Milk and Its Derivatives as Sources of Components and Microorganisms with Health-Promoting Properties: Probiotics and Bioactive Peptides.

Milk is a source of many valuable nutrients, including minerals, vitamins and proteins, with an important role in adult health. Milk and dairy products naturally containing or with added probiotics have healthy functional food properties. Indeed, probiotic microorganisms, which beneficially affect the host by improving the intestinal microbial balance, are recognized to affect the immune response and other important biological functions. In addition to macronutrients and micronutrients, biologically active peptides (BPAs) have been identified within the amino acid sequences of native milk proteins; hydrolytic reactions, such as those catalyzed by digestive enzymes, result in their release. BPAs directly influence numerous biological pathways evoking behavioral, gastrointestinal, hormonal, immunological, neurological, and nutritional responses. The addition of BPAs to food products or application in drug development could improve consumer health and provide therapeutic strategies for the treatment or prevention of diseases. Herein, we review the scientific literature on probiotics, BPAs in milk and dairy products, with special attention to milk from minor species (buffalo, sheep, camel, yak, donkey, etc.); safety assessment will be also taken into consideration. Finally, recent advances in foodomics to unveil the probiotic role in human health and discover novel active peptide sequences will also be provided.

In-house validation of an LC-MS method for the multiplexed quantitative determination of total allergenic food in chocolate.

Mass spectrometry has been widely accepted as a confirmatory tool for the sensitive detection of undeclared presence of allergenic ingredients. Multiple methods have been developed so far, achieving different levels of sensitivity and robustness, still lacking harmonization of the analytical validation and impairing comparability of results. In this investigation, a quantitative method has been validated in-house for the determination of six allergenic ingredients (cow's milk, hen's egg, peanut, soybean, hazelnut, and almond) in a chocolate-based matrix. The latter has been produced in a food pilot plant to provide a real and well-characterized matrix for proper assessment of method performance characteristics according to official guidelines. In particular, recent considerations issued by the European Committee for Standardization have been followed to guide a rigorous single-laboratory validation and to feature the main method performance, such as selectivity, linearity, and sensitivity. Synthetic surrogates of the peptide markers have been used both in native and labelled forms in matrix-matched calibration curves as external calibrants and internal standards, respectively. A two-order of magnitude range was investigated, focusing on the low concentration range for proper assessment of the detection and quantification limits (LOD and LOQ) by rigorous calibration approach. Conversion factors for all six allergenic ingredients have been determined for the first time to report the final quantitative information as fraction of total allergenic food protein (TAFP) per mass of food (µgTAFP/gfood), since such a reporting unit is exploitable in allergenic risk assessment plans. The method achieved good sensitivity with LOD values ranging between 0.08 and 0.2 µgTAFP/gfood, for all ingredients besides egg and soybean, whose quantitative markers reported a slightly higher limit (1.1 and 1.2 µgTAFP/gfood, respectively). Different samples of chocolate bar incurred at four defined concentration levels close to the currently available threshold doses have been analyzed to test the quantitative performance of the analytical method, with a proper estimate of the measurement uncertainty from different sources of variability. The sensitivity achieved resulted in compliance with the various threshold doses issued or recommended worldwide.

Standardization of a Mass Spectrometry-Based Workflow for Food Allergen Quantification.

In this chapter, the analytical workflow typically used for the development and validation of an analytical method tailored to food allergen detection and quantification is presented. The main steps defining the workflow are herein described and commented with specific notes about the critical issues that can be faced and common solutions to be adopted. References to guidelines and/or recommendation available from official bodies, as well as main papers from international consortia operating on the specific research field, are also reported, whenever possible. As such, this chapter may represent a practical guide to drive method development in the standardization of analytical methods for food allergen detection.

Reactivity to allergenic food contaminants: A study on products on the market.

