Oleosin Cor a 15 is a novel allergen for Italian hazelnut allergic children.

BACKGROUND: Hazelnut allergy, which is characterized by symptoms that range from mild to severe, is one of the most common allergies in children throughout Europe, and an accurate diagnosis of this allergy is therefore essential. However, lipophilic allergens, such as oleosins, are generally underrepresented in diagnostic tests. We therefore sought to characterize the IgE reactivity of raw and roasted hazelnut oleosins, using the sera of hazelnut-allergic pediatric patients. METHODS: Raw and roasted hazelnut oil body-associated proteins were analyzed by means of 1D and 2D electrophoresis and MS. Oleosin IgE reactivity was assessed by immunoblotting with the sera of 27 children who have confirmed hazelnut allergies and from 10 tolerant subjects. A molecular characterization of the oleosins was performed by interrogating the C. avellana cv. Jefferson and cv. TGL genomes, and through expression and purification of the recombinant new allergen. RESULTS: A proteomic and genomic investigation allowed two new oleosins to be identified, in addition to Cor a 12 and Cor a 13, in hazelnut oil bodies. One of the new oleosins was registered as a new allergen, according to the WHO/IUIS Allergen Nomenclature Subcommittee criteria, and termed Cor a 15. Cor a 15 was the most frequently immunorecognized oleosin in our cohort. Oleosins resulted to be the only immunorecognized allergens in a subgroup of allergic patients who showed low ImmunoCAP assay IgE values and positive OFC and PbP. Hazelnut roasting resulted in an increase in oleosin immunoreactivity. CONCLUSION: A novel hazelnut oleosin, named Cor a 15, has been discovered. Cor a 15 could play a role in eliciting an allergic reaction in a subgroup of pediatric patients that exclusively immunorecognize oleosins. The high prevalence of hazelnut oleosin sensitization here reported further confirms the need to include oleosins in routine diagnostic procedures.

Donkey Milk Fermentation by Lactococcus lactis subsp. cremoris and Lactobacillus rhamnosus Affects the Antiviral and Antibacterial Milk Properties.

BACKGROUND: Milk is considered an important source of bioactive peptides, which can be produced by endogenous or starter bacteria, such as lactic acid bacteria, that are considered effective and safe producers of food-grade bioactive peptides. Among the various types of milk, donkey milk has been gaining more and more attention for its nutraceutical properties. METHODS: Lactobacillus rhamnosus 17D10 and Lactococcus lactis subsp. cremoris 40FEL3 were selected for their ability to produce peptides from donkey milk. The endogenous peptides and those obtained after bacterial fermentation were assayed for their antioxidant, antibacterial, and antiviral activities. The peptide mixtures were characterized by means of LC-MS/MS and then analyzed in silico using the Milk Bioactive Peptide DataBase. RESULTS: The peptides produced by the two selected bacteria enhanced the antioxidant activity and reduced E. coli growth. Only the peptides produced by L. rhamnosus 17D10 were able to reduce S. aureus growth. All the peptide mixtures were able to inhibit the replication of HSV-1 by more than 50%. Seventeen peptides were found to have 60% sequence similarity with already known bioactive peptides. CONCLUSIONS: A lactic acid bacterium fermentation process is able to enhance the value of donkey milk through bioactivities that are important for human health.

Back to the past: "find the guilty bug-microorganisms involved in the biodeterioration of archeological and historical artifacts".

Microbial deterioration accounts for a significant percentage of the degradation processes that occur on archeological/historical objects and artworks, and identifying the causative agents of such a phenomenon should therefore be a priority, in consideration of the need to conserve these important cultural heritage items. Diverse microbiological approaches, such as microscopic evaluations, cultural methods, metabolic- and DNA-based techniques, as well as a combination of the aforementioned methods, have been employed to characterize the bacterial, archaeal, and fungal communities that colonize art objects. The purpose of the present review article is to report the interactions occurring between the microorganisms and nutrients that are present in stones, bones, wood, paper, films, paintings, and modern art specimens (namely, collagen, cellulose, gelatin, albumin, lipids, and hydrocarbons). Some examples, which underline that a good knowledge of these interactions is essential to obtain an in depth understanding of the factors that favor colonization, are reported. These data can be exploited both to prevent damage and to obtain information on historical aspects that can be decrypted through the study of microbial population successions.

