Streamlining assays of glycosyltransferases activity using in vitro GT-array (i-GT-ray) platform: Application to family GT37 fucosyltransferases.

Numerous putative glycosyltransferases (GTs) have been identified using bioinformatic approaches. However, demonstrating the activity of these GTs remains a challenge. Here, we describe the development of a rapid in vitro GT-array screening platform for activity of GTs. GT-arrays are generated by cell-free in vitro protein synthesis and binding using microplates precoated with a N-terminal Halo- or a C-terminal GST-tagged GT-encoding plasmid DNA and a capture antibody. These arrays are then used for screening of transferase activities and the reactions are monitored by a luminescence GLO assay. The products formed by these reactions can be analyzed directly from the microplates by mass spectrometry. Using this platform, a total of 280 assays were performed to screen 22 putative fucosyltransferases (FUTs) from family GT37 (seven from Arabidopsis and 15 from rice) for activity toward five acceptors: non-fucosylated tamarind xyloglucan (TXyG), arabinotriose (Ara3), non-fucosylated rhamnogalacturonan I (RG-I), and RG-II from the mur1-1 Arabidopsis mutant, and the celery RG-II monomer lacking Arap and MeFuc of chain B and l-Gal of chain A. Our screen showed that AtFUT2, AtFUT5, and AtFUT10 have activity toward RG-I, while AtFUT8 was active on RG-II. Five rice OsFUTs have XyG-FUT activity and four rice OsFUTs have activity toward Ara3. None of the putative OsFUTs were active on the RG-I and RG-II. However, promiscuity toward acceptors was observed for several FUTs. These findings extend our knowledge of cell wall polysaccharide fucosylation in plants. We believe that in vitro GT-array platform provides a valuable tool for cell wall biochemistry and other research fields.

Divergent evolution of extreme production of variant plant monounsaturated fatty acids.

Metabolic extremes provide opportunities to understand enzymatic and metabolic plasticity and biotechnological tools for novel biomaterial production. We discovered that seed oils of many Thunbergia species contain up to 92% of the unusual monounsaturated petroselinic acid (18:1Δ6), one of the highest reported levels for a single fatty acid in plants. Supporting the biosynthetic origin of petroselinic acid, we identified a Δ6-stearoyl-acyl carrier protein (18:0-ACP) desaturase from Thunbergia laurifolia, closely related to a previously identified Δ6-palmitoyl-ACP desaturase that produces sapienic acid (16:1Δ6)-rich oils in Thunbergia alata seeds. Guided by a T. laurifolia desaturase crystal structure obtained in this study, enzyme mutagenesis identified key amino acids for functional divergence of Δ6 desaturases from the archetypal Δ9-18:0-ACP desaturase and mutations that result in nonnative enzyme regiospecificity. Furthermore, we demonstrate the utility of the T. laurifolia desaturase for the production of unusual monounsaturated fatty acids in engineered plant and bacterial hosts. Through stepwise metabolic engineering, we provide evidence that divergent evolution of extreme petroselinic acid and sapienic acid production arises from biosynthetic and metabolic functional specialization and enhanced expression of specific enzymes to accommodate metabolism of atypical substrates.

Phl p 5 levels more strongly associated than grass pollen counts with allergic respiratory health.

BACKGROUND: Studies have linked daily pollen counts to respiratory allergic health outcomes, but few have considered allergen levels. OBJECTIVE: We sought to assess associations of grass pollen counts and grass allergen levels (Phl p 5) with respiratory allergic health symptoms in a panel of 93 adults with moderate-severe allergic rhinitis and daily asthma hospital admissions in London, United Kingdom. METHODS: Daily symptom and medication scores were collected from adult participants in an allergy clinical trial. Daily counts of asthma hospital admissions in the London general population were obtained from Hospital Episode Statistics data. Daily grass pollen counts were measured using a volumetric air sampler, and novel Phl p 5 levels were measured using a ChemVol High Volume Cascade Impactor and ELISA analyses (May through August). Associations between the 2 pollen variables and daily health scores (dichotomized based on within-person 75th percentiles) were assessed using generalized estimating equation logistic models and with asthma hospital admissions using Poisson regression models. RESULTS: Daily pollen counts and Phl p 5 levels were each positively associated with reporting a high combined symptom and medication health score in separate models. However, in mutually adjusted models including terms for both pollen counts and Phl p 5 levels, associations remained for Phl p 5 levels (odds ratio [95% CI]: 1.18 [1.12, 1.24]), but were heavily attenuated for pollen counts (odds ratio [95% CI]: 1.00 [0.93, 1.07]). Similar trends were not observed for asthma hospital admissions in London. CONCLUSIONS: Grass allergen (Phl p 5) levels are more consistently associated with allergic respiratory symptoms than grass pollen counts.

