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Human heparanase (HPSE) is an enzyme that degrades the extracellular matrix. It is implicated in a multiplicity of physiological and pathological processes encouraging angiogenesis and tumor metastasis. The protein is a heterodimer composed of a subunit of 8 kDa and another of 50 kDa. The two protein subunits are noncovalently associated. The cloning and expression of the two protein subunits in Escherichia coli and their subsequent purification to homogeneity under native conditions result in the production of an active HPSE enzyme. The substrate specificity of the HPSE was studied by docking of a putative substrate that is a designed oligosaccharide with the minimum recognition backbone, with the additional 2-N-sulfate and 6-O-sulfate groups at the nonreducing GlcN and a fluorogenic tag at the reducing extremity GlcN. To develop a quantitative fluorescence assay with this substrate would be extremely useful in studies on HPSE, as the HPSE cleavage of fluorogenic tag would result in a measurable response.

Ovine milk samples, from 240 half udders, with low (1000-2000 x 103/ml) were obtained from a flock of 120 primiparous Sarda ewes. Chemical analysis, immuno-electrophoretic and HPLC-ESI/MS techniques were carried out in order to evaluate the influence of SCC on ewe’s milk composition. Dry matter, fat, true protein, casein (CN), non protein nitrogen and urea contents were not affected by SCC level up to 2000 x 103/ml. The pH value of the milk and the whey protein content significantly increased together with the SCC while lactose content resulted lower in milk with high SCC. By electrophoresis at alkaline pH and immunoblotting with polyclonal antibodies against s1, s2, and -CN, the electrophoretic patterns of milk samples with high SCC showed a different heterogeneity at level of each casein fraction respect to ones with low SCC. In the former case, the casein heterogeneity was due to a synergic action of two phenomena: proteolysis by plasmin action on s2 and -casein, mainly, as well as alterations of phosphorylation degree, close related to an increased alkaline phosphatase activity, with a concomitant loss of phosphate groups.

Two types of lamb rennet pastes, traditional (A) and industrial (B), were used for making two types of PDO Fiore Sardo cheese (FSA and FSB) to ascertain the influence of their use on cheese characteristics. Traditional rennet was produced from the stomachs of lambs fed only milk and slaughtered, after suckling, on a full stomach. Industrial rennet was produced from the stomachs of lambs weaned and slaughtered on an empty stomach. The cheeses were significantly different (Po0.01) in pH values and pH 4.6-soluble N/total N throughout ripening. FSA cheese had a higher rate of proteolytic activity. Within 240 d of ripening, as1-CN was degraded completely whereas b-CN was partly hydrolysed by plasmin, irrespective of the rennet paste preparation. A number of phosphopeptides were detected in fresh cheeses, which were progressively shortened and dephosphorylated during ripening. In FSA cheese, greater amounts of free fatty acids were released, in particular butyric acid.

The main elements of milk proteins can be summarized as follows: variable phosphorylation degree of the four caseins, variable glycosylation of κ-CN and α-La, frequent presence of casein peptides generated by the action of endogenous and bacterial enzymes, wide molecular mass range whey proteins, easy thermodenaturation of whey proteins. With more knowledge of the primary structure, the location of the phosphate groups on the casein chains, and intra-and inter-protein disulphide bond exchanges during milk processing, particularly during digestion by proteolytic enzyme, the function and the role of most importantmilk proteins are now better understood. Further insight into the basic properties of milk proteins should lead to a greater understanding of the natural function of milk and the roles its components play in human nutrition. In this paper milk proteins are reviewed in the perspective of preventing milk allergies in newborns.

