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Research demonstrates production of fermented milks containing angiotensin-I-converting-enzyme-inhibitory peptides by two species of Lactobacillus species. Data on ACE-inhibitory peptide sequences, chemical synthesis, and biological activity are presented.

Background: Casein phosphopeptides (CPPs) are phosphorylated casein-derived peptides produced synthetically by proteolytic digestion of αs1-, αs2- and β-casein. The anticariogenic activity of CPPs is due to their ability to stabilize high levels of amorphous calcium phosphate (ACP) on tooth surface, preventing demineralization and enhancing remineralization of enamel caries. The aim of this study was to test the in vitro ability of natural CPPs (contained in yogurt) to prevent demineralization and promote remineralization of dental enamel. Methods: Eighty human molars were used. After standardizing an in vitro demineralization procedure for producing artificial caries (Group 1: pH 4.8; Group 2: pH 3.97), this procedure was used on teeth, but with the addition of natural CPPs (Group 3: pH 4.8; Group 4: pH 3.97). The effects of these procedures were evaluated by quantitative analysis (change in weight and calcium titration) and qualitative analysis (SEM). Statistical analysis of the results was performed using ANOVA. Results: Statistical analysis showed significant differences in weight changes between the groups with and without natural CPPs. The SEM observation showed the protective effects of natural CPPs. Conclusions: The results demonstrated that CPPs contained in yogurt have an inhibitory effect on demineralization and promote the remineralization of dental enamel. [ABSTRACT FROM AUTHOR]

In crime investigations, the unambiguous identification of biological traces can be decisive for framing the events. In this study, we applied proteomics to analyze scant amounts of biological residues in the context of an alleged rape case, focusing on the detection of traces of vomit. We used high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and two distinct proteomic workflows to identify proteins and possible proteolytic peptides in biological residues from clothing, bedding, and car upholstery from the alleged crime scene. Specifically, a fragment of pillowcase contained a protein pattern indicative of human saliva and a complex panel of peptides resulting from extensive hydrolysis of salivary proteins. The presence of partly digested proteins from bovine meat, wheat, and eggs, along with salivary and gastric enzymes, demonstrated the presence of vomit on the alleged victim's trousers, also providing insights into the recently consumed meal. A drop of cow's milk on the seat of the suspect's car was likely irrelevant to the criminal act. Other fabric samples showed only common contaminants, excluding significant biological traces or food-derived proteins. These findings support the judicial decision regarding consent to sexual intercourse, for which DNA individualization lacks evidentiary power, and establish a reference for annotating saliva and vomit traces in forensic investigations., Competing Interests: Declarations. Ethics approval and consent to participate: It was not necessary to request any approval from the ethics committee since the analyses were performed at the Court’s request and authorization. The experiments reported in this study did not directly involve human subjects but were conducted on fabric fragments. Sensitive details have been omitted in the text to respect the privacy of the people involved. Furthermore, the study complies with the ethical principles for medical research involving human participants established by the Declaration of Helsinki. Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

To evaluate the potential differences in the propensity of β-casein A1 (β-CNA1) and A2 (β-CNA2) from bovine milk to release health-relevant β-casomorphins (BCMs), food-derived peptides were monitored over time in the blood of eight human volunteers who consumed milk containing both protein variants. Liquid chromatography coupled with high resolution tandem mass spectrometry revealed interindividual variability of milk peptidomic profiles in human blood. BCMs were not detected, whereas BCM precursors originating from both β-CNA1 and β-CNA2 were ascertained, with β-CNA2-derived peptides showing a slightly greater susceptibility to proteolysis. Ten synthetic peptides mimicking circulating BCM precursors from β-CNA1 and β-CNA2, which were incubated ex vivo with the blood of two volunteers, showed comparable potential to generate BCMs. The formation of BCMs seemed to depend mainly on the size of the BCM precursors and less on the presence of His 67 or Pro 67 . These findings challenge the belief that BCMs are released exclusively from β-CNA1 and support the nutritional safety of conventional milk, informing health policies regarding milk consumption., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Pink/red discoloration encompasses a series of relatively common spoilage defects of commercial dairy products. In this study, we used shotgun proteomics to identify the microorganism responsible for the production of intensely red-coloured slimes found on the surface of freshly opened commercial spreadable cheese and yogurt samples. Proteome-wide characterization of microbial proteins allowed to identify 1042 and 687 gene products from Rhodotorula spp. in spreadable cheese and yogurt samples, respectively, while no significant protein scores from other microorganisms were recorded. Subsequent microbiological analyses and sequencing of the 26S rRNA gene region supported the proteomic results demonstrating that the microorganism involved was Rhodotorula mucilaginosa, a carotenoid - producing basidiomycetous that can be potentially pathogenic to humans, especially for immunocompromised individuals. This is the first time that shotgun proteomics has been used to identify a microorganism responsible for spoilage in dairy products, proposing it as a relatively fast, sensitive, and reliable alternative or complement to conventional methods for microbial identification., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