Wheat-Based Glues in Conservation and Cultural Heritage: (Dis)solving the Proteome of Flour and Starch Pastes and Their Adhering Properties.

Plant-based adhesives, such as those made from wheat, have been prominently used for books and paper-based objects and are also used as conservation adhesives. Starch paste originates from starch granules, whereas flour paste encompasses the entire wheat endosperm proteome, offering strong adhesive properties due to gluten proteins. From a conservation perspective, understanding the precise nature of the adhesive is vital as the longevity, resilience, and reaction to environmental changes can differ substantially between starch- and flour-based pastes. We devised a proteomics method to discern the protein content of these pastes. Protocols involved extracting soluble proteins using 0.5 M NaCl and 30 mM Tris-HCl solutions and then targeting insoluble proteins, such as gliadins and glutenins, with a buffer containing 7 M urea, 2 M thiourea, 4% CHAPS, 40 mM Tris, and 75 mM DTT. Flour paste's proteome is diverse (1942 proteins across 759 groups), contrasting with starch paste's predominant starch-associated protein makeup (218 proteins in 58 groups). Transformation into pastes reduces proteomes' complexity. Testing on historical bookbindings confirmed the use of flour-based glue, which is rich in gluten and serpins. High levels of deamidation were detected, particularly for glutamine residues, which can impact the solubility and stability of the glue over time. The mass spectrometry proteomics data have been deposited to the ProteomeXchange, Consortium (http://proteomecentral.proteomexchange.org) via the MassIVE partner repository with the data set identifier MSV000093372 (ftp://MSV000093372@massive.ucsd.edu).

Ratiometric Fluorescence Aptasensor of Allergen Protein Based on Multivalent Aptamer-Encoded DNA Flowers as Fluorescence Resonance Energy Transfer Platform.

Life-threatening allergic reactions to food allergens, particularly peanut protein Ara h1, are a growing public health concern affecting millions of people worldwide. Thus, accurate and rapid detection is necessary for allergen labeling and dietary guidance and ultimately preventing allergic incidents. Herein, we present a novel ratiometric fluorescence aptasensor based on multivalent aptamer-encoded DNA flowers (Mul-DNFs) for the high-stability and sensitive detection of allergen Ara h1. The flower-shaped Mul-DNFs were spontaneously packaged using ultralong polymeric DNA amplicons driven by a rolling circle amplification reaction, which contains a large number of Ara h1 specific recognition units and has excellent binding properties. Furthermore, dual-color fluorescence-labeled Mul-DNFs probes were developed by hybridizing them with Cy3- and Cy5-labeled complementary DNA (cDNA) to serve as a ratiometric fluorescence aptasensor platform based on fluorescence resonance energy transfer. Benefiting from the combined merits of the extraordinary synergistic multivalent binding ability of Mul-DNFs, the excellent specificity of the aptamer, and the sensitivity of the ratiometric sensor to avoid exogenous interference. The developed ratiometric aptasensor showed excellent linearity (0.05-2000 ng mL-1) with a limit of detection of 0.02 ng mL-1. Additionally, the developed ratiometric fluorescence aptasensor was utilized for quantifying the presence of Ara h1 in milk, infant milk powder, cookies, bread, and chocolate with recoveries of 95.7-106.3%. The proposed ratiometric aptasensor is expected to be a prospective universal aptasensor platform for the rapid, sensitive, and accurate determination of food and environmental hazards.

Holy Grail possibly made from clay.

