Anna Maria Guastella

Bioactive peptides in dairy products

Abstract Bioactive peptides are specific protein fragments that have a positive impact on body functions and conditions and may ultimately influence health. Most of the biological activities are encrypted within the primary sequence of the native protein and can be released by enzymatic hydrolysis and proteolysis or by food processing. Milk is a rich source of bioactive peptides which may contribute to regulate the nervous, gastrointestinal and cardiovascular systems as well as the immune system, confirming the added value of dairy products that, in certain cases, can be considered functional foods. The main biological activities of these peptides and their bioavailability in dairy products are reviewed. The natural concentration of these biomolecules is quite low and, to date one of the main goals has been to realize products enriched with bioactive peptides that have beneficial effects on human health and proven safety. Even though several health-enhancing products have already been launched and their integration in the diet could help in the prevention of chronic diseases such as hypertension, cancer and osteoporosis, more clinical trials are required in order to develop a deeper understanding of the activity of biopeptides on the human physiological mechanisms and also to assess the efficacy of their effects in a long term view. New scientific data are also needed to support their commercialisation in compliance with current regulations.

Goat milk with different αS2-casein content: analysis of allergenic potency by REAST-inhibition assay

Abstract In goat at CSN1S2 locus, seven alleles have been observed, characterized by different levels of expression, and, in particular, a null allele CSN1S2 O that significantly influences protein composition of milk. In a local goat breed reared in the North East of Sicily, “Argentata dell’Etna”, the combined use of electrophoretic milk analysis (SDS-PAGE) and 11th exon genomic analysis (PCR-RFLP) made it possible to identify three genotypes respectively normal (CSN1S2 NN ), heterozygote for the null allele (CSN1S2 NO ) and homozygote for the null allele (CSN1S2 OO ). In 214 “Argentata dell’Etna” goats, CSN1S2 O allele has shown a frequency of 0.033. An immunological test, REAST-inhibition assay (REAST, reverse enzyme allergo sorbent test), was carried out on three lyophilized milk samples (casein and seroprotein fractions) produced by goats with different genotypes at CSN1S2 locus, in order to assess their allergenic potency. By comparing the C 50 , the casein fraction CSN1S2 NN turned out to be the most allergenic, followed by the fraction CSN1S2 OO with 60% potency compared to the first one, while the fraction CSN1S2 NO presented 54% potency compared to the first one.

A simple model to predict the herbage intake of grazing dairy ewes in semi-extensive Mediterranean systems

The objective of this study was to develop a model to predict herbage intake of dairy lactating ewes during grazing in semi-extensive Mediterranean systems. For this purpose a total of 670 individual intakes from 210 Comisana and Pinzirita lactating ewes were collected in Sicily over 8 years of trials. The relationships between herbage intake and characteristics of the pasture (biomass availability, DM, CP and NDF content, herbage height), the animal (live weight, milk production) and the supplement (grams of DM and CP supplied daily to each animal) were investigated. Processing of data resulted in the formulation of two linear equations diversified for two qualitative levels of the pasture recognizable from the CP content of the herbage (CP >16% DM and CP ≤16% DM). Of the variables analysed, biomass, herbage height, herbage DM content, milk production and feed supplement, expressed as grams of CP individually offered each day, were those significantly correlated with ingestion. Inclusion of these variables in the model provides good reliability in predicting ingestion. The r2 values were 0.45 for CP ≤16% and 0.54 for CP >16%.

Diet selection and milk production and composition in Girgentana goats with different αs1-casein genotype

In goats, alpha s1-casein polymorphism is related to different rates of protein synthesis. Two genetic variants, A and F, have been identified as strong and weak alleles based on a production of 3.5 and 0.45 g/l of alpha s1-casein per allele. The aim of the trial was to test whether goats can select their diet as a function of their genetic aptitude to produce milk at different casein levels and whether this selection can influence milk production or composition. Two groups of 8 animals, homozygous for strong (AA) or weak (FF) alleles were housed in individual pens. Using a manger subdivided into five separate containers, the goats were offered daily for 3 weeks: 1.5 kg of alfalfa pelleted hay, 0.7 kg of whole barley, 0.7 kg of whole maize, 0.7 kg of whole faba bean and 0.7 kg of pelleted sunflower cake. Total dry matter intake was similar between groups and resulted in nutrient inputs much higher than requirements. On average, goats selected 86% of maize plus barley and only 46% of faba bean plus sunflower. Indeed, AA goats selected less faba bean compared with FF goats (37.2 v. 56.7% of the available amount; P=0.01); during week 2 and week 3 they significantly increased maize selection (respectively for week 2 and week 3: 94.9 and 99.1% v. 85.3 and 87.3%) thus increasing the ratio between the high-energy feeds and the high-protein feeds (2.41 v. 1.81, P=0.023). As for true protein, the high soluble fraction (B1) and the indigestible fraction (C) were lower in the diet selected by AA goats (respectively in AA and FF groups: B1, 7.85 v. 9.23% CP, P<0.01; C, 6.07 v. 6.30% CP, P<0.001); these diet characteristics can be associated with lower losses of protein. Milk production, being similar in AA and FF groups when goats were fed with a mixed diet, significantly increased in AA group, when free-choice feeding was given (mean productions: 1198 v. 800 g/d, P<0.01). Casein content was higher in AA group than in FF group (2.70 v. 2.40%, P<0.01) whereas milk urea was higher in FF group (59.7 v. 48.8 mg/dl, P<0.01). In conclusion, when the animals were free to select their diet, their higher genetic aptitude to produce casein seemed to adjust their energy and protein dietary input in qualitative terms, thus leading to an increase in milk production and a decrease in milk urea. These results seem to demonstrate that interactions probably occurred between genetic polymorphism at the alpha s1-casein locus, diet selection and the efficiency of nutrient transformation into milk.

