Time maters: Exploring the dynamics of bioactive compounds content, bioaccessibility and antioxidant activity during Lupinus angustifolius germination.

Germination is a process that enhances the content of health-promoting secondary metabolites. However, the bioaccessibility of these compounds depends on their stability and solubility throughout the gastrointestinal tract. The study aimed to explore how germination time influences the content and bioaccessibility of γ-aminobutyric acid and polyphenols and antioxidant capacity of lupin (Lupinus angustifolius L.) sprouts during simulated gastrointestinal digestion. Gamma-aminobutyric acid showed a decrease following gastrointestinal digestion (GID) whereas phenolic acids and flavonoids exhibited bioaccessibilities of up to 82.56 and 114.20%, respectively. Although the digestion process affected the profile of phenolic acids and flavonoids, certain isoflavonoids identified in 7-day sprouts (G7) showed resistance to GID. Germination not only favored antioxidant activity but also resulted in germinated samples exhibiting greater antioxidant properties than ungerminated counter parts after GID. Intestinal digests from G7 did not show cytotoxicity in RAW 264.7 macrophages, and notably, they showed an outstanding ability to inhibit the production of reactive oxygen species. This suggests potential benefit in mitigating oxidative stress. These findings contribute to understand the dynamic interplay between bioprocessing and digestion in modulating the bioaccessibility of bioactive compounds in lupin, thereby impacting health.

Consequences of pollen defense compounds for pollinators and antagonists in a pollen-rewarding plant.

Plants produce an array of defensive compounds with toxic or deterrent effects on insect herbivores. Pollen can contain relatively high concentrations of such defense compounds, but the causes and consequences of this enigmatic phenomenon remain mostly unknown. These compounds could potentially protect pollen against antagonists but could also reduce flower attractiveness to pollinators. We combined field observations of the pollen-rewarding Lupinus argenteus with chemical analysis and laboratory assays to test three hypotheses for the presence of pollen defense compounds: (1) these compounds are the result of spillover from adjacent tissues, (2) they protect against pollen thieves, and (3) they act as antimicrobial compounds. We also tested whether pollen defense compounds affect pollinator behavior. We found a positive relationship between alkaloid concentrations in pollen and petals, supporting the idea that pollen defense compounds partly originate from spillover. However, pollen and petals exhibited quantitatively (but not qualitatively) distinct alkaloid profiles, suggesting that plants can adjust pollen alkaloid composition independently from that of adjacent tissues. We found no relationship between pollen alkaloid concentration and the abundance of pollen thieves in Lupinus flowers. However, pollen alkaloids were negatively associated with bacterial abundance. Finally, plants with more alkaloids in their pollen received more pollinator visits, but these visits were shorter, resulting in no change in the overall number of flowers visited. We propose that pollen defense compounds are partly the result of spillover from other tissues, while they also play an antimicrobial role. The absence of negative effects of these compounds on pollinator visitation likely allows their maintenance in pollen at relatively high concentrations. Taken together, our results suggest that pollen alkaloids affect and are mediated by the interplay of multiple interactions.

Quantitation of Lupinus spp. Quinolizidine Alkaloids by qNMR and Accelerated Debittering with a Resin-Based Protocol.

Quinolizidine alkaloids (QAs) are toxic secondary metabolites of the Lupinus species, the presence of which limits the expansion of lupin beans consumption, despite their high protein content. Evaluation of the level of alkaloids in edible Lupinus species is crucial from a food safety point of view. However, quantitation of QAs is complicated by the fact that not all important alkaloids used for quantitation are commercially available. In this context, we developed a method for the simultaneous quantitation of eight major lupin alkaloids using quantitative NMR spectroscopy (qNMR). Quantitation and analysis were performed in 15 different seed extracts of 11 Lupinus spp. some of which belonged to the same species, with different geographical origins and time of harvest, as well as in all aerial parts of L. pilosus. The mature seeds of L. pilosus were found to be a uniquely rich source of multiflorine. Additionally, we developed a protocol using adsorption or ionic resins for easy, fast, and efficient debittering of the lupine seeds. The protocol was applied to L. albus, leading to a decrease of the time required for alkaloids removal as well as water consumption and to a method for QA isolation from the debittering wastewater.