The main goal of the work was to find biochemical protein markers specific for grapes and wine in ancient amphorae shards and fermentation pools. Grape-specific proteins are more reliable markers than tartaric acid and other small organic acids (tartaric acid natural source are not only grape but also apple, mango, and other plants). The Yavne winery (located in the Central District of Israel) is stated to be the largest known wine production complex from the Byzantine period (ca. 1500 years ago). The site has been excavated recently, and a number of wine jar have been recovered. We have applied our ethylene vinyl acetate (EVA) (EVA studded with strong cation and anion exchangers) diskettes to the inner surface of a number of jars, thus capturing residual grape proteins therein. Via mass spectrometry analyses, we have been able to identify four grape and three yeast proteins. This has been possible because the EVA films, applied to such surfaces, are able to harvest and concentrate any trace species, rendering them amenable to instrumental analysis. Our analysis makes it possible to propose an explanation for the Holy Grail phenomenon as a dish in which wine or water begins to smell pleasant. We attribute this to the slow release of terpenes, aldehydes, and ketones from the clay walls of pottery. After digital modeling, we identified that "scallop-shaped" niches in winery were used for the condensation of high percentage alcohol by passive evaporation from fermentation tanks.

An ABHD17-like hydrolase screening system to identify de-S-acylation enzymes of protein substrates in plant cells.

Protein S-acylation is an important post-translational modification in eukaryotes, regulating the subcellular localization, trafficking, stability, and activity of substrate proteins. The dynamic regulation of this reversible modification is mediated inversely by protein S-acyltransferases and de-S-acylation enzymes, but the de-S-acylation mechanism remains unclear in plant cells. Here, we characterized a group of putative protein de-S-acylation enzymes in Arabidopsis thaliana, including 11 members of Alpha/Beta Hydrolase Domain-containing Protein 17-like acyl protein thioesterases (ABAPTs). A robust system was then established for the screening of de-S-acylation enzymes of protein substrates in plant cells, based on the effects of substrate localization and confirmed via the protein S-acylation levels. Using this system, the ABAPTs, which specifically reduced the S-acylation levels and disrupted the plasma membrane localization of five immunity-related proteins, were identified respectively in Arabidopsis. Further results indicated that the de-S-acylation of RPM1-Interacting Protein 4, which was mediated by ABAPT8, resulted in an increase of cell death in Arabidopsis and Nicotiana benthamiana, supporting the physiological role of the ABAPTs in plants. Collectively, our current work provides a powerful and reliable system to identify the pairs of plant protein substrates and de-S-acylation enzymes for further studies on the dynamic regulation of plant protein S-acylation.

A co-fractionation mass spectrometry-based prediction of protein complex assemblies in the developing rice aleurone-subaleurone.

Multiprotein complexes execute and coordinate diverse cellular processes such as organelle biogenesis, vesicle trafficking, cell signaling, and metabolism. Knowledge about their composition and localization provides useful clues about the mechanisms of cellular homeostasis and system-level control. This is of great biological importance and practical significance in heterotrophic rice (Oryza sativa) endosperm and aleurone-subaleurone tissues, which are a primary source of seed vitamins and stored energy. Dozens of protein complexes have been implicated in the synthesis, transport, and storage of seed proteins, lipids, vitamins, and minerals. Mutations in protein complexes that control RNA transport result in aberrant endosperm with shrunken and floury phenotypes, significantly reducing seed yield and quality. The purpose of this study was to broadly predict protein complex composition in the aleurone-subaleurone layers of developing rice seeds using co-fractionation mass spectrometry. Following orthogonal chromatographic separations of biological replicates, thousands of protein elution profiles were subjected to distance-based clustering to enable large-scale multimerization state measurements and protein complex predictions. The predicted complexes had predicted functions across diverse functional categories, including novel heteromeric RNA binding protein complexes that may influence seed quality. This effective and open-ended proteomics pipeline provides useful clues about system-level posttranslational control during the early stages of rice seed development.

The Arabidopsis PeptideAtlas: Harnessing worldwide proteomics data to create a comprehensive community proteomics resource.