BACKGROUND: The frequency and severity of reactions in food-allergic consumers exposed to unintentional food allergen contamination during production is unknown. To warn allergic consumers, it has been suggested for pre-packaged foods to be precautionary labelled when the food allergen contamination may exceed the amount to which 1%-5% of the population could react (ED01-ED05). ED01 for hazelnut and milk have been estimated at 0.1 and 0.2 mg, respectively, by the Voluntary Incidental Trace Allergen Labelling (VITAL) initiative. The respective reference doses recommended by the FAO/WHO Codex consultation are 3 and 2 mg. We evaluated the reactivity to potential traces of milk and hazelnut allergens in allergen-free pre-packaged products by children affected by severe allergies to milk and hazelnuts. METHODS: Oral Food Challenges with commercially available hazelnut-free wafer biscuits and milk-free chocolate pralines were administered to patients with severe food allergies to hazelnut and cow's milk, respectively. Contamination levels of milk or hazelnut allergens were measured using chromatographic separation interfaced with triple quadrupole mass spectrometry. RESULTS: No hazelnut allergic patient showed allergic reactions to exposure to biscuits, nor any milk allergic patient displayed allergic reactions to the dark chocolate praline. While no hazelnut trace was detected in biscuits, the praline was found to be contaminated by milk at concentrations ranging between 8 and 35 mg total protein/kg food. In our dose model, these amounts exceeded 1.5-10 times the VITAL ED01 and reached the threshold suggested by the FAO/WHO Codex consultation. CONCLUSIONS: Upon the consumption of food products available on the market, many patients with severe food allergies tolerate significantly higher doses of allergen than reference doses indicated in the VITAL system used for precautionary allergen labelling. These doses support the safety of the FAO/WHO recommended reference doses.

Mass spectrometry-based approaches to assess the botanical authenticity of dietary supplements.

Dietary supplements are legally considered foods despite frequently including medicinal plants as ingredients. Currently, the consumption of herbal dietary supplements, also known as plant food supplements (PFS), is increasing worldwide and some raw botanicals, highly demanded due to their popularity, extensive use, and/or well-established pharmacological effects, have been attaining high prices in the international markets. Therefore, botanical adulteration for profit increase can occur along the whole PFS industry chain, from raw botanicals to plant extracts, until final PFS. Besides the substitution of high-value species, unintentional mislabeling can happen in morphologically similar species. Both cases represent a health risk for consumers, prompting the development of numerous works to access botanical adulterations in PFS. Among different approaches proposed for this purpose, mass spectrometry (MS)-based techniques have often been reported as the most promising, particularly when hyphenated with chromatographic techniques. Thus, this review aims at describing an overview of the developments in this field, focusing on the applications of MS-based techniques to targeted and untargeted analysis to detect botanical adulterations in plant materials, extracts, and PFS.

Alternative Protein Sources and Novel Foods: Benefits, Food Applications and Safety Issues.

The increasing size of the human population and the shortage of highly valuable proteinaceous ingredients has prompted the international community to scout for new, sustainable, and natural protein resources from invertebrates (e.g., insects) and underutilized legume crops, unexploited terrestrial and aquatic weeds, and fungi. Insect proteins are known for their nutritional value, being rich in proteins with a good balance of essential amino acids and being a valuable source of essential fatty acids and trace elements. Unconventional legume crops were found rich in nutritional, phytochemical, and therapeutic properties, showing excellent abilities to survive extreme environmental conditions. This review evaluates the recent state of underutilized legume crops, aquatic weeds, fungi, and insects intended as alternative protein sources, from ingredient production to their incorporation in food products, including their food formulations and the functional characteristics of alternative plant-based proteins and edible insect proteins as novel foods. Emphasis is also placed on safety issues due to the presence of anti-nutritional factors and allergenic proteins in insects and/or underutilized legumes. The functional and biological activities of protein hydrolysates from different protein sources are reviewed, along with bioactive peptides displaying antihypertensive, antioxidant, antidiabetic, and/or antimicrobial activity. Due to the healthy properties of these foods for the high abundance of bioactive peptides and phytochemicals, more consumers are expected to turn to vegetarianism or veganism in the future, and the increasing demand for such products will be a challenge for the future.

Multi-Target Detection of Nuts and Peanuts as Hidden Allergens in Bakery Products through Bottom-Up Proteomics and High-Resolution Mass Spectrometry.