Effect of different oligomerization assemblies of γ-conglutin on its interaction behavior with vitexin.

BACKGROUND: Several different factors underlie the molecular mechanisms of phenolic compound-protein interactions. They include the environmental conditions. In the case of γ-conglutin, pH conditions translate directly into the adoption of two distinct oligomeric assemblies, i.e. hexameric (pH 7.5) or monomeric (pH 4.5). This paper reports research on the pH-dependent oligomerization of γ-conglutin in terms of its ability to form complexes with a model flavonoid (vitexin). RESULTS: Fluorescence-quenching thermodynamic measurements indicate that hydrogen bonds, electrostatic forces, and van der Waals interactions are the main driving forces involved in the complex formation. The interaction turned out to be a spontaneous and exothermic process. Assessment of structural composition (secondary structure changes and arrangement/dynamics of aromatic amino acids), molecular size, and the thermal stability of the different oligomeric forms showed that γ-conglutin in a monomeric state was less affected by vitexin during the interaction. CONCLUSION: The data show precisely how environmental conditions might influence phenolic compound-protein complex formation directly. This knowledge is essential for the preparation of food products containing γ-conglutin. The results can contribute to a better understanding of the detailed fate of this unique health-promoting lupin seed protein after its intake. © 2023 Society of Chemical Industry.

A not-glycosylated isoform of γ-conglutin, a hexameric glycoprotein of Lupinus albus seed, participates in the oligomerization equilibrium.

γ-conglutin (γ-C) is a hexameric glycoprotein accumulated in lupin seeds and has long been considered as a storage protein. Recently, it has been investigated for its possible postprandial glycaemic regulating action in human nutrition and for its physiological role in plant defence. The quaternary structure of γ-C results from the assembly of six monomers in reversible pH-dependent association/dissociation equilibrium. Our working hypothesis was that the γ-C hexamer is made up of glycosylated subunits in association with not-glycosylated isoforms, that seem to have 'escaped' the correct glycosylation process in the Golgi. Here we describe the isolation of not-glycosylated γ-C monomers in native condition by two in tandem lectin-based affinity chromatography and the characterization of their oligomerization capacity. We report, for the first time, the observation that a plant multimeric protein may be formed by identical polypeptide chains that have undergone different post-translational modifications. All obtained considered, the results strongly suggest that the not-glycosylated isoform can also take part in the oligomerization equilibrium of the protein.

Chemical and biological characterization of the DPP-IV inhibitory activity exerted by lupin (Lupinus angustifolius) peptides: From the bench to the bedside investigation.

Dipeptidyl peptidase IV (DPP-IV) is considered a key target for the diabetes treatment, since it is involved in glucose metabolism. Although lupin protein consumption shown hypoglycemic activity, there is no evidence of its effect on DPP-IV activity. This study demonstrates that a lupin protein hydrolysate (LPH), obtained by hydrolysis with Alcalase, exerts anti-diabetic activity by modulating DPP-IV activity. In fact, LPH decreased DPP-IV activity in a cell-free and cell-based system. Contextually, Caco-2 cells were employed to identify LPH peptides that can be intestinally trans-epithelial transported. Notably, 141 different intestinally transported LPH sequences were identified using nano- and ultra-chromatography coupled to mass spectrometry. Hence, it was demonstrated that LPH modulated the glycemic response and the glucose concentration in mice, by inhibiting the DPP-IV. Finally, a beverage containing 1 g of LPH decreased DPP-IV activity and glucose levels in humans.

Conventional solid-state fermentation impacts the white lupin proteome reducing the abundance of allergenic peptides.