he possibility of detecting extraneous milk in singles species cheese-milk has been explored. A mass spectrometry (MS)-based procedure has been developed to detect 'signature peptides', corresponding to the predefined subset of 'proteotypic peptides', as matchless analytical surrogates of the parent caseins. Tryptic digests of skimmed milk samples from four species were analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS. Amongst the candidate signature peptides that are able to differentiate milks from the four species, the alpha(s1)-casein (CN) f8-22 peptide was selected as a convenient marker for bovine, ovine and water buffalo milk while the f4-22 peptide was selected as a marker for the two caprine alpha(s1)-CN A and B variants, which differ by a Pro(16) (B)->Leu(16) (A) substitution. MALDI analysis of the digest allowed the detection of alpha(s1)-CN f8-22 and caprine f4-22. The accurate evaluation of caprine milk in a quaternary mixture required the development of a liquid chromatography/electrospray ionization (LC/ESI)-MS procedure. Five synthetic signature peptide analogues, which differed from their natural counterparts by a single amino acid substitution, were used as internal standards to quantify the alpha(s1)-CN, which was chosen as a reference milk protein, from the different species. The limits of detection were 0.5% (1% for caprine) for either the MALDI or the LC/ESI-MS method. The isotopic-label-free quantification of isoform- or variant-specific signature peptides has disclosed a convenient approach for targeting proteins in complex mixtures.

A dedicated two-step purification procedure prior to nanoliquid chromatography-electrospray-tandem mass spectrometry analysis enabled the identification of bovine milk-derived peptides absorbed and circulating in the plasma of three healthy volunteers who received 250 mL of pasteurized milk after a 10-days washout. The appearance and clearance of milk peptides in plasma were monitored at various time points. Overall, 758, 273 and 212 unique peptides derived from 15, 15 and 18 bovine milk proteins, respectively, were identified in the plasma of these volunteers, evidencing a substantial inter-individual variability. Peptides encrypting possible bioactive and/or immunogenic molecules originating from caseins, β-lactoglobulin and minor milk proteins were detected. Peptide representation data revealed the combined action of endoproteases involved in primary hydrolysis during gastroduodenal digestion and exopeptidases that hydrolyse peptides in the small intestine. It remains to be established whether the half-life and concentration ranges of circulating milk-derived peptides may have any impacts on human health.

Plant polyphenols have beneficial antioxidant effects on human health, practices aimed at preserving their content in foods and/or reusing food by-products are encouraged. The impact of the traditional practice of the water curing procedure of chestnuts, which prevents insect/mould damage during storage, was studied to assess the release of polyphenols from the fruit. Metabolites extracted from pericarp and integument tissues or released in the medium from the water curing process were analyzed by matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and electrospray-quadrupole-time of flight-mass spectrometry (ESI-qTOF-MS). This identified: (i) condensed and hydrolyzable tannins made of (epi)catechin (procyanidins) and acid ellagic units in pericarp tissues, (ii) polyphenols made of gallocatechin and catechin units condensed with gallate (prodelphinidins) in integument counterparts, (iii) metabolites resembling those reported above in the wastewater from the chestnut curing process. Comparative experiments were also performed on aqueous media recovered from fruits treated with processes involving: (i) tap water, (ii) tap water containing an antifungal Lb. pentosus strain, (iii) wastewater from a previous curing treatment. These analyses indicated that the former treatment determines a 6–7-fold higher release of polyphenols in the curing water with respect to the other ones. This event has a negative impact on the luster of treated fruits but qualifies the corresponding wastes as a source of antioxidants. Such a phenomenon does not occur in wastewater from the other curing processes, where the release of polyphenols was reduced, thus preserving the chestnut’s appearance. Polyphenol profiling measurements demonstrated that bacterial presence in water hampered the release of pericarp metabolites. This study provides a rationale to traditional processing practices on fruit appearance and qualifies the corresponding wastes as a source of bioactive compounds for other nutraceutical applications.

Many Trichoderma spp. are successful plant beneficial microbial inoculants due to their ability to act as biocontrol agents with direct antagonistic activities to phytopathogens, and as biostimulants capable of promoting plant growth. This work investigated the effects of treatments with three selected Trichoderma strains (T22, TH1, and GV41) to strawberry plants on the productivity, metabolites and proteome of the formed fruits. Trichoderma applications stimulated plant growth, increased strawberry fruit yield, and favored selective accumulation of anthocyanins and other antioxidants in red ripened fruits. Proteomic analysis of fruits harvested from the plants previously treated with Trichoderma demonstrated that the microbial inoculants highly affected the representation of proteins associated with responses to stress/external stimuli, nutrient uptake, protein metabolism, carbon/energy metabolism and secondary metabolism, also providing a possible explanation to the presence of specific metabolites in fruits. Bioinformatic analysis of these differential proteins revealed a central network of interacting molecular species, providing a rationale to the concomitant modulation of different plant physiological processes following the microbial inoculation. These findings indicated that the application of Trichoderma-based products exerts a positive impact on strawberry, integrating well with previous observations on the molecular mechanisms activated in roots and leaves of other tested plant species, demonstrating that the efficacy of using a biological approach with beneficial microbes on the maturing plant is also able to transfer advantages to the developing fruits.