Due to the growing global incidence of allergy to nuts and peanuts, the need for better protection of consumers sensitive to those products is constantly increasing. The best strategy to defend them against adverse immunological reactions still remains the total removal of those products from their diet. However, nuts and peanuts traces can also be hidden in other food products, especially processed ones, such as bakery products, because of cross-contamination occurring during production. Precautionary labelling is often adopted by producers to warn allergic consumers, usually without any evaluation of the actual risk, which would require a careful quantification of nuts/peanuts traces. In this paper, the development of a multi-target method based on liquid chromatography-tandem high resolution mass spectrometry (LC-MS, MS/MS), able to detect traces of five nuts species (almonds, hazelnuts, walnuts, cashews and pistachios) and of peanuts in an in-house incurred bakery product (cookie) through a single analysis is described. Specifically, allergenic proteins of the six ingredients were used as the analytical targets, and the LC-MS responses of selected peptides resulting from their tryptic digestion, after extraction from the bakery product matrix, were exploited for quantification, following a bottom-up approach typical of proteomics. As a result, nuts/peanuts could be detected/quantified down to mg·kg-1 levels in the model cookie, thus opening interesting perspectives for the quantification of hidden nuts/peanuts in bakery products and, consequently, for a more rational use of precautionary labelling.

Microwave-Assisted Extraction of Bioactive Compounds from Lentil Wastes: Antioxidant Activity Evaluation and Metabolomic Characterization.

The recovery of industrial by-products is part of the zero-waste circular economy. Lentil seed coats are generally considered to be a waste by-product. However, this low-value by-product is rich in bioactive compounds and may be considered an eco-friendly source of health-promoting phytochemicals. For the first time, a sustainable microwave-assisted extraction technique was applied, and a solvent screening was carried out to enhance the bioactive compound content and the antioxidant activity of green and red lentil hull extracts. With respect to green lentil hull extracts that were obtained with different solvents, the aqueous extract of the red lentil seed coats showed the highest total phenolic and total flavonoid content (TPC = 28.3 ± 0.1 mg GAE/g dry weight, TFC = 1.89 ± 0.01 mg CE/100 mg dry weight, respectively), as well as the highest antioxidant activity, both in terms of the free radical scavenging activity (ABTS, 39.06 ± 0.73 mg TE/g dry weight; DPPH, IC50 = 0.39 µg/mL) and the protection of the neuroblastoma cell line (SH-SY5Y, IC50 = 10.1 ± 0.6 µg/mL), the latter of which has never been investigated so far. Furthermore, a metabolite discovery analysis was for the first time performed on the aqueous extracts of both cultivars using an HPLC separation which was coupled with an Orbitrap-based high-Resolution Mass Spectrometry technique.

Rational Discovery of Antiviral Whey Protein-Derived Small Peptides Targeting the SARS-CoV-2 Main Protease.

In the present work, and for the first time, three whey protein-derived peptides (IAEK, IPAVF, MHI), endowed with ACE inhibitory activity, were examined for their antiviral activity against the SARS-CoV-2 3C-like protease (3CLpro) and Human Rhinovirus 3C protease (3Cpro) by employing molecular docking. Computational studies showed reliable binding poses within 3CLpro for the three investigated small peptides, considering docking scores as well as the binding free energy values. Validation by in vitro experiments confirmed these results. In particular, IPAVF exhibited the highest inhibitory activity by returning an IC50 equal to 1.21 µM; it was followed by IAEK, which registered an IC50 of 154.40 µM, whereas MHI was less active with an IC50 equal to 2700.62 µM. On the other hand, none of the assayed peptides registered inhibitory activity against 3Cpro. Based on these results, the herein presented small peptides are introduced as promising molecules to be exploited in the development of "target-specific antiviral" agents against SARS-CoV-2.

Tree Nuts and Peanuts as a Source of Beneficial Compounds and a Threat for Allergic Consumers: Overview on Methods for Their Detection in Complex Food Products.