Back to the past-forever young: cutting-edge biochemical and microbiological tools for cultural heritage conservation.

Ancient documents and milestones of human history such as manuscripts and textiles are fragile and during aging undergo chemical, physical, and biological deterioration. Among the different causes of damage, also human intervention plays a role since some restoration strategies proved to be transient and/or they generated further damage. Outdoor monuments undergo deterioration since they are exposed to pollution, weathering, microbial attack (giving rise to undesired pigmentation, discoloration or true dissolution, corrosion, and overall decay), as well as man-made damage (i.e., graffiti). This review article reports the best-fitting strategies used to restore wall paintings, outdoor monuments, textiles, and paper documents to their ancient beauty by employing "soft" biobased approaches such as viable bacteria or suitable enzymes.

Back to the past: deciphering cultural heritage secrets by protein identification.

The present review article reports the most innovative methods to detect proteins in historical and archeological samples as well as to characterize proteins used as binders in artworks. Difficulties to ascribe proteins to a certain animal species are often due to post-translational modifications originated by chemical or microbial deterioration during aging. Combining different techniques such as peptide mass fingerprinting and tandem mass spectrometry can solve some of these problems and also allow discrimination between taxonomically related species like sheep and goat. The most studied proteins in bones and textile samples are osteocalcin, collagen and keratin, whereas egg yolk and white proteins, casein and collagen are the most relevant for binders used in old paintings. With the suitable approaches (immune-based methods, DOT-blot, etc…) it is also possible to obtain in situ characterization or analyze the samples directly in the museum laboratories, with the advantage of avoiding artwork damage and expensive external commitments. Recent cutting-edge strategies allowed detection of proteinaceous infection markers that, for instance, were used to establish the cause of death of old Inca mummies and also proved the presence of Yersinia pestis in old documents dating from the period in 17th century in which the plague ravaged Europe.

Effects of two different domestic boiling practices on the allergenicity of cow's milk proteins.

BACKGROUND: The sale of raw drinking milk through automatic dispensers is permitted in some EU member states, but consumers are usually advised to boil the milk before consumption. The present study has been conducted to evaluate the effects of two common domestic boiling techniques on the proteins of raw milk and, in particular, on their potential allergenicity. RESULTS: Native one-dimensional electrophoresis, N-terminal amino acid sequencing and immunoblotting have been used to characterize the protein pattern and to evaluate the possible changes in the allergenic properties of the processed milk. The main result of this investigation is that heating induces the aggregation of ß-lactoglobulin in higher-molecular-weight products, while caseins seem to be more resistant to the treatments. ß-Lactoglobulin aggregates have been found to be non-immunoreactive with the sera of subjects suffering from cow's milk protein allergy. CONCLUSION: Domestic boiling modifies the milk protein profile, causing a minor reduction in milk allergenicity. © 2017 Society of Chemical Industry.

Mite-Induced Asthma and IgE Levels to Shrimp, Mite, Tropomyosin, Arginine Kinase, and Der p 10 Are the Most Relevant Risk Factors for Challenge-Proven Shrimp Allergy.