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 CSN1S1B bt 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.

Introduction: This paper aims to theoretically and empirically investigate the concept of digital capital in the Italian context. Digital Capital can be conceived as independent individual capital whose lack within a population can be a cause of digital inequality. Our paper draws from recent works that have measured the Digital Capital as a combination of digital access and digital competences, and have tested this operational definition through an online survey on a UK sample. The results of such research proved the construct validity of the operational definition, thus showing that Digital Capital could be empirically measured. However, a measurement model needs to be tested and validated over time and in different socio-cultural contexts in order to be refined and strengthened, and eventually disseminated on a large scale., Method: This is the reason why this paper will show the results of a funded research project (named DigiCapItaly) carried out to test the validity of the Digital Capital measure in a different country, i.e., Italy. The data were collected with an online survey using a representative sample (by age, gender and geographical area) of individuals living in Italy aged 18 years or more. The creation of a composite index to measure Digital Capital followed a two-stage Principal Component Analysis approach., Results: First, the paper provides a methodological framework for facing challenges and pitfalls in operationalizing and assessing a complex concept in social research. Secondly, results show that Digital Capital operational definition works in Italy as well as in the UK, thus legitimizing its recognition as an independent capital., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Addeo, D'Auria, Delli Paoli, Punziano, Ragnedda and Ruiu.)

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., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Multiple analytical techniques were combined to achieve a detailed characterization of organic residues in different typologies of funerary pottery, which were found at two separate archeological sites in the Campania Region (Italy) and both dated back to the first millennium BC. Gas chromatography-mass spectrometry (GC-MS) analysis of lipids provided inconclusive results. The attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectra of encrustation on two glazed bowls of the 3rd to 4th century BC were comparable to those of fresh bone, revealing the presence of hydroxyapatite and proteins, which were identified as bovine collagen chains by liquid chromatography coupled to high-resolution tandem mass spectrometry (LC-MS/MS)-based proteomics. This finding confirmed that Italic populations used to inhume the dead along with votive meat offerings. Proteomics was decisive for identifying bovine milk in an unusually shaped amphora unearthed from a grave that belonged to a woman at the necropolis of the Greek colony in Cuma (7th century BC). Peptidomic analysis demonstrated that the genetic variant A 1 of β-casein was already present in the southern Mediterranean area at least 2500 years ago. Overall, these results depict an agropastoral system of Italic populations at the age of Magna Graecia based on a significant role of domesticated cattle.