Consumption of tree nuts and peanuts has considerably increased over the last decades due to their nutritional composition and the content of beneficial compounds. On the other hand, such widespread consumption worldwide has also generated a growing incidence of allergy in the sensitive population. Allergy to nuts and peanuts represents a global relevant problem, especially due to the risk of the ingestion of hidden allergens as a result of cross-contamination between production lines at industrial level occurring during food manufacturing. The present review provides insights on peanuts, almonds, and four nut allergens-namely hazelnuts, walnuts, cashew, and pistachios-that are likely to cross-contaminate different food commodities. The paper aims at covering both the biochemical aspect linked to the identified allergenic proteins for each allergen category and the different methodological approaches developed for allergens detection and identification. Attention has been also paid to mass spectrometry methods and to current efforts of the scientific community to identify a harmonized approach for allergens quantification through the detection of allergen markers.

Development of incurred chocolate bars and broth powder with six fully characterised food allergens as test materials for food allergen analysis.

The design and production of incurred test materials are critical for the development and validation of methods for food allergen analysis. This is because production and processing conditions, together with the food matrix, can modify allergens affecting their structure, extractability and detectability. For the ThRAll project, which aims to develop a mass spectrometry-based reference method for the simultaneous accurate quantification of six allergenic ingredients in two hard to analyse matrices. Two highly processed matrices, chocolate bars and broth powder, were selected to incur with six allergenic ingredients (egg, milk, peanut, soy, hazelnut and almond) at 2, 4, 10 and 40 mg total allergenic protein/kg food matrix using a pilot-scale food manufacturing plant. The allergenic activity of the ingredients incurred was verified using food-allergic patient serum/plasma IgE, the homogeneity of the incurred matrices verified and their stability at 4 °C assessed over at least 30-month storage using appropriate enzyme-linked immunosorbent assays (ELISA). Allergens were found at all levels from the chocolate bar and were homogenously distributed, apart from peanut and soy which could only be determined above 4 mg total allergenic ingredient protein/kg. The homogeneity assessment was restricted to analysis of soy, milk and peanut for the broth powder but nevertheless demonstrated that the allergens were homogeneously distributed. All the allergens tested were found to be stable in the incurred matrices for at least 30 months demonstrating they are suitable for method development.

In Vivo and In Vitro Assessment and Proteomic Analysis of the Effectiveness of Physical Treatments in Reducing Allergenicity of Hazelnut Proteins.

Hazelnut is a widespread nut species, especially present in Europe, that can be consumed raw or roasted thanks to its pleasant taste and nutritional properties. In addition to renowned beneficial properties hazelnuts contain several proteins capable of inducing food allergy in sensitized individuals, including Cor a 2 (a profilin), Cor a 8 (a lipid transfer protein), Cor a 9 (an 11S seed storage globulin, legumin-like), and Cor a 11 (a 7S seed storage globulin, vicilin-like). In the present paper we investigated the effectiveness of autoclave-based treatments in decreasing the allergic potential of hazelnut as assessed by submitting the treated material to an in vivo skin prick test and an in vitro immunoblot analysis, with sera of allergic individuals exposed to the treated food material. This preliminary analysis showed that autoclave treatment preceded by hydration and/or followed by drying seems to be a promising approach and appears to be effective in reducing the allergenicity of hazelnuts in most patients, probably due to the denaturation of most major and minor allergenic proteins. This work opens up the opportunity to produce hypoallergenic hazelnut derivatives that can be tolerated by allergic subjects.

Are Physicochemical Properties Shaping the Allergenic Potency of Plant Allergens?

