The problem the researchers set out to solve was stark. Published half-lives for the same protein, the major birch pollen allergen Bet v 1, ranged from two to sixteen hours across studies that used endolysosomal proteins from the same murine dendritic cell line, JAWSII. Such inconsistency undermines the assay’s core promise: a quantitative readout, the endolysosomal half-life, that links protein stability during antigen processing to immunogenicity. The underlying logic is that proteins with moderate stability are processed optimally for presentation on MHC class II molecules, whereas proteins that are either extremely stable or extremely fragile fail to generate the right peptides at the right time, resulting in lower immunogenicity.

Two culprits emerged as the main sources of variability. The first is pH. Endolysosomal maturation is tied to acidification, with the pH dropping from about 6.8 in early endosomes to 4.8 in late endolysosomal compartments, and protease activity is exquisitely sensitive to this gradient. Previous work showed that Bet v 1’s half-life changed roughly fourteen-fold when the assay pH shifted from 5.2 to 4.5. The Salzburg team found that even the way the citrate buffer stock is prepared matters: a one-molar citrate buffer adjusted to pH 4.8 drifted up to pH 5.1 when diluted to the working concentration of 0.1 molar, whereas a buffer prepared by mixing trisodium citrate and citric acid in a precise three-to-two ratio, without prior pH adjustment, landed exactly at pH 4.8 upon dilution. The polyanionic nature of citrate at high concentrations distorts electrostatic interactions and pH measurements, so the protocol now prescribes an exact preparation route.

The second culprit is the protein buffer in which the test protein is dissolved. Buffer simulation experiments showed that sodium phosphate at pH 6 preserved the required pH 4.8 across all tested concentrations and protein amounts, while buffers at pH 7 or above only maintained the correct pH at low molarities or high protein concentrations. Because a higher pH dampens proteolytic activity, an innocent-looking difference in the sample buffer can silently skew degradation kinetics. The optimized protocol therefore includes recommendations for buffer exchange or dilution, and advises researchers to verify the pH of a simulated degradation mixture whenever conditions fall outside the tested range.

The more fundamental fix, however, targets the enzyme preparation itself. The isolated endolysosomal fraction is a complex protein mixture in which active proteases are just one component, and its composition varies between batches. Using a fixed mass of endolysosomal protein, as earlier protocols did, therefore delivered wildly different catalytic doses. The team quantified cathepsin activity using the fluorogenic substrate Z-FR-AMC, which releases a fluorescent AMC group upon cleavage, and normalized measurements against an AMC standard curve. They then defined an endolysosomal constant of 10 micromolar cathepsin activity for degrading 5 micrograms of test protein, and derived a simple formula for calculating how much endolysosomal protein to add based on each batch’s measured activity.

The results were striking. When endolysosomal activity was normalized to the 10 micromolar constant, seven independent Bet v 1 measurements yielded a consistent half-life of one hour, plus or minus 0.1 hours. In contrast, following the old protocol with a fixed 7.5 micrograms of endolysosomal protein from a batch with 2.7-fold higher cathepsin activity compressed the half-life to 0.6 hours. The same pattern held for the second model allergen, Aes h 1, a recently identified 5.8-kilodalton plant defensin from horse chestnut stabilized by four disulfide bonds: normalization produced a reproducible half-life of 16 hours, whereas the unadjusted protocol skewed it to 12 hours. In practical terms, a 2.1-fold difference in cathepsin activity had been producing a 1.7-fold difference in measured half-life, an error the new constant eliminates.

The team also addressed practical workflow concerns. Endolysosomal proteins had always been isolated fresh, one day before each experiment, a time-consuming requirement. Storage tests at minus 70 degrees Celsius showed that cathepsin activity remained stable for up to 63 days, with degradation kinetics, half-lives, and peptide profiles indistinguishable from freshly prepared material. Half-life calculations were likewise standardized by fitting densitometric gel data to a one-phase association model constrained to start at zero and plateau at complete degradation, replacing the earlier practice of eyeballing a linear segment crossing the fifty percent mark, which could not properly model the underlying kinetics.

