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Characterization and bioactivity of oligopeptides in Asiago d'Allevo Cheese
Full text linkThe present Ph.D. thesis concerns the identification and the investigation of biological functions of oligopeptides extracted from the italian PDO Asiago cheese. Using High Performance Liquid Chromatography coupled on electrospray mass spectrometry (HPLC/ESI-MS), several peptides were identified, most of them arose from alfas1- and beta-casein and some of them never previously reported, suggesting a contribution of non starter bacteria enzymes. PCA analysis based on peptide profile allowed to differentiate the cheeses on account of the ripening time and of the use of starter. Therefore, peptides may be used as potential molecular markers as a tool of authenticity of PDO Asiago. The potential antihypertensive activity of Asiago water-soluble extracts (WSEs) was evaluated. Six-months-aged cheeses had a significantly higher inhibitory potency than 12 and 18 months ones and the 3 kDa fraction made a more considerable contribution to the ACE inhibitory activity than 10kDa fraction. Using HPLC/ESI-MS antihypertensive peptides mainly derived from alfas1-casein and beta-casein were identified. The assess of potential antimicrobial activity of WSEs was carried out because the naturally presence of antimicrobial peptides in cheesse may be a useful tool to control the growth of pathogenic bacterial or to determine their death. Six-months-aged cheeses showed higher inhibition against Listeria innocua and slighter higher inhibition against Listeria monocytogenes than 12 or 18 aged cheeses. However, this weak inhibition disappeared after 24 h of incubation suggesting a bacteriostatic activity of Asiago water-soluble peptides. A few antibacterial peptides were identified through HPLC/ESI-MS.La presente tesi di dottorato si è focalizzata sull'identificazione e valutazione di attività biologiche di oligopeptidi estratti d aun formaggio italian Asiago DOP. Mediante la cromatogriafia accoppiata a spettrometria di massa (HPLC/ESI-MS), sono stati identificati molti peptidi, molti dei quali derivanti dall'alfa s1-caseina e dalla beta-caseina e alcuni peptidi finora mai riportati, suggerendo un sostanziale contributo ad opera degli enzimi di batteri non starter. L'analisi PCA basata sul profilo peptidico ha consentito di discriminare i formaggi in base all'epoca di stagionatura ed all'utilizzo dello starter. Quindi, i peptidi risultano utili marker molecolari per determinare l'autenticità del formaggio Asiago. E' stata in seguito valutata l'attività anti-ipertensiva di oligopeptidi solubuli in acqua presenti nel formaggio Asiago. Formaggi stagionati 6 mesi presenttavano una maggiore capacità inibitoria rispetto ai formaggi stagionati 12 e 18 mesi, inoltre, frazioni con peso molecolare inferiore a 3 kDa aveva una maggiore potenza inibitoria rispetto alla frazione con peso molecolare inferiore a 10 kDa. Mediante HPLC/ESI-MS, sono stati identificati peptidi anti-ipertensivi derivanti principalmente dall'alfas1-caseina e dalla beta-caseina. Infine è stata valutata l'attività antimicrobica di estratti in quanto peptidi antimicrobici naturalmente presenti nel formaggio possono risultare molto utili al fine del controllo della crescita di batteri patogeni o addirittura a determinarne la morte. Formaggi stagionati 6 mesi presentavano una maggiore capacità antimicrobica verso Listeria innocua e Listeria monocytogenes rispetto ai formaggio stagionati 12 e 18 mesi. Tuttavia, questa debole inibizione scompariva dopo 24 ore suggerendo che i peptidi presenti nell'Asiago abbiano una azione batteriostatica. Alcuni peptidi antimicrobci sono stati identificati mediante HPLC/ESI-M
Anti-listeria effect of water-soluble extracts of Asiago cheese
No full textCheese ripening involves a complex series of biochemical events that leads to the characteristics
taste, aroma and texture of each cheese variety. The most complex of these biochemical events,
proteolysis, is caused by milk, milk-clotting, starter and secondary flora enzymes. During cheese
ripening, peptides generation mainly results from caseinolysis. Some of these peptides are
bioactive: they exert biological activities such as immunomodulatory, antithrombotic and
antibacterial activities.
Asiago is a “Protected Denomination of Origin” (PDO) cheese of the North-Eastern region of Italy
produced in two different varieties according to the length of ripening. Asiago d’Allevo is the
variety produced with skimmed raw milk and ripened for periods varying from 6 to 18 months.
The main goal of this research was to evaluate the presence of antibacterial peptides in Asiago
d’Allevo cheese toward two bacterial strains. The samples analysed were produced in alpine farms
of Asiago plateau (Vicenza, Italy).
The presence of antimicrobial peptides was assessed in water-soluble extracts (WSE) of Asiago
cheese ultrafiltrated onto 10 kDa cut-off membranes to remove proteins and dialysed in 100 Da cutoff
dialysis bags to remove salt and organic acids. The antimicrobial activity of these WSEs was
tested on Listeria innocua first. The samples that presented an inhibition were subsequently tested
on Listeria monocytogenes. Antimicrobial activity was assayed by a micro-method using 96-well
microplates and a microplate reader to determine microbial growth inhibition
Crosstalk between the mesothelium and lymphomatous cells: insight into the mechanisms involved in the progression of body cavity lymphomas.
No full textFatty acids profile of cow’s milk produced from alpine grazing.