We developed a resource, the Arabidopsis PeptideAtlas (www.peptideatlas.org/builds/arabidopsis/), to solve central questions about the Arabidopsis thaliana proteome, such as the significance of protein splice forms and post-translational modifications (PTMs), or simply to obtain reliable information about specific proteins. PeptideAtlas is based on published mass spectrometry (MS) data collected through ProteomeXchange and reanalyzed through a uniform processing and metadata annotation pipeline. All matched MS-derived peptide data are linked to spectral, technical, and biological metadata. Nearly 40 million out of ∼143 million MS/MS (tandem MS) spectra were matched to the reference genome Araport11, identifying ∼0.5 million unique peptides and 17,858 uniquely identified proteins (only isoform per gene) at the highest confidence level (false discovery rate 0.0004; 2 non-nested peptides ≥9 amino acid each), assigned canonical proteins, and 3,543 lower-confidence proteins. Physicochemical protein properties were evaluated for targeted identification of unobserved proteins. Additional proteins and isoforms currently not in Araport11 were identified that were generated from pseudogenes, alternative start, stops, and/or splice variants, and small Open Reading Frames; these features should be considered when updating the Arabidopsis genome. Phosphorylation can be inspected through a sophisticated PTM viewer. PeptideAtlas is integrated with community resources including TAIR, tracks in JBrowse, PPDB, and UniProtKB. Subsequent PeptideAtlas builds will incorporate millions more MS/MS data.

Contributions of the N-terminal flanking residues of an antigenic peptide from the Japanese cedar pollen allergen Cry j 1 to the T-cell activation by HLA-DP5.

Cry j 1 is a major allergen present in Japanese cedar (Cryptomeria japonica) pollens. Peptides with the core sequence of KVTVAFNQF from Cry j 1 ('pCj1') bind to HLA-DP5 and activate Th2 cells. In this study, we noticed that Ser and Lys at positions -2 and -3, respectively, in the N-terminal flanking (NF) region to pCj1 are conserved well in HLA-DP5-binding allergen peptides. A competitive binding assay showed that the double mutation of Ser(-2) and Lys(-3) to Glu [S(P-2)E/K(P-3)E] in a 13-residue Cry j 1 peptide (NF-pCj1) decreased its affinity for HLA-DP5 by about 2-fold. Similarly, this double mutation reduced, by about 2-fold, the amount of NF-pCj1 presented on the surface of mouse antigen-presenting dendritic cell line 1 (mDC1) cells stably expressing HLA-DP5. We established NF-pCj1-specific and HLA-DP5-restricted CD4+ T-cell clones from HLA-DP5 positive cedar pollinosis (CP) patients, and analyzed their IL-2 production due to the activation of mouse TG40 cells expressing the cloned T-cell receptor by the NF-pCj1-presenting mDC1 cells. The T-cell activation was actually decreased by the S(P-2)E/K(P-3)E mutation, corresponding to the reduction in the peptide presentation by this mutation. In contrast, the affinity of NF-pCj1·HLA-DP5 for the T-cell receptor was not affected by the S(P-2)E/K(P-3)E mutation, as analyzed by surface plasmon resonance. Considering the positional and side-chain differences of these NF residues from previously reported T-cell activating sequences, the mechanisms of enhanced T-cell activation by Ser(-2) and Lys(-3) of NF-pCj1 may be novel.

Small-scale integrated farming systems can abate continental-scale nutrient leakage.

Beef is the most resource intensive of all commonly used food items. Disproportionate synthetic fertilizer use during beef production propels a vigorous one-way factory-to-ocean nutrient flux, which alternative agriculture models strive to rectify by enhancing in-farm biogeochemical cycling. Livestock, especially cattle, are central to these models, which advocates describe as the context most likely to overcome beef's environmental liabilities. Yet the dietary potential of such models is currently poorly known. Here, I thus ask whether nitrogen-sparing agriculture (NSA) can offer a viable alternative to the current US food system. Focusing on the most common eutrophication-causing element, N, I devise a specific model of mixed-use NSA comprising numerous small farms producing human plant-based food and forage, the latter feeding a core intensive beef operation that forgoes synthetic fertilizer and relies only on locally produced manure and N fixers. Assuming the model is deployed throughout the high-quality, precipitation-rich US cropland (delimiting approximately 100 million ha, less than half of today's agricultural land use) and neglecting potential macroeconomic obstacles to wide deployment, I find that NSA could produce a diverse, high-quality nationwide diet distinctly better than today's mean US diet. The model also permits 70%-80% of today's beef consumption, raises today's protein delivery by 5%-40%, and averts approximately 60% of today's fertilizer use and approximately 10% of today's total greenhouse gas emissions. As defined here, NSA is thus potentially a viable, scalable environmentally superior alternative to the current US food system, but only when combined with the commitment to substantially enhance our reliance on plant food.