On the peptide side, the researchers introduced two innovations. First, for proteins stabilized by disulfide bonds, they added a reduction and alkylation step after degradation, using TCEP and iodoacetamide to prevent artificial disulfide scrambling that renders cysteine-containing peptides invisible to mass spectrometry. For Aes h 1, this was decisive: without treatment, only a single peptide lacking cysteine was identified, whereas reduction and alkylation revealed 28 peptides, many of them cysteine-containing. Second, they built PeptideStudio, an open-source, browser-based visualization tool that maps peptides onto the protein sequence with color-coded intensity values, displays each time point on separate tracks, and allows users to annotate validated T cell epitopes, filter by length and intensity, and export publication-quality figures. The application runs locally, so no sequence or intensity data leaves the user’s machine.

The biological readouts validate the approach. Bet v 1’s degradome contained ten peptide clusters spanning the entire protein, and experimentally validated T cell epitopes fell within the dominant clusters, including the immunodominant epitope Bet v 1 142-156, whose peptides appeared early and persisted for several hours, consistent with the idea that early and sustained peptide availability enhances MHC class II loading. Aes h 1, analyzed for the first time, yielded two prominent clusters, one encompassing the region homologous to the immunodominant T cell epitope of the mugwort allergen Art v 1, with peptides peaking at 24 hours and long fragments covering up to 92 percent of the tiny protein persisting to 72 hours, mirroring its high proteolytic stability.

The authors acknowledge limitations, including the use of a murine cell line rather than human antigen-presenting cells, although prior work showed similar kinetics between the two, and the semi-quantitative nature of mass spectrometric peptide intensities. Still, the payoff is a robust, reproducible assay that can compare proteins across laboratories and batches, distinguishing high- and low-allergenic variants within protein families and flagging potential T cell epitopes before any clinical exposure. For novel foods, protein-based immunotherapeutics, and synthetic proteins where a priori safety knowledge is scarce, a standardized window into how the endolysosomal system will process them could prove an invaluable early warning system.

Subject of Research: Optimization and standardization of the endolysosomal degradation assay for predicting protein immunogenicity and T cell epitopes

Article Title: The optimized endolysosomal degradation assay with standardized enzyme activity represents an orthogonal method for predicting protein immunogenicity and T cell epitopes

Article References: Öztemiz, E., Regl, C., Brandstetter, H., Huber, C. G., Verhoeckx, K., & Gadermaier, G. (2026). The optimized endolysosomal degradation assay with standardized enzyme activity represents an orthogonal method for predicting protein immunogenicity and T cell epitopes. Frontiers in Allergy, 7, Article 1936335. https://doi.org/10.3389/falgy.2026.1936335

Image Credits: AI Generated

DOI: 10.3389/falgy.2026.1936335

Keywords: endolysosomal degradation, protein immunogenicity, T cell epitopes, cathepsin activity, allergens, Bet v 1, Aes h 1, mass spectrometry, antigen processing, PeptideStudio, allergy, novel foods

The research, published in Frontiers in Allergy, comes from the EU-funded ALLPreT project, a Marie Sklodowska-Curie network spanning institutions in Germany, Denmark, Luxembourg, the United Kingdom, and the Netherlands. The team, led by Thomas Holzhauser of the Paul-Ehrlich-Institut, set out to solve a fundamental technical challenge that has plagued allergenicity risk assessment for years: the lack of standardized protein preparations that allow results from different laboratories and different testing methods to be meaningfully compared.

Under EU Regulation 2015/2283, any food not significantly consumed before May 1997 must undergo rigorous safety assessment before market authorization. Common food allergens are proteins, so any novel food containing proteins is considered potentially allergenic. Yet while methods for assessing nutritional, microbiological, and toxicological risks are well established, the allergenicity assessment of novel foods remains a weak link. Current approaches rely on a weight-of-evidence framework originally designed for genetically modified organisms, focusing on sequence homology to known allergens, stability to gastrointestinal digestion, and the capacity of proteins to bind Immunoglobulin E, the antibody central to allergic reactions.