Protein expression from the berry skin of four red grape biotypes with varying hybrid character was compared at a proteome-wide level to identify the metabolic pathways underlying divergent patterns of secondary metabolites. A bottom-up shotgun proteomics approach with label-free quantification and MaxQuant-assisted computational analysis was applied. Red grapes were from (i) purebred Vitis vinifera (Aglianico cv .); (ii) V. vinifera (local Sciascinoso cv .) grafted onto an American rootstock; (iii) interspecific hybrid ( V. vinifera × V. labrusca , Isabel), and (iv) uncharacterized grape genotype with hybrid lineage, producing relatively abundant anthocyanidin 3,5- O -diglucosides. Proteomics supported the differences between hybrids and purebred V. vinifera grapes, consistently with distinct phenotypic metabolite assets. Methanol O -anthraniloyltransferase, which catalyses the synthesis of methyl anthranilate, primarily responsible for the "foxy" odour, was exclusive of the Isabel hybrid grape. Most of the proteins with different expression profiles converged into coordinated biosynthetic networks of primary metabolism, while many possible enzymes of secondary metabolism pathways, including 5-glucosyltransferases expected for hybrid grapes, remained unassigned due to incomplete protein annotation for the Vitis genus. Minor differences of protein expression distinguished V. vinifera scion grafted onto American rootstocks from purebred V. vinifera skin grapes, supporting a slight influence of the rootstock on the grape metabolism.

The aim of this study was to evaluate the dynamics of proteolysis during dough fermentation started with different lactic acid bacteria species, through the identification of intermediate and small-sized peptides generated during fermentation. Single-strain cultures of Levilactobacillus brevis, Fructilactobacillus sanfranciscensis, Companilactobacillus alimentarius, and Leuconostoc pseudomesenteroides were assayed as sourdough starters. Assays were carried out at lab-scale for 48 h of fermentation, using both unstarted and yeast-leavened dough as controls. Physicochemical and microbiological analyses were combined with peptidomic and proteomic profiling, identifying several hundreds of peptides mainly released from the water-soluble wheat proteins, including β-amylase, triticin, and serpins. Both α- and γ-gliadins were hydrolyzed, though only at the N-terminal domain, while the central protein region - encrypting celiac disease epitopes- remained unaffected. The bacterial-mediated consumption of sugars and the concomitant hydrolysis of starch degrading β-amylase could underlie improved digestibility and several nutritionally beneficial effects of sourdough baked products., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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.

Donkey's milk (DM) has been extensively investigated as a valuable substitute of breast milk, often suitable to manage cow's milk protein allergy in infants. DM exhibits potent inhibitory properties against numerous microbial species. Although oligosaccharides and lipids might contribute to the antimicrobial potential, the current inventory of proteins is not able to justify the low count of microorganisms generally observed in DM. The shotgun proteomic analysis of fractionated DM disclosed a set of 94 gene products, 41% of which have documented antimicrobial activity or are involved in transferring the passive immunity to the donkey offspring. The concerted action of lysozyme, lactoferrin, immunoglobulins provides the molecular basis for part of the DM antibacterial potential. The pH -4.6 insoluble fraction contained significant levels of L-amino acid oxidase, identified with 11 unique peptides matching the horse homologue gene product. This enzyme catalyses the oxidative deamination of amino acids into ketoacids, producing ammonia and H 2 O 2 . κ-casein, likely occurring as a fully O-glycosylated protein, may concur to inhibit the adhesion of pathogenic microorganisms, along with other glycoproteins. Proteomics supports the alimentary use of DM not only as a substitute of human milk in early infancy, but also for growing children, convalescent, elderly people and general population. SIGNIFICANCE: Donkey's milk (DM) is acquiring increasing popularity because it is a suitable substitute of the human milk, when breastfeeding is not possible and infants suffer from cow's milk allergy. DM is characterized by a much lower microbial load compared to ruminants' milk. This feature has been traditionally attributed to the high content of lysozyme. DM exhibits potent activity against a broad range of bacteria, viruses and fungi, suggesting that other protein components can be responsible of the antimicrobial potential. The gel-free proteomic analysis of pH 4.6-insoluble and soluble (whey) fractions demonstrated that DM contains a large number of gene products involved in antimicrobial mechanisms and in transferring passive immunity to the donkey offspring. DM contains relatively high levels of L-amino acid oxidase that catalyses the oxidative deamination of amino acid substrates into ketoacids, with production of ammonia and H 2 O 2 . In combination with lysozyme, lactoferrin and immunoglobulins, the presence of L-amino acid oxidase provides the molecular basis of the antibacterial potential observed for DM. Considered the low microbial load, DM can be sanitated at mild conditions, thereby preserving many of the native nutritional traits. Thus, DM can be considered a safe and nutritionally valid alimentary resource for growing children, convalescent, elderly people and general population. Data of this study represent the largest inventory of proteins identified in Equidae milk, so far., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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 fatty acid (FA) composition of human milk (HM) from N = 9 Italian healthy donors following a free diet exhibited FA-dependent ranges of variability, as assessed by GC-FID. The possible short-term changes in the FA profile were monitored in the milk of lactating mothers (three) collected at five time points over a 6 h period, following an oral load (200 mL) of bovine milk. An array of techniques was exploited, including UHPLC-ESI-MS/MS of intact lipids and MALDI-TOF MS before and after chemical hydrogenation or bromination, in addition to MALDI-TOF MS analysis of FA after saponification, to monitor short-chain and odd-chain FA in HM as markers of bovine milk fat. A single administration of bovine milk did not appreciably modify the lipid pattern, suggesting that the maternal diet could induce not detectable short-term changes on the lipid composition of HM. Diet-induced increase of butyric acid was also excluded by 13 C NMR. The functions that HM FA exert in infant physiology appear finely regulated through maternal metabolism., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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.