No full textMilk from alpine grazing shows a different fatty acids profile (FA) from that produced in lowlands farms. In particular, it seems to have an increased content in Mono- and Polyunsaturated Fatty Acids (MUFA and PUFA) and a better n-6 to n-3 ratio. Conjugated linoleic acid compounds (CLA) content also seems to be enriched. The present study aimed at evaluating the effect of alpine grazing (AG), in comparison to a Total Mixed Ration (TMR) feeding system, on fatty acids profile of milk. The study took place from May to September 2006 and involved a farm which during summer move to alpine grazing (Novegno Mountain - Vicenza, North Italy). During the lowland period (P1, May, 25) animals were fed a TMR diet, whereas during the alpine periods (P2, July, 18; P3, September, 5) animals were kept at pasture and received a concentrate supplement based on maize and barley. Bulked milk samples (mix of evening and morning milking) were collected 3 times throughout one week of each experimental periods (n=9). FA profile was assessed by gas-chromatography and the obtained data (% of the total detected FAME) were submitted to a one-way ANOVA; the 3 levels of fixed effect Period/Feeding system were used to calculate two orthogonal contrasts (P1 vs P2+P3 and P2 vs P3). Results showed a significant decrease of Saturated Fatty Acids (from P1: 62.9 to P3: 56.6%; P<0.001) and an increase of MUFA (from P1: 25.3 to P3: 31.1; P<0.001) and PUFA (from P1: 4.2 to P3: 5.5; P<0.01), determining also a better n-6 to n-3 ratio (from P1: 3.6 to P3: 2.6; P<0.01), Thrombogenic (from P1: 3.1 to P3: 2.3; P<0.001) and Atherogenic (from P1: 2.6 to P3: 1.9; P<0.001) indexes and CLA content (from P1: 0.8 to P3: 2.2; P<0.001). For all of these variables contrast analysis showed a significant difference (P<0,01) between TMR (P1) and Alpine grazing (P2+P3). These results could be explained by the higher intake of unsaturated FA when cows grazed on alpine pasture. Moreover, the increase of Unsaturated Fatty Acids during P2 and P3 periods was probably due to the high mobilization of body fat, as a result of the deficiency in energy intake and/or of the advanced stage of lactation. In conclusion, alpine grazing seems to enhance fatty acid profile of milk, with particular regard to those fractions of interest in human nutrition
Angiotensin I converting enzyme-inhibitory peptides in Asiago d'Allevo cheese.
No full textAsiago d’Allevo (AA) is an Italian PDO cheese produced with raw skimmed milk and ripened from 6 to 18 months. This study aimed at evaluating the Angiotensin-I-converting enzyme (ACE)-inhibitory activity (IA) of water-soluble extracts (WSEs) from AA cheeses. The samples analysed were obtained from milk produced by cows fed with a total mixed ration or alpine pasture plus concentrate and ripened for 6, 12 or 18 months. WSEs were ultrafiltrated onto 10000 Da cut-off membrane and subsequently onto 3000 Da cut-off to obtain small peptides which probably make a considerable contribution to ACE-IA. This bioactivity was determined with a rapid in vitro enzymatic test. Values of ACE-inhibition were submitted to ANOVA considering also the peptide concentration tested as covariate. Preliminary results showed no differences among ripening groups while the concentration of peptides resulted statistically significant (P<0.05). In addition, the interaction between ripening and feeding system was also statistically significant. This finding suggests that alpine grazing condition influences microbial enzymes activity and the derived biopeptides. Furthermore gastro-intestinal digestion will be simulated in order to evaluate whether digestive enzymes generate peptides with high ACE-I
Use of bovine lymphocytes to assess the immunomodulatory effect of natural extracts
Full text linkA simple and rapid method based on the in vitro bovine lymphocyte proliferation was developed to
assess the immunomodulatory activity of natural compounds extracted from plant and foods. The proliferation of
lymphocytes was measured by the MTT [3-(4, 5- dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide] assay. This
test was used to measure the immuno-enhancing activity of 2 plant extracts and 2 samples of fermented milk after
48 h of incubation. To evaluate the effect of the cow physiological phase on the assay response, the tests were repeat-
ed using lymphocytes taken from non-pregnant dry, pregnant dry and post partum cows. The assay was not affect-
ed by the cow physiological phase and the results obtained were comparable. Both plant extracts enhanced lym-
phocyte growth, in particular the extract 2 was slightly more potent than extract 1. The fermentation of milk with
proteolytic starter cultures, such as FAIR E-63 and LA 2, can generate peptidic compounds from milk protein with
potential immunostimulating activity. These samples tested in cell culture inhibited lymphocyte proliferation
because they showed an acidic pH of 4.5. However, this bioassay was enough sensitive to detect biological activity
of different compounds also at low concentrations
Use of bioassay to assess functional properties of fermented milks
No full textThe fermentation of milk with proteolytic starter cultures, such as Enterococcus faecalis (FAIR E-63) and Lactobacillus delb. bulgaricus (LA 2), can generate peptidic compounds from milk proteins with potential immunomodulating and ACE-inhibitory activity implicated in the regulation of blood pressure. The fractions obtained from centrifugation of fermented milks with Centricon Amicon Ultra 15 filters (cut off 5000Da) were used to assess the ACE-inhibitory activity using a rapid enzymatic test. In addition, fraction aliquots were freeze-dried, re-suspended in culture medium and used at different concentrations to assess the immunomodulatory activity by a simple method based on in vitro bovine lymphocyte proliferation. The proliferation of lymphocytes obtained from non-pregnant dry cows was measured by the MTT assay after 48 h of incubation. The immunomodulating effects of the two different strains were analysed by ANOVA (SPSS 14.0). FAIR E-63 showed a greater ACE-inhibitory potency (72%) than LA2 (59%). Both fermented milks inhibited concanavaline-A lymphocyte proliferation and FAIR E-63 was more potent than LA-2. This effect was statistically significant (P<0.05) at low concentrations (5-25 μg/ml)
Alpine farm scale investigations of the relationships between productive system and quality of dairy products
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