Polyphenol Profiling of Chestnut Pericarp, Integument and Curing Water Extracts to Qualify These Food By-Products as a Source of Antioxidants.

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.

Cloning and bacterial expression systems for recombinant human heparanase production: Substrate specificity investigation by docking of a putative heparanase substrate.

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.

Differential representation of liver proteins in obese human subjects suggests novel biomarkers and promising targets for drug development in obesity.

The proteome of liver biopsies from human obese (O) subjects has been compared to those of nonobese (NO) subjects using two-dimensional gel electrophoresis (2-DE). Differentially represented proteins were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS)-based peptide mass fingerprinting (PMF) and nanoflow-liquid chromatography coupled to electrospray-tandem mass spectrometry (nLC-ESI-MS/MS). Overall, 61 gene products common to all of the liver biopsies were identified within 65 spots, among which 25 ones were differently represented between O and NO subjects. In particular, over-representation of short-chain acyl-CoA dehydrogenase, Δ(3,5)-Δ(2,4)dienoyl-CoA isomerase, acetyl-CoA acetyltransferase, glyoxylate reductase/hydroxypyruvate reductase, fructose-biphosphate aldolase B, peroxiredoxin I, protein DJ-1, catalase, α- and ß-hemoglobin subunits, 3-mercaptopyruvate S-transferase, calreticulin, aminoacylase 1, phenazine biosynthesis-like domain-containing protein and a form of fatty acid-binding protein, together with downrepresentation of glutamate dehydrogenase, glutathione S-transferase A1, S-adenosylmethionine synthase 1A and a form of apolipoprotein A-I, was associated with the obesity condition. Some of these metabolic enzymes and antioxidant proteins have already been identified as putative diagnostic markers of liver dysfunction in animal models of steatosis or obesity, suggesting additional investigations on their role in these syndromes. Their differential representation in human liver was suggestive of their consideration as obesity human biomarkers and for the development of novel antiobesity drugs.

Simultaneously tracing the geographical origin and presence of bovine milk in Italian water buffalo Mozzarella cheese using MALDI-TOF data of casein signature peptides.

Water buffalo (WB) casein (CN) and curd samples from indigenous Italian and international breeds were examined with the objective of identifying signature peptides that could function as an indicator to determine the origin of their milk products. CN in complex mixtures were digested with trypsin, and peptide fragments were subsequently identified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS). The unique presence of a ß-CN A variant and an internally deleted αs1-CN (f35-42) variant in international WB milk samples was ascertained by identifying signature tryptic peptides from either dephosphorylated or native CN. Four signature unphosphorylated peptides derived from ß-CN A, i.e. (f49-68) Asn(68) (2223.6 Da), (f1-28) Ser(10) (3169.4 Da), (f1-29) Ser(10) (3297.4 Da) and (f33-48) Thr(41) (1982 Da) and two from αs1-CN (f35-42) deleted fragments, i.e. (f23-34) Met(31) (1415.7 Da) and (f43-58) Val(44) (1752.7 Da), were identified. Two signature casein phosphopeptides (CPPs), i.e. ß-CN (f1-28) 4P (3489.1 Da) and ß-CN (f33-48) 1P (2062.0 Da), were identified in the tryptic hydrolysate of native casein or curd and cheese samples using in-batch hydroxyapatite (HA) chromatography. All these fragments functioned as analytical surrogates of two αs1- and ß-casein variants that specifically occur in the milk of international WB breeds. Furthermore, the bovine peptide ß-CN (f1-28) 4P had a distinct and lower molecular mass compared with the WB counterpart and functioned as a species-specific marker for all breeds of WB. Advantages of this analytical approach are that (i) peptides are easier to separate than proteins, (ii) signature peptide probes originating from specific casein variants allow for the targeting of all international WB milk, curd and cheese samples and (iii) bovine and WB casein in mixtures can be simultaneously determined in protected designation of origin (PDO) "Mozzarella di Bufala Campana" cheese. This analytical method enabled the specific detection of international WB and bovine casein with a sensitivity threshold of 2 and 0.78 %, respectively. Graphical Abstract Monitoring of prototypic tryptic CPPs by MALDI-TOF analysis in Mediterranean (A), Romanian (B), Indian (C), Polish (D) and Canadian (E) curd samples to guarantee the authenticity of the PDO "Mozzarella di Bufala Campana" cheese.

Use of phytochemomics to evaluate the bioavailability and bioactivity of antioxidant peptides of soybean ß-conglycinin.