This review searched for published evidence that could explain how different physicochemical properties impact on the allergenicity of food proteins and if their effects would follow specific patterns among distinct protein families. Owing to the amount and complexity of the collected information, this literature overview was divided in two articles, the current one dedicated to protein families of plant allergens and a second one focused on animal allergens. Our extensive analysis of the available literature revealed that physicochemical characteristics had consistent effects on protein allergenicity for allergens belonging to the same protein family. For example, protein aggregation contributes to increased allergenicity of 2S albumins, while for legumins and cereal prolamins, the same phenomenon leads to a reduction. Molecular stability, related to structural resistance to heat and proteolysis, was identified as the most common feature promoting plant protein allergenicity, although it fails to explain the potency of some unstable allergens (e.g. pollen-related food allergens). Furthermore, data on physicochemical characteristics translating into clinical effects are limited, mainly because most studies are focused on in vitro IgE binding. Clinical data assessing how these parameters affect the development and clinical manifestation of allergies is minimal, with only few reports evaluating the sensitising capacity of modified proteins (addressing different physicochemical properties) in murine allergy models. In vivo testing of modified pure proteins by SPT or DBPCFC is scarce. At this stage, a systematic approach to link the physicochemical properties with clinical plant allergenicity in real-life scenarios is still missing.

Are Physicochemical Properties Shaping the Allergenic Potency of Animal Allergens?

Key determinants for the development of an allergic response to an otherwise 'harmless' food protein involve different factors like the predisposition of the individual, the timing, the dose, the route of exposure, the intrinsic properties of the allergen, the food matrix (e.g. lipids) and the allergen modification by food processing. Various physicochemical parameters can have an impact on the allergenicity of animal proteins. Following our previous review on how physicochemical parameters shape plant protein allergenicity, the same analysis was proceeded here for animal allergens. We found that each parameter can have variable effects, ranging on an axis from allergenicity enhancement to resolution, depending on its nature and the allergen. While glycosylation and phosphorylation are common, both are not universal traits of animal allergens. High molecular structures can favour allergenicity, but structural loss and uncovering hidden epitopes can also have a similar impact. We discovered that there are important knowledge gaps in regard to physicochemical parameters shaping protein allergenicity both from animal and plant origin, mainly because the comparability of the data is poor. Future biomolecular studies of exhaustive, standardised design together with strong validation part in the clinical context, together with data integration model systems will be needed to unravel causal relationships between physicochemical properties and the basis of protein allergenicity.

Threshold of Reactivity and Tolerance to Precautionary Allergen-Labelled Biscuits of Baked Milk- and Egg-Allergic Children.

Extremely sensitive food-allergic patients may react to very small amounts of allergenic foods. Precautionary allergen labelling (PAL) warns from possible allergenic contaminations. We evaluated by oral food challenge the reactivity to a brand of PAL-labelled milk- and egg-free biscuits of children with severe milk and egg allergy. We explored the ability of proteomic methods to identify minute amounts of milk/egg allergens in such biscuits. Traces of milk and/or egg allergens in biscuits were measured by two different liquid-chromatography-mass spectrometry methods. The binding of patient's serum with egg/milk proteins was assessed using immunoblotting. None of the patients reacted to biscuits. Egg and milk proteins were undetectable with a limit of detection of 0.6 µg/g for milk and egg (method A), and of 0.1 and 0.3 µg /g for milk and egg, respectively (method B). The immunoblots did not show milk/egg proteins in the studied biscuits. Milk/egg content of the biscuits is far lower than 4 µg of milk or egg protein per gram of product, the minimal doses considered theoretically capable of causing reactions. With high sensitivity, proteomic assessments predict the harmlessness of very small amount of allergens in foods, and can be used to help avoiding unnecessary PAL.

Food labeling issues for severe food allergic patients.

Food allergy carries high importance and responsibility, affecting an estimated 220 million people worldwide. It is a frequent cause of food-induced anaphylaxis, a life-threatening condition requiring a toll of about one death per 50 million people a year worldwide. In order to help patients to identify allergenic foods and thus avoid anaphylactic reactions, 66 countries over the 5 continents require by law that allergenic ingredients must be declared when used in prepackaged foods. Unfortunately, the mandatory allergen list is not uniform, but varies among different countries. The widespread adoption of Precautionary Allergen Labeling (PAL) results in a proliferation of unregulated PALs with different informative statements. In this situation, the need of a scientific consensus on the definition of food allergy and the identification of a tolerable risk with routinely used detection assays, considering not only the eliciting dose but also the food source, is urgent. The aim of this manuscript is: 1) to draw a picture of the global situation in terms of PALs, and 2) to highlight new approaches that could aid in tackling the problem of regulating the labeling of allergens. These include the Voluntary Incidental Trace Allergen Labelling (VITAL) system, which intersects reference doses and labelling decisions, and a direct quantification of trace amounts of allergens at lower limit of detection (LOD) levels in the food itself through proteomics. We here highlight how, although with some limitations, the steady advances in proteomic approaches possess higher sensitivity than the recommended VITAL reference doses, allowing the identification of allergens at much lower LOD levels than VITAL. Considering that each assay used to detect allergen in food products carries method-specific issues, a more comprehensive and harmonized approach implementing both quantitative and qualitative methods could help overcoming the risk stratification approach and the overuse of PALs, offering promise as the field moves forward towards improving consumers' quality of life.