The demand for high-quality and sustainable protein sources is on the rise. Lupin is an emerging plant-based source of protein with health-enhancing properties; however, the allergenic potential of lupins limits their widespread adoption in food products. A combination of discovery and targeted quantitative proteome measurements was used to investigate the impact of solid-state fermentation induced by Rhizopus oligosporus on the proteome composition and allergenic protein abundances of white lupin seed. In total, 1,241 proteins were uniquely identified in the fermented sample. Moreover, the effectiveness of the solid-state fermentation in reducing the abundance of the tryptic peptides derived from white lupin allergens was demonstrated. Comparably, a greater decrease was noted for the major white lupin allergen based on ß-conglutin peptide abundances. Hence, conventional solid-state fermentation processing can be beneficial for reducing the potential allergenicity of lupin-based foods. This finding will open new avenues for unlocking the potential of this under-utilised legume.

Characteristics of N-Glycosylation and Its Impact on the Molecular Behavior of Lupinus angustifolius γ-Conglutin.

γ-Conglutin, a lupin seed protein, is an intriguing protein both in terms of the complexity of its molecular structure and a broad spectrum of unique health-promoting properties manifested in animal and human trials. Moreover, this protein is an evolutionary cornerstone whose physiological significance for the plant has not been determined yet. Herein, a comprehensive characterization of γ-conglutin glycosylation is presented and includes (i) the identification of the N-glycan-bearing site, (ii) the qualitative and quantitative composition of glycan-building saccharides, as well as (iii) the effect of oligosaccharide removal on structural and thermal stability. The obtained results indicate the presence of glycans belonging to different classes attached to the Asn98 residue. In addition, the detachment of the oligosaccharide significantly affects secondary structure composition, which disturbs the oligomerization process. The structural changes were also reflected in biophysical parameters, i.e., at a pH value of 4.5, an increase in γ-conglutin thermal stability was observed for the deglycosylated monomeric form. Collectively, the presented results provide evidence of the high complexity of the post-translational maturation and suggest the possibility of a functional effect that glycosylation might have on γ-conglutin structure integrity.

Impact of Germination on the Protein Solubility and Antinutritive Compounds of Lupinus angustifolius and Vicia faba in the Production of Protein-Rich Legume-Based Beverages.

Lupines and faba beans are protein-rich legumes, which can be utilized as a plant-based substitute for animal proteins in human nutrition in general and in the beverage industry in particular. However, their application is hampered by low protein solubility in the acidic pH range and by antinutrients such as flatulence-causing "raffinose family oligosaccharides" (RFOs). Germination is known in the brewing industry for increasing enzymatic activities and mobilizing storage compounds. Therefore, germinations of lupines and faba beans were performed at different temperatures, and their impact on protein solubility, the concentration of free amino acids, and degradation of RFOs, alkaloids, and phytic acid was evaluated. In general, changes were comparable for both legumes but less pronounced in faba beans. Germination depleted the RFOs entirely in both legumes. The protein size distribution was found to be shifted toward smaller fractions, the concentrations of free amino acids multiplied, and protein solubility increased. No substantial reductions in the binding capacity of phytic acid toward iron ions were observed, but a release of free phosphate in lupines was detected. The results prove that germination is an applicable process for refining lupines and faba beans for use not only in refreshing beverages or milk alternatives but also in other food applications.

Impact of environmental factors on the presence of quinolizidine alkaloids in lupins: a review.

Lupin seeds have a high potential as an alternative for animal proteins in feed and food. However, the possible presence of alkaloids hinders the usage of lupins in human diets. This review aims to identify the main factors that influence the presence of alkaloids in lupins. A literature study covering English-published scientific papers in Scopus from 1980 to 2022 was performed. Biotic, abiotic, and genotypic factors influence the production of these toxic secondary metabolites by lupines. In particular, sweet cultivars with high 13-hydroxylupanine and 13-tigloyloxylupanine concentrations, abundant light exposure and standard diurnal cycles, well-watering procedures, relatively cold environment, N-deficient fertilizer with 240 mg K kg-1 and 60 mg P kg-1, high soil pH, and organic growing system conditions, are the best options to avoid high global alkaloid content. Results of this study can be used to develop predictive mechanistic models, although there is still the necessity to collect additional data by performing multi-variate studies.

