Infant milk formula, produced by membrane filtration, promotes mucus production in the upper small intestine of young pigs.

Human breast milk promotes maturation of the infant gastrointestinal barrier, including the promotion of mucus production. In the quest to produce next generation infant milk formula (IMF), we have produced IMF by membrane filtration (MEM-IMF). With a higher quantity of native whey protein, MEM-IMF more closely mimics human breast milk than IMF produced using conventional heat treatment (HT-IMF). After a 4-week dietary intervention in young pigs, animals fed a MEM-IMF diet had a higher number of goblet cells, acidic mucus and mucin-2 in the jejunum compared to pigs fed HT-IMF (P < 0.05). In the duodenum, MEM-IMF fed pigs had increased trypsin activity in the gut lumen, increased mRNA transcript levels of claudin 1 in the mucosal scrapings and increased lactase activity in brush border membrane vesicles than those pigs fed HT-IMF (P < 0.05). In conclusion, MEM-IMF is superior to HT-IMF in the promotion of mucus production in the young gut.

Allergenic Shrimp Tropomyosin Distinguishes from a Non-Allergenic Chicken Homolog by Pronounced Intestinal Barrier Disruption and Downstream Th2 Responses in Epithelial and Dendritic Cell (Co)Culture.

BACKGROUND: Tropomyosins (TM) from vertebrates are generally non-allergenic, while invertebrate homologs are potent pan-allergens. This study aims to compare the risk of sensitization between chicken TM and shrimp TM through affecting the intestinal epithelial barrier integrity and type 2 mucosal immune activation. METHODS: Epithelial activation and/or barrier effects upon exposure to 2-50 µg/mL chicken TM, shrimp TM or ovalbumin (OVA) as a control allergen, were studied using Caco-2, HT-29MTX, or HT-29 intestinal epithelial cells. Monocyte-derived dendritic cells (moDC), cocultured with HT-29 cells or moDC alone, were exposed to 50 µg/mL chicken TM or shrimp TM. Primed moDC were cocultured with naïve Th cells. Intestinal barrier integrity (TEER), gene expression, cytokine secretion and immune cell phenotypes were determined in these human in vitro models. RESULTS: Shrimp TM, but not chicken TM or OVA exposure, profoundly disrupted intestinal barrier integrity and increased alarmin genes expression in Caco-2 cells. Proinflammatory cytokine secretion in HT-29 cells was only enhanced upon shrimp TM or OVA, but not chicken TM, exposure. Shrimp TM enhanced the maturation of moDC and chemokine secretion in the presence or absence of HT-29 cells, while only in the absence of epithelial cells chicken TM activated moDC. Direct exposure of moDC to shrimp TM increased IL13 and TNFα secretion by Th cells cocultured with these primed moDC, while shrimp TM exposure via HT-29 cells cocultured with moDC sequentially increased IL13 expression and IL4 secretion in Th cells. CONCLUSIONS: Shrimp TM, but not chicken TM, disrupted the epithelial barrier while triggering type 2 mucosal immune activation, both of which are key events in allergic sensitization.

A glimpse into plant-based fermented products alternative to animal based products: Formulation, processing, health benefits.

Fermented foods and beverages are increasingly being included in the diets of people around the world, as they significantly contribute to flavor and interest in nutrition and food consumption. Plant sources, like cereals and pulses, are employed to produce vegan fermented foods that are either commercially available or the subject of ongoing scientific investigation. In addition, the inclination towards nutritionally healthy, natural, and clean-label products amongst consumers has encouraged the development of vegan fermented products alternative to animal-based products for industrial-scale production. However, as the vegan diet is more restrictive than the vegetarian diet, manufacturing food products for vegans presents a significant problem due to the limited availability of many raw materials. So further research is required on this topic. This paper aims to review the formulation, quality, microbial resources, health benefits, and safety of foods that can be categorised as vegan fermented foods and beverages.

Membrane filtration processing of infant milk formula alters protein digestion in young pigs.