Optimization of an Untargeted DART-HRMS Method Envisaging Identification of Potential Markers for Saffron Authenticity Assessment.

Saffron is one of the most expensive agricultural products in the world and as such, the most commonly adulterated spice, with undeclared plant-based surrogates or synthetic components simulating color and morphology. Currently, saffron quality is certificated in the international trade market according to specific ISO guidelines, which test aroma, flavor, and color strength. However, it has been demonstrated that specific adulterants such as safflower, marigold, or turmeric up to 20% (w/w) cannot be detected under the prescribed approach; therefore, there is still a need for advanced and sensitive screening methods to cope with this open issue. The current investigation aims to develop a rapid and sensitive untargeted method based on an ambient mass spectrometry ionization source (DART) and an Orbitrap™high-resolution mass analyzer to discriminate pure and adulterated saffron samples with either safflower or turmeric. The metabolic profiles of pure and adulterated model samples prepared at different inclusion levels were acquired. Unsupervised multivariate analysis was carried out based on hierarchical cluster analysis and principal component analysis as first confirmation of the discriminating potential of the metabolic profile acquired under optimized DART-HRMS conditions. In addition, a preliminary selection of potential markers for saffron authenticity was accomplished, identifying compounds able to discriminate the type of adulteration down to a concentration level of 5%.

Milk Ingredients in Meat Products: Can Autoclaving and In Vitro Gastroduodenal Digestion Mitigate Their IgE-Binding Capacity?

The food industry commonly uses milk ingredients as technological aids in an uncounted number of products. On the other hand, milk contains allergenic proteins causing adverse allergic reactions in sensitized/allergic individuals. This work intends to evaluate the effect of autoclaving and in vitro digestion on the allergenicity of milk proteins incurred in meat products. Protein profiles of raw and autoclaved sausages without and with the addition of 10% of milk protein concentrates were analyzed by gel electrophoresis and liquid chromatography-mass spectrometry. Additionally, residual IgE-reactivity was evaluated by immunoblot analysis using pooled sera of cow's-milk-allergic individuals followed by bioinformatic analysis. Results showed that autoclaving led to an increase in protein fragmentation (higher number of short peptides) and consequently to a higher digestion rate, that was found to be more pronounced in ß-casein. The IgE-binding capacity of milk proteins seems to be reduced after autoclaving prior to digestion, with a residual reactivity in caseins, but was eliminated following digestion. This study highlights the importance of autoclaving as a processing strategy to produce hypoallergenic formulas.

Epitope mapping of the major allergen 2S albumin from pine nut.

The epitopes of the major allergen of pine nut, Pin p 1, were analyzed using a peptide library and sera from patients with clinical allergy to pine nut in order to deepen into the allergenic characteristics of Pin p 1. Analyses of epitope similarities and epitopes location in a 3D-model were also performed. Results showed that three main regions of Pin p 1 containing 5 epitopes were recognized by patient sera IgE. The epitopes of Pin p 1 had important similarities with epitopes of allergenic 2S albumins from peanut (Ara h 2 and 6) and Brazil nut (Ber e 1). The epitopes of Pin p 1 were found in α-helices and coils in the 3D protein structure. Interestingly, all epitopes were found to be well-exposed in the protein surface, which suggests facile access for IgE-binding to the structure of Pin p 1 which is known to be highly resistant.