A comprehensive review on pulse protein fractionation and extraction: processes, functionality, and food applications.

The increasing world population requires the production of nutrient-rich foods. Protein is an essential macronutrient for healthy individuals. Interest in using plant proteins in foods has increased in recent years due to their sustainability and nutritional benefits. Dry and wet protein fractionation methods have been developed to increase protein yield, purity, and functional and nutritional qualities. This review explores the recent developments in pretreatments and fractionation processes used for producing pulse protein concentrates and isolates. Functionality differences between pulse proteins obtained from different fractionation methods and the use of fractionated pulse proteins in different food applications are also critically reviewed. Pretreatment methods improve the de-hulling efficiency of seeds prior to fractionation. Research on wet fractionation methods focuses on improving sustainability and functionality of proteins while studies on dry methods focus on increasing protein yield and purity. Hybrid methods produced fractionated proteins with higher yield and purity while also improving protein functionality and process sustainability. Dry and hybrid fractionated proteins have comparable or superior functionalities relative to wet fractionated proteins. Pulse protein ingredients are successfully incorporated into various food formulations with notable changes in their sensory properties. Future studies could focus on optimizing the fractionation process, improving protein concentrate palatability, and optimizing formulations using pulse proteins.

Determination of nut varieties and their detection in festive cookies by MALDI-TOF mass spectrometry.

RATIONALE: Nuts contain a large amount of essential fatty acids, amino acids, and whole range of minerals and vitamins valuable for human health, yet certain risks are associated with their consumption, of which allergic reaction is the most important. Considering the growing number of people suffering from allergies caused by allergens of protein origin, the aim of this work is to find out whether nuts can be distinguished from each other on the basis of contained proteins. METHODS: A total of eleven raw and subsequently heat-treated nuts (almonds, Brazil nuts, cashews, coconuts, hazelnuts, macadamia nuts, peanuts, pecans, pine nuts, pistachios, and walnuts) were analyzed using MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry with the subsequent finding of characteristic m/z values for each analyzed nut. No previous method for protein extraction was used. RESULTS: The characteristic values were used to verify the composition of seven types of festive cookies - six commercial products and one "unknown" cookie, where it was not known in advance, which nut it was made from. CONCLUSIONS: The procedure, together with the found characteristic m/z values, could serve to rapidly identify the plant origin of nut products.

Evaluation of PAC and FASP Performance: DIA-Based Quantitative Proteomic Analysis.

The aim of this study was to compare filter-aided sample preparation (FASP) and protein aggregation capture (PAC) starting from a three-species protein mix (Human, Soybean and Pisum sativum) and two different starting amounts (1 and 10 µg). Peptide mixtures were analyzed by data-independent acquisition (DIA) and raw files were processed by three commonly used software: Spectronaut, MaxDIA and DIA-NN. Overall, the highest number of proteins (mean value of 5491) were identified by PAC (10 µg), while the lowest number (4855) was identified by FASP (1 µg). The latter experiment displayed the worst performance in terms of both specificity (0.73) and precision (0.24). Other tested conditions showed better diagnostic accuracy, with specificity values of 0.95-0.99 and precision values between 0.61 and 0.86. In order to provide guidance on the data analysis pipeline, the accuracy diagnostic of three software was investigated: (i) the highest sensitivity was obtained with Spectronaut (median of 0.67) highlighting the ability of Spectronaut to quantify low-abundance proteins, (ii) the best precision value was obtained by MaxDIA (median of 0.84), but with a reduced number of identifications compared to Spectronaut and DIA-NN data, and (iii) the specificity values were similar (between 0.93 and 0.99). The data are available on ProteomeXchange with the identifier PXD044349.