The problem is that these methods may not predict de novo sensitization for unknown proteins. In vivo animal testing and in vitro cellular models have been proposed to fill this gap, but here is where the technical bottleneck emerges: every published study has relied on its own study-specific protein preparations, extracted using different buffers, different pH values, and different processing conditions. The result is a patchwork of data with limited comparability across studies, making it nearly impossible to distinguish between weakly and strongly allergenic proteins with confidence.

The ALLPreT team tackled this by testing four different extraction buffers across thirteen legume species, including common dietary legumes like peanut, soybean, lupine, pea, lentil, chickpea, white bean, and field bean, as well as small-seeded legumes like fenugreek, alfalfa, and red and white clover, which serve as examples of potential novel foods. The buffers ranged from pH 7.1 to 9.6 and included Tris-buffered saline, phosphate-buffered saline, ammonium carbonate, and sodium carbonate. Seeds were frozen in liquid nitrogen, ground to a fine powder using a cryogenic mill, and then extracted under controlled conditions of suspension, incubation, centrifugation, filtration, and lyophilization.

The winner was ammonium carbonate buffer at pH 9.0. This buffer extracted significantly more protein than the lower-pH alternatives, with mean concentrations of 5.8 milligrams per milliliter compared to 3.4 for Tris-buffered saline and 2.8 for phosphate-buffered saline. But the real genius lies in a property that seems almost too elegant: ammonium carbonate is an unstable salt that completely decomposes into gaseous ammonia, carbon dioxide, and water during freeze-drying. This means that after lyophilization, no buffer components remain in the final protein preparation. The extracts can then be reconstituted in any buffer required for downstream testing, making them compatible with virtually any in vitro cellular assay, in vivo animal model, or diagnostic IgE screening method.

Mass spectrometric analysis confirmed that the optimized protocol recovered proteins from all the important allergenic protein families in legumes, including 11S globulins, 7S globulins, 2S albumins, seed albumins, pathogenesis-related 10 proteins, profilins, lipid transfer proteins, oleosins, defensins, cyclophilins, annexins, and late embryogenesis abundant proteins. The estimated extraction efficiency ranged from 33.6 percent for roasted peanut to 96.7 percent for alfalfa, with a median of 62.3 percent across all species. Notably, the team found that roasting dramatically altered allergen composition, with roasted peanuts containing higher levels of 11S globulins but nearly four times less 2S albumins than unroasted peanuts.

Perhaps the most striking finding was the team’s decision to skip a defatting step. While treating seed powder with n-hexane increased protein yield from roasted peanut threefold, from 14.4 to 45.9 milligrams per milliliter, Western blot analysis revealed that the defatted extracts showed weaker immunostaining with anti-peanut antibodies, suggesting that the solvent may negatively affect the immunological properties of the proteins. This is particularly relevant because conventional defatting procedures frequently exclude lipophilic proteins such as oleosins, which have been recognized as clinically relevant allergens associated with severe peanut allergy. By preserving the natural oil body fraction, the optimized protocol ensures a broader allergenic profile.

The functional validation was equally impressive. All extracts bound IgG from polyclonal peanut or soybean antisera and IgE from legume-sensitized human subjects. In a humanized mediator release assay using rat basophilic leukemia cells transfected with the human IgE receptor alpha-chain, all extracts demonstrated functional IgE-crosslinking capacity after passive sensitization with allergen-specific human IgE. The sera also showed mediator release with the small-seeded legume extracts, comparable to the common legumes, demonstrating some degree of IgE cross-binding between dietary legumes and potential novel foods like alfalfa and clover.

This harmonized panel of well-characterized legume allergen preparations now provides the ALLPreT consortium with standardized study materials for use across all in silico, in vitro, and in vivo tools being developed within the project. Once validated across multiple experimental platforms, this protocol may be proposed for future allergenicity studies of novel legume foods, potentially transforming how regulators evaluate the safety of the next generation of plant-based products before they reach consumers.