Proteomics was exploited to assess the nature of possible traces of vomit found on the scene of an alleged sexual assault. In the case in point, a woman reported to the police to be raped five days before by a cousin of hers in his car. The woman declared she had vomited in the car before fainting definitely, due to alcohol or possible drugs covertly slipped in her drinks. The suspect confirmed the sexual intercourse, but he claimed consensual sex while the woman was fully conscious. To establish consent and hence subsistence of the crime, the Magistrate requested toxicological analyses on items sampled from the car and from woman's boots. Negative results obtained from toxicological analyses could not exclude the actual assumption of psychoactive substances by the alleged victim, due to sample aging. On the contrary, proteomic analysis disclosed a pattern of 249 gene products including signature endogenous and food-derived proteins along with a multitude of peptide digests, clearly indicative of vomit, thereby supporting the victim's report in the case under examination. Proteomics also provided detailed information about the nature of meal, which might contribute to frame the crime scene in similar cases. SIGNIFICANCE: The identification of traces of vomit supported the report of the victim's report according to which she vomited before definitely losing consciousness, so providing key contribution to establish consent for the sexual intercourse. This is the first time that proteomics is used to identify traces of vomit for forensic purposes. In spite of the scantiness of the biological specimen available, proteomics was successful to define a panel of characteristic endogenous proteins as well as to identify partly digested food-proteins arising from a complex meal. Proteomics is increasingly used as a forensic technique, well complementing the existing tools. In general, assessing traces of vomit in biological specimens and characterizing the nature of food ingested at the molecular level could afford probative elements to frame a crime scene., (Copyright © 2019. Published by Elsevier B.V.)

Analyzing an in vitro gastroduodenal digest of whey proteins by high-performance liquid chromatography (HPLC) coupled to high-resolution/high-sensitivity tandem mass spectrometry (MS/MS), we sought to evaluate if state-of-art peptidomics provide comprehensive peptide coverage of food "digestomes". A multitude of small-sized peptides derived from both α-lactalbumin and β-lactoglobulin as well as disulfide cross-linked hetero-oligomers remained unassigned, even when the digests were compared before and after S-S reduction. The precipitation with 12% trichloroacetic acid demonstrated the occurrence of large-sized polypeptides that escaped the bioinformatic identification. The analysis of a HPLC-MS/MS run with different proteomic search engines generated dissimilar peptide subsets, thus emphasizing the demand of refined searching algorithms. Although the MS/MS fragmentation of monocharged ions with exclusion of non-peptide-interfering compounds enlarged the inventory of short peptides, the overall picture of the "digestome" was still incomplete. These findings raise relevant implications for the identification of possible food-derived bioactive peptides or allergenic determinants.

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 α s1 -CN hepta-phosphorylated species as well as of still unknown β-CN A hexa-phosphorylated and N-terminally-nicked β-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., (Published by Elsevier Ltd.)