New Pipeline for Analysing Fruit Proteolytic Products Used as Digestive Health Nutraceuticals.

Proteolytic products are extensively used in the nutraceutical sector to improve protein digestion and muscle quality in target populations (e.g., athletes or elderly). These products are processed using techniques that often lead to low purity but competitive pricing. Despite their widespread use and well-established production methods, the industry lacks standardized analytical methods for assessing these products and detecting potential fraud. This study proposes a comprehensive and harmonized pipeline for their analysis, which includes quantifying total soluble protein and proteolytic activity, as well as the determination of product stability and protein profile using SDS-PAGE and proteomic techniques. Despite the fact that protease extracts from pineapple had the highest protein content, most of the bromelain remained inactive, unlike in kiwi and papaya. SDS-PAGE revealed partial protein degradation of pineapple extracts, whereas kiwi extracts reflected a lower purification level but a higher protein integrity. The application of proteomic approaches strengthened the identification and origin tracing of the proteases. This study contributes to the development of a robust framework for analyzing proteolytic extracts, spanning from soluble protein quantification to protein profiling and activity determination. It may also ensure reliable supplier selection, high-quality manufacturing practices, and the implementation of optimal storage and formulation strategies in the nutraceutical industry.

Rapid Tryptophan Assay as a Screening Procedure for Quality Protein Maize.

Tryptophan is an essential amino acid deficient in cereals, especially maize. However, maize (Zea mays L.) is the main source of protein in some developing countries in Africa and Latin America. In general, the nutritional profile of cereals is poor, because they are deficient in essential amino acids such as tryptophan and lysine due to a relatively higher proportion of alcohol-soluble proteins. Quality protein maize (QPM) has been developed through genetic manipulation for the nutritional enrichment of maize to address these problems. Nevertheless, methods for protein, lysine and tryptophan are time-consuming and require relatively large amounts of samples. Therefore, we have advanced here a simple, cheap, fast, reliable and robust procedure for the determination of protein and tryptophan in the same biuret supernatant, which can also be used for chemical characterization of other cereals. Samples of 50 mg maize ground to pass through a 0.1 mm screen were sonicated for 5 min. in eppendorf vials with 1.5 mL of a biuret reagent each. After centrifugation and protein determination by biuret, 0.2 mL of supernatant was treated with 0.8 mL of a tryptophan reagent. Both total protein and tryptophan can be determined in microplates at 560 nm to speed up the measurements. The main advantage of the new micro-method is the rapid estimation of the nutrient quality of maize samples by a single weighing of a small amount of valuable plant materials.

Comprehensive Mapping of Cyclotides from Viola philippica by Using Mass Spectrometry-Based Strategy.

Cyclotides are plant cyclic peptides with exceptional stability and diverse bioactivity, making them promising candidates for biomedical applications. Therefore, the study of cyclotides has attracted increasing attention in recent years. However, the existing cyclotide detection methods face limitations in sensitivity, accuracy, and reliability. To address these challenges, we developed an integrated strategy using a combination of strong cation exchange chromatography techniques for removing interfering small molecules, Orbitrap Exploris 480 mass spectrometry (OEMS); this is a detection and database searching-based method for cyclotide verification, which greatly improved the sensitivity, accuracy, and reliability of cyclotide identification. This strategy was subsequently employed for cyclotide mapping in Viola with a minute amount of starting tissue, resulting the identification of 65 known and 18 potentially novel cyclotides, which is the largest dataset of cyclotides for Viola philippica. This strategy provided valuable insights into the cyclotide diversity and distribution in V. philippica, with potential applications in drug discovery and other biomedical fields.

Chemical Composition and Nutritive Value of Sea Buckthorn Leaves.