Effects of shearing and supplemental level on intake of dry ewes grazing on barley stubble

Twenty-eight Comisana dry ewes were utilised to evaluate the effect of shearing (14 shorn and 14 unshorn ewes) and supplemental level (100 and 300g of concentrate) on intake of barley stubble. Shorn ewes showed a small increase in dry matter intake (at the end of August: 1.32 vs. 1.10kg/day; p<0.01). Intake of barley stubble decreased as supplement level increased (mean: 1.18 vs. 1.29; p<0.01). On average, substitution effect was 0.63. Total lamb weight per ewe was greater in the group receiving 300g of concentrate (7.38 vs. 5.85; p<0.05). Shearing induced a small, but not significant decrease in total lamb weight per ewe (6.18 vs. 7.05kg/ewe). Results seem to indicate that unshorn ewes react better to environmental stress and that 300g improve reproductive performance.

Molecular characterization and genetic structure of the Nero Siciliano pig breed

Nero Siciliano is an autochthonous pig breed that is reared mainly in semi-extensive systems in northeastern Sicily. Despite its economic importance and well-appreciated meat products, this breed is currently endangered. Consequently, an analysis of intra-breed variability is a fundamental step in preserving this genetic resource and its breeding system. In this work, we used 25 microsatellite markers to examine the genetic composition of 147 unrelated Nero Siciliano pigs. The total number of alleles detected (249, 9.96 per locus) and the expected heterozygosity (0.708) indicated that this breed had a high level of genetic variability. Bayesian cluster analysis showed that the most likely number of groups into which the sample could be partitioned was nine. Based on the proportion of each individuals genome derived from ancestry, pigs with at least 70% of their genome belonging to one cluster were assigned to that cluster. The cluster size ranged from 7 to 17 (n = 108). Genetic variability in this sub-population was slightly lower than in the whole sample, genetic differentiation among clusters was moderate (FST 0.125) and the FIS value was 0.011. NeighborNet and correspondence analysis revealed two clusters as the most divergent. Molecular coancestry analysis confirmed the good within-breed variability and highlighted the clusters that retained the highest genetic diversity.

Melanocortin 1 receptor (MC1R) gene polymorphisms in three Italian cattle breeds

The Melanocortin 1 receptor (MC1R) is one of the main genes implicated in the determination of the coat colour in mammals. This locus showed a relevant genetic variation between breeds that can be exploited for breed traceability of the animal productions. Modicana, Cinisara and Sardo-Modicana are three Italian endangered cattle breeds. Genetic characterisation by molecular markers is a fundamental prerequisite for managing genetic resources and for developing potential genetic traceability protocols. In order to improve the knowledge on Modicana, Cinisara and Sardo-Modicana breeds and to evaluate the possibility to develop DNA-based protocols for their mono-breeds products traceability, the genetic structure of MC1R gene was analysed. Four main alleles were observed in a representative sample of 162 animals. In the black coated Cinisara breed (n = 42), the ED and E+ alleles segregated with a frequency of 0.93 for ED allele. In the red coated Modicana (n = 60) and Sardo-Modicana (n = 60) breeds the E+ and E1 alleles segregated with frequencies of 0.42, 0.57 and 0.52, 0.47, respectively. The recessive allele e showed a low frequency (0.01) in both breeds. Sequencing a subsample of 34 animals the rare E2 allele was found only in Modicana and Sardo-Modicana at a good frequency (0.50). A new PCR-RFLP test, based on BstOI restriction endonuclease, was devised to assay for this allele. Results of the work indicate that red coat in Modicana and Sardo-Modicana cattle is genetically determined by the E+ and E1 alleles instead of the e allele at homozygote status, as occurs in other red European breeds. In these three Italian breeds of local importance, MC1R polymorphisms can be used to discriminate Cinisara from Modicana and Sardo-Modicana, but it was not able to distinguish between the two red coat populations.