Nutritional composition and biological activity of narrow-leafed lupins (Lupinus angustifolius L.) hydrolysates and seeds.

Lupins are an interesting source of nutrients, part of the Fabaceae family. More specifically, narrow-leafed lupin (Lupinus angustifolius L.) is a legume, largely produced in Australia, which is used both for human food and animal fodder. There is a growing interest in plant proteins-derived products due to benefits for the ecosystem and lower production costs compared to traditional animal sources of protein. This review aimed to summarize major and minor chemical components in Lupinus angustifolius L., and potential health benefits of this plant and product thereof. In particular, the protein fraction of Lupinus and their biological properties are described. L. angustifolius seed and proteins by-products can be used as a valuable source of high value-added compounds for diverse food products with the goal to maximize its economic value.

Ultrasensitive determination of ß-conglutin food allergen by means an aptamer assay based on inductively coupled plasma mass spectrometry detection.

The overall objective of this work is the evaluation of different competitive aptamer assays based on inductively coupled plasma mass spectrometry (ICP-MS) detection for the determination of ß-conglutin (food protein allergen from lupin) in flour samples. To this end, two competitive aptamer assay schemes were developed using either thiolated aptamers chemisorbed onto gold nanoparticles (AuNPs) or biotinylated aptamers linked to streptavidin-AuNPs. The influence of ICP-MS detection mode (i.e., conventional vs single particle) on assay performance was explored. In the case of the thiolated aptamer, the limit of detection (LoD) obtained using the single particle mode was improved 2-fold as compared to the LoD provided by the conventional mode. With regards to the biotinylated aptamer, the use of the conventional mode provided a 5-fold improvement of LoD as compared to that obtained for the single particle one. Using the optimized conditions, the best LoD of 2 pM was obtained with the biotinylated aptamer operating with conventional ICP-MS detection. When compared to previous reports using the same aptamer in a competitive assay, the developed method significantly improved the LoD by at least an order of magnitude. Different flour samples containing lupin were successfully analyzed according to European Conformity guidelines for the analysis of food contaminants.

Development of an HPLC-MS/MS Method for the Determination of Alkaloids in Lupins.

Lupin alkaloids (LAs) represent a class of toxic secondary metabolites in plants, in particular in Lupinus spp.; they are produced as a defense mechanism due to their strong bitter taste and are very dangerous for human and animals. In this work, a sensitive and reliable high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analytical method for the identification and quantification of thirteen lupin alkaloids was developed and validated according to FDA guidelines. Efficient extraction and clean-up steps, carried out by solid-phase extraction, were finely tuned on the basis of the characteristics of the analytes and lupin samples, providing good selectivity with minimized matrix interference. The effectiveness of the method was proven by the satisfactory recovery values obtained for most of the analytes and a matrix effect ≤23% for all tested levels. In addition, a sensitive and reliable determination of the target compounds was obtained; LOQs were between 1 and 25 µg Kg-1, i.e., below the requested maximum levels (<200 mg Kg-1). The method was applied to evaluate the LAs profile in different batches of raw L. albus L. samples, varying in size and across farming treatments.

Narrow Leafed Lupin (Lupinus angustifolius L.) ß-Conglutin Seed Proteins as a New Natural Cytotoxic Agents against Breast Cancer Cells.

Breast cancer (BC) is the most widespread tumor in women and the second type of most common cancer worldwide. Despite all the technical and medical advances in existing therapies, between 30 and 50% of patients with BC will develop metastasis, which contributes to the failure of existing treatments. This situation urges the need to find more effective prevention and treatment strategies like the use of plant-based nutraceutical compounds. In this context, we purified three Narrow Leafed Lupin (NLL) ß-conglutins isoforms using affinity-chromatography and evaluated their effectiveness in terms of viability, proliferation, apoptosis, stemness properties, and mechanism of action on both BC cell lines and a healthy one. NLL ß-conglutins proteins have very promising effects at the molecular level on BC cells at very low concentrations, emerging as a potential natural cytotoxic agent and preserving the viability of healthy cells. These proteins could act through a dual mechanism involving tumorigenic and stemness-related genes such as SIRT1 and FoxO1, depending on the state of p53. More studies must be carried out to completely understand the underlying mechanisms of action of these nutraceutical compounds in BC in vitro and in vivo, and their potential use for the inhibition of other cancer cell types.

