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.

Viruses and Human Milk: Transmission or Protection?

Human milk (HM) is considered the best source of nutrition for infant growth and health. This nourishment is unique and changes constantly during lactation to adapt to the physiological needs of the developing infant. It is also recognized as a potential route of transmission of some viral pathogens although the presence of a virus in HM rarely leads to a disease in an infant. This intriguing paradox can be explained by considering the intrinsic antiviral properties of HM. In this comprehensive and schematically presented review, we have described what viruses have been detected in HM so far and what their potential transmission risk through breastfeeding is. We have provided a description of all the antiviral compounds of HM, along with an analysis of their demonstrated and hypothesized mechanisms of action. Finally, we have also analyzed the impact of HM pasteurization and storage methods on the detection and transmission of viruses, and on the antiviral compounds of HM. We have highlighted that there is currently a deep knowledge on the potential transmission of viral pathogens through breastfeeding and on the antiviral properties of HM. The current evidence suggests that, in most cases, it is unnecessarily to deprive an infant of this high-quality nourishment and that the continuation of breastfeeding is in the best interest of the infant and the mother.

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.

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.

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.

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.

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.

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.

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.

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.

Investigation of the protein profile of silkworm (Bombyx mori) pupae reared on a well-calibrated artificial diet compared to mulberry leaf diet.

BACKGROUND: Silkworm pupae is the main by-product of the sericulture industry with an interesting nutritional profile, especially in terms of proteins. In consideration of its possible use as a food or food ingredient in Western countries, a comparative proteomic experiment has been performed to investigate the differences of the protein profile of male and female silkworm pupae reared on mulberry leaves or on an artificial diet. METHODS: The nutritional profile of lyophilized silkworm pupae in terms of dry matter and ash was evaluated according to the AOAC procedures, the total nitrogen content was determined by a nitrogen analyzer and the silkworm pupae gross energy value was measured using an adiabatic calorimetric bomb. The comparative proteomic analysis was performed on male and female silkworm pupae reared on mulberry leaves or on the artificial diet. Proteins were separated by two-dimensional electrophoresis and, after a multivariate statistical analysis, the differentially expressed proteins were identified by LC-MS/MS. RESULTS: The comparative proteomic approach highlighted 47 silkworm pupae proteins differentially expressed comparing diet and gender. PCA analysis showed that seven proteins were more effective in discriminating the sex and five were more effective in discriminating the diet type. In spite of the above-mentioned differences in the silkworm pupae protein profile, no strong alteration of the pupa physiological traits have been demonstrated, suggesting a general silkworm pupae flexibility to adapt to a well-balanced artificial diet. Differences in lipid transport and metabolism were found among the experimental groups, that might have a relevant effect on the timing and on hormone secretion. This aspect may also affect silk production, as univoltine strains are the most productive. The proteomic data provided in this work, may offer a contribution in understanding also the influence of gender and farming strategy on the allergen profile of Bombyx mori, when used as food or as a food ingredient. Female silkworm pupae reared on mulberry leaves seemed to contain lower levels of known allergens than those reared in the other experimental conditions; these findings will have to be taken into account when farming B. mori for food production purposes. However, our results need to be supported by further characterization of the allergenic potential of B. mori.

Enhanced arginine biosynthesis and lower proteolytic profile as indicators of Saccharomyces cerevisiae stress in stationary phase during fermentation of high sugar grape must: A proteomic evidence.

A strain of Saccharomyces (S) cerevisiae (ISE19), which displayed an initial good adaptation to a high sugar medium with increased acetate and glycerol production but weak overall growth/fermentation performances, was selected during the alcoholic fermentation of Cortese grape must. To obtain insights into the metabolic changes that occur in the must during growth in particular conditions (high ethanol, high residual sugars and low nitrogen availability) leading to a sluggish fermentation or even fermentation arrest, comparative in-gel proteomic analyses were performed on cells grown in media containing 200g/L and 260g/L of glucose, respectively, while the YAN (Yeast Assimilable Nitrogen) concentration was maintained as it was. Two post-translationally different arginine synthases (pIs 5.6 and 5.8) were found in higher abundances in the high glucose-grown cells, together with an increased abundance of a glycosyltransferase involved in cell-wall mannans synthesis, and of two regulatory proteins (K7_Bmh1p and K7_Bmh2p) that control membrane transport. In parallel, a proteinase K-like proteolytic enzyme and three other protein fragments (Indolepyruvate decarboxylase 1, Fba1p and Eno1p) were present in lower abundances in the high glucose condition, where oxidative stress and cell cycle involved enzymes were also found to be less abundant. The overall results suggest that in stationary phase stress conditions, leading to stuck fermentation, S. cerevisiae ISE19 decreases cell replication, oxidative stress responses and proteolytic activity, while induces other metabolic modifications that are mainly based on cell-wall renewal, regulation of the solute transport across the cell membrane and de novo arginine synthesis.