This research investigates how in vitro digestion contributes to the release of antioxidant peptides crypted in soybean ß-conglycinin (7S) and its deglycosylated form (D7S). It also investigates the uptake of the bioactive peptides by human intestinal Caco-2 cells using a bicameral system, and their effect on the antioxidant cell defense. Phytochemomics is used as a tool for achieving this goal. The peptides are obtained by mimicking human physiological gastrointestinal digestion conditions. The antioxidant capacity of the peptides is tested by ABTS•(+) radical cation decolorization (2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS)) and oxygen radical absorbance capacity assays. The antioxidant power of the peptides recovered from the basolateral chamber is also evaluated by an analysis of biomarkers of cellular oxidative stress such as cell proliferation, alkaline phosphatase, and secretion of nitric oxide, lipid peroxidation, superoxide dismutase and catalase. Peptides from D7S were more active than those of 7S in the modulation of the cell proliferation, oxidative status and differentiation of Caco-2 cells treated with H2 O2 . Differences in the bioactivity of the peptides of both proteins can be explained by analysis of the structural data obtained by mass spectrophotometry. Our findings support the bioavailability of antioxidant peptides of 7S. The antioxidant properties of 7S soy protein were influenced by events such as glycosylation, digestion, and absorption. Deglycosylation seems to be an innovative strategy for improving the properties of 7S. Deglycosylation might enhance 7S antioxidant power and reduce its immunoreactivity. The combined use of advanced analytical techniques and biochemical analyses (phytochemomics) has been a key part of this study.

Faox enzymes inhibited Maillard reaction development during storage both in protein glucose model system and low lactose UHT milk.

Fructosamines, also known as Amadori products, are formed by the condensation of glucose with the amino group of amino acids or proteins. These compounds are precursors of advanced glycation end products (AGEs) that can be formed either endogenously during aging and diabetes, and exogenously in heat-processed food. The negative effects of dietary AGEs on human health as well as their negative impact on the quality of dairy products have been widely described, therefore specific tools able to prevent the formation of glycation products are needed. Two fructosamine oxidase enzymes isolated from Aspergillus sp. namely, Faox I and Faox II catalyze the oxidative deglycation of Amadori products representing a potential tool for inhibiting the Maillard reaction in dairy products. In this paper, the ability of recombinant Faox I and II in limiting the formation of carboxy-methyl lysine (CML) and protein-bound hydroxymethyl furfurol (b-HMF) in a commercial UHT low lactose milk and a beta-lactoglobulin (ß-LG) glucose model system was investigated. Results show a consistent reduction of CML and b-HMF under all conditions. Faox effects were particularly evident on b-HMF formation in low lactose commercial milk. Peptide analysis of the ß-LG glucose system identified some peptides, derived from cyanogen bromide hydrolysis, as suitable candidates to monitor Faox action in milk-based products. All in all data suggested that non-enzymatic reactions in dairy products might be strongly reduced by implementing Faox enzymes.

Myrtucommulone production by a strain of Neofusicoccum australe endophytic in myrtle (Myrtus communis).

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.

A Genotyping Method for Detecting Foreign Buffalo Material in Mozzarella di Bufala Campana Cheese Using Allele-Specific- and Single-Tube Heminested-Polymerase Chain Reaction.

Mozzarella di Bufala Campana (MdBC) cheese is a Protected Designation of Origin (PDO) product that is important for the economy and cultural heritage of the Campania region. Food fraud can undermine consumers' trust in this dairy product and harm the livelihood of local producers. The current methods for detecting adulteration in MdBC cheese due to the use of buffalo material from foreign countries could exhibit limitations associated with the required use of expensive equipment, time-consuming procedures, and specialized personnel. To address these limits here, we propose a rapid, reliable, and cost-effective genotyping method that can detect foreign buffalo milk in a counterpart from the PDO area and in MdBC cheese, ensuring the quality and authenticity of the latter dairy product. This method is based on dedicated allele-specific and single-tube heminested polymerase chain reaction procedures. By using allele-specific primers that are designed to detect the nucleotide g.472G>C mutation of the CSN1S1Bbt allele, we distinguished an amplicon of 330 bp in the amplification product of DNA when extracted from milk and cheese, which is specific to the material originating from foreign countries. By spiking foreign milk samples with known amounts of the counterpart from the PDO area, the sensitivity of this assay was determined to be 0.01% v/v foreign to PDO milk. Based on a rough estimate of its simplicity, reliability, and cost, this method could be a valuable tool for identifying adulterated buffalo PDO dairy products.

Lactosylated casein phosphopeptides as specific indicators of heated milks.

