 24 h nakon hlađenja polovica); otpuštanje mesnog soka (“metodom vrećice”); boja (izmjerena Minolta kolorimetrom i izražena kao Hunter L, a i b vrijednosti); tvrdoća/nježnost mesa te udio vlage, masti, proteina i kolagena (na skuhanom odsječku MLD). Umnoženi produkti CAST gena podvrgnuti su restrikciji HinfI endonukleazom te su utvrđena tri genotipa (AA, AB i BB). Statističkom analizom utvrđeno je da su svinje AB genotipa imale najpoželjnije vrijednosti tvrdoće/nježnosti mesa te najviši udio proteina u MLD-u, dok su za tehnološka svojstva kvalitete mesa statistički značajne razlike utvrđene između genotipa AA i BB te AB za završne vrijednosti električne provodljivosti u MLD-u, kao i između BB te AA i AB za vrijednosti otpuštanja mesnog soka

12-plex highly polymorphic microsatellite marker set for parentage analysis in Banija spotted pigs

Microsatellites (MS) have been, for the last two decades, widely used for parentage analysis in all types of livestock, including pigs. Conservation efforts on Banija spotted pigs included genetic characterization of the breed with MS markers. Since recent comparison of pedigree and MS data revealed some inconsistencies, the aim of this study was to develop a set of highly polymorphic and heterozygous MS markers, which could be used for parentage analysis and to prevent pedigree errors. 12 MS markers with the polymorphic information content (PIC) above 0.62 were chosen and combined into a single multiplex polymerase chain reaction (PCR). The combined non-exclusion probability for one candidate parent (NE-1P) was 0.00246149, the combined NE-1P given the genotype of a known parent of the opposite sex (NE-2P) was 0.00003739 and combined non-exclusion probability for a candidate parent pair (NE-PP) was 0.00000003. Due to a high information content of selected MS markers it was possible to obtain high accuracy in parentage assignment, which was confirmed by analyzing actual data with known genetic relationship

Genetic diversity of the Banija spotted pig breed using microsatellite markers

Banija spotted pig breed was created at the end of 19th century by crossing domestic white sows with drooping ears and/or Turopolje pig sows with black Berkshire. After a period of stagnation in breeding, the population is newly recognized only few years ago and is currently in the process of breed valorisation. The aim of this study was to assess genetic diversity and structure of Banija spotted pig (BS) in comparison with geographically close populations, and to assess the differentiation level of Banija spotted pig. Based on the genotypes obtained for 24 ISAG-FAO recommended pig microsatellite markers, basic genetic diversity indicators were determined for 30 samples of BS, 20 samples of Turopolje and Crna Slavonska pig each, and 17 samples of Landrace population. Observed heterozygosity ranged from 0.36 to 0.6 and was high in BS population (0.58). Average allelic richness (range 2 – 5.2) and the rarefacted number of private alleles (range 9 to 27) were the highest in BS. Inbreeding coefficient in BS was estimated 0.06 (bootstrapping confidence interval 0.022 to 0.123). Structure analysis showed that there are five clusters in the four analysed populations, where BS are clearly distinguished from other populations and substructured. Wright’s fixation index estimates are in accordance with the clustering results. In conclusion, the population of Banija spotted pig shows great genetic diversity and is genetically different from neighbouring (geographically close) pig populations

A simple DNA based method for determination of pure Black Slavonian pigs

The aim of this work was to determine the MC1R genotype of Black Slavonian pigs and to find an efficient and simple PCR-RFLP method, based on differences in MC1R genotype, to distinguish between purebred Black Slavonian pigs and their crossings with commercial pig breeds and Wild Boars. Sequencing of the MC1R exon was performed to determine the genotype of MC1R in Black Slavonian pig breed, which was shown to be MC1R*2. Digestion reactions of both PCR products representing the majority of MC1R exon revealed presence of the BspHI restriction site at position 121 and absence of the AccII and CrfI restriction site at position 240, which is characteristic for the MC1R*2 genotype. A simple PCR-RFLP method, based on different coat colour MC1R gene genotypes was determined by which it is possible to detect potential crossings of autochthonous Black Slavonian pig with commercial pig breeds and also with Wild Boars

Genetic diversity of autochthonous pig breeds analyzed by microsatellite markers and mitochondrial DNA D-loop sequence polymorphism

<p>The evaluation of the genetic structure of autochthonous pig breeds is very important for conservation of local pig breeds and preservation of diversity. In this study, 18 microsatellite loci were used to detect genetic relationship between autochthonous pig breeds [Black Slavonian (BS), Turopolje pig (TP), and Croatian wild boar] and to determine phylogenetic relationship among Croatian autochthonous pig breeds and certain Asian and European pigs using the mitochondrial DNA (mtDNA) D-loop sequence polymorphism. Relatively high degree of genetic variation was found between the observed populations. The analysis of mtDNA showed that haplotypes of the studied pig populations are different from the other European and Chinese haplotypes. BS pigs showed some similarities with Mangalitsa and Duroc breeds. The genetic distances of TP can be explained by high degree of inbreeding during the past century. Despite the European origin of Croatian pig breeds with some impact of Chinese breeds in the past, the results of present study show that genetic diversity is still pronounced within investigated breeds. Furthermore, the genetic diversity is even more pronounced between Croatian breeds and other European and Chinese pig breeds. Thus, conservation of Croatian pig breeds will contribute to overall genetic diversity preservation of pig breeds.</p

Genetic diversity and population structure of six autochthonous pig breeds from Croatia, Serbia, and Slovenia

Background: The importance of local breeds as genetic reservoirs of valuable genetic variation is well established. Pig breeding in Central and South-Eastern Europe has a long tradition that led to the formation of several local pig breeds. In the present study, genetic diversity parameters were analysed in six autochthonous pig breeds from Slovenia, Croatia and Serbia (Banija spotted, Black Slavonian, Turopolje pig, Swallow-bellied Mangalitsa, Moravka and Krskopolje pig). Animals from each of these breeds were genotyped using microsatellites and single nucleotide polymorphisms (SNPs). The results obtained with these two marker systems and those based on pedigree data were compared. In addition, we estimated inbreeding levels based on the distribution of runs of homozygosity (ROH) and identifed genomic regions under selection pressure using ROH islands and the integrated haplotype score (iHS). Results: The lowest heterozygosity values calculated from microsatellite and SNP data were observed in the Turopolje pig. The observed heterozygosity was higher than the expected heterozygosity in the Black Slavonian, Moravka and Turopolje pig. Both types of markers allowed us to distinguish clusters of individuals belonging to each breed.The analysis of admixture between breeds revealed potential gene fow between the Mangalitsa and Moravka, and between the Mangalitsa and Black Slavonian, but no introgression events were detected in the Banija spotted and Turopolje pig. The distribution of ROH across the genome was not uniform. Analysis of the ROH islands identifed genomic regions with an extremely high frequency of shared ROH within the Swallow-bellied Mangalitsa, which harboured genes associated with cholesterol biosynthesis, fatty acid metabolism and daily weight gain. The iHS approach to detect signatures of selection revealed candidate regions containing genes with potential roles in reproduction traits and disease resistance. Conclusions: Based on the estimation of population parameters obtained from three data sets, we showed the existence of relationships among the six pig breeds analysed here. Analysis of the distribution of ROH allowed us to estimate the level of inbreeding and the extent of homozygous regions in these breeds. The iHS analysis revealed genomic regions potentially associated with phenotypic traits and allowed the detection of genomic regions under selection pressure