BACKGROUND: Shrimp sensitization is common in the general population, but the presence of symptoms is only moderately related to sensitization. A point still at issue is which in vivo and/or in vitro tests (food challenge, component-resolved diagnosis, house dust mite [HDM] sensitization) can help in distinguishing shrimp-allergic subjects from subjects that are sensitized but tolerant. METHODS: The aim of this study was to evaluate the role of IgE to the different shrimp and mite allergens in distinguishing shrimp challenge-positive from challenge-negative patients. Subjects with suspected hypersensitivity reactions to shrimp, positive skin prick tests (SPTs), and/or anti-shrimp IgE were submitted to open and double-blind placebo-controlled food challenges (DBPCFC). Specific IgE to shrimp, mites, and the recombinants rPen a 1, rDer p 1, 2, and 10 were tested using ImmunoCAP-FEIA. IgE immunoblotting was performed to identify the patients' allergenic profiles. RESULTS: In total, 13 out of 51 (25.5%) patients with reported reactions to shrimp were truly shrimp allergic (7 DBPCFC positive and 6 with documented severe reactions). These patients had significantly higher skin test wheal diameters than nonallergic patients, as well as higher levels of IgE to rPen a 1 and rDer p 10. HDM-induced asthma and the simultaneous presence of anti-nDer p 1, 2, and 10 IgE levels increased the risk of true shrimp allergy. CONCLUSION: Food challenge tests are mandatory for the diagnosis of shrimp allergy. Tropomyosin is associated with clinical reactivity. HDM-induced asthma and anti-mite IgE are risk factors for shrimp allergy.

Reduction of whey protein concentrate antigenicity by using a combined enzymatic digestion and ultrafiltration approach.

The global interest in saving food resources is leading to recycle wasted-food materials to extract useful nutrients. In dairy industry, the recycling of whey proteins determines their utilization in the healthy-addressed foods, which, however, can cause immunological responses in allergic subjects. In this work, a whey protein concentrate (WPC) was alternatively hydrolyzed with pepsin, papain, trypsin and rennin in order to attenuate or abolish the ß-lactoglobulin (BLG) antigenicity. The electrophoretic profiles of both pepsin and papain WPC hydrolysates proved the disappearance of the BLG band, even though a slight antigenicity was still found by ELISA. Pepsin hydrolysates, filtered through a 10-kDa cut-off membrane, did not produce immunological response. A deeper investigation carried out on pepsin digested and ultrafiltered samples by LC-MS/MS showed the disappearance of the immunoreactive BLG-fragment IVTQMKGLDIQKVAGTW. The remaining peptides, partially overlapped to major IgE binding epitopes, were not able to give immunoreactivity response. The combined WPC pepsin digestion with ultrafiltration confirmed to be a user-friendly strategy to reduce markedly the WPC antigenicity. The improvement of this two-steps process could be used to produce novel hypoallergenic infant food formulas.

Identification of Equine Lactadherin-derived Peptides That Inhibit Rotavirus Infection via Integrin Receptor Competition.

Human rotavirus is the leading cause of severe gastroenteritis in infants and children under the age of 5 years in both developed and developing countries. Human lactadherin, a milk fat globule membrane glycoprotein, inhibits human rotavirus infection in vitro, whereas bovine lactadherin is not active. Moreover, it protects breastfed infants against symptomatic rotavirus infections. To explore the potential antiviral activity of lactadherin sourced by equines, we undertook a proteomic analysis of milk fat globule membrane proteins from donkey milk and elucidated its amino acid sequence. Alignment of the human, bovine, and donkey lactadherin sequences revealed the presence of an Asp-Gly-Glu (DGE) α2ß1 integrin-binding motif in the N-terminal domain of donkey sequence only. Because integrin α2ß1 plays a critical role during early steps of rotavirus host cell adhesion, we tested a minilibrary of donkey lactadherin-derived peptides containing DGE sequence for anti-rotavirus activity. A 20-amino acid peptide containing both DGE and RGD motifs (named pDGE-RGD) showed the greatest activity, and its mechanism of antiviral action was characterized; pDGE-RGD binds to integrin α2ß1 by means of the DGE motif and inhibits rotavirus attachment to the cell surface. These findings suggest the potential anti-rotavirus activity of equine lactadherin and support the feasibility of developing an anti-rotavirus peptide that acts by hindering virus-receptor binding.

Milk Fat Globule Proteins Are Relevant Bovine Milk Allergens in Patients with α­Gal Syndrome.