Casein phosphopeptides (CPP) were identified in small amounts in milks heated at various intensities by using matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. CPP selectively concentrated on hydroxyapatite (HA) were regenerated using phosphoric acid mixed in the matrix. Unphosphorylated peptides not retained by HA were removed by buffer washing. This procedure enhanced the MALDI signals of CPP that are ordinarily suppressed by the co-occurrence of unphosphorylated peptides. CPP, belonging to the ß-casein (CN) family, i.e., (f1-29) 4P, (f1-28) 4P, and (f1-27) 4P, and the α(s2)-CN family, i.e., (f1-21) 4P and (f1-24) 4P, were observed in liquid and powder milk. The lactosylated counterparts were specific to intensely heated milks, but absent in raw and thermized/pasteurized milk. Most CPP with C-terminal lysines probably arose from the activity of plasmin; an enzyme most active in casein hydrolysis. A CPP analogue was used as the internal standard. The raw milk signature peptide ß-CN (f1-28) 4P constituted ~4.3% of the total ß-CN. Small amounts of lactosylated peptides, which varied with heat treatment intensity, were detected in the milk samples. The limit of detection of ultra-high-temperature milk adjunction in raw or pasteurized milk was ~10%.

In vivo absorptomics: Identification of bovine milk-derived peptides in human plasma after milk intake.

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, ß-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.

Toward milk speciation through the monitoring of casein proteotypic peptides.

The 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 alpha(s1)-CN 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.

Proteolysis and Process-Induced Modifications in Synbiotic Yogurt Investigated by Peptidomics and Phosphopeptidomics.

Probiotic and synbiotic yogurt preparations were manufactured at the semi-industrial pilot scale with Lactobacillus acidophilus and Bifidobacteria strains without inulin or fortified with 1 and 3% (w/w) inulin. The pathway of casein breakdown was determined in probiotic, synbiotic, conventional yogurt, and nonstarted milk base using HPLC-ESI-MS/MS-based peptidomics and phosphopeptidomics; in the latter case, casein phosphorylated peptides (CPPs) were previously enriched by hydroxyapatite chromatography. Compared with traditional yogurt, casein proteolysis increased in probiotic and even more in synbiotic yogurt with 1% inulin. Fortification with 3% inulin greatly modified the proteolytic pattern, indicating a characteristic contribution of probiotics to proteolysis. The enhanced proteolysis in synbiotic yogurt exposed the neo-formed peptides to progressively increase enzymatic or chemical modifications, such as dephosphorylation of CPPs, methionine oxidation, and formation of N-terminal pyroglutamic acids. These modifications might constitute molecular signature descriptors of metabolic processes mediated by complex bacterial communities, with technological, nutritional, and sensorial significance.

Influence of the Casein Composite Genotype on Milk Quality and Coagulation Properties in the Endangered Agerolese Cattle Breed.

The aim of this study was the characterization of CSN1S1, CSN2 and CSN3 genetic variability in Agerolese cattle, and the investigation of the effect of casein composite genotypes (CSN1S1, CSN2 and CSN3) on quality and coagulation traits of the corresponding milk. To these purposes, blood and milk from 84 cows were sampled and analysed. Allele frequencies at CSN2 and CSN3 revealed no Hardy-Weinberg equilibrium in the population with a prevalence of allele A2 for CSN2 and allele B for CSN3. BBA1A2AB and BBA2A2AB composite genotypes were the most common in the population. BBA1A2AB showed a higher total solids and fat content (12.70 ± 0.16 and 3.93 ± 0.10, respectively), while BBA2A2BB showed the best coagulation properties (RCT 12.62 ± 0.81; k20 5.84 ± 0.37; a30 23.72 ± 1.10). Interestingly, the A2 allele of CSN2 was very widespread in the population; thus, it will be intriguing to verify if A2A2 Agerolese cattle milk and the derived cheese may have better nutraceutical characteristics.

Trichoderma Applications on Strawberry Plants Modulate the Physiological Processes Positively Affecting Fruit Production and Quality.

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.

Olive oil from the 79 A.D. Vesuvius eruption stored at the Naples National Archaeological Museum (Italy).

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).

Effect of Trichoderma Bioactive Metabolite Treatments on the Production, Quality, and Protein Profile of Strawberry Fruits.

Fungi of the genus Trichoderma produce secondary metabolites having several biological activities that affect plant metabolism. We examined the effect of three Trichoderma bioactive metabolites (BAMs), namely, 6-pentyl-α-pyrone (6PP), harzianic acid (HA), and hydrophobin 1 (HYTLO1), on yield, fruit quality, and protein representation of strawberry plants. In particular, 6PP and HA increased the plant yield and number of fruits, when compared to control, while HYTLO1 promoted the growth of the roots and increased the total soluble solids content up to 19% and the accumulation of ascorbic acid and cyanidin 3-O-glucoside in red ripened fruits. Proteomic analysis showed that BAMs influenced the representation of proteins associated with the protein metabolism, response to stress/external stimuli, vesicle trafficking, carbon/energy, and secondary metabolism. Results suggest that the application of Trichoderma BAMs affects strawberry plant productivity and fruit quality and integrate previous observations on deregulated molecular processes in roots and leaves of Trichoderma-treated plants with original data on fruits.