Subject of Research: ALLPreT standardized legume allergen extracts for novel food allergenicity testing

Article Title: The ALLPreT panel of legume allergen preparations: characterization of optimized protein extracts for allergen composition, IgE-binding and IgE-crosslinking properties

Article References: Olivares-Martínez, A. L., Yue, S. R., Randow, S., Vestergaard Lukassen, M., Assugeni, I., Oliver, M., Kuehn, A., Verhoeckx, K., Bøgh, K. L., & Holzhauser, T. (2026). The ALLPreT panel of legume allergen preparations: characterization of optimized protein extracts for allergen composition, IgE-binding and IgE-crosslinking properties. Frontiers in Allergy, 7, Article 1938923. https://doi.org/10.3389/falgy.2026.1938923

Image Credits: AI Generated

DOI: 10.3389/falgy.2026.1938923

Keywords: food allergy, legumes, allergenicity, novel foods, IgE, ALLPreT, protein extraction, mass spectrometry, mediator release assay, EU regulation, panel, legume

The review begins with a sober assessment of sustainability claims. Insects such as Tenebrio molitor, Acheta domesticus, Locusta migratoria, Alphitobius diaperinus, Bombyx mori and Hermetia illucens offer high feed conversion efficiency, short growth cycles and lower land requirements than conventional livestock, and many species contain all essential amino acids in nutritionally significant amounts. Yet the authors stress that environmental advantages only materialise when the entire production chain is optimised, including rearing substrate, energy use for temperature control, killing method and drying. Insect proteins should therefore be treated as conditional alternatives whose benefits depend on well-controlled systems rather than assumed by default.

Even measuring how much protein an insect contains turns out to be surprisingly tricky. The conventional nitrogen-to-protein conversion factor of 6.25 can overestimate protein content because insect biomass contains nitrogen from chitin and other non-protein compounds. Janssen and colleagues proposed a lower factor of approximately 4.76 for mealworm, lesser mealworm and black soldier fly larvae. Reported values also depend on whether composition is expressed on a fresh-weight or dry-matter basis, and on the developmental stage, rearing diet and processing history of the material. Digestibility adds another layer: in vitro studies show mealworm and cricket proteins can achieve digestibility figures of 79 to 99 percent and favourable DIAAS scores, but these outcomes shift with heat treatment, defatting and chitin reduction, making digestibility a processing-dependent property rather than a fixed species trait.

Processing is where the review’s central framework comes into play. Pretreatments such as killing, blanching, drying, grinding and defatting establish the initial state of the biomass, and each involves trade-offs. Blanching improves microbial and enzyme control but can unfold proteins and promote aggregation that reduces extraction efficiency. Freeze-drying limits thermal damage but is costly, while microwave and hot-air drying scale better yet risk Maillard reactions, lipid oxidation and protein denaturation. Defatting enriches protein and limits oxidation, since residual lipids rich in polyunsaturated fatty acids generate rancid aldehydes such as hexanal and nonanal, but solvent contact and duration can alter protein conformation and downstream recovery. Quantitatively, defatting raised Tenebrio molitor flour from 57.8 to 64.6 percent protein while cutting fat to 2.8 percent, and lifted Hermetia illucens flour from 34.7 to 44.9 percent protein.

Protein recovery routes then diverge sharply. Dry fractionation by milling and sieving is simple and low in water and chemical use, but yields fractions still loaded with lipids, chitin, ash and pigments, making them better suited to bakery and snack applications than to refined functional roles. Alkaline solubilisation followed by isoelectric precipitation boosts purity and yield, but extreme pH can trigger denaturation, browning and unwanted structural changes. Assisted technologies offer further options: ultrasound cavitation disrupts tissues and aggregates, improving extraction yields by 35 to 94 percent in sonicated silkworm pupae, while high-pressure processing increased protein solubility in Protaetia brevitarsis seulensis up to an optimum of 200 megapascals before the benefit declined. No single technology wins outright; the authors insist processing should be designed backwards from the target food.

Post-recovery modification tailors proteins to specific functions, and here the quantitative evidence is striking. Enzymatic hydrolysis of migratory locust protein raised solubility from as low as 10 percent to 55 percent and foamability to 326 percent at pH 3. Cricket hydrolysates reached emulsifying activity indices of up to 32 square metres per gram, compared with 6.23 for defatted cricket flour. But hydrolysis illustrates what the authors call the protein-functional modification dilemma: excessive cleavage weakens interfacial films, releases bitter hydrophobic peptides and, crucially, does not reliably eliminate allergenic epitopes unless proteolysis is extensive, on the order of 60 to 85 percent degree of hydrolysis needed to disrupt major allergens such as tropomyosin. Fermentation offers another route, with fungal fermentation of cricket powder increasing solubility by roughly 20 percent and water-holding capacity from 2.17 to about 2.62 grams per gram, though it demands careful microbiological control.

