Maria Feligini

Quantification of bovine casein fractions by direct chromatographic analysis of milk. Approaching the application to a real production context.

The ability to quantify the casein content by an exact and cost-effective approach represents an issue of crucial importance in the dairy industry as the natural variations in milk protein concentration can markedly affect the yield of the cheesemaking processes, thus causing a direct and significant economic impact on the producers. In this work, the separation and quantification of alpha(s1)-, alpha(s2)-, kappa- and beta-casein was carried out by direct RP-HPLC analysis of milk. The identification of each casein was established by electrospray ionization mass spectrometry. The data show that this method is able to effectively separate the bovine casein fractions, it provides simplified analytical conditions (with special regard to mobile phase composition and gradient profile) and faster separation while ensuring adequate precision to achieve reliable quantifications in milk samples from dairy production.

Identification and Quantification of αS1, αS2, β, and κ-Caseins in Water Buffalo Milk by Reverse Phase-High Performance Liquid Chromatography and Mass Spectrometry

A method for the simultaneous quantitation of alpha(S1), alpha(S2), beta, and kappa-caseins in water buffalo (Bubalus bubalis) milk using reverse phase high-performance liquid chromatography was developed. The molecular masses of the peaks separated by the described chromatographic protocol were determined by ESI-MS. alpha(S1)- and kappa-caseins were found to be heteromorphic in several individual milk samples. In particular, alpha(S1)-casein showed two peaks with a molecular mass of 23,490 Da and 23,516 Da, and kappa-casein showed three peaks with molecular masses of 19,165 Da, 19,177 Da, and 19,247 Da. Only one form for beta-casein (24,033 Da) and alpha(S2)-casein (22,741 Da) were detected. The mean values of casein fraction concentration observed throughout the individual samples were 8.89 gL(-1) with a relative standard deviation (RSD) of 20% for alpha(S1)-casein, 5.08 gL(-1) with a RSD of 25% for alpha(S2)-casein, 20.91 gL(-1) with a RSD of 16% for beta-casein, and 4.13 gL(-1) with a RSD of 24% for kappa-casein. Linear and second-order polynomial correlations with total nitrogen were calculated for all casein fractions.

Identification of Microbiota Present on the Surface of Taleggio Cheese Using PCR–DGGE and RAPD–PCR

UNLABELLED Microbial DNA from 9 batches of Taleggio PDO cheese sampled at various times during ripening, brines, swabs of wooden shelves used for cheese dry-salting, and 13 commercial cheeses were analyzed by denaturing gradient gel electrophoresis (PCR-DGGE) and/or random amplification of polymorphic DNA (RAPD-PCR). Sequencing allowed the detection of 12 genera, 27 species, and 2 unclassified bacteria. Molecular analysis allowed for the detection of microorganisms not previously associated with Taleggio such as Lactobacillus paracasei, Carnobacterium maltaromaticum, Bacillus licheniformis, Corynebacterium variabile, Psychrobacter cibarius, and Staphylococcus carnosus. For the first time Massilia spp. was detected in a dairy ecosystem. PRACTICAL APPLICATION Indigenous species and strains of bacteria identified by this study could be used for the selection of dairy cultures to be employed routinely by manufacturers to control the Taleggio cheese production. The new cultures may give the bases for driving dairy processes and, consequently, control the typical flavor resulting from metabolic actions of environmental microorganisms.

Water Buffalo (Bubalus bubalis): Complete Nucleotide Mitochondrial Genome Sequence

In this work, we report the whole sequence of the water buffalo (Bubalus bubalis) mitochondrial genome. The water buffalo mt molecule is 16.355 base pair length and shows a genome organization similar to those reported for other mitochondrial genome. These new data provide an useful tool for many research area, i.e. evolutionary study and identification of food origin.

Internal Transcribed Spacer as a Target to Assess Yeast Biodiversity in Italian Taleggio PDO Cheese

UNLABELLED Three batches of soft smear-ripened Taleggio PDO cheese were made in Northern Italy during the summertime 2010. A total of 129 isolates cultured from cheese surface were examined by using PCR-based methods and sequencing of both the ITS1 region and D1 and D2 domains of the 26S rRNA gene. Sequence analysis of isolates brought to the identification of 6 species: Debaryomyces hansenii, Kluyveromyces lactis, Kluyveromyces marxianus, Yarrowia lipolytica, Pichia guilliermondii, and Torulaspora delbrueckii. Analysis of DNA directly extracted from 45 cheese surfaces permitted to detect 2 additional species Candida sake and Candida etchellsii. D. hansenii was predominant and widespread whereas the other yeast species were detected less frequently. To determine the relationships between yeast community and the environment, 39 isolates from wooden boxes used for dry salting of cheese were analyzed as well. Sequencing of ITS1 region allowed to identify D. hansenii, T. delbrueckii, and K. lactis. ITS1 multiple sequence alignments of D. hansenii detected in wooden boxes showed an in-del polymorphism at position 169. ITS1 secondary structures of yeasts were modeled to explore new applications of this region for molecular identification purposes. PRACTICAL APPLICATION This study used molecular analysis to identify adventitious yeast population present in the surface of Taleggio smear-ripened cheese. D. hansenii was found predominant in pasteurized milk, in dry salting equipment, and in all cheese samples until the end of ripening.

Diversity of fungal flora in raw milk from the Italian Alps in relation to pasture altitude

The present paper explores the diversity of mycobiota inhabiting raw milk sampled at different altitudes (1400 m, 1800 m, 2200 m) from cows grazing Alpine pastures of Valle d’Aosta (North-Western Italian Alps). To this aim, multilocus sequencing was performed at barcodes commonly used for fungal identification (ITS1, D1/D2 domains of the 26S rRNA gene, and part of the β-tubulin gene). A total of 31 species were detected, most of them yeasts, followed by moulds and by 2 sequences of macroscopic fungi. Several yeasts and moulds were well-characterized inhabitants of the dairy environment, known to positively contribute to cheesemaking. Among these, Candida was the most represented genus with a tendency to cluster at the highest altitudes (6 over 8 observations at ≥ 1800 m), and Kluyveromyces marxianus the most abundant single species, retrieved at all altitudes. The environmental ascomycetous Atrotorquata lineata, never put in relation with food nor described outside North-America, was another species among those most frequently retrieved and was detected in 6 milks at 1400 and 1800 m. The remaining fungi, in general never reported in milk, were mostly environmental. Many of them resulted associated with plants as pathogens or symbionts. Finally, the highest sampled altitude yielded a significant fungal diversity (17 species). This work enlarges the knowledge of fungal consortia inhabiting raw milk and introduces microbial ecology among the altitude-dependent factors, in the composition of Alpine pastures, with the potential of shaping the properties of milks and cheeses, together with the already described physical, chemical and botanical variables.

The complete coding region sequence of river buffalo (Bubalus bubalis) SRY gene.

The Y-linked SRY gene is responsible for testis determination in mammals. Mutations in this gene can lead to XY Gonadal Dysgenesis, an abnormal sexual phenotype described in humans, cattle, horses and river buffalo. We report here the complete river buffalo SRY sequence in order to enable the genetic diagnosis of this disease. The SRY sequence was also used to confirm the evolutionary divergence time between cattle and river buffalo 10 million years ago.