The antimicrobial peptides casocidins I and II: Solution structural studies in water and different membrane-mimetic environments.

Antimicrobial peptides (AMPs) represent crucial components of the natural immune defense machinery of different organisms. Generally, they are short and positively charged, and bind to and destabilize bacterial cytoplasmic membranes, ultimately leading to cell death. Natural proteolytic cleavage of αs2-casein in bovine milk generates the antimicrobial peptides casocidin I and II. In the current study, we report for the first time on a detailed structure characterization of casocidins in solution by means of Nuclear Magnetic Resonance spectroscopy (NMR). Structural studies were conducted in H2O and different membrane mimetic environments, including 2,2,2-trifluoroethanol (TFE) and lipid anionic and zwitterionic vesicles. For both peptides, results indicate a mainly disordered conformation in H2O, with a few residues in a partial helical structure. No wide increase of order occurs upon interaction with lipid vesicles. Conversely, peptide conformation becomes highly ordered in presence of TFE, with both casocidins presenting a large helical content. Our data point out a preference of casocidins to interact with model anionic membranes. These results are compatible with possible mechanisms of action underlying the antimicrobial activity of casocidins that ultimately may affect membrane bilayer stability.

A non-canonical phosphorylation site in ß-casein A from non-Mediterranean water buffalo makes quantifiable the adulteration of Italian milk with foreign material by combined isoelectrofocusing-immunoblotting procedures.

The need of controlling illegal addition of water buffalo (WB) milk from foreign countries to the Italian counterpart devoted to the production of Protected Denomination of Origin (PDO) Mozzarella di Bufala Campana (MBC) cheese has promoted the development of simple, fast and cheap isoelectrofocusing (IEF) methods for evaluating the nature of the raw material to be used according to a high-throughput sample multiplexing format, avoiding the use of dedicated mass spectrometry-based procedures. Thus, combined proteomic methods were here integrated with optimized western blotting protocols in solving the complex IEF pattern of casein (CN) mixtures observed when Italian and foreign WB milk are mixed together. Identification of internally deleted αs1-CN hepta-phosphorylated species as well as of still unknown ß-CN A hexa-phosphorylated and N-terminally-nicked ß-CN A phosphorylated forms present uniquely in foreign WB milk samples, allowed recognizing these molecules as adulteration markers to be assayed in combined IEF-immunoblotting procedures; the latter ones showing optimal migration characteristics to be used in routine assays. A linear relationship between detected area of specific immunorecognized gel bands and percentage of international WB milk added to the Italian counterpart was verified, demonstrating that this method has an adulteration detection limit close to 3% v/v. Based on these results, this analytical procedure is here proposed as optimal one for evaluating the authenticity of PDO MBC cheese products.

On the interaction of the highly charged peptides casocidins with biomimetic membranes.

Casocidin I and II (CI and CII) are structurally related antimicrobial peptides made of 39 and 31 amino acids, respectively, which derive from natural proteolytic processing of αs2-casein and adopt an ordered α-helical structure in biomimetic membranes. Their putative membrane-permeabilizing activity was investigated at Hg-supported self-assembled monolayers (SAMs) and at tethered bilayer lipid membranes (tBLMs); the latter consisted of a monolayer of 2,3,di-O-phytanyl-sn-glycerol-1-tetraethylene-glycol-d,l-α lipoic acid ester thiolipid (DPTL), with a dioleoylphosphatidylcholine (DOPC) or dioleoylphosphatidylserine (DOPS) monolayer on top of it. Interaction of CI and CII with these biomimetic membranes was studied by four electrochemical techniques at pH 3, 5.4 and 6.8. Peptide incorporation in tBLMs was attempted via scans of electrochemical impedance spectra. Experiments demonstrated that CI and CII penetrate SAMs as well as the distal DOPC monolayer of DPTL/DOPC tBLMs, but not the proximal phytanyl monolayer, with the only exception of CII at pH 5.4. Conversely, CII permeabilized DPTL/DOPS tBLMs to a moderate extent at all investigated pH values by forming holes across the membrane, but not ion channels. Structural distribution of charged residues seemed to prevent CII from having a hydrophobic side of the α-helix capable of stabilizing a regular ion channel in the lipid matrix.

Eventual limits of the current EU official method for evaluating milk adulteration of water buffalo dairy products and potential proteomic solutions.

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 γ2- and γ3-CN fragments after corresponding plasminolysis. We here used proteomics to characterise false positive results occurring in the ERM as being due to WB ß-CN(f100-209), which is also formed after plasminolysis of genuine WB milk/dairy products and comigrates in IEF with B γ2-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 ß-CN(f1-25)4P and WB ß-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.