Chemical Characterization of White Lupin (Lupinus albus) Flour Treated by Extrusion Cooking and Aqueous Debittering Processes.

Lupin is a very nutritious legume with high levels of protein and fiber, but it also contains quinolizidine alkaloids which, depending on the species, can accumulate to toxic levels. The objective of this work was to evaluate the white lupin chemical composition, due to the effects of different processes (aqueous debittering, extrusion cooking, and reactive extrusion), aiming at reducing total alkaloids, preserving fibers, and increasing in vitro protein digestibility. Regarding raw material, the aqueous process reduced significantly total alkaloids (-93.87%), increased dietary fiber (+22.03%), and increased protein digestibility (+6.73%), whereas the extrusion processes were inefficient to reduce alkaloids (< -3.70%) and reduced the dietary fiber content, the reduction being more severe during reactive extrusion (-75.36%). Protein digestibility was improved by extrusion cooking (+3.07%), while the reactive extrusion reduced digestibility (-12.50%). Electrophoresis and quantification of soluble proteins and aromatic amino acids confirmed the high digestibility index, staying only the γ-conglutin fraction in the digested samples evaluated by SDS-PAGE. The aqueous process proved to be the best option, as it reduces the alkaloid content to safe levels and improves the protein digestion of white lupin flour.

Source-Supported Suspect Screening (4S) of Phytotoxins in Terrestrial and Aquatic Environments: A Field Study of Lupinus angustifolius L. (Blue Lupin).

Phytotoxins (PTs) are bioactive secondary metabolites produced by plants. More recently, they have been recognized as important aquatic micropollutants. Despite that, only a few PTs have been detected and reported in terrestrial and aquatic environments, while their source and leaching pathways remain largely unclear. Herein, we established a novel approach named source-supported suspect screening (4S) to discover PTs in different environments, investigate their environmental occurrences, identify their sources, and initiate discussions on their leaching mechanisms. The 4S-approach was demonstrated on a five-month Lupinus angustifolius L. (L. angustifolius) crop field experiment, where plant, topsoil, drainage water, and surface water were sampled and analyzed. As a result, 72 PTs (flavonoids and alkaloids) were identified at high confidence, with 10 PTs fully confirmed. Fifty-three PTs detected in soil or water were linked to L. angustifolius, among which 26 PTs were coherently detected in all three environmental compartments. The occurrence and abundance of PTs in terrestrial soil and aquatic environments were influenced by the plant growth stage and precipitation. Soil served as an intermedium when PTs leached from L. angustifolius to the drainage water, while the degree of retardation and eventual occurrence in the aquatic environment depended on both PTs and soil physico-chemical properties.

Bioactive Peptides from Lupin (Lupinus angustifolius) Prevent the Early Stages of Atherosclerosis in Western Diet-Fed ApoE-/- Mice.

We have previously reported the in vitro hypocholesterolemic, anti-inflammatory, and antioxidant effects of Alcalase-generated lupin protein hydrolysate (LPH). Given that lipoprotein deposition, oxidative stress, and inflammation are the main components of atherogenesis, we characterized the LPH composition, in silico identified LPH-peptides with activities related to atherosclerosis, and evaluated the in vivo LPH effects on atherosclerosis risk factors in a mouse model of atherosclerosis. After 15 min of Alcalase hydrolysis, peptides smaller than 8 kDa were obtained, and 259 peptides out of 278 peptides found showed biological activities related to atherosclerosis risk factors. Furthermore, LPH administration for 12 weeks reduced the plasma lipids, as well as the cardiovascular and atherogenic risk indexes. LPH also increased the total antioxidant capacity, decreased endothelial permeability, inflammatory response, and atherogenic markers. Therefore, this study describes for the first time that LPH prevents the early stages of atherosclerosis.