Reducing heat treatment (HT) during processing of infant milk formula (IMF) is desirable to produce a product that more closely resembles breast milk. By employing membrane filtration (MEM), we produced an IMF (60:40 whey to casein ratio) at pilot scale (250 kg). MEM-IMF had a significantly higher content of native whey (59.9 %) compared to HT-IMF (4.5 %) (p < 0.001). Pigs, at 28 days old, were blocked by sex, weight and litter origin and assigned to one of two treatments (n = 14/treatment): (1) starter diet containing 35 % of HT-IMF powder or (2) starter diet containing 35 % of MEM-IMF powder for 28 days. Body weight and feed intake were recorded weekly. Pigs at day 28 post weaning were sacrificed 180 min after their final feeding, for the collection of gastric, duodenal, jejunum and ileal contents (n = 10/treatment). MEM-IMF diet resulted in more water-soluble proteins and higher levels of protein hydrolysis in the digesta at various gut locations compared to HT-IMF (p < 0.05). In the jejunal digesta, a higher concentration of free amino acids were present post MEM-IMF consumption (247 ± 15 µmol g-1 of protein in digesta) compared to HT-IMF (205 ± 21 µmol g-1 of protein). Overall, average daily weight gain, average dairy feed intake and feed conversion efficiency were similar for pigs fed either MEM-IMF or HT-IMF diets, but differences and trends to difference of these indicators were determined in particular intervention periods. In conclusion, reducing heat treatment during processing of IMF influenced protein digestion and revealed minor effects on growth parameters providing in vivo evidence that babies who are fed with IMF processed by MEM are likely to have different protein digestion kinetics but minimal effect on overall growth trajectories as babies fed IMF processed by traditional thermal processing.

Critical features of an in vitro intestinal absorption model to study the first key aspects underlying food allergen sensitization.

New types of protein sources will enter our diet in a near future, reinforcing the need for a straightforward in vitro (cell-based) screening model to test and predict the safety of these novel proteins, in particular their potential risk for de novo allergic sensitization. The Adverse Outcome Pathway (AOP) for allergen sensitization describes the current knowledge of key events underlying the complex cellular interactions that proceed allergic food sensitization. Currently, there is no consensus on the in vitro model to study the intestinal translocation of proteins as well as the epithelial activation, which comprise the first molecular initiation events (ME1-3) and the first key event of the AOP, respectively. As members of INFOGEST, we have highlighted several critical features that should be considered for any proposed in vitro model to study epithelial protein transport in the context of allergic sensitization. In addition, we defined which intestinal cell types are indispensable in a consensus model of the first steps of the AOP, and which cell types are optional or desired when there is the possibility to create a more complex cell model. A model of these first key aspects of the AOP can be used to study the gut epithelial translocation behavior of known hypo- and hyperallergens, juxtaposed to the transport behavior of novel proteins as a first screen for risk management of dietary proteins. Indeed, this disquisition forms a basis for the development of a future consensus model of the allergic sensitization cascade, comprising also the other key events (KE2-5).

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.

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.

Thermal or membrane processing for Infant Milk Formula: Effects on protein digestion and integrity of the intestinal barrier.

Infant Milk Formula (IMF) is designed as a breastmilk substitute to satisfy the nutritional requirements during the first months of life. This study investigates the effects of two IMF processing technologies on cow milk protein digestion using an infant static in vitro gastrointestinal model. The degree of protein hydrolysis at the end of the gastric phase was 3.7-fold higher for IMF produced by high temperature (IMF-HT), compared to IMF produced by cascade membrane filtration (IMF-CMF), as assessed by free N-terminal group analysis. The processing type also influenced the panel of bioavailable peptides detected in basolateral compartments of Caco-2 monolayers exposed to gastrointestinal digested IMFs. In addition, IMF-CMF significantly increased tight junction protein, claudin 1, whilst IMF-HT significantly reduced tight junction integrity. In conclusion, producing IMF by CMF may preserve intestinal barrier integrity and can deliver its own unique inventory of bioavailable peptides with potential bioactivity.

Modulation of Milk Allergenicity by Baking Milk in Foods: A Proteomic Investigation.