Nanoflow-HPLC-tandem mass spectrometry (MS/MS) was used to analyze the peptide fraction of breast milk samples collected from a single non-atopic donor on different days (10 samples) after receiving an oral load of cow's milk (by drinking 200 mL of bovine milk). In addition, breast milk was sampled from the same lactating mother over a 6-h period at five time points after drinking cow's milk. We aimed to trace the intra-individual variability and to define a time profile of the excretion of dietary peptides into breast milk. Overall, 21 peptides exclusively originating from both bovine caseins and whey proteins with no match within the human milk proteome were identified in the breast milk samples. These peptides were missing in the breast milk obtained from the mother after a prolonged milk- and dairy-free diet (three samples). The time course of cow's milk-derived β-Lg f(125-135) and β-casein f(81-92) in breast milk was determined from the MS ion intensity of the peptide signals. No intact cow's milk gene products were detected by HPLC-MS/MS analysis and Western blotting with anti-β-Lg antibody, but dot-blot analysis confirmed the occurrence of β-Lg fragments in the enriched peptide fraction of breast milk. These data suggest shifting the analytical perspective for the detection of dietary food allergens in breast milk from intact proteins to digested peptide fragments. The possible sensitization and elicitation potential or the tolerogenic properties of such low amounts of dietary peptides for the breastfed newborns remain to be explored.

Enzymatic dough improvers (DIs) are increasingly used as baking co-adjuvants. Herein, an array of techniques, including Western blotting, PCR, electrophoresis-based and shotgun proteomics, was addressed to identify the enzymes in six commercial DI preparations. In particular, this work sought to exclude the possible undeclared use of amylolytic enzymes from porcine (or other animal origin) pancreas in DIs. PCR-amplified mitochondrial cytochrome b (mt cyt b) gene region and porcine pancreatic α-amylase were the targets of DNA-based and protein methods, respectively, both assuring a limit of detection lower than 0.5-0.1% (w/w). Aspergillum oryzae α-amylase and Hordeum vulgare (barley) β-amylase were the most represented enzymes in all DI samples. Although one sample was PCR-positive, none among the DIs contained porcine pancreatic enzymes. Comparative gas chromatographic analysis of fatty acids suggested that the porcine contamination might arise from hard fats of porcine origin (lard), emphasizing the need of performing analyses at the protein level when the targets are enzymes or proteins., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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 γ 2 - and γ 3 -CN fragments after corresponding plasminolysis. We here used proteomics to characterise false positive results occurring in the ERM as being due to WB β-CN(f100-209), which is also formed after plasminolysis of genuine WB milk/dairy products and comigrates in IEF with B γ 2 -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 β-CN(f1-25)4P and WB β-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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The fatty acid composition of pomegranate (Punica granatum L.) seed oil (PSO) is dominated by punicic acid, a conjugated linolenic acid (18:3ω-5). As a free fatty acid, punicic acid is rapidly oxidized in air and extensively isomerizes upon acid-catalyzed methylation at 90 °C. In contrast, triacylglycerol-bound punicic acid in PSO was unchanged by simulated gastric conditions and was degraded by 5-7% by severe heating (up to 170 °C for 4 h), as herein assessed by gas chromatography, attenuated total reflectance-Fourier transform infrared spectroscopy, 1 H and 13 C NMR, and high-resolution electrospray ionization mass spectrometry. Total polar compounds of PSO were slightly affected by thermal stress, accounting for 5.71, 6.35, and 9.53% (w/w) in the unheated, heated at mild temperature (50 °C, 2 h), and heated at frying temperature (170 °C, 4 h) PSO, respectively. These findings support from a structural standpoint the potential use of PSO as a health-promoting edible oil.

Exclusively breast-fed infants can exhibit clear signs of IgE or non IgE-mediated cow's milk allergy. However, the definite characterization of dietary cow's milk proteins (CMP) that survive the maternal digestive tract to be absorbed into the bloodstream and secreted into breast milk remains missing. Herein, we aimed at assessing possible CMP-derived peptides in breast milk. Using high performance liquid chromatography (HPLC)-high resolution mass spectrometry (MS), we compared the peptide fraction of breast milk from 12 donors, among which 6 drank a cup of milk daily and 6 were on a strict dairy-free diet. We identified two bovine β-lactoglobulin (β-Lg, 2 out 6 samples) and one αs1-casein (1 out 6 samples) fragments in breast milk from mothers receiving a cup of bovine milk daily. These CMP-derived fragments, namely β-Lg (f42-54), (f42-57) and αs1-casein (f180-197), were absent in milk from mothers on dairy-free diet. In contrast, neither intact nor hydrolyzed β-Lg was detected by western blot and competitive ELISA in any breast milk sample. Eight additional bovine milk-derived peptides identified by software-assisted MS were most likely false positive. The results of this study demonstrate that CMP-derived peptides rather than intact CMP may sensitize or elicit allergic responses in the neonate through mother's milk. Immunologically active peptides from the maternal diet could be involved in priming the newborn's immune system, driving a tolerogenic response.

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 β-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 β-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. β-CN (f1-28) 4P (3489.1 Da) and β-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 β-casein variants that specifically occur in the milk of international WB breeds. Furthermore, the bovine peptide β-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.

Scope: This study aimed to assess the impact of the "often neglected" intestinal brush border membranes (BBMs) hydrolases on dietary peptides, exploring the possibility that the disintegration of proteins progressed in the small intestine up to a "core" of intrinsically stable oligopeptides, persisting independently on the up-stream breakdown., Methods and Results: Samples of sodium caseinate, skim milk powder, and whey protein isolate were submitted to in vitro simulated gastropancreatic digestion using two different procedures: (i) a simplified model involving the main compartmental specific proteases; (ii) a static digestion method based on a frameset of parameters inferred from in vivo. The gastroduodenal digesta were further hydrolyzed with peptidases from porcine jejunal BBM. The peptidomes arising from the two digestion models, characterized by combined HPLC and MS techniques, differed to some extent. However, only specific protein domains survived digestion, among which are potential bioactive or immunogenic (food allergy) peptides. The degree of hydrolysis (DH) after BBM digestion (70-77%) practically did not differ between the digestion models and significantly increased the DH after duodenal steps., Conclusion: Any in vitro digestion model should be supplemented with a jejunal phase to realistically determine the bioaccessibility and bioavailability of dietary peptides., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Wheat beer is a traditional light-colored top-fermenting beer brewed with at least 50% malted (e.g., German Weissbier) or unmalted (e.g., Belgian Witbier) wheat (Triticum aestivum) as an adjunct to barley (Hordeum vulgare) malt. For the first time, we explored the proteome of three Weissbier samples, using both 2D electrophoresis (2DE)-based and 2DE-free strategies. Overall, 58 different gene products arising from barley, wheat, and yeast (Saccharomyces spp.) were identified in the protein fraction of a representative Weissbier sample analyzed in detail. Analogous to all-barley-malt beers (BMB), barley and wheat Z-type serpins and nonspecific lipid transfer proteins dominated the proteome of Weissbier. Several α-amylase/trypsin inhibitors also survived the harsh brewing conditions. During brewing, hundreds of peptides are released into beer. By liquid chromatography-electrospray tandem mass spectrometry (LC-ESI MS/MS) analysis, we characterized 167 peptides belonging to 44 proteins, including gliadins, hordeins, and high- and low-molecular-weight glutenin subunits. Because of the interference from the overabundant yeast-derived peptides, we identified only a limited number of epitopes potentially triggering celiac disease. However, Weissbier samples contained 374, 372, and 382 ppm gliadin-equivalent peptides, as determined with the competitive G12 ELISA, which is roughly 10-fold higher than a lager BMB (41 ppm), thereby confirming that Weissbier is unsuited for celiacs. Western blot analysis demonstrated that Weissbier also contained large-sized prolamins immunoresponsive to antigliadin IgA antibodies from the pooled sera of celiac patients (n = 4).

The aim of the present study was to identify and characterize the celiacogenic/immunogenic proteins and peptides released during digestion of pasta (Triticum durum semolina). Cooked pasta was digested using a harmonized in vitro static model of oral-gastro-duodenal digestion. The course of pasta protein digestion was monitored by SDS-PAGE, and gluten proteins were specifically analyzed by Western blot using sera of celiac patients. Among the allergens, nonspecific lipid-transfer protein was highly resistant to gastro-duodenal hydrolysis, while other digestion-stable allergens such as α-amylase/trypsin inhibitors were not detected being totally released in the pasta cooking water. To simulate the final stage of intestinal degradation, the gastro-duodenal digesta were incubated with porcine jejunal brush-border membrane hydrolases. Sixty-one peptides surviving the brush-border membrane peptidases were identified by liquid chromatography-mass spectrometry, including several gluten-derived sequences encrypting different motifs responsible for the induction of celiac disease. These results provide new insights into the persistence of wheat-derived peptides during digestion of cooked pasta samples.

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., (Copyright © 2014 Elsevier B.V. All rights reserved.)

For the purpose of a varietal assessment, the berry skin anthocyanin profiles of 11 ancient native red grape varieties, sampled within the Irpinian area (Southern Italy), were compared to those of three reference Vitis vinifera cultivars and of a Kober 5BB rootstook hybrid (Vitisberlandieri×Vitisriparia). The 3,5-O-diglucoside anthocyanins and their acylated derivatives were monitored as signature compounds of non-V. vinifera grapes, using both reversed phase-high performance liquid chromatography (RP-HPLC) and matrix assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometry (MS). One variety (i.e. Tenta) was demonstrated to be an interspecific hybrid cross. Three additional varieties, namely Lacrima Nera, Aglianicone and a yet-unnamed variety, were classified as "late generation hybrids" (or non-V. vinifera×V. vinifera hybrids) because of a very diluted hybrid character, that was revealed only by MS methods. Five cultivars, i.e. Aglianico Lasco, Cannella, Coda di Volpe Rossa, Mentuonico, Olivella Nera, were catalogued as purebred V. vinifera. Due to the peculiar anthocyanin profile one variety (Tuccanese) remained unassigned. The methodology is of general applicability to support the process of varietal discrimination on a molecular basis with the objective of classifying autochthonous old grapevine varieties., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

A novel genetic variant at the αs1-casein locus of water buffalo (WB), 8-residue shorter than its wild-type has been found and sequenced. The internal deletion of the peptide E(35)KVNELsT(42) was confirmed by the isolation of the junction peptide. The 8-residue deletion mutant has a molecular weight that is 919 Da less than that of the wild-type. The novel isoform with a unique f35-42 deletion could be the result of the skipping of exon 6, generating an exon 6-deleted variant of αs1-casein. The wild-type and its shortened αs1-casein forms were found to co-exist in many individual milk samples. In contrast, the 8-residue, internally deleted αs1-casein variant did not occur in water buffaloes of the Mediterranean breed reared in Italy. Wild-type αs1-casein has 6 to 8 phosphate groups (P) while the internally deleted form 6 and 7P per molecule., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The entire panel of peptides produced from caseins (CN) and whey proteins (WP) that survive in vitro sequential gastro-pancreatic digestion and translocate across monolayers of Caco-2 cells, used as a model of the intestinal epithelium, has been characterised by HPLC and mass spectrometry. Among the milk-derived bioactive peptides, only minor amounts of mono-phosphorylated peptides arising from αs1- and β-CN were detected. The absorption behaviour of two resistant β-lactoglobulin (β-Lg) domains, β-Lg 125-135 and β-Lg 40-60, was studied in detail using synthetic peptides. The IgE-binding properties of the digests recovered from the apical and basolateral monolayer compartments were evaluated by dot-blot, using the sera of milk allergic children (N=5). Outcomes indicated β-Lg 127-135 as a possible "immune sensitising factor"in vivo. The almost complete loss of the IgE-affinity of CN and WP after digestion points out the need to design in vivo experiments to track the metabolic fate of dietary proteins., (Copyright © 2013 Elsevier Ltd. All rights reserved.)