Structural Characterization of Dietary Fiber from Different Lupin Species (Lupinus sp.).

Dietary fiber fractions of whole seeds from different lupin species were structurally characterized. The low-molecular-weight soluble dietary fiber fraction contains mainly stachyose and verbascose. The soluble dietary fiber fraction is dominated by homogalacturonan and rhamnogalacturonan type I (RGI), with (arabino-)galactans and to a lesser portion arabinans as neutral RGI side chains. Arabinans are preferentially branched in position O2 as demonstrated by methylation analysis and an arabinan profiling approach. Insoluble dietary fiber is mainly composed of cellulose and pectins, but xylans and xyloglucans are present, too. Application of an enzymatic xyloglucan profiling approach demonstrated a substitution degree of 75% and proved the existence of fucosylated xyloglucans. Lignin of all lupin species was analyzed as being rich in guaiacyl units; however, the degree of lignification is low. Alcohol-insoluble residue polysaccharides from both seed coat and embryo/endosperm were analyzed separately, demonstrating tissue-related differences in the portions of cellulose and RGI.

Composition and Antifungal Activity of the Alkaloidal Fraction of Lupinus mirabilis Leaves: A Biochemometrics-Based Exploration.

Lupinus plants are well-recognized due to their significant alkaloid content, which has made them the subject of several studies. However, the lack of chemical and biological information on the Colombian Lupinus species remains a fact. Therefore, the alkaloidal fractions from the leaves of L. mirabilis obtained by conventional solvent and ultrasound-assisted extraction (CSE and UAE, respectively) at different time frames were analyzed. Sparteine (2) was the main component in all cases; however, its relative abundance showed large variability, ranging from 64.7% to 80.6%. Minor constituents were also affected by the extraction conditions. In general, prolonged times gave a higher proportion of alkaloids under CSE, while only a slight decrease was observed under UAE. Both the method and extraction time appeared to equally affect the ratios of particular alkaloids, leading to variations in their effect on the mycelial growth of Fusarium oxysporum. Holistic analysis through multiple-covariate statistical methods as an approach to integrating chemical and bioactivity datasets allowed inferring the compounds most likely responsible for the changes in mycelial growth inhibition. 13α-Hydroxylupanine (12) might represent a promising compound to be included in further studies against this phytopathogen.

Effect of substrate properties and phosphorus supply on facilitating the uptake of rare earth elements (REE) in mixed culture cropping systems of Hordeum vulgare, Lupinus albus and Lupinus angustifolius.

This study presents how phosphate (P) availability and intercropping may influence the migration of rare earth elements (REEs) in legume-grass associations. In a replacement model, Hordeum vulgare was intercropped with 11% Lupinus albus and 11% Lupinus angustifolius. They were cultivated on two substrates, A (pH = 7.8) and B (pH = 6.6), and treated with 1.5 g P m-2 or 3 g P m-2. Simultaneously, a greenhouse experiment was conducted to quantify carboxylate release. There, one group of L. albus and L. angustifolius was supplied with either 200 µmol L-1 P or 20 µmol L-1 P. L. albus released higher amounts of carboxylates at low P supply than L. angustifolius, while L. angustifolius showed the opposite response. Plants cultivated on substrate B accumulated substantially higher amounts of nutrients and REE, compared to substrate A. Higher P supply did not influence the leaf and stem P concentrations of H. vulgare. Addition of P decreased REE accumulation in barley monocultures on alkaline soil A. However, when H. vulgare was cultivated in mixed culture with L. angustifolius on alkaline substrate A with high P supply, the accumulation of REE in H. vulgare significantly increased. Conversely, on acidic substrate B, intercropping with L. albus decreased REE accumulation in H. vulgare. Our findings suggest a predominant effect of soil properties on the soil-plant transfer of REEs. However, in plant communities and within a certain soil environment, interspecific root interactions determined by species-specific strategies related to P acquisition in concert with the plant's nutrient supply impact REE fluxes between neighbouring plants.