Cow's milk is considered the best wholesome supplement for children since it is highly enriched with micro and macro nutrients. Although the protein fraction is composed of more than 25 proteins, only a few of them are capable of triggering allergic reactions in sensitive consumers. The balance in protein composition plays an important role in the sensitization capacity of cow's milk, and its modification can increase the immunological response in allergic patients. In particular, the heating treatments in the presence of a food matrix have demonstrated a decrease in the milk allergenicity and this has also proved to play a pivotal role in developing tolerance towards milk. In this paper we investigated the effect of thermal treatment like baking of cow's milk proteins that were employed as ingredients in the preparation of muffins. A proteomic workflow was applied to the analysis of the protein bands highlighted along the SDS gel followed by western blot analyses with sera of milk allergic children in order to have deeper information on the impact of the heating on the epitopes and consequent IgE recognition. Our results show that incorporating milk in muffins might promote the formation of complex milk-food components and induce a modulation of the immunoreactivity towards milk allergens compared to milk baked in the oven at 180 °C for ten minutes. The interactions between milk proteins and food components during heating proved to play a role in the potential reduction of allergenicity as assessed by in vitro tests. This would help, in perspective, in designing strategies for improving milk tolerance in young patients affected from severe milk allergies.

Investigation on the allergen profile of the soluble fraction of autoclaved peanuts and its interaction with Caco-2 cells.

Peanuts are a source of proteins and fats but they are also considered a harmful food for individuals who are allergic to them due to their ability to trigger severe and life-threatening reactions. Strict avoidance of peanuts is the most effective means to prevent the development of an allergic reaction. Physical or chemical strategies employing autoclaving can represent a valid alternative to produce a final food with a decreased allergenic power as in the case of peanuts. Thermal processing might induce protein modifications in foods and affect protein digestibility or absorption of nutrients across the intestinal mucosa. Besides, the type of processing could also alter food protein allergenicity thus influencing the interplay with the biological system at the gut level. In this paper, we investigated the influence of autoclaving based treatments on the proliferation of epithelial cells at the intestinal level. Extractable proteins of raw and autoclaved peanuts were analysed by SDS-PAGE and untargeted LC-high resolution-MS/MS to investigate the peptide composition. Our findings show that when raw peanuts were assayed on Caco 2 cell lines, an antiproliferative effect was observed. By contrast, peanuts subjected to hydration and autoclaving did not show an inhibition of proliferation on Caco-2 cells. In parallel, extensive fragmentation induced by autoclaving treatments on the original peanut proteins was also recorded by LC-MS/MS analysis with a consequent increase in the number of peptides detected. These results indicate that the processing applied to peanuts can have an influence on both the nutritional and allergological sides, and more investigations will be required on this issue to understand the alteration of inflammatory mediators induced by the treatment applied.

Heat and Pressure Treatments on Almond Protein Stability and Change in Immunoreactivity after Simulated Human Digestion.

Almond is consumed worldwide and renowned as a valuable healthy food. Despite this, it is also a potent source of allergenic proteins that can trigger several mild to life-threatening immunoreactions. Food processing proved to alter biochemical characteristics of proteins, thus affecting the respective allergenicity. In this paper, we investigated the effect of autoclaving, preceded or not by a hydration step, on the biochemical and immunological properties of almond proteins. Any variation in the stability and immunoreactivity of almond proteins extracted from the treated materials were evaluated by total protein quantification, Enzyme Linked Immunosorbent Assay (ELISA), and protein profiling by electrophoresis-based separation (SDS-PAGE). The sole autoclaving applied was found to weakly affect almond protein stability, despite what was observed when hydration preceded autoclaving, which resulted in a loss of approximately 70% of total protein content compared to untreated samples, and a remarkable reduction of the final immunoreactivity. The final SDS-PAGE protein pattern recorded for hydrated and autoclaved almonds disclosed significant changes. In addition, the same samples were further submitted to human-simulated gastro-intestinal (GI) digestion to evaluate potential changes induced by these processing methods on allergen digestibility. Digestion products were identified by High Pressure Liquid Chromatography-High Resolution Tandem Mass Spectrometry (HPLC-HRMS/MS) analysis followed by software-based data mining, and complementary information was provided by analyzing the proteolytic fragments lower than 6 kDa in size. The autoclave-based treatment was found not to alter the allergen digestibility, whereas an increased susceptibility to proteolytic action of digestive enzymes was observed in almonds subjected to autoclaving of prehydrated almond kernels. Finally, the residual immunoreactivity of the GI-resistant peptides was in-silico investigated by bioinformatic tools. Results obtained confirm that by adopting both approaches, no epitopes associated with known allergens survived, thus demonstrating the potential effectiveness of these treatments to reduce almond allergenicity.