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.

Validation of a mass spectrometry-based method for milk traces detection in baked food.

A simple validated LC-MS/MS-based method was set up to detect milk contamination in bakery products, taking the effects of food processing into account for the evaluation of allergen recovery and quantification. Incurred cookies were prepared at eight levels of milk contamination and were cooked to expose all milk components, including allergenic proteins, to food processing conditions. Remarkable results were obtained in term of sufficiently low LOD and LOQ (1.3 and 4 mg/kg cookies, respectively). Precision was calculated as intra-day repeatability (RSD in the 5-20% range) and inter-day repeatability (4 days; RSD never exceeded 12%). The extraction recovery values ranged from 20% to 26%. Method applicability was evaluated by analysing commercial cookies labelled either as "milk-free" or "may contain milk". Although the ELISA methodology is considered the gold standard for detecting allergens in foods, this robust LC-MS/MS approach should be a useful confirmatory method for assessing and certifying "milk-free" food products.

Different protein expression profiles in cheese and clinical isolates of Enterococcus faecalis revealed by proteomic analysis.

The use of Enterococcus faecalis in the food industry has come under dispute because of the pathogenic potential of some strains of this species. In this study, we have compared the secretome and whole-cell proteome of one food isolate (E. faecalis DISAV 1022) and one clinical isolate (E. faecalis H1) by 2-DE and iTRAQ analyses, respectively. Extracellular protein patterns differed significantly, with only seven proteins common to both strains. Notably, only the clinical isolate expressed various well-characterized virulence factors such as the gelatinase coccolysin (GelE) and the extracellular serine proteinase V8 (SprE). Moreover, various other putative virulence factors, e.g. superoxide dismutase, choline- and chitin-binding proteins and potential moonlighting proteins, have been detected exclusively in the secretome of the clinical isolate, but not in the food isolate. The iTRAQ analysis of whole-cell proteins of the two strains highlighted a stronger expression of pathogenic traits such as an endocarditis-specific antigen and an adhesion lipoprotein in the pathogenic strain E. faecalis H1. Subsequently, six food isolates (including E. faecalis DISAV 1022) and six clinical isolates (including E. faecalis H1) were tested for the presence of gelatinase and protease activity in the culture supernatants. Both enzymatic activities were found in the clinical as well as the food isolates which clearly indicates that protease expression is strain specific and not representative for pathogenic isolates. Genetic analyses revealed that not only the gelatinase and serine protease genes but also the regulatory fsr genes must be present to allow protease expression.

Engineering new metabolic capabilities in bacteria: lessons from recombinant cellulolytic strategies.

Cellulose waste biomass is the most attractive substrate for 'biorefinery strategies' producing high-value products (e.g. fuels or plastics) by fermentation. However, traditional biomass bioconversions are economically inefficient multistep processes. Thus far, no microorganisms able to perform single-step fermentation into products (consolidated bioprocessing; CBP) have been isolated. Metabolic engineering is currently employed to develop recombinant microorganisms suitable for CBP. The heterologous expression of extracellular proteins (e.g. cellulases or hemicellulases) is the key feature of recombinant cellulolytic strategies, conferring cellulolytic ability to microorganisms exhibiting high product yields and titers. Although more molecular tools are becoming available, efficient heterologous expression of secreted proteins is still a challenge. The present review summarizes both bottlenecks and solutions of organism engineering for biomass biorefinery strategies.

Proteomic characterization of a selenium-metabolizing probiotic Lactobacillus reuteri Lb26 BM for nutraceutical applications.

Selenium (Se), Se-cysteines and selenoproteins have received growing interest in the nutritional field as redox-balance modulating agents. The aim of this study was to establish the Se-concentrating and Se-metabolizing capabilities of the probiotic Lactobacillus reuteri Lb26 BM, for nutraceutical applications. A comparative proteomic approach was employed to study the bacteria grown in a control condition (MRS modified medium) and in a stimulated condition (4.38 mg/L of sodium selenite). The total protein extract was separated into two pI ranges: 4-7 and 6-11; the 25 identified proteins were divided into five functional classes: (i) Se metabolism; (ii) energy metabolism; (iii) stress/adhesion; (iv) cell shape and transport; (v) proteins involved in other functions. All the experimental results indicate that L. reuteri Lb26 BM is able to metabolize Se(IV), incorporating it into selenoproteins, through the action of a selenocysteine lyase, thus enhancing organic Se bioavailability. This involves endo-ergonic reactions balanced by an increase of substrate-level phosphorylation, chiefly through lactic fermentation. Nevertheless, when L. reuteri was grown on Se a certain degree of stress was observed, and this has to be taken into account for future applicative purposes. The proteomic approach has proven to be a powerful tool for the metabolic characterization of potential Se-concentrating probiotics.