Sea buckthorn leaves (SBT_LVs) form notable by-product during harvesting and post-harvest management of the berries. It is already known that sea buckthorn berries are important for their chemical composition and based on this, they occupy a wide field in nutrition. SBT_LVs also have a rich chemical composition, like the berries. The aim of this study was to describe these by-products in the context of protein and complex carbohydrates-dietary fiber fractions, including qualitative and quantitative composition of amino acids. Proximate composition, amino acids, nutritional values of the protein, and dietary fiber fractions of SBT_LVs of four cultivars (cvs.) Ascola, Habego, Hergo, and Leikora were assessed. SBT_LVs from different years of the study had statistically different levels of crude protein, ether extract, crude ash, and nitrogen-free extract (NFE), confirming that the quality of the raw material (leaves) can be significantly modified by habitat conditions. The largest fraction of dietary fiber was neutral detergent fiber (NDF), including the sum of hemicellulose, cellulose, and lignin, followed by the acid detergent fiber fraction (ADF), consisting of lignin and cellulose. The content of essential amino acids in SBT_LV protein was high. Overall, this study confirms that SBT_LVs hold promise as a valuable resource for use as a food ingredient, functional food, and dietary supplement for both humans and animals.

Evaluation of Alternative Sources of Proteins and Other Nutrients with Potential Applications in Fish Nutrition.

The European Union's (EU) agricultural self-sufficiency is challenged by its reliance on imported plant proteins, particularly soy from the Americas, contributing to deforestation and greenhouse gas emissions. Addressing the EU's protein deficit, this study evaluates alternative protein sources for aquaculture, focusing on their nutritional value, elemental content, and polycyclic aromatic hydrocarbons (PAHs). Protein flours from gastropods (Helix pomatia, Arion lusitanicus, Arion vulgaris) and their hepatopancreas, along with plant-based proteins from food industry by-products (oilcakes, coffee grounds, spent brewer's yeast), were analyzed. Results revealed that snail flour contained the highest protein content at 59.09%, significantly outperforming hepatopancreas flour at 42.26%. Plant-based proteins demonstrated substantial nutritional value, with coffee grounds flour exhibiting a remarkable protein content of 71.8% and spent brewer's yeast flour at 57.9%. Elemental analysis indicated high levels of essential minerals such as magnesium in hepatopancreas flour (5719.10 mg/kg) and calcium in slug flour (48,640.11 mg/kg). However, cadmium levels in hepatopancreas flour (11.45 mg/kg) necessitate caution due to potential health risks. PAH concentrations were low across all samples, with the highest total PAH content observed in hepatopancreas flour at 0.0353 µg/kg, suggesting minimal risk of PAH-related toxicity. The analysis of plant-based protein sources, particularly oilcakes derived from sunflower, hemp, flax, and pumpkin seeds, revealed that these by-products not only exhibit high protein contents but present a promising avenue for enhancing the nutritional quality of feed. This study underscores the potential of utilizing gastropod and plant-based by-products as sustainable and nutritionally adequate alternatives to conventional feeds in aquaculture, contributing to the EU's environmental sustainability goals.

Characteristics of Selected Bioactive Compounds and Malting Parameters of Hemp (Cannabis sativa L.) Seeds and Malt.

Hemp (Cannabis sativa L.) seeds are an interesting raw material for malting regarding its relatively high bioactive compounds concentration and proven advantageous properties in different food products and dietary supplements. In the first stage of the study, important seeds properties relevant to the malting process including moisture content, seed viability, and water absorption capacity were determined. However, a few parameters determining the seeds' usability for malt preparation, such as germination ability and water sensitivity, are different in comparison to typical malting raw materials such as barley or wheat. However, they make it possible to obtain high-quality hemp malt. In the next stage of research, spectroscopic and chromatographic analyses, including measurements of antioxidant activity and protein separation by SEC-HPLC, were conducted. The results showed that the malting process improved the total antioxidant potential of hemp seeds by 15%, leading to an increase in the concentration of lower molecular weight proteins and oligopeptides-below molecular mass of 10 kDa-responsible for this high antioxidant activity. The processing of hemp seeds reduced the phytate content while increasing phosphate fractions with fewer phosphate groups, which may have a beneficial effect on nutritional value. These results suggest that malting hemp seeds needs optimalization of the process but can increase its nutritional value as a promising raw material in the food industry.