Effect of thermal/pressure processing and simulated human digestion on the immunoreactivity of extractable peanut allergens.

Peanut allergy is one of the most widespread types of food allergies especially affecting developed countries. To reduce the risk of triggering allergic reactions, several technological strategies have been devised to modify or remove allergens from foods. Herein we investigated the combination of high temperature and pressure on the modulation of peanuts immunoreactivity after simulated gastro-duodenal digestion. Extractable proteins of raw and autoclaved peanuts were separated on SDS-PAGE and immunogenicity was assessed by ELISA and Western Blot analyses. Proteins surviving the heat treatment and reacting towards allergic patients' sera were analysed and attributed to Ara h 3 and Ara h 1 proteins by untargeted LC-high resolution-MS/MS. A progressive reduction in the intensity of the major allergen proteins was also highlighted in the protein fraction extracted from autoclaved peanuts, with a total disappearance of the high molecular allergens when samples were preliminary exposed to 2 h hydration although the lower molecular weight fraction was not investigated in the present work. Furthermore, raw and processed peanuts underwent simulated digestion experiments and the IgE binding was assessed by using allergic patients' sera. The persistence of an immunoreactive band was displayed around 20 kDa. In conclusion, the synergistic effects of heat and pressure played a pivotal role in the disappearance of the major peanut allergens also contributing to the significant alteration of the final immunoreactivity. In addition, the surviving of allergenic determinants in peanuts after gastrointestinal breakdown provides more insights on the fate of allergenic proteins after autoclaving treatments.

Isolation, Characterization, and Selection of Molds Associated to Fermented Black Table Olives.

Table olives are one of the most important fermented food in the Mediterranean countries. Apart from lactic acid bacteria and yeasts that mainly conduct the olive fermentation, molds can develop on the brine surface, and can have either deleterious or useful effects on this process. From the food safety point of view, occurring molds could also produce mycotoxins, so, it is important to monitor and control them. In this respect, identification of molds associated to two Italian and two Greek fermented black table olives cultivars, was carried out. Sixty strains were isolated and molecularly identified as Penicillium crustosum (21), P. roqueforti (29), P. paneum (1), P. expansum (6), P. polonicum (2), P. commune (1). A group of 20 selected isolates was subjected to technological (beta-glucosidase, cellulolytic, ligninolytic, pectolytic, and xylanolytic activities; proteolytic enzymes) and safety (biogenic amines and secondary metabolites, including mycotoxins) characterization. Combining both technological (presence of desired and absence of undesired enzymatic activities) and safety aspects (no or low production of biogenic amines and regulated mycotoxins), it was possible to select six strains with biotechnological interest. These are putative candidates for future studies as autochthonous co-starters with yeasts and lactic acid bacteria for black table olive production.

Peanut digestome: Identification of digestion resistant IgE binding peptides.

Stability to proteolytic degradation in the digestive tract is considered a general feature shared by most food allergens. Current digestibility models exclusively utilize purified allergen proteins, neglecting the relevant effects of matrix that occur for foodstuff systems. In the present study, we investigated digestion stability of the major peanut allergens directly in the natural matrix using an in vitro static model that simulates the gastrointestinal digestion including the oral, gastric, duodenal and intestinal (brush border membrane enzymes) phases. Immunogenicity was evaluated by Western Blot using N=8 pooled sera of peanut allergic pediatric subjects. Immunoreactive, large-sized and fragments of Ara h 2, Ara h 6 and Ara h 3 survived hydrolysis as assessed by SDS-PAGE. Smaller resistant peptides mainly arising from Ara h 3 and also Ara h 1 were detected and further identified by LC-high resolution-MS/MS. RP-HPLC purification followed by dot-blot analysis and MS/MS-based identification demonstrated that stable IgE-binding peptides derived from Ara h 3. These results provide a more realistic picture of the potentially allergenic determinants of peanuts that survived the human digestion, taking into account the role of the food matrix, which may significantly affect gastrointestinal breakdown of peanut allergens.

Application of the adverse outcome pathway (AOP) concept to structure the available in vivo and in vitro mechanistic data for allergic sensitization to food proteins.