Using a range of chromatographic, spectroscopic, and mass spectrometric analytical techniques, we characterized one of the “edible items” found at the Vesuvius archeological sites and guarded at the National Archaeological Museum of Naples (MANN) in Naples, Italy. We authenticated the specimen contained in a glass bottle (Mann-S1 sample) as originally olive oil and mapped the deep evolution throughout its 2000 years of storage. Triacylglycerols were completely hydrolyzed, while the resulting (hydroxy) fatty acids had partly condensed into rarely found estolides. A complex pattern of volatile compounds arose mainly from breakdown of oleic acid. With excellent approximation, radiocarbon dating placed the find at the time of the Plinian Mount Vesuvius eruption in 79 A.D., indicating that Mann-S1 is probably the oldest residue of olive oil in the world found in bulk amount (nearly 0.7 L).

The European reference method (ERM) recognises the fraudulent addition of bovine (B) milk in water buffalo (WB) milk/dairy products based on concomitant isoelectric focusing (IEF) detection of B Beta2- and Beta3-CN fragments after corresponding plasminolysis. We here used proteomics to characterise false positive results occurring in the ERM as being due to WB Beta-CN(f100-209), which is also formed after plasminolysis of genuine WB milk/dairy products and comigrates in IEF with B Beta2-CN. These ERM limitations were overcome by a dedicated proteomic procedure based on loading of B/WB milk/cheese CN extracts on a hydroxyapatite column, in situ trypsinolysis and elution of B Beta-CN(f1-25)4P and WB Beta-CN(f1-28)4P proteotypic peptides. Based on their similar ionisation properties and resolution in MALDI-TOF-MS, these phosphopeptides were identified as suitable markers for detection of B material in WB milk/dairy products to a detection limit of 0.8% v/v. This proteomic procedure is here proposed as integrative/alternative to the ERM.

A "virtual two-dimensional (2D)-like" approach based on pH 2.5-6.0 gel isoelectrofocusing coupled to MALDI-TOF mass spectrometry (MS) was addressed for challenging the ovine casein heterogeneity due to polymorphism and post-translational modifications. The procedure enabled the identification of the main casein components including i) the four casein families at different degrees of phosphorylation, ii) nonallelic variants of either alpha(s1)- or alpha(s2)-CN, iii) differently glycosylated kappa-CN, and iv) high molecular mass proteolytic peptides. Protein assignment was confirmed by MALDI-TOF (MS) mass mapping of the tryptic digests, while phosphorylation sites were localized by tandem mass spectrometry. A straightforward identification was hindered by unexpected molecular mass shifts due to the oxidization of alpha(s1)- and the beta-CN. Indeed, using nano-ESI-MS, the casein sub-types were proved to contain a variable number of oxidized methionine residues. Data were additionally substantiated using polyclonal antibodies raised against the single casein families for immunolabelling purposes. The outcomes herein demonstrate the potentiality of the "virtual 2D-like" approach and provide indications useful for the inter-laboratory discrepancies in casein identification. They also greatly facilitate the comparison of data and the establishment of multi-user image-based isoelectrofocusing gels. (C) 2012 Elsevier Ltd. All rights reserved.

A study on casein genetic polymorphism in water buffalo milk of Mediterranean breed, reared in Southern Italy, was carried out by means of immunoelectrophoretic and chromatographic techniques coupled with mass spectrometry analysis. When compared with reference as1-CN variant A, the results showed the occurrence of a new as1-casein (as1-CN) B genetic variant having a single silent amino acid substitution Leu 178 (A)/ Ser 178 (B). In Mediterranean buffalo milks the allelic frequency of new as1-CN B was very similar to that determined in Mozzarella di bufala campana (MBC), a Protected Denomination of Origin (PDO) cheese, made exclusively from whole raw buffalo milk of this breed. The relative percentage of the two as1-CN variants, either in bulk milk or MBC cheese, can be used in defining the authenticity of PDO MBC cheese. Published by Elsevier Ltd.