Protein fraction heterogeneity in donkey’s milk analysed by proteomic methods

Abstract Donkey’s milk is often well tolerate by patients affected by cow’s milk protein allergy, probably thanks to its protein composition. This empiric evidence, confirmed by some clinical trials, needs to be better investigated. A preliminary survey on the protein fraction of donkey’s milk was carried out: fifty-six individual milk samples have been collected and analysed by IEF and SDS-PAGE. Five different IEF patterns have been identified, showing a marked heterogeneity both in casein and whey protein fractions. A single IEF pattern showed an apparent reduced amount of casein fraction highlighted by SDS. Three of the five IEF patterns have been further investigated by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS).

Mitochondrial DNA control region variation in Sanfratellano horse and two other Sicilian autochthonous breeds

Abstract Mitochondrial D-loop hypervariable region was analysed in 20 Sanfratellano and two other Sicilian autochthonous horse breeds (20 Sicilian Oriental Purebred and 20 Sicilian Indigenous) in order to investigate matrilineal genetic diversity. A total of 20 different haplotypes were identified sequencing a fragment of 397 bp; overall, haplotypes showed 31 polymorphic sites (7.8%). High diversity was detected in Sanfratellano (11 haplotypes) and Sicilian Indigenous (13 haplotypes), whereas only one haplotype was found in Sicilian Oriental Purebred. Sanfratellano sequences were compared with those belonging to the other Sicilian autochthonous horses and 118 sequences selected from the GenBank database in order to calculate the statistics of molecular diversity. Six haplotypes were exclusive of Sanfratellano which shares haplotype C, D, H, and O with the Sicilian Indigenous and haplotype U with the Sicilian Oriental Purebred; not significant differentiation was found between Sanfratellano and Sicilian Indigenous. BLAST search showed Sicilian haplotypes overlap with the database sequences but for three. Phylogenetic analysis did not show monophyletic group for Sanfratellano samples or the other breeds included in this analysis.

Genetic diversity and relationship among the three autochthonous Sicilian donkey populations assessed by microsatellite markers

Abstract In the developed countries donkey has lost its main function as draft animal because of the mechanization in agri-culture; as a consequence donkey population was greatly reduced. According to SAVE monitoring institute, three of the eight Italian endangered donkey breeds are native of Sicily (Ragusano, Pantesco, Grigio Siciliano). Urgent safeguard plans are required. The aim of this work is to investigate the distribution of genetic diversity and the relationships among the three Sicilian autochthonous donkey breeds using a set of microsatellite markers. A total of 116 blood samples (61 Ragusano, 39 Pantesco, 16 Grigio Siciliano) were collected in 9 herds all over Sicily. Representative samples of Ragusano and Grigio donkey populations consist of unrelated individuals, whereas the sample of Pantesco represents nearly the entire studbook-registered population managed by “Ispettorato Ripartimentale delle Foreste di Erice (TP)” in the “Azienda S. Matteo”. Genomic DNA was amplified at 11 microsatellite loci (HTG10, VHL20, HTG7, HTG4, AHT5, AHT4, HMS3, HMS6, HMS7, HMS2 and HTG6), located on different chromosomes in two PCR multiplex reactions. The PCR products were mixed with GeneScan 350 ROX internal size standard and ran on an ABI Prism 377 DNA Sequencer equipped with Genescan and Genotyper softwares. MICROSATELLITES ANALYSER, FSTAT and MICROSAT softwares had been used in order to calculate the main parameters of genetic diversity and the genetic distances. Phylogeny trees were obtained using PHYLIP 3.66 and SPLITSTREE 4 4.6 software packages. Clustering analysis has been carried out using STRUCTURE 2.0 program. All microsatellites were polymorphic in each breed, generating a total of 76 alleles (from 3 to 11 in HTG4 and HTG7, respectively). The number of observed alleles was higher in Ragusano (72, 6.55 per locus), than in Grigio (55, 5 per locus) and Pantesco (45 alleli, 4.09 per locus). The number of effective alleles did not reproduce the same trend (2.62 in Grigio, 2.53 in Ragusano and 2 in Pantesco). A total of 13 private alleles (17% of the generated alleles), with frequencies always lower than 0.10, have been detected (13 in Ragusano, 2 in Pantesco, 2 in Grigio Siciliano). The genetic differentiation coefficient was low (Gst = 0.065). According to the Gst value (0.163) HMS3 was the most discriminating marker in the set, whereas HTG4 was the less one. The populations were not at genetic equilibrium, showing a significant deficit of heterozygotes. The average expected heterozygosity (He) over all population was 0.612. He was lower in Pantesco (0.500) than in Ragusano and Grigio (0.605 and 0.618, respectively). Fis value, which indicates the degree of departure from random mating, was particularly high in Pantesco (0.230), in which the sample represent the whole population, than in the other two breeds. Genetic distances estimated by different methods (Da and Ds) and the related NJ and UPGMA dendrograms, as well as the clustering analysis, agree with the evidence that Pantesco endangered breed shows the higher degree of genetic distinctness. Ragusano and Grigio result closely related (Da=0.058), probably because of their common origin: in fact before 1953 in Sicily local donkey with bay and grey coat were usually managed as a single population.