Alpha-gal syndrome (AGS) is a mammalian meat allergy associated with tick bites and specific IgE to the oligosaccharide galactose-α-1,3-galactose (α-gal). Recent studies have shown that 10-20% of AGS patients also react to the dairy proteins. Considering the already described role of the meat lipid fraction in AGS manifestations, the aim of this work has been to investigate whether the milk fat globule proteins (MFGPs) could be involved in AGS. The MFGPs are extracted and their recognition by the IgE of AGS patients is proved through immunoblotting experiments. The identification of the immunoreactive proteins by LC-HRMS analysis allows to demonstrate for the first time that butyrophillin, lactadherin, and xanthine oxidase (XO) are α-gal glycosylated. The role of xanthine oxidase seems to be prevalent since it is highly recognized by both the anti-α-gal antibody and AGS patient sera. The results obtained in this study provide novel insights in the characterization of α-Gal carrying glycoproteins in bovine milk, supporting the possibility that milk, especially in its whole form, may give reactions in AGS patients. Although additional factors are probably associated with the clinical manifestations, the avoidance of milk and milk products should be considered in individuals with AGS showing symptoms related to milk consumption.

Rice allergy demonstrated by double-blind placebo-controlled food challenge in peach-allergic patients is related to lipid transfer protein reactivity.

BACKGROUND: The risk factors for sensitisation to rice and the involved allergens are still partially unknown. In this study we evaluated the clinically relevant aspects of rice allergy in DBPCF-positive patients, the major rice allergens, the severity of peach- and rice-induced symptoms in respect to Pru p 3 sensitisation and the role of anti-rPru p 3 IgE levels as a risk factor for rice allergy. METHODS: In 148 peach-allergic subjects, patients with allergic reactions to rice and rice-positive serum IgE were selected. Symptoms were verified by double-blind placebo-controlled food challenges (DBPCFCs), performed at a maximum dosage of 25 g. Rice allergens, identified by IgE immunoblotting, were characterised by N-terminal amino acid sequencing. The relationship between anti-rPru p 3, 1 and 4 IgE levels and rice symptoms were statistically analysed. RESULTS: Eight out of 10 recruited rice-allergic patients had positive DBPCFCs, while 2 patients were not challenged due to their previously documented severe reactions. All patients with rice-induced symptoms were Pru p 3 positive and presented with higher anti-rPru p 3 levels than the rice-sensitised but tolerant patients. A 9-kDa lipid transfer protein, which was highly homologous to Pru p 3, was identified as the major rice allergen and elicited a positive response in all of the patients. Five patients reacted to a putative 15- to 17-kDa rice allergenic protein, and 3 patients reacted to an [alpha]-amylase/subtilisin inhibitor that was approximately 20 kDa. CONCLUSION: Rarely, allergic reactions to rice can arise in patients with peach allergies who are sensitised to Pru p 3, particularly in patients with high anti-rPru p 3 IgE levels.

A2 Milk and BCM-7 Peptide as Emerging Parameters of Milk Quality.

Beta-casein makes up about 30% of the total protein contained in milk and can be present in cows' milk in two distinct forms (A1 or A2) or as a combination of the two. The only difference between these two variants of ß-casein (ß-CN) is a single amino acid substitution. This results in a different behavior of the protein upon enzymatic cleavage, following human consumption or due to microbial action. In most of the commercially available milk containing A1 or A1/A2 ß-CN variants, the ß-casomorphin-7 peptide (BCM-7) is released upon digestion and during cheese manufacturing/ripening, while this does not happen with A2 milk. BCM-7 is a known µ-opioid receptor agonist that may influence the gastro-intestinal physiology directly and may also exert effects elsewhere in the body, such as on the cardiovascular, neurological and endocrine systems. The present article is aimed at a revision of prior review papers on the topic, with a focus on the impact of ingestion of A1 ß-CN milk and A2 ß-CN milk on any health-related outcomes and on the impact of A1 or A2 ß-CN variant on technological properties of cows' milk. When systematic reviews were considered, it was possible to conclude that A2 ß-CN exerts beneficial effects at the gastrointestinal level compared with A1 ß-CN, but that there is no evidence of A1 ß-CN having negative effects on human health. Physicochemical differences among cows' milk containing either ß-CN A2 or ß-CN A1 and their effects on technological properties are discussed.

Citron-N is a neuronal Rho-associated protein involved in Golgi organization through actin cytoskeleton regulation.