Four Lactobacillus helveticus strains were studied for proteolytic capacity and general aminopeptidase (AP) and X-Pro dipeptidyl aminopeptidase (DAP) activity. The rate of hydrolysis and the activity against synthetic substrates with N-terminal residues of Arg, Lys, Leu, Glu or Pro, varied markedly among the strains. The X-Pro DAP activity was consistently high. The crude cell-wall and cytoplasm extracts from strain Lb. helveticus ISLC59 were analysed thoroughly for their proteolysis ability by using four synthetic peptide substrates, including αs1-CN(f1-23). Peptides formed during in vitro hydrolysis of the synthetic substrates by cell wall and cytoplasm preparations were identified by LC-ESI/MS. In doing so, it was possible to infer a prevalent endopeptidase activity splitting Lys7-His8 and Gln13-Glu14 bonds in the cytoplasm, and to deduce a secondary activity, which hydrolysed Glu14-Val15, Leu16-Asn17, Glu18-Asn19 and Lys3-His4 bonds lacking in the cell-wall. The presence of exopeptidases, as mainly AP, DAP, and carboxypeptidase (CPase) was deduced from the formation of several N- and C-terminally truncated peptides sets. The AP activity was higher in the cell-wall layer, where CPase activity was absent. The in vitro assays with cell extracts of the Lb. helveticus ISLC59 strain revealed extensive exopeptidase and endopeptidase activities. In several cases, the hydrolytic system of Lb. helveticus that splits in vitro αs1-CN(f1-23) peptide bonds was similar to that of Lactococcus lactis. The effects were also compared with those occurring in vivo in hard cheese such as Grana Padano.

Myrtucommulones are acylphloroglucinol compounds reported from myrtle (Myrtus communis) and a few more plant species belonging in the Myrtaceae that have recently attracted the attention of pharmacologists for their anti-oxidant, anti-inflammatory and anti-tumor properties. An endophytic strain of Neofusicoccum australe recovered from a myrtle branch was selected based on the bioactivity of its culture extracts, and found to produce myrtucommulones A and D. A mixture of these compounds induced anti-proliferative effects on the human prostatic cancer cell lines DU145 and PC3, with a IC₅₀ of respectively 4.64 and 3.11 mg/l. Along the lines of recent evidences of the ability by endophytic fungi to produce bioactive compounds originally extracted from their host plants, this is the first report of myrtucommulones as secondary metabolites of an endophytic fungal strain. The availability of a microbial strain to be cultured in vitro may provide access to more substantial amounts of these products for further investigations in view of their possible pharmaceutical use.

The comprehensive analysis of natural lipid mixtures is often challenging due to the simultaneous occurrence of functionally and structurally heterogeneous compounds. Modern analytical approaches in system-wide lipidomics essentially rely on mass spectrometry (MS). The overabundant amount of triacylglycerols (TAGs) in most samples hinders the direct detection of phospholipids (PLs) or other low-abundance lipids; therefore, a fractionation step is most often required to reduce sample complexity prior to MS analysis. In this work, we explore the use of hydroxyapatite (HAP) as a chromatographic stationary phase (gel HAP) or chemo-affinity sorbent material (ceramic HAP) to selectively enrich PLs and polar lipids from chicken egg yolk and buttermilk. Due to the affinity of phosphate-containing compounds for HAP, both in-column and in-batch HAP-based chromatography were effective to deplete TAGs before releasing PLs with an eluting ternary system containing sodium phosphate as the displacing agent. Buttermilk gangliosides and PLs co-eluted, indicating that HAP also exhibits a high affinity for sialylated glycolipids and that polar lipids are retained through a combined mechanism of chemo-affinity and hydrogen bonding. To the best of our knowledge, this is the first report in which HAP is proposed as an alternative stationary phase for separating both the PLs and sialylated glycolipids from TAGs in complex lipidomes. The HAP-based chromatography has potential to be improved for the separation of the polar lipid classes. (C) 2014 Elsevier B.V. All rights reserved.