BACKGROUND: The introduction of whole new foods in a population may lead to sensitization and food allergy. This constitutes a potential public health problem and a challenge to risk assessors and managers as the existing understanding of the pathophysiological processes and the currently available biological tools for prediction of the risk for food allergy development and the severity of the reaction are not sufficient. There is a substantial body of in vivo and in vitro data describing molecular and cellular events potentially involved in food sensitization. However, these events have not been organized in a sequence of related events that is plausible to result in sensitization, and useful to challenge current hypotheses. The aim of this manuscript was to collect and structure the current mechanistic understanding of sensitization induction to food proteins by applying the concept of adverse outcome pathway (AOP). MAIN BODY: The proposed AOP for food sensitization is based on information on molecular and cellular mechanisms and pathways evidenced to be involved in sensitization by food and food proteins and uses the AOPs for chemical skin sensitization and respiratory sensitization induction as templates. Available mechanistic data on protein respiratory sensitization were included to fill out gaps in the understanding of how proteins may affect cells, cell-cell interactions and tissue homeostasis. Analysis revealed several key events (KE) and biomarkers that may have potential use in testing and assessment of proteins for their sensitizing potential. CONCLUSION: The application of the AOP concept to structure mechanistic in vivo and in vitro knowledge has made it possible to identify a number of methods, each addressing a specific KE, that provide information about the food allergenic potential of new proteins. When applied in the context of an integrated strategy these methods may reduce, if not replace, current animal testing approaches. The proposed AOP will be shared at the www.aopwiki.org platform to expand the mechanistic data, improve the confidence in each of the proposed KE and key event relations (KERs), and allow for the identification of new, or refinement of established KE and KERs.

Insight into the gastro-duodenal digestion resistance of soybean proteins and potential implications for residual immunogenicity.

Soy is an important component of the human diet thanks to its nutritional value and high protein content; however, it also represents a risk for allergenic consumers due to its potential to trigger adverse reactions in sensitized individuals. The putative correlation between immunoreactivity and resistance to the human gastrointestinal (GI) digestion has drawn attention to investigating soybean proteins digestibility. In this work, we provided further insights into this field by performing in vitro simulated GI digestion experiments directly on ground soybean seeds, to provide more realistic results obtained from the digestion of the whole food matrix. Soybean digestion products were analyzed by SDS-PAGE followed by untargeted HPLC-MS/MS analysis and the final data were software treated to enable protein/peptide identification. The latter allowed monitoring the proteolytic degradation of the main soybean proteins during the gastric and duodenal phases. In particular, ß-conglycinin and trypsin inhibitors showed the highest resistance to the combined activity of GI enzymes, showing a partial degradation at the end of the duodenal phase as ascertained by the strong electrophoretic bands displayed at 50 kDa and 20 kDa, respectively. Glycinin subunits also presented, even if to a lower extent, resistance to the complete proteolytic degradation, as demonstrated by polypeptide fragments with molecular weight lower than 20 kDa displayed in the gel at the end of duodenal digestion. In addition, by bioinformatics analysis it was demonstrated that the GI resistant fragments of the allergenic proteins, ß-conglycinin and glycinin, retained in their primary structure linear epitopes potentially able to trigger an immunoreaction when exposed to the intestinal mucosa. Moreover, such resistant peptides also presented a structural homology with epitope sequences recognized in other legume species, presenting a potential risk of adverse cross-reaction for a larger category of allergic consumers.

Fungal isolates and metabolites in locally processed rice from five agro-ecological zones of Nigeria.

This study reports the distribution of fungal isolates and fungal metabolites that naturally contaminate locally processed rice from five agro-ecological zones of Nigeria. The fungal species were isolated by the dilution plate technique and identified by appropriate diagnostics, while metabolites were determined by a liquid chromatographic tandem mass spectrometric method. Aspergillus and Penicillium species were the predominant isolates found in the rice samples while Fusarium spp. were not isolated. The mean fungal count differed significantly (p < 0.05) across the zones and ranged from 9.98 × 102 cfu g-1 in the Southern Guinea Savannah to 96.97 × 102 cfu g-1 in the Derived Savannah. For 16 fungal metabolites, selected from 63 positively identified fungal metabolites based on their concentration and spread across the zones, an occurrence map was constructed. The Northern Guinea Savannah recorded the highest contamination of fungal metabolites while the Sudan Savannah zone recorded the least.