The actin cytoskeleton is best known for its role during cellular morphogenesis. However, other evidence suggests that actin is also crucial for the organization and dynamics of membrane organelles such as endosomes and the Golgi complex. As in morphogenesis, the Rho family of small GTPases are key mediators of organelle actin-driven events, although it is unclear how these ubiquitously distributed proteins are activated to regulate actin dynamics in an organelle-specific manner. Here we show that the brain-specific Rho-binding protein Citron-N is enriched at, and associates with, the Golgi apparatus of hippocampal neurons in culture. Suppression of the whole protein or expression of a mutant form lacking the Rho-binding activity results in dispersion of the Golgi apparatus. In contrast, high intracellular levels induce localized accumulation of RhoA and filamentous actin, protecting the Golgi from the rupture normally produced by actin depolymerization. Biochemical and functional analyses indicate that Citron-N controls actin locally by assembling together the Rho effector ROCK-II and the actin-binding, neuron-specific, protein Profilin-IIa (PIIa). Together with recent data on endosomal dynamics, our results highlight the importance of organelle-specific Rho modulators for actin-dependent organelle organization and dynamics.

Multi-target detection of egg-white and pig gelatin fining agents in Nebbiolo-based aged red wine by means of nanoHPLC-HRMS.

The presence of residues from fining agents in wines may represent a risk for allergic consumers and a source of discomfort for others, such as vegans. Even though ELISA is the official detection method for such residues, this technique may be hindered by cross-reactivity issues, or by matrix-molecule interference due to a high polyphenol content, especially in red wines. An HRMS-based method has been developed to detect pig gelatin and egg white in experimental five-year aged Nebbiolo-based red wine. Biomarker peptides were selected, after tryptic digestion, and quantified by multitarget nanoHPLC-HRMS analysis. The method resulted in an LLOQs of 5 µg/mL in the experimental wine, and between 1 and 2 µg/mL in the buffer. This method allowed both gelatin and egg white proteins to be detected and quantified in aged red wine, while whereas the commercial ELISA kit was instead unable to detect egg white in the same samples.

Effect of hot air and infrared roasting on hazelnut allergenicity.

Roasting is known to affect the protein profile and allergenicity of hazelnuts (Corylus avellana cv TGL). The aim of the study was to investigate whether roasting techniques based on different heat transfer methods (hot air and infrared), differently affect the protein solubility and the IgE-binding capacities of both the soluble and insoluble hazelnut protein fractions. The immune-reactivity of the Cor a 9, Cor a 11 and Cor a 14 allergens resulted to be stable after roasting at 140 °C, for both types of treatment, while roasting at 170 °C caused a reduction in IgE-binding, which was particularly noticeable after infrared processing, that led to an almost complete disappearance of allergenicity. Microscopical analyses showed that roasting caused cytoplasmic network disruption, with a loss of lipid compartmentalization, as well as an alteration of the structure of the protein bodies and of the cell wall organization.

Thermal processing of insect allergens and IgE cross-recognition in Italian patients allergic to shrimp, house dust mite and mealworm.

Edible insects are considered as a promising and sustainable alternative protein source for humans, although risk assessments, with particular reference to the allergic potential of insect proteins, are required. Considering that insects are likely to be consumed after processing, it is crucial to assess how processing can influence allergenicity. In our study, we investigated how boiling and frying affect the IgE cross-recognition of proteins from five edible insects (mealworm, buffalo worm, silkworm, cricket and grasshopper). We considered three groups of Italian patients allergic to shrimps and to house dust mites, who had never consumed insects before and two subjects with occupational allergy and food sensitization to mealworm. Our data suggest that thermal processing may change the solubility of proteins, thereby resulting in a protein shift from water-soluble fractions to water-insoluble fractions. Immunoblot and LC-MS/MS analyses have shown that tropomyosin may play an important role as a cross-allergen for house dust mite and shrimp allergic patients, while larval cuticle protein seems to play a major role in the cross-reactivity of patients primarily sensitized to mealworm. On the basis of our results, the effects of processing appear to be protein-, species- and treatment-specific. Therefore, house dust mite, shrimp and mealworm allergic patients should consume insects with caution, even after thermal processing.