Application studies show that ingredient form must match product structure. Bread is highly sensitive because insect flour dilutes the gluten network that traps fermentation gases, producing compact crumbs and darker colour, so insects work best as supplementary ingredients. Biscuits, crackers, muffins and protein bars tolerate higher inclusion levels because their quality depends less on gas retention. Extruded snacks sit in between: cricket powder at 12.5 to 15 percent could qualify products as protein sources, but 7.5 percent was recommended to preserve expansion and texture. In meat analogues, insect proteins perform best as complementary fractions within hybrid systems. High-moisture extrusion of insect and soy protein concentrates showed that around 15 to 40 percent insect protein could match the cutting properties of a soy control, while substituting 10 percent of soy protein isolate with cricket powder actually enhanced fibrous anisotropy, although higher levels produced brittle, incoherent extrudates. Three-dimensional printing studies found that pellet fractions of cricket and mealworm at 15 percent gave the best shape fidelity and post-print texture, underlining that fractionation, not the insect species alone, is the controlling design variable.

Liquid foods impose the strictest demands. Beverages require high solubility at acidic pH to avoid sedimentation and cloudiness, making whole insect flours largely unsuitable and pushing formulators toward soluble concentrates and hydrolysates. Consumer testing of mealworm protein shakes identified a rejection threshold of 14.6 percent mealworm content only among blind-tasting participants; health- and sustainability-framed groups showed no comparable threshold, suggesting communication can shift tolerance, though it cannot rescue poor sensory quality. Bioactive peptides add another dimension: cricket hydrolysates produced by microwave-assisted enzymatic hydrolysis showed ACE-inhibitory IC50 values of 0.096 milligrams per millilitre and DPP-IV-inhibitory values of 0.27, better than conventional hydrolysis. The authors caution, however, that these results come from chemical and in vitro enzyme assays and cannot be equated with proven antihypertensive or glycaemic effects in humans.

Safety and regulation complete the picture. Insect biomass can carry Enterobacteriaceae, Salmonella, Listeria, Staphylococcus aureus and spore-forming bacteria, so hurdle-based strategies combining blanching, drying, water-activity control and hygienic handling are essential. Chemical hazards such as mycotoxins and heavy metals depend on rearing substrate, meaning circular production using food-industry side streams requires rigorous contaminant monitoring. Allergenicity remains a critical concern because insects share conserved pan-allergens, including tropomyosin and arginine kinase, with crustaceans, molluscs and house dust mites; one observational study found 73 percent of crustacean- and mite-sensitised patients showed IgE reactivity to recombinant black soldier fly tropomyosin. In the European Union, novel food authorisations are species- and process-specific, as illustrated by the approved partially defatted house cricket powder, which carries mandatory allergen labelling. The review’s conclusion is clear: no single insect ingredient will suit every product. Commercial success will depend on standardised, application-specific ingredients whose processing history, functional performance, safety profile and sensory character are validated together, from raw material to finished food.

Subject of Research: Processing-structure-function relationships in edible insect proteins for food applications

Article Title: Designing edible insect proteins for food applications: Processing-Structure-Function relationships, product performance and safety trade-offs

Article References: Nguyen, C. H., & Truong, D. H. (2026). Designing edible insect proteins for food applications: Processing-Structure-Function relationships, product performance and safety trade-offs. Journal of Agriculture and Food Research, 31, Article 103340. https://doi.org/10.1016/j.jafr.2026.103340

Image Credits: AI Generated

DOI: 10.1016/j.jafr.2026.103340

Keywords: edible insects, insect protein, food processing, protein functionality, allergenicity, food safety, meat analogues, protein hydrolysates, novel foods, sustainable protein, techno-functional properties, consumer acceptance