Glutamate-induced metabolic changes in Lactococcus lactis NCDO 2118 during GABA production: combined transcriptomic and proteomic analysis.

GABA is a molecule of increasing nutraceutical interest due to its modulatory activity on the central nervous system and smooth muscle relaxation. Potentially probiotic bacteria can produce it by glutamate decarboxylation, but nothing is known about the physiological modifications occurring at the microbial level during GABA production. In the present investigation, a GABA-producing Lactococcus lactis strain grown in a medium supplemented with or without glutamate was studied using a combined transcriptome/proteome analysis. A tenfold increase in GABA production in the glutamate medium was observed only during the stationary phase and at low pH. About 30 genes and/or proteins were shown to be differentially expressed in glutamate-stimulated conditions as compared to control conditions, and the modulation exerted by glutamate on entire metabolic pathways was highlighted by the complementary nature of transcriptomics and proteomics. Most glutamate-induced responses consisted in under-expression of metabolic pathways, with the exception of glycolysis where either over- or under-expression of specific genes was observed. The energy-producing arginine deiminase pathway, the ATPase, and also some stress proteins were down-regulated, suggesting that glutamate is not only an alternative means to get energy, but also a protective agent against stress for the strain studied.

Antimicrobial Potential of Food Lactic Acid Bacteria: Bioactive Peptide Decrypting from Caseins and Bacteriocin Production.

Lactic acid bacteria (LAB) potential in the food industry and in the biotechnological sector is a well-established interest. LAB potential in counteracting especially food-borne infections has received growing attention, but despite being a road full of promises is yet poorly explored. Furthermore, the ability of LAB to produce antimicrobial compounds, both by ribosomal synthesis and by decrypting them from proteins, is of high value when considering the growing impact of multidrug resistant strains. The antimicrobial potential of 14 food-derived lactic acid bacteria strains has been investigated in this study. Among them, four strains were able to counteract Listeria monocytogenes growth: Lactococcus lactis SN12 and L. lactis SN17 by high lactic acid production, whereas L. lactis 41FLL3 and Lactobacillus sakei I151 by Nisin Z and Sakacin P production, respectively. Strains Lactococcus lactis MG1363, Lactobacillus rhamnosus 17D10 and Lactobacillus helveticus 4D5 were tested and selected for their potential attitude to hydrolyze caseins. All the strains were able to release bioactive peptides with already known antimicrobial, antihypertensive and opioid activities. These features render these strains or their bioactive molecules suitable for use in food as biocontrol agents, or as nutraceutical supplements to treat mild disorders such as moderate hypertension and children insomnia. These results highlight once again that LAB potential in ensuring food safety, food nutraceutical value and ultimately in favoring human health is still underexplored and underexploited.

Extracellular Vesicles in Human Preterm Colostrum Inhibit Infection by Human Cytomegalovirus In Vitro.

Breast milk is a complex biofluid that nourishes infants, supports their growth and protects them from diseases. However, at the same time, breastfeeding is a transmission route for human cytomegalovirus (HCMV), with preterm infants being at a great risk of congenital disease. The discrepancy between high HCMV transmission rates and the few reported cases of infants with severe clinical illness is likely due to the protective effect of breast milk. The aim of this study was to investigate the anti-HCMV activity of human preterm colostrum and clarify the role of colostrum-derived extracellular vesicles (EVs). Preterm colostrum samples were collected and the EVs were purified and characterized. The in vitro anti-HCMV activity of both colostrum and EVs was tested against HCMV, and the viral replication step inhibited by colostrum-purified EVs was examined. We investigated the putative role EV surface proteins play in impairing HCMV infection using shaving experiments and proteomic analysis. The obtained results confirmed the antiviral action of colostrum against HCMV and demonstrated a remarkable antiviral activity of colostrum-derived EVs. Furthermore, we demonstrated that EVs impair the attachment of HCMV to cells, with EV surface proteins playing a role in mediating this action. These findings contribute to clarifying the mechanisms that underlie the protective role of human